Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02197673 2002-04-22
1
DRUGS TO IMPROyE SYNAPTIC TRANSMISSION
This invention was made with support under Grant Nos.
NS25928 and NS27341, awarded by the National Institute
of Health. The United States Government has certain rights
in this invention.
BACRGROVND OF THE IANSNPION
1. Field of the Inventjon
This invention relates to drugs for enhancing learning
potential and to methods of their use for improving
learning capacity. More particularly, this invention
relates to drugs that induce long term potentiation (LTP)
and enhance cognitive function in mammals.
2. Descriptjon of Related Art
The hippocampal formation, which is a specialized region
of the limbic cortex located in the temporal lobe, is
also known as the cornu ammonia from which come the names
of its two major divisions, CA1 and CA3. Neurons in the
entorhinal cortex relay incoming information through a
bundle of axons called the perforant path to neurons in
the dentate gyrus, another region of the hippocampal
formation. All association areas of the brain send
information to, and receive information from, the
hippocampal formation, via the entorhinal cortex. Thus,
the hippocampal formation is in a position to know -- and
to influence--what is going on in the rest of the brain.
Several neurotransmitters, including glutamate, gamma-
aminobutyric acid (GABA), noradrenalin, serotonin, and
acetylcholine have a profound effect on the activity of
the hippocampal formation and, undoubtedly, on its
functions.
LTP is a use-induced increase in the magnitude of the
postsynaptic response (i.e., the strengthening of the
synaptic connection between two neurons) caused by
wo 96/09299 2I97/ 7 3 PCTlUS95/10454
v 0
2
binding of sJlutamate to receptors in the postsynaptic
membrane. However, the maintenance of LTP may involve
both presynaptic and postsynaptic mechanisms (for review,
see Bliss & Collingridge, Nature, 361:31-39, 1993) . This
increase is understood to signify that LTP is associated
with enhanced cognitive function. LTP was first
discovered in 1966 to result from a response to high
frequency electrical stimulation (tetanus) of the axons
in the perforant path with a burst of approximately one
hundred pulses of electrical stimulation, delivered
within a few seconds. Evidence that LTP has occurred is
obtained by periodically delivering single pulses to the
perforant path and recording the response in the dentate
gyrus. . If, for example, the population EPSP is larger
than it was before tetanus, LTP has taken place. LTP can
also be produced in fields CA3 and CA1 as well as in the
neocortex (Perkins and Teyler, Brain Research, 439:25-47,
1988; Brown, et al., in Neural Models of Plasticity:
Experimental and Theoretical Approaches, ed. J.H. Byrne
and W.O. Berry, San Diegoc AcademicBzeas, 1989). It can
last for severalmonths (Bliss and LOmo, J. Physiol.,
2~U:331, 1973). Even more importantly, LTP can involve
the interaction between different synapses on a
particular neuron. That is, when -weak and strong
synapses on a single neuron are stimulated at
approximately the same time, the weak synapse becomes
strengthened, and this strengthening of the synapse has
been proposed to be the basis of long-term learning
(Hebb, The Organization of Behavior, New York: Wiley-
Interscience, 1949). This phenomenon, produced by the
association (in time) between the activity of two
synapses, ia_called associative LTP because it resembles
what happens during classical conditioning for learned
responses.
Many experiments have demonstrated that associative LTP
can take place in hippocampal tissue slices, which are
WO 96/09299 219 76 73 PCT/US95/10454
3
maintained in a temperature-controlled chamber filled
with a liquid that resembles interstitial fluid and
remain alive for up to 40 hours. For example, Chatterji,
Stanton, and Sejnowski (Brain Research, 9a:145-150, 1989)
stimulated two sets of axons: the axons that connect the
dentate gyrus with field CA3 (strong input) and a set of
collateral axons arising from other pyramidal cells in
CA3 (weak input). The researchers found that when the
weak input and the strong input were stimulated together,
the response of the CA3 pyramidal cells to the weak input
increased.
LTP occurs when a sufficient amount of calcium enters the
post-synaptic neuron. When a hippocampal slice is placed
in a solution that contains very little calcium, LTP does
not take place (Dunwiddie and Lynch, Brain Res., 169:103-
110, 1979). It was found that induction of LTP could be
blocked by the intracellular injection of the calcium
chelatoY (EDTA) (Lynch, et al., Nature, 305:719-721,
1983). These results suggested that calcium signaling is
involved in the induction of LTP. Several calcium-
sensitive enzymes have been proposed to play a role in
converting the initial calcium signal into persistent
modifications of synaptic strength. For example, entry
of calcium into the dendritic spine causes structural
changes in the spine by activation of the enzyme calpain,
which is instrumental in break-down of spectrin, a
protein that appears to serve as a framework support for
the structure of the spine. These changes decrease the
electrical resistance between the spine and the rest of _-
the dendrite, thus increasing the effect of EPSPs on the
dendritic membrane potential. Staubli, et al. (Brain
Res., 444=:153-158, 1988) found that proteolytic activity
of calpain is blocked by infusion of a drug called
leupeptin into lateral ventricles of rats with the result
that electrical stimulation of the hippocampus no longer
produces LTP.
w 96/09299 2 19 76 73 PCT/US95/10454
fb
4
Some sort of additive effect is required to produce LTP.
A series of electrical pulses delivered at a high rate
all in one burst will produce LTP, but the same number of
pulses given at a slow rate will not. Apparently, each
pulse produces an aftereffect that dissipates with time.
If the next pulse comes before the aftereffect fades
away, its own effect will be amplified. Thus, each pulse
"primes" the following one. if the interval between
pulses is chosen carefully, very little stimulation is
required.
Experiments have shown that the priming effect consists
of a depolarization of the postsynaptic membrane: the
depolarization caused by one pulse primes the synapse for
the next one_ The N-methyl-D-aspartate (NMDA) receptor,
a type of glutamate receptor named for its agonist, are
glutamate activated calcium permeable ion channels, which
are normally blocked by magnesium ions to prevent calcium
ions from entering the cell. When the postsynaptic
membrane is depolarized the magnesium ion is ejected from
the ion channel, and in the presence of glutamate, the
channels will open, allowing calcium ions to enter the
dendritic spine where they effect the physical changes
responsible for LTP. Collingridge, et al. (J. Physiol.,
2~4:33-46, 1983) discovered that drugs, such as APV (2-
amino-5-phosphonopentanoate), that block N-methyl-D-
aspartate (NMDA) receptors prevent LTP from taking place
in the CAl field and the dentate gyrus. However, such
drugs had no effect on any LTP that had already been
established, indicating that the NMDA receptor is not
involved in the maintenance of LTP.
While LTP is a strong cellular model for learning and
memory, whether LTP participates in memory formation is
still unresolved. In general, treatments that afFect LTP
also have consequences for the acquisition of certain.
learning tasks. When rats are exposed to novel, complex
WO 96109299 PCT/US95110454
! 21975673
environments, the extracellular population spike in the
dentate gyrus increases, just as it does when the
entorhinal cortex is subjected to high-frequency
stimulation, and treatments that interfere with LTP also
interfere with the learning of tasks in which the
hippocampus is involved. For instance Morris, et al.
(Nature, 319:774-776, 1986) trained rats in manoeuvreing
a"milk maze", a spatially guided task in separate
experiments. When APV was infused into the animals'
lateral ventricles, the animals were prevented from
learning the task. Physical damage to the hippocampus
results in the same inability to learn a spatially guided
task. Perhaps the strongest support for a role for LTP
in learning and memory will come from the discovery of a
drug that induces LTP in vivo and enhances at least one
form of learning in mammals.
Heretofore, no drug has been discovered that will
chemically stimulate the hippocampus so as to cause LTP
in vivo when administered peripherally. Drugs that
induce LTP may be effective in the treatment of cognitive
impairment such as memory loss and learning dysfunction
associated with Alzheimer's Disease, stroke and other
neurological disorders. They may also be useful for
enhancing learning potential in the healthy mammalian
brain. The practical utility of such a drug will depend
upon its ability to cross the blood-brain barrier without
inducing deleterious effects, such as seizures and
convulsions, since injection into the lateral ventricals
of the brain is not a practical method of enhancing
learning in humans and animals. Brief application in
vitro to slices of rat hippocampus of K' channel blockers
(tetraethylammonium, mast-cell-degranulating peptide) or
the metabotropic glutamate receptor agonist
aminocyclopentane-1S,3R-decarboxylate (iS,3R-ACPD)
induces a long lasting potentiation of synaptic responses
(Cherubini, et al., Nature, 328:70-73, 1987; Aniksztejn
WO 96/09299 L 19767 3 PCr/US95110454
6 =.
and Ben-Ari, Nature, 349:67-69, 1991; and Bashir, et a1.,
Nature, 363:347-350, 1993). However, when mast-cell-
degranulating peptide or 1S,3R-ACPD are administered
directly into the brain, both compounds produce seizures
5and convulsions in experimental animals (Cherubini, et
al., Nature, 12$:70-73, 1987; Bidard, et al., Brain Res.,
9~@:235-244, 1987; Sacaan and Schoepp, Neurosci. Lett.,
1,U:77-82, 1992). Therefore, the need exists for the
discovery of-new compounds capable of inducing LTP in
vivo and for-methods of utilizing them so as to enhance
learning ability in humans and animals.
BIIMMARY OF THE INVENTION
Novel compounds, such as analogs and derivatives of
Brefeldin A (BA), are provided that safely induce LTP and
enhance cognitive function when administered peripherally
to a mammal. These compounds cross the blood-brain
barrier and increase the efficiency of synaptic
transmission in the hippocampus region of the brain to
induce LTP. Heretofore, all compounds that induce LTP in
vitro and have been tested for in vivo induction of LTP
have caused seizures and convulsions in laboratory
animals. By contrast, the compounds of this invention
can be safely administered in vivo to mammals without
inducing ill._effects in the subject treated.,
Methods are provided for using compounds such as BA and
analogs and derivatives of BA to enhance learning and
memory storage in subjects with healthy brains wishing to
perform at a higher learning level as well as in subjects
suffering from learning and memory dysfunction associated
with a decrease in the efficiency of synaptic
transmission or loss of functioning synapses such as is
found in the early stages of Alzheimer's disease.
WO 96/09299 2 i 9 7 6 7 3 PCT/US95/10454
~ 7
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE lA is a schematic diagram showing positions of
stimulation and recording electrodes in a hippocampal
slice. Recording electrodes were placed in the pyramidal
cell layer of CAl region and the stratum radiatum for
extracellular recordings of the population spike (PS) and
field excitatory postsynaptic potential (EPSP),
respectively. The experiments were carried out with over
30 slices.
FIGURE iB is a graph showing the PS recorded in a slice
of hippocampal tissue before (dotted line) and 150
minutes after (solid line) application of BA. The PS
amplitude increases 150 minutes after application of BA.
FIGURE 1C is a graph showing field EPSPs recorded in a
slice of hippocampal tissue before (dotted line) and 150
minutes after (solid line) application of BA. The
amplitude and slope of field EPSP increase 150 minutes
after application of BA.
FIGURE 2A is a graph showing the time course of the
changes in amplitude of the PS recorded in a control
hippocampal slice exposed to vehicle solution without BA.
Fluctuations of PS amplitude were within t20t of baseline
during the recording. The PS amplitude was measured as
indicated by the arrow in Figure 1B.
FIGURE 2B is a graph showing the time course of the
changes in amplitude of the PS recorded in a slice of
hippocampal tissue upon administration of tetanus
(electric shock) at t=15 minutes, as indicated by the
' arrowhead.
WO 96ro9299 2 19 7 6 73 PCTIUS95/10454
a
FIGURE 2C is a graph showing the time course of the
changes in amplitude of the PS and the slope of the field
EPSP recorded in a hippocampal slice upon administration
of BA. After induction of LTP, tetanus of 100 Hz for one
second applied at t=240 minutes caused an additional
increase in the amplitude of PS and the slope of the
field EPSP, indicating additional LTP. The slope of the
field EPSP was measured as indicated by the arrow in
Figure 1C.
FIGURE 3 is a graph showing the results of recordings in
a hippocampal slice administered a N-methyl-D-aspartate
(NMDA) receptor blocker prior to and during
administration of BA (0.l g/ml). The results indicate
induction of LTP without the involvement of NMDA
receptors.
FIGURE 4A is a graph showing the results of recordings at
time points a, b, c, and d of the PS in the hippocampus
of an experimental rat injected intraperitoneally with BA
to induce LTP.
FIGURE 4B is a graph showing the time course of the
change in population spike amplitude induced in the
hippocampus of an experimental rat injected
intraperitoneally with BA (3 mg/kg) to induce LTP at the
time point indicated by the arrow in bold.
FIGURE 4C is a graph showing the results of recordings at
time points x, y and z of the PS in the hippocampus of a
control rat injected peripherally witki saline vehicle.
FIGURE 4D is a graph showing the time course of the
change in population spike amplitude in the hippocampus
of a control rat injected peripherally with saline
vehicle at the time point indicated by the arrow in bold.
WO 96/09299 219 7 6 7 3 PCT1US95/10454
~ 9
FIGURE 5 is a schematic - drawing illustrating the
apparatus used for testing acquisition of inhibitory
avoidance tasks in mice.
FIGURE 6A is a dose-response histogram showing mean
retention latencies in seconds of experimental mice
administered BA two hours before training in acquisition
of inhibitory avoidance tasks.
FIGURE 6B is a histogram showing the mean number of
trials for acquisition of inhibitory avoidance tasks in
test mice administered BA two hours before training
sessions.
FIGURE 7 is a schematic drawing illustrating the Y maze
apparatus used for testing inhibitory avoidance in mice.
FIGURE 8 is a dose-response histogram showing the mean
number of errors on a reversal discrimination task as a
function of the dose of BA administered two hours prior
to training in the Y maze illustrated in Figure 7. The
control group of 12 mice was administered saline
(vehicle). The higher the mean number of errors, the
greater is the indication of acquisition of the task.
All three groups of mice (12 mice per group) receiving
the BA exhibited a higher mean number of errors than the
control group did.
FIGURE 9 is a time course histogram showing the mean
retention latencies in groups of mice (n=12) trained in
the apparatus illustrated in Figure 5 that have been
injected intraperitoneally with BA 7 days or 120 minutes
before training sessions, immediately before training, or
two hours after training sessions.
WO 96109299 PCT/US95/10454
2197673
FIGURE 10 is a time course histogram comparing the mean
retention latencies in groups of mice (n=12) trained in
the Y maze apparatus illustrated in Figure 7 that have
been injected intraperitoneally with BA or vehicle either
5 7 days or- 120 minutes before training sessions,
immediately before training, or two or six hours after
training sessions.
10 A DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds that cross the
blood brain barrier when administered peripherally, which
increase the efficiency of synaptic transmission in the
hippocampal region of the brain. Such compounds increase
LTP of such synapses without involving NMDA receptors.
They have utility for enhancing learning and memory
storage in mammalian subjects with healthy brains as well
as in subjects suffering from learning and memory
dysfunction due to a decrease in the efficiency of
synaptic transmission or loss of functioning synapses as
occurs in Alzheimer's disease.
The preferred compounds are BA and analogs or derivatives
thereof, which produce significant dose and time-
dependent effects on memory in learning tasks.
Performance in retention tasks is also enhanced in
animals that were administered the drugs as much as two
hours after learning of the task was terminated,
indicating that the effects of the drug are not due to
influences on non-associational processes, such as
sensory, motivational and motor processes, that might
directly affect acquisition or retention of learning.
Derivatives and analogs of BA are screened and monitored
to determine their usefulness in inducing LTP using
utilizing hippocampal slice preparations. A bipolar
stimulation electrode is positioned in the stratum
WO 96/09299 2197673 PLTI1JS95/10454
~
11 -
radiatum to stimulate-. Schaffer collateral/commissural
fibers. Recording electrodes are placed in the pyramidal
cell layer of the CAl region and in the stratum radiatum
as shown in Figure lA to record the population spike (PS)
and' field excitatory postsynaptic potential (EPSP),
respectively. PS are recorded before administration of
the drug to be tested and at timed intervals after
administration of the drug, for instance at about 2
minute * intervals for up to 250 minutes after
administration of BA in artificial cerebrospinal fluid at
a concentration of 0.1 g/ml. To evaluate the effect of
BA, control slices of hippocampal tissue were tested with
vehicle. In addition, the slope of the field EPSP is
monitored as an indication of induction of LTP as
illustrated in Figure 1C.
The chemical structure of BA is as follows:
H OH
O ~
HO ~=== O 3
H
BA
As used herein the term "derivatives and analogs of BA"
are those compounds that produce at least 50!k of the
population spike or field excitatory postsynaptic
potential in slices of hippocampal tissue generated by
administration of BA. Preferred compounds have the
chemical formula I or II:
i I
CA 02197673 2002-04-22
12
R
CH0
3
A 0 3 p ~
R
R*.
R*
R R
Cli3 q..
R O q O
q...
R R' _C: NRl CH3
R
OR' ~3
A CC! 0 OH
FORMULA I
or
CA 02197673 2002-04-22
13
R A
0
0 CH 0 0 crt,
R
R*R*
q
N CH, R
0 R A.~
R 0
A...
q R=
A O NR' H3
0
R
R D o R~ CH3
q OH
FORMULA II
wherein each R is independently selected from -OH, -OR',
-SH, -SR1, -NR2R2, and a carbonyl oxygen; each. R' is
independently a C, to C4 alkyl; each R'' is independently
selected from hydrogen, -OH, -OR', and -NRZRZ; wherein R'll is
independently selected from -CHO,
OH 0
I 11 -COOH, -CR1, and -CONRZRZ; and R* is independently hydrogen or
-OH; and wherein R" is a C, to C4 alky.l ; and R2 is hydrogen or
a C, to C4 alkyl.
CA 02197673 2002-04-22
14
The novel compounds of this invention have the chemical
formula:
R q
O
q 0 ~' p O ~3
q
R*R*
R R
R ~~~O CH3 R 0 Qi3
R RR qs
R NR' ~
R ~ ~
R R
q O . OR' oooA-CH3 o OR'CH3
R OH OH
wherein each R is independently selected from -OH, ORl,
-SH, -SRl, -NRZRz, and a carbonyl oxygen; each R' is
independently a C1 to C4 alkyl; each R* is independently
hydrogen or -OH; and wherein R' is a C1 to C4 alkyl; and R2 is
hydrogen or a C1 to C4 alkyl.
CA 02197673 2002-04-22
14a
This invention also provides the use of Brefeldin A and
the above-described analogs thereof for enhancing learning or
for treatment of memory dysfunction in a mammal and, for
preparation of medicaments for such purposes. The
medicaments may be for administration to the peripheral
circulation of the mammal, including intraperitoneal
administration.
WO 96/09299 2 1 9 7 U 7 3 pCT1US95/10454
Methods for synthesis of BA and its derivatives and
analogs are well known in the art (Corey and Wollenberg,
Tetrahedron Lett., pp.4701-4704, 1976; Kitahara, et al.,
Tetrahedron Lett., pp_3021-3024, 1979).
5
In behavioral experiments using mice, evidence has been
obtained indicating that BA enhances retention in two
learning tasks: inhibitory avoidance (IA) and Y maze
discrimination (YNID). When administered peripherally by
10 intraperitoneal injection two hours before training, the
drug produced dose-dependent effects on retention tests
administered 48 hours after training. In both tasks
significant effects were obtained at doses of 1.0 and 3.0
mg/kg but a dose of 10 mg/kg was most effective in the Y
15 maze task. Furthermore, in both tasks, the drug was also
effective in enhancing retention when injected either one
week prior to training or immediately post-training. In
the Y maze task significant effects were obtained when
the drug was administered two hours, but not six hours
after training. Drug injections administered two hours
post-training were ineffective in the IA task.
Thus, the evidence clearly indicates that BA produces
significant dose and time-dependent effects in mammals on
48 hour memory in learning tasks. The fact that
retention is enhanced by post-training injections
indicates that the drug effects are not due to influences
on non-associational processes (e.g. sensory,
motivational and motor processes) directly affecting
acquisition or retention performance. Consequently, the
findings indicate that the drug affects retention by
altering memory storage processes. The memory enhancing
effects obtained with BA are similar to those obtained
with drugs affecting noradrenergic agonists, opiate and
GABAergic antagonists, as well as cholinergic agonists.
WO 96/09299 PCT/US95/10454
2197673 ~
16
The two tasks used in the experiments described in this
application provide very different measures of memory.
In the IA task, mice indicated memory of footshock
training by inhibiting a high probability response
(leaving a lighted compartment of an alley to enter a
darkened compartment). In the Y maze task, on the other
hand, mice indicated memory of training on how to escape
footshock even though entrances to the escape alley had
been punished with footshock during retention test
trials. Thus, the two behavioral measures, response
inhibition and alley choice, provide evidence that drug
injections administered either before or shortly after
training significantly improved memory on retention as
assessed two days after training.
LTP induced-by BA differs in several ways from that
produced by other art methods utilizing a train of high-
frequency electrical stimulations. First, electrical
stimulations induce LTP immediately, whereas induction of
LTP by BA usually occurs 60 to 120 minutes after drug
application. Second, in the case of tetanus-induced LTP,
activation of the NMDA receptor is essential for
triggering LTP. In contrast, induction of LTP by BA
appears to be by a process independent of NMDA receptors
because BA causes LTP in the presence of an inhibitor of
NMDA receptors known as 2 -amino- 5-phosphonopentanoate
(APV).
BA induces LTP in the CA1 area of the hippocampus of
anesthetized rats after peripheral administration,
indicating that BA crosses the blood-brain barrier and
acts on target molecules in the brain that lead to
induction of -LTP. This observed actionof BA is novel.
There is evidence to suggest that synthesis of new
proteins is necessary for LTP. Expression of the
immediate early genes zif/268 and c-fos is induced by
WO 96/09299 21Q767' 3 PCT/US95/10454
C
~ 17
tetanic stimulation, and is blocked by NNIDA receptor
antagonists. These results indicate that calcium
signalling is required for the induction of immediate
early genes. Furthermore, after tetanization, protein
kinase C mRNA is down regulated, whereas mRNA encoding
Ca''/calmodium-dependent protein kinase is up-regulated.
Thus, altered gene expression may play a role in
induction, expression and/or maintenance of LTP. Using
in situ and Northern blot hybridization techniques, the
effects of BA on the expression of genes (immediate-early
genes, growth factor genes, neuroactive peptide genes,
protein kinase genes, and receptor and ion channel genes)
can be measured in specific brain regions. It can be
determined whether detected changes in gene expression
are accompanied by changes in levels of encoded protein
by using antibodies to the protein encoded by the gene of
interest.
Neurotransmitter receptors and ion channels are
fundamental elements required for signaling in the brain.
In particular, the involvement of several
neurotransmitter receptors in the induction of LTP by a
train of high-frequency electrical stimulations has been
demonstrated by the use of receptor antagonists. Through
its action on such molecules, BA affects signal
transduction processes involved in LTP. Thus, by
expression of a variety of receptors and ion channels in
Xenopus oocytes or cultured cells, and by using a variety
of biochemical and electrophysiological techniques, it
can be determined whether binding of BA modulates the
function of a receptor (for example, glutamate,
acetylcholine, or GABA receptors) or an ion channel (for
example sodium, potassium or calcium ion channels) , or
whether.it activates second messenger pathways. Since
many different subtypes of receptors and ion channels
have already been cloned, a cDNA coding for a specific
subtype of a particular receptor or ion channel can be
WO 96/09299 21 9 7 6 7 3 PCT/US95/10454
18
obtained relatively easily utilizing the polymerase chain
reaction in standard techniques. Receptor cDNAs can be
expressed incultured cells for studying the interaction
of the receptors or ion channels with BA with standard
electrophysiological techniques.
To determine the drug binding protein, conventional
techniques for characterization of drug binding proteins
known to one of skill in the art can be used. These
techniques involve first the purification of the desired
protein by monitoring the binding activity of radioactive
drugs in fractions of broken cell preparations after each
purification step. In an alternative embodiment, the
gene expressing the protein sequence to which BA binds is
determined by preparation of a cDNA library in an RNA
expression vector from size-fractionated hippocampus
mRNA, and an mRNA mixture is synthesized in vitro and
assayed after injection into oocytes using a well known
technique described by J.B. Gurdon, et al. (Nature,
2U:177-82, 1971) and Sumikawa, et al. (Methods in
Neuroscience, Vol 1, 30-45, 1989). The expression of BA
binding protein is then tested by measurement of
electrophysiological responses or_Ca" signals in response
to application of BA in vitro. When a positive response
is observed, the library is subdivided serially until a
single positive clone is identified. From the clone, the
BA binding protein can be obtained and reproduced either
synthetically or recombinantly using technigues well
known in the art. Resolution of the structure of the
binding protein allows for the designing and screening of
new drugs.
One embodiment of this invention is a method for
enhancing synaptic efficacy by inducing LTP in subjects
by peripheral administration of a therapeutic amount of
the compounds of this invention, such as BA and its
analogs and derivatives. Another embodiment of this
WO 96109299 219 7 6 7 3 PCT/US95/10454
~ 19
invention is a method for ameliorating learning
deficiencies in a subject in need thereof by peripheral
administratiori of a therapeutic amount of a compound of
this invention. About 5t of the general population over
the age of 65 suffer from Alzheimer's disease, and the
prevalence of this disease increases with increasing
aging. The progressive aging of our population means
that more people are likely to suffer from Alzheimer's
disease. There is no effective treatment for Alzheimer's
disease. Thus, development of treatments for this
disease is an important issue. Synaptic connections in
the hippocampus, a brain region known to be important for
learning and memory, are especially vulnerable to
Alzheimer's disease, a fact presumed to account,for the
loss of memory characteristic of early stages of
Alzheimer's disease. Therefore, one method for reducing
the memory loss associated with Alzheimer's disease is to
enhance synaptic efficacy at synapses remaining on the
slowly atrophying neurons by peripheral injection of a
therapeutic amount of a compound of this invention.
Yet another embodiment of this invention is a method for
enhancing learning in mammals by peripheral
administration of an amount of BA sufficient to induce
LTP in the brains of such mammals. As used herein a
"therapeutic amount" of BA, or an analog or derivative
thereof, is an amount calculated to achieve and maintain
a blood'level in the brain of a human or animal over the
period of time desired sufficient to enhance LTP. These
amounts vary with the potency of each analog or
derivative, the amount required for the desired
therapeutic or other effect, the rate of elimination or
breakdown of the substance by the body once it has
entered the bloodstream, the blood-brain barrier
transport mechanism involved in transport of the drug
into the brain, and the amount of BA or its analog or
derivative in the formulation.
CA 02197673 2004-04-07
One skilled in the art of treatment of the conditions
described herein will know how to titrate the dosage to
achieve the desired therapeutic effect. In accordance
with conventional prudent formulating practices, a dosage
5 near the lower end of the useful range of a particular
agent is usually employed initially and the dosage
increased or decreased as indicated from the observed
response, as in the routine procedure of the physician.
In general, however, the amount of the BA, its analog or
10 derivatives, is in the range from about 1.0 to 15.0,
preferably 3.0 to 10.0 mg/kg of body weight of the
subject to be treated for enhanced learning and memory
retention in accordance with this invention.
15 Preferably and conveniently, the combined or single drug
is administered to the subject to be treated by
injection, for instance, intravenously or
intraperitoneally in combination with a physiologically
acceptable carrier. The carrier may comprise any
20 conventional diluting agent for injections such as those
described in Remington's "Pharmaceutical Sciences," 17th
Edition (Mack Publishing Co., Pa),
For instance, the BA
can be dissolved in or suspended in an aqueous or
nonaqueous sterile vehicle that meets the test for
pyrogenicity, such as sterile water or sterile aqueous
solutions of electrolytes and/or dextrose. One skilled
in the art can readily provide additional suitable
vehicles for the preparation and administration of
therapeutic dosages of the drugs disclosed herein.
The following examples illustrate the manner in which the
invention can be practiced. It is understood, however,
that the examples are for the purpose of illustration and
the invention is not to be regarded as limited to any of
the specific materials or conditions therein.
WO 96/09299 PCT/[7S95/10454
~ 21~17673
EBAMPLE 1
Transverse slices (500 m) were cut from the hippocampi of
4-8 week-old rats (or Guinea pigs weighing 300-400g),
submerged, and continuously perfused at 2-3 ml/min with
oxygenated artificial cerebrospinal fluid (10mM NaCl; 5mM
KC1; 1.3 mM NaH2PO4; 1.9 mM MgSO4= 7 H20; 22mM NaHCO3) at
30 C. Excitatory post-synaptic potentials were elicited
every 5 seconds by stimulation of Schaffer
collateral/commissural fibers with bipolar tungsten
stimulating electrodes. Stimulation and recording
electrodes were placed as shown in Figure 1A into the
pyramidal cell layer of CA1 region and the stratum
radiatum for extracellular recordings of the population
spike (PS) and field excitatory postsynaptic potential
(EPSP), respectively. A bipolar stimulation electrode
positioned in the stratum radiatum was used to stimulate
Schaffer collateral/commissural fibers.
PS were recorded from the CA1 pyramidal cell layer before
and after BA (0.05 - 0.5 g/ml; MW = 280.37) application
with glass microelectrodes filled with the artificial
cerebrospinal fluid. A stock solution of BA in methanol
(1 mg/ml) was diluted with oxygenated artificial
cerebrospinal fluid and applied to the hippocampal slices
at 2-3 ml/min for 2 to 10 minutes. To evaluate the
effect of BA, the PS amplitude and slope of EPSP was
monitored beforeand after application of BA.
As shown in Figure 1B, PS were recorded before and at 150
minutes after application of BA in a concentration of 0.1
g/ml. As shown in Figure 1C, field EPSPs were recorded
before and 150 minutes after application of BA in a
concentration of 0.1 .g/ml. After induction of LTP the
amplitude and the slope of the field EPSP increase.
Thus, to evaluate the effect of BA, the slope of the EPSP
was measured as indicated in the control record.
WO 96/09299 PC3YUS95/10454
2197673
22
The BA-induced potentiation lasted for several hours or
even longer__ The degrees of BA-induced potentiation in
individual slices varied largely, probably due to
differences in conditions of hippocampus slices, drug
concentrations, and/or duration of drug application.
E7CAMPLS 2
Studies were.conducted to determine the time course of
induction of LTP by BA in hippocampal tissue. The
amplitude of PS was recorded in a several control
hippocampal slices as shown in Figure 2A. Fluctuations
of PS amplitude were within t20t of baseline during the
recording. Upon application of tetanus of 100 Hz for 1
sec at t=15, the amplitudes of PS recorded increased from
about 2mV to about 6mV (Fig. 2B). In tetanic-induced
LTP, the PS amplitude increased immediately after tetanus
of 100 Hz for one second and reached a plateau level
within several minutes which was higher than that before
tetanus.
As shown in Figure 2C, when BA was administered to
hippocampal tissue at t=50 minutes the PS amplitude and
slope of EPSP began to increase approximately 60 minutes
after BA application at t=10 minutes. About a four-fold
increase (400% that of the control slice) in PS occurred
by t=240 minutes, and a small increase in the slope of
the iield EPSP was observed. Upon administration of
tetanus of 100 hz for 1 second at about t=240 to this
slice of hippocampal tissue in which LTP had already been
induced by BA, a further increase in PS and slope of the
field EPSP was observed. Figure shown is a
representative result. Similar results were obtained
from over 30 slices. -
WO 96/09299 2 19 7 6 7 J PCT/US95/10454
23
E]CAMPLE 3
An experiment was conducted to measure BA-induced (0.1
g/ml) increase in LTP in a hippocampal slice prepared as
in Example 1 in the presence of APV, a N-methyl-D-
aspartate (NMDA) receptor blocker. Administration of a
50 M solution of APV was commenced at t=10 minutes and
continued until t=50 minutes. As shown in Figure 3,
administration of 0.i g/ml of BA in the artificial
cerebralspinal fluid of Example 1 at about t=25 minutes
resulted in a three-fold increase in the amplitude of PS
over that of the same slice before BA application. This
demonstrates NMDA receptor independent induction of LTP
by BA.
EXAMPLE 4
Induction of LTP in vivo by Brefeldtn A.
In this experiment LTP was induced in the CA1 area of the
hippocampus of anesthetized male Sprague Dawley (250-275
g) rats aged about two months after peripheral
administration of BA, showing that BA crosses the blood
brain barrier in sufficient concentration to induce LTP.
Recordings of the PS (Figure 4A) and the time course of
the change in PS amplitude (Figures 4B) were made just
before and at intervals after intraperitoneal
administration of BA at a concentration of 3mg/kg.
Figures 4C and 4D show the recordings of the PS and the
time course of the change in PS amplitude, respectively,
from a control rat which received injection the saline
vehicle without BA. Similar results were obtained with
four additional animals. As can be seen by comparing the
results summarized in Figures 4A and 4D, the rat
receiving peripheral BA showed a 200%- increase in the
amplitude of PS over the course of about 170 minutes,
while the amplitude of the PS in the control rat declined
slightly.
WO 96,09299 _ 219 7 6 7 3 PCd'/US95/10454
24
ExAMPLE 5
SYfect of BA upon learning of inhibitory avoidance.
For testing of learning in an inhibitory avoidance (IA)
task, five groups of 12 male, CD-1 mice (Charles River
Labs, Wilmington, MA) (27-30 g, 3-4 weeks) were tested to
determine the effect upon learning of peripheral
administration two hours before training sessions of
either saline vehicle (3%-ethanol in physiological saline)
(as control) or 0.3, 1.0, 3.0, or 10.0 mg/kg of BA in the
saline vehicle. The test apparatus used was a divided
lucite box illustrated schematically in Figure 5. One
side of the box was illuminated, while the other side,
which is slightly larger than the lighted side remained
dark. In the dark arm the flood has two plates separated
by less than a centimeter connected to a positive and a
negative termini so as to deliver a shock of 0.6 mA
through the floor bars of the box in the dark compartment
to the foot of the animal.
Animals were injected either 7 days or 120 minutes prior
to training, or 120 min. post training and tested 48 h.
later. For training the mouse was placed facing the
experimenter_-iaway from the door to the dark chamber) in
the lighted side of the box. The door dividing the two
chambers was lifted, and the time transpiring before the
mouse entered the dark chamber (the latency) was
measured. Upon receiving the mild shock, the mouse
returned to the lighted side, at which time the clock was
reset. Then a second latency was measured and if the
latency was greater than 60 seconds but less than 300
seconds it was recorded. All animals were required to
show a minimum of 60 seconds latency to enter the dark
chamber, at which point they were deemed to have acquired
the inhibitory avoidance task (acquisition).
CA 02197673 2003-06-11
Forty eight hours after training, a retention test was
conducted in which the animal was placed in the
illuminated chamber and the latency to enter the dark
chamber was recorded. All experiments were run. as double
5 blind studies. Statistical significance was calculated
according to analysis of variance (ANOVA) and Mann-
Whitney non-parametric testing.
As shown in Figure 6A, mice treated with 1.0 and with 3.0
10 mg/kg doses experienced statistically significant
increase (p( .05) in mean retention latencies, as compared
with those of control mice, indicating that these animals
stayed in the illuminated chamber longer before re-
entering the dark chamber, where they had previously
15 experienced shock, than did control mice. Increased
retention latencies are indicative of increased memory
retention.
The mean number of trials required by each group to reach
20 the 60 second criterion level (desigraated acquisition) is
shown in Figure 6B. There was no significant di.fference
in acquisition among the groups, indicating that the drug
does not affect the initial achievement of the 60 second
latency.
ERAMPLE6
Acquisition of reverssl discrim,fnaticn in a Y maze taak.
The Y maze apparatus as illustrated by Figure "7 consists
of a lucite chamber with three arms, each approximately
one foot long. The floors of the arms were similar to
those of the IA apparatus of Example 5, having two plates
separated by less than a centimeter connected to a
positive and a negative terminal so as to deliver a 0.35
mA shock to the foot of the test animal. The Y maze is
used to test the animal's capacity to remember which of
the two arms delivered a shock draring the preliminary
WO96109299 2 1 9 767 3 PCT/US95/10451
26
learning phase. Therefore, during the testing phase the
safe-arm is reversed from the one that was safe during
the learning,phase. The more the animal remembers from
the learning phase, the more errors it will make during
the test phase. Consequently, a high error score
indicates learning of the task.
The training phase of the Y maze discrimination reversal
task was as follows. Animals were placed in a three arm
lucite Y maze apparatus, with each arm separated by a
sliding door (Figure 7). The left arm was illuminated
during testing as during training, while the center arm
and the right arm remained dark. Animals were placed in
the darkened center (start) arm (at the base of the Y);
after 10 seconds, a footshock (0.35 mA, 60 Hz) was
delivered to the start arm and to the intersection of all
three arms, but not to the illuminated arm until the
animal entered the lighted arm. Mice that failed to
enter the lighted arm within 60 seconds were removed and
placed again into the start arm. Those that had fully
entered the illuminated arm were lifted and returned to
the start arm. The interval between training trials was
40 seconds. The final stage of training required that,
when given the choice between the lighted and dark arms,
the mice successfully chose the lighted arm three
consecutive times (the criterion) . All mice were then
subjected to a forced entry into the dark arm where they
received a 5 second footshock to ensure that all animals
experienced footshock in the dark arm.
Testing was conducted 48 hours later, but with the dark
arm as the safe arm and the illuminated arm providing a
foot shock. All mice were given six test trials.
Records were made of the first arm chosen by the animal
during testing (L=lit or D=dark) and of the latency
period to reach the dark (safe) arm. The number of
errors in six trials was recorded. As shown by the data
WO 96/09299 2 1 9 7 U/ 3 PCTIUS95/10454
27
in Figure 8, administration of BA in the saline vehicle
described above to three groups of mice (n=12) at a
dosage of 1.0, 3.0 or 10.0 mg/kg of body weight all
resulted in a statistically significant increase of
errors over that of the control group (n=24), which
received administration only of the saline vehicle (3%
ethanol in physiological saline), indicating that the
drug facilitates memory for this task.
EXAMPLE 7
To determine the retention latencies of BA, the drug was
injected peripherally into groups of mice (n=12) at a
dosage of -3mg/kg intraperitoneally at various time
intervals prior to or following training and performance
of theacquired learning task described in Example 6 of
this application. Four sets of control and test groups
of mice (12 mice per group) were injected with BA at a
dosage of 3mg/kg of body weight 7 days prior to training,
120 minutes prior to training, immediately prior to
training, or 120 minutes after training. As shown by the
results summarized in Figure 9, the mean retention
latencies in test animals were significantly higher than
control when the drug was injected 7 days prior to
training, 2 hours prior to training, and immediately
post-training. However, injection of BA two hours post
training did not result in an increase in retention
latency over that of the control group. No adverse
health effects that could be attributed to the
administration of BA were observed in the test mice.
EICAMPLF3 8
To determine the retention latencies of BA, the drug was
injected intraperitoneally into groups of mice at a
dosage of 3mg/kg at various time intervals prior to or
following training and performance of the acquired
WO 96'09299 2 1 9 7 6 7 3 PCVUS95/10454
28
learning in -the Y maze task described in Example 7 of
this application. Four sets of control and test groups
of mice (12 mice per group) were injected with BA at a
dosage of 3mgJkg of body weight 7 days prior to training,
120 minutes prior to training, immediately post training,
or 120 minutes after training. As shown by the results
summarized in Figure 10, the number of errors on reversal
in test animals was significantly higher than in the
parallel control group when the drug was injected up to
7 days prior to training and up to 2 hours post training.
However, injection of BA six hours post training did not
result in_an increase in the number of errors over that
of the control group. No adverse health effects that
could be attributed to the administration of BA were
observed in the test mice.
The foregoing description of the invention is exemplary
for purposes of illustration and explanation. It should
be understood that various modifications can be made
without departing from the spirit and scope of the
invention. Accordingly, the following claims are
intended to be interpreted to embrace all such
modifications.
