U.S. patent application number 13/279636 was filed with the patent office on 2012-02-16 for epigenetic mechanisms re-establish access to long-term memory after neuronal loss.
This patent application is currently assigned to The General Hospital Corporation d/b/a Massachusetts General Hospital, The General Hospital Corporation d/b/a Massachusetts General Hospital. Invention is credited to Andre Fischer, Stephen Haggarty, Stuart L. Schreiber, Weiping Tang, Li-Huei Tsai.
Application Number | 20120039909 13/279636 |
Document ID | / |
Family ID | 40088987 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120039909 |
Kind Code |
A1 |
Tsai; Li-Huei ; et
al. |
February 16, 2012 |
EPIGENETIC MECHANISMS RE-ESTABLISH ACCESS TO LONG-TERM MEMORY AFTER
NEURONAL LOSS
Abstract
The invention relates to methods and products for enhancing and
improving recovery of lost memories. In particular the methods are
accomplished through the increase of histone acetylation.
Inventors: |
Tsai; Li-Huei; (Cambridge,
MA) ; Fischer; Andre; (Goettingen, DE) ;
Haggarty; Stephen; (Dorchester, MA) ; Tang;
Weiping; (Middleton, WI) ; Schreiber; Stuart L.;
(Boston, MA) |
Assignee: |
The General Hospital Corporation
d/b/a Massachusetts General Hospital
Boston
MA
Massachusetts Institute of Technology
Cambridge
MA
President and Fellows of Harvard College
Cambridge
MA
|
Family ID: |
40088987 |
Appl. No.: |
13/279636 |
Filed: |
October 24, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11998834 |
Nov 30, 2007 |
8088951 |
|
|
13279636 |
|
|
|
|
60861883 |
Nov 30, 2006 |
|
|
|
Current U.S.
Class: |
424/158.1 ;
514/357; 514/359; 514/415; 514/44A; 514/44R; 514/456; 514/49;
514/557; 514/575; 514/615; 514/616; 514/617; 514/628 |
Current CPC
Class: |
A61K 31/164 20130101;
A61K 31/4192 20130101; A61K 31/7052 20130101; A61K 31/19 20130101;
A61K 31/404 20130101; A61K 31/215 20130101; A61K 31/7068 20130101;
A61P 25/00 20180101; A61P 25/28 20180101 |
Class at
Publication: |
424/158.1 ;
514/575; 514/628; 514/359; 514/616; 514/615; 514/415; 514/617;
514/456; 514/44.R; 514/44.A; 514/49; 514/557; 514/357 |
International
Class: |
A61K 31/167 20060101
A61K031/167; A61K 31/404 20060101 A61K031/404; A61K 31/166 20060101
A61K031/166; A61K 31/353 20060101 A61K031/353; A61K 48/00 20060101
A61K048/00; A61P 25/00 20060101 A61P025/00; A61K 39/395 20060101
A61K039/395; A61K 31/7068 20060101 A61K031/7068; A61K 31/19
20060101 A61K031/19; A61K 31/4406 20060101 A61K031/4406; A61P 25/28
20060101 A61P025/28; A61K 31/4192 20060101 A61K031/4192; A61K
31/7088 20060101 A61K031/7088 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under NIH
NS051874. Accordingly, the Government has certain rights in this
invention.
Claims
1. (canceled)
2. A method for accessing long-term memory in a subject having
diminished access to a long-term memory comprising administering an
HDAC2 inhibitor to the subject in an amount effective to
reestablish access to long-term memory in the subject.
3. The method of claim 2, wherein the long-term memory is
impaired.
4. The method of claim 3, wherein the long-term memory impairment
is age related.
5. The method of claim 3, wherein the long-term memory impairment
is injury related.
6. The method of claim 2, wherein a synaptic network in the subject
is re-established.
7. The method of claim 6, wherein re-establishing the synaptic
network comprises an increase in the number of active brain
synapses.
8. The method of claim 6, wherein re-establishing the synaptic
network comprises reversing neuronal loss.
9.-11. (canceled)
12. The method of claim 10, further comprising administering a
second HDAC inhibitor.
13. The method of claim 10, wherein the second HDAC inhibitor
comprises a compound of the formula: ##STR00004## wherein R1 is
selected from either an aryl ring system optionally substituted, or
--N.dbd.R4; R2 is selected from either carbon or a heteroatom that
is optionally substituted with at least one hydrogen, alkyl,
alkenyl, aryl, halogen, and heteroatomic groups; R3 is absent or it
is a carbon or heteroatom that is optionally substituted with at
least one hydrogen, alkyl, alkenyl, aryl, halogen, and heteroatomic
groups; R4 comprises a carbon atom bound to an optionally
substituted aryl, heteroaromatic, or biaryl ring system; and L is 1
to 12 carbons optionally bonded to heteroatoms, alkyl, alkenyl, or
aryl groups; or a pharmaceutically acceptable salt thereof.
14. The method of claim 13, wherein R2 and R3 are adjacent atoms
within an aromatic or heteroaromatic ring system that is optionally
substituted with at least one hydrogen, alkyl, alkenyl, aryl,
halogen, and heteroatomic groups.
15. The method of claim 13, wherein the structure is:
##STR00005##
16. The method of claim 13, wherein the structure is:
##STR00006##
17. The method of claim 13, wherein the structure is:
##STR00007##
18. The method of claim 13, wherein the structure is:
##STR00008##
19. The method of claim 13, wherein the structure is:
##STR00009##
20. The method of claim 13, wherein the structure is:
##STR00010##
21. The method of claim 13, wherein the structure is:
##STR00011##
22. The method of claim 2, further comprising altering the
methylation level of one or more genes.
23. The method of claim 22, further comprising administering a DNA
methylation inhibitor.
24. The method of claim 23, wherein the DNA methylation inhibitor
is selected from the group consisting of 5-azacytidine,
5-aza-2'deoxycytidine, 5,6-dihydro-5-azacytidine,
5,6-dihydro-5-aza-2'deoxycytidine, 5-fluorocytidine,
5-fluoro-2'deoxycytidine, and short oligonucleotides containing
5-aza-2'deoxycytosine, 5,6-dihydro-5-aza-2'deoxycytosine, and
5-fluoro-2'deoxycytosine, and procainamide, Zebularine, and
(-)-egallocatechin-3-gallate.
25. The method of claim 2, wherein the HDAC2 inhibitor is
administered orally, intravenously, cutaneously, subcutaneously,
nasally, imtramuscularly, intraperitoneally or
intracerebroventricularly.
26.-39. (canceled)
40. A method for recapturing a memory comprising administering an
HDAC2 inhibitor, in an amount effective to re-establish access to a
lost memory, in a subject having Alzheimer's disease.
41. A method for re-establishing a synaptic network in a subject
comprising: administering an HDAC2 inhibitor to a human subject
having chronic memory loss, in an amount effective reestablish a
synaptic network in the human subject having chronic memory
loss.
42. The method of claim 41, wherein the subject has Alzheimer's
disease.
43. The method of claim 41, wherein the subject has age related
memory loss.
44. The method of claim 41, wherein the subject has injury related
memory loss.
45. The method of claim 41, wherein the subject has Parkinson's
disease.
46. The method of claim 41, wherein re-establishing the synaptic
network comprises an increase in the number of active brain
synapses.
47. A method comprising: administering an HDAC2 inhibitor to a
human subject having Alzheimer's disease, in an amount effective
reestablish a synaptic network in the human subject.
Description
RELATED APPLICATION
[0001] This application is a continuation application and claims
the benefit under 35 U.S.C. .sctn.120 of U.S. application Ser. No.
11/998,834, entitled "EPIGENETIC MECHANISMS RE-ESTABLISH ACCESS TO
LONG-TERM MEMORY AFTER NEURONAL LOSS" filed on Nov. 30, 2007, which
is herein incorporated by reference in its entirety. application
Ser. No. 11/998,834 claims priority under 35 U.S.C. .sctn.119(e) to
U.S. Provisional Application Ser. No. 60/861,883, entitled
"EPIGENETIC MECHANISMS RE-ESTABLISH ACCESS TO LONG-TERM MEMORY
AFTER NEURONAL LOSS" filed Nov. 30, 2006, which is herein
incorporated by reference in its entirety.
BACKGROUND OF INVENTION
[0003] Brain atrophy occurs during normal aging and is an early
feature of neurodegenerative diseases associated with impaired
learning and memory. Only recently have mouse models with extensive
neurodegeneration in the forebrain been reported (1-3). One of
these models is the bi-transgenic CK-p25 Tg mice where expression
of p25, a protein implicated in various neurodegenerative diseases
(4), is under the control of the CamKII promoter and can be
switched on or off with a doxycycline diet (3,5). Post-natal
induction of p25 expression for 6 weeks caused learning impairment
that was accompanied by severe synaptic and neuronal loss in the
forebrain. However, pre-clinical research has not yet explored
strategies to recover lost memories after substantial neuronal loss
had taken place.
SUMMARY OF INVENTION
[0004] Neurodegenerative diseases of the central nervous system are
often associated with impaired learning and memory, eventually
leading to dementia. An important aspect that has not been
addressed extensively in pre-clinical research, is the loss of
long-term memories and the exploration of strategies to
re-establish access to those memories. In some embodiments the
current invention provides methods for restoring access to
long-term memory after synaptic and neuronal loss has already
occurred. Environmental enrichment (EE) has been shown to reinstate
learning behavior and re-establish access to long-term memories
after significant brain atrophy and neuronal loss has already
occurred. Also shown herein is a correlation between EE and
epigenetic changes. EE increases histone-tail acetylation and
changes the level of methylation. The increase in acetylation and
change in level of methylation is observed in hippocampal and
cortical histone 3 (H3) and histone 4 (H4). In turn, elevated
histone H3 and H4 acetylation initiate rewiring of the neural
network.
[0005] In some embodiments the invention provides methods for
inducing an increase in histone acetylation. In some aspects the
invention provides methods for the change in level of methylation
in histones. One aspect of the invention is the increase in histone
acetylation through the administration of inhibitors of
histone-deacetylases. The administration of inhibitors of
histone-deacetylases induced sprouting of dendrites, an increased
number of synapses, and reinstated learning behavior and access to
long-term memories. In some embodiments the invention provides
epigenetic approaches as a therapeutic avenue for neurodegenerative
diseases associated with learning and memory impairment including
the recovery of long-term memories in patients with dementia.
[0006] In some aspects the invention provides a method for
recapturing a memory comprising increasing histone acetylation in
an amount effective to re-establish access to a memory in a subject
having memory loss. In some embodiments the invention provides a
method for accessing long-term memory in a subject having
diminished access to a long-term memory comprising increasing
histone acetylation in an amount effective to reestablish access to
long-term memory in the subject.
[0007] In some aspects of the invention the long-term memory is
impaired. In some embodiments the impairment may be age-related or
injury-related. In some embodiments of the invention a synaptic
network in the subject is re-established. In some embodiments
re-establishing the synaptic network comprises an increase in the
number of active brain synapses. In some embodiments
re-establishing the synaptic network comprises a reversal of
neuronal loss.
[0008] In some embodiments of the invention histone acetylation
comprises acetylation of H3 or H4. A further embodiment comprises
increasing histone acetylation by administering a therapeutically
effective amount of HDAC inhibitor to the subject. In some
embodiments the HDAC inhibitor is trichostatin A, trichostatin B,
trichostatin C, trapoxin A, trapoxin B, chlamydocin, sodium salts
butyrate, butyric acid, sodium salts of phenylbutyrate,
phenylbutyric acid, scriptaid, FR901228, depudecin, oxamflatin,
pyroxamide, apicidin B, apicidin C, Helminthsporium carbonum toxin,
2-amino-8-oxo-9,10-epoxy-decanoyl,
3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide, suberoylanilide
hydroxamic acid, [valproic acid], FK228 or m-carboxycinnamic acid
bis-hydroxamide. In some embodiments a second HDAC inhibitor is
administered. In some embodiments the HDAC inhibitor is
administered orally, intravenously, cutaneously, to subcutaneously,
nasally, imtramuscularly, intraperitoneally or
intracerebroventricularly.
[0009] In some embodiments the method comprises altering the
methylation level of one or more genes. In some embodiments
altering the methylation level of one or more genes comprises
administering a DNA methylase inhibitor. In some embodiments the
DNA methylation inhibitor is 5-azacytidine, 5-aza-2'deoxycytidine,
5,6-dihydro-5-azacytidine, 5,6-dihydro-5-aza-2'deoxycytidine,
5-fluorocytidine, 5-fluoro-2'deoxycytidine, and short
oligonucleotides containing 5-aza-2'deoxycytosine,
5,6-dihydro-5-aza-2'deoxycytosine, and 5-fluoro-2'deoxycytosine,
and procainamide, Zebularine, or (-)-egallocatechin-3-gallate.
[0010] In some aspects the invention provides a pharmaceutical
composition comprising an HDAC inhibitor and a pharmaceutically
acceptable carrier in a formulation for delivery to brain
tissue.
[0011] In some aspects the invention provides a method for
recapturing a memory comprising increasing histone acetylation, by
a method other than an HDAC inhibitor, in an amount effective to
re-establish access to a lost memory in a subject having
Alzheimer's disease.
[0012] In yet other aspects the invention provides a method for
recapturing a memory comprising administering an HDAC inhibitor, in
an amount effective to re-establish access to a lost memory, in a
subject having Alzheimer's disease and monitoring the subject to
identify recapture of a memory that was previously lost.
[0013] In some embodiments of the methods and compositions
described herein the histone acetylase inhibitor is a binding
peptide such as an antibody or antibody fragment. In other
embodiments the histone acetylase inhibitor is an antisense
molecule. In yet other embodiments the histone acetylase inhibitor
is an siRNA. The histone acetylase inhibitor in other embodiments
is an HDAC inhibitor such as for instance a compound of the
formula:
##STR00001##
[0014] wherein R1 is selected from either an aryl ring system
optionally substituted, or --N.dbd.R4; R2 is selected from either
carbon or a heteroatom that is optionally substituted with at least
one hydrogen, alkyl, alkenyl, aryl, halogen, and heteroatomic
groups; R3 is absent or it is a carbon or heteroatom that is
optionally substituted with at least one hydrogen, alkyl, alkenyl,
aryl, halogen, and heteroatomic groups; R4 comprises a carbon atom
bound to an optionally substituted aryl, heteroaromatic, or biaryl
ring system; and L is 1 to 12 carbons optionally bonded to
heteroatoms, alkyl, alkenyl, or aryl groups or a pharmaceutically
acceptable salt thereof.
[0015] In some embodiments R2 and R3 are adjacent atoms within an
aromatic or heteroaromatic ring system that is optionally
substituted with at least one hydrogen, alkyl, alkenyl, aryl,
halogen, and heteroatomic groups.
[0016] The histone acetylase inhibitor may be one or more of the
following structures:
##STR00002##
[0017] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways. Also, the
phraseology and terminology used herein is for the purpose of
description and should not be regarded as limiting. The use of
"including," "comprising," or "having," "containing", "involving",
and variations thereof herein, is meant to encompass the items
listed thereafter and equivalents thereof as well as additional
items.
BRIEF DESCRIPTION OF DRAWINGS
[0018] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0019] FIG. 1 shows that enrichment reinstates learning in CK-p25
Tg mice after neurodegeneration. a. Experimental design. b.
Enriched and non-enriched CK-p25 Tg mice displayed similar brain
atrophy. c. Fear memories (tested 24 h after the training). d.
Brain weight plotted against freezing behavior. e. Escape latency
in water maze test. f. Representative immunoblots from control
mice, enriched and non-enriched CK-p25 Tg mice. g. Representative
pictures of the hippocampal region showing staining for the
synaptic marker protein synaptophysin (SVP).
[0020] FIG. 2 shows that enrichment reestablishes the access to
long-term memories. a. Experimental design I. b. the loss of
consolidated long-term memories. c. to Experimental design II. d.
Memory test: freezing behavior. e. Representative images and plots
showing NeuN staining and brain atrophy. f. Representative images
of the anterior cingulate cortex region showing staining and plots
of the synaptic marker protein synaptophysin (SVP). Scale bar: 20
.mu.m. *P<0.05 vs. non enriched group, n=3. cg, cingulated
cortex; M2, motor cortex 2; cc, corpus callosum.
[0021] FIG. 3 shows that enrichment induces epigenetic changes and
histone-deacetylase inhibitors facilitate learning behavior. a.
Acetylation changes upon EE. b. Quantification of A. c.+d. C57BL/6J
were injected [ip] daily with SB (1.2 g/kg) or saline for 4 weeks,
which resulted in increased acetylation of H3 and H4 in hippocampal
lysates. e.+f. SB injected mice showed significantly facilitated
associative and spatial learning.
[0022] FIG. 4 shows that sodiumbutyrate facilitates learning and
reestablishes the access to long-term memories in CK-p25 Tg mice.
a. Experimental design. b. Freezing behavior. c. water maze
paradigm. d. Representative images showing increased hippocampal
SVP-IR in CK-p25 mice injected with SB, when compared to
vehicle-injected mice (P<0.0001). Scale bar: 20 .mu.m. Analysis
was performed as described in FIG. 1. e. Quantification of (D).
f-i. Sodiumbutyrate treatment reinstates access to long-term
memories in CK-p25 Tg mice. f. Experimental design. g. Freezing
behavior. h. Representative images showing SVP-IR in the ACC
(P<0.0001). Scale bar: 20 .mu.m. i. Quantification of (h). py,
pyramidal cell layer; cg, cingulate cortex.
[0023] FIG. 5 shows that enrichment facilitates learning. a. Images
showing a home cage and a cage used for environmental enrichment
(EE). b. EE and freezing behavior. c. EE and the Morris water maze
paradigm.
[0024] FIG. 6 shows that enrichment facilitates synaptic
plasticity. a. Representative images showing MAP-2 immunoreativitiy
(IR) in the hippocampus and anterior cingulate cortex (b) of
enriched mice. c. Representative images of the hippocampal and
anterior cingulate cortex (d) region of enriched and non-enriched
mice showing staining of the synaptic marker protein synaptophysin
(SVP). e. Representative immunoblots from the hippocampus and
cortex of enriched and non-enriched mice. **P<0.001. py,
pyramidal cell layer; oriens; rad, stratum radiatum; Lmol,
molecular layer; mol, lower molecular layer; GrDG, granula cell
layer of the dentate gyrus, DG, dentate gyrus; CA1, hippocamapal
regions CA1; cg, cingulate cortex.
[0025] FIG. 7 shows that effects of enrichment or sodiumbutyrate
treatment on plasticity factors in CK-p25 Tg mice that developed
severe neurodegeneration. a-d. Enrichment of CK-p25 TG mice. a.
Representative pictures showing immunostaining for NeuN in the
hippocampal region. b. Brain sections immunostained for the
dendritic marker protein MAP-2. c. Staining of the anterior
cingulated cortex. d. Representative immunoblots from cortical
lysates of control mice, enriched and non-enriched CK-p25 Tg mice.
e-h. Sodiumbutyrate treatment of CK-p25 Tg mice. e. Representative
images showing hippocampal NeuN staining confirming that SB and
vehicle injected CK-p25 mice displayed hippocampal neuronal loss to
the same degree. f. Representative images showing hippocampal MAP-2
staining. g. Representative images showing NeuN staining of the
ACC. h. Representative immunoblots from the hippocampus and cortex
of all groups. *P<0.05 vs. vehicle group, n=3. py, pyramidal
cell layer; rad, stratum radiatum; Lmol, molecular layer; mol,
lower molecular layer; GrDG, granula cell layer of the dentate
gyrus, DG, dentate gyrus; CA1, hippocamapal regions CA1; cg,
cingulate cortex.
[0026] FIG. 8 shows that the effect of sodiumbutyrate injection on
learning, basal anxiety, explorative behavior and brain plasticity.
a. Freezing behavior during the memory test, ip administration. b.
Freezing behavior during the memory test, icy administration. c.
Elevated plus maze and open field test. d. Representative images
showing increased MAP-2 immunoreativitiy (IR) in the hippocampus.
e. Representative immunoblots (n=3/group) from hippocampal lysates.
f. Representative images showing increased SVP IR in the
hippocampus. py, pyramidal cell layer; or, oriens; rad, stratum
radiatum; Lmol, molecular layer; mol, lower molecular layer; GrDG,
granula cell layer of the dentate gyrus, DG, dentate gyrus; CA1,
hippocamapal regions CA1; cg, cingulate cortex.
[0027] FIG. 9 shows that evidence that enrichment and
sodiumbutyrate injection lead to the recovery of spatial memories.
a. Training of CK-p25 Tg mice in which p25 expression was repressed
and control mice were trained in the water maze paradigm. b. Mean
escape rate. c. Mean escape rate after EE. d. Mean escape rate
after SB injection.
[0028] FIG. 10 shows that the over expression of HDAC2, but not
that of HDAC1, impairs fear conditioning without affecting
sensory-motor functions. a. Experimental design. b. Freezing
behavior during the during the context- and tone-dependent memory
test. c. Distance traveled during the initial 3 min exposure to the
training box. d. Average activity during the training and the
increased activity during electoral stimulation.
[0029] FIG. 11 shows that the over expression of HDAC2 impairs
spatial learning. a. Escape latencies of control mice that were
subjected to the water maze paradigm for nine consecutive days,
with 2 trials everyday. b. The probe test, performed after 5 days
of training trials.
[0030] FIG. 12 shows that the HDAC2 gene knockout enhances
associative learning. a. Freezing behavior of HDAC2 knockout (KO)
mice and control mice during the contextual dependent memory test.
b. Distance traveled during the initial 3 minutes exposure to the
training box. c. The occurrence of average activity during training
and increased activity during electoral stimulation.
[0031] FIG. 13 shows that HDAC 2 over expression decreases newly
generated neurons in dentate gyrus of adult mice. a. Representative
images of hippocampal brain sections from two month-old--mice group
(n=3/group) immunostained for the newborn neuron marker
Doublecortin (DCX). b. Representative pictures show BrdU
immunoreativitiy (light regions) in the hippocampus dentate
gyrus.
[0032] FIG. 14 HDAC 2 Tgs show reduced CREB expression in
hippocampus. a. Representative images of hippocampal brain sections
from two-month-old mice group immunostained for the CREB in CA1 and
CA3 regions. b. Protein levels were analyzed in forebrain lysates
of all experimental groups by immunoblotting.
[0033] FIG. 15 shows that HDAC2 directly binds to the promoter
region of specific genes and regulates gene expression. Whole
brains from two-month-old experiment groups were fixed and
homogenized. Lysates of all experimental groups are
immune-precipitated with HDAC2 antibody and subjected to
semi-quantities PCR analysis.
[0034] FIG. 16 shows that chronic treatment with SAHA or sodium
butarate, but not MS-275, facilitates contextual fear conditioning
learning. a. After chronic treatment for 10-21 days, we found that
SAHA is more potent than SB in facilitating associative learning
using the contextual fear conditioning paradigm. b. Conversely,
MS-275, a class 1 HDAC inhibitor, does not facilitate associative
learning in mice. c. Brain lysates from SAHA and MS-275 treated
mice exhibit increased acetylation of lys9lys 14 on histone 3 and
lys5 on histone 4 compared to saline treated mice demonstrating
that both drugs affect HDAC activity in the brain.
[0035] FIG. 17 shows that HDAC inhibitors regulate neuronal
activity and neurogenesis in dentate gyrus. a. In agreement with
its beneficial effect on associative learning, SAHA treatment
increased BrdU positive cells in the subgranule zone of the dentate
gyrus. b. In addition, DCX, a marker for newly generated neurons,
labeled more cells in SAHA treated but not MS-275 treated dentate
gyms. These observations suggest that SAHA upregulates adult
neurogenesis in the dentate gyrus. c. Furthermore, SAHA treated
mice showed higher number of c-fos positive cells after fear
conditioning training but MS-275 treated mice showed lower number
of c-fos positive cells compared to saline treated group. d. Data
is presented in bar graph format demonstrating significant
enhancement by SAHA. Thus, activity induced c-fos expression may
also be used to evaluate the effect of HDAC inhibitors on
hippocampus dependent learning.
[0036] FIG. 18 are data from cell-based and biochemical assays for
optimizing HDAC inhibitor potency and selectivity. Images of (a)
untreated (DMSO) and (b) trichostatin A-treated rat neural
progenitor cells cultured in a 384-well plate after staining with
antibodies for acetylated histones (light regions). c. Comparison
of the histone modification signatures of two HDAC inhibitors,
trichostatin (bars on the left) and valproate (bars on the right)
using a human neuroblastoma cell line and a panel of antibodies
toward different histone modifications. d. Example of an in vitro
deacetylase assay for HDAC inhibitor screening using recombinant,
full-length, human HDAC5 (0.5 .mu.g). HDAC5 deacetylase activity
(AFU; change in arbitrary fluorescence units/min) as a function of
substrate concentration. e. IC50 values (nM) of SAHA, trichostain A
and the non-hydroxamate apicidin against human HDAC3 (class I) and
HDAC5 (class II). f. Structures of potential novel class
II-selective biasing elements for HDAC inhibitor design. g. HDAC
inhibition of a representative benzohydrazide (BCB-001) and
triazole carboxylate (BCB-002) as measured using recombinant HDAC2
(class I) and HDAC10 (class II).
[0037] FIG. 19 depicts chemical analogs and the SAHA lead
structure. Based upon the lead structure of SAHA (Compound 1), and
the "cap-linker-chelator" model for the design of HDAC inhibitors,
analogs of SAHA are developed to improve potency and selectivity
for class II HDACs.
[0038] FIG. 20 is a diagram of the structure of two new molecules
UW_WT-I.sub.--06 and UW_WT-I.sub.--05.
[0039] FIG. 21 is a bar graph demonstrating that UW_WT-I.sub.--06
significantly and UW_WT-I.sub.--05 to a lesser extent enhanced
associative learning. These compounds show strong inhibitory
activity toward HDAC 1,2, and 10 with UW_WT-I.sub.--06 also
inhibiting HDAC 6 and 8.
DETAILED DESCRIPTION
[0040] The invention relates in some aspects to therapeutics for
enhancing and/or retrieving memories. A "memory" as used herein
refers to the ability to recover information about past events or
knowledge. Memories include short-term memory (also referred to as
working or recent memory) and long-term memory. Short-term memories
involve recent events, while long-term memories relate to the
recall of events of the more distant past. Enhancing or retrieving
memories is distinct from learning. However, in some instances in
the art learning is referred to as memory. The present invention
distinguishes between learning and memory and is focused on
enhancing memories. Learning, unlike memory enhancement, refers to
the ability to create new memories that had not previously existed.
Thus in order to test the ability of a therapeutic agent to effect
the ability of a subject to learn rather than recall old memories,
the therapeutic would be administered prior to or at the same time
as the memory is created. In order to test the ability of a
therapeutic to effect recall of a previously created memory the
therapeutic is administered after the memory is created and
preferably after the memory is lost.
[0041] In some instances the invention relates to methods for
recapturing a memory in a subject. In order to recapture the memory
the memory has been lost. A lost memory is one which cannot be
retrieved by the subject without assistance, such as the
therapeutic of the invention. In other words the subject cannot
recall the memory. As used herein the term "recapture" refers to
the ability of a subject to recall a memory that the subject was
previously unable to recall. Generally, such a subject has a
condition referred to as memory loss. A subject having memory loss
is a subject that cannot recall one or more memories. The memories
may be short term memories or long term memories. Methods for
assessing the ability to recall a memory are known to those of
skill in the art and may include routine cognitive tests.
[0042] In other instances the invention relates to a method for
accessing long-term memory in a subject having diminished access to
a long-term memory. A subject having diminished access to a memory
is a subject that has experienced one or more long term memory
lapses. The long-term memory lapse may be intermittent or
continuous. Thus, a subject having diminished access to a long term
memory includes but is not limited to a subject having memory loss,
with respect to long term memories.
[0043] In some instances the long-term memory of the "subject
having diminished access" may be impaired. An impaired long-term
memory is one in which a physiological condition of the subject is
associated with the long-term memory loss. Conditions associated
with long-term memory loss include but are not limited to age
related memory loss and injury related memory loss.
[0044] As used herein "age related memory loss" refers to refers to
any of a continuum of conditions characterized by a deterioration
of neurological functioning that does not rise to the level of a
dementia, as further defined herein and/or as defined by the
Diagnostic and Statistical Manual of Mental Disorders: 4th Edition
of the American Psychiatric Association (DSM-IV, 1994). This term
specifically excludes age-related dementias such as Alzheimer's
disease and Parkinson's disease, and conditions of mental
retardation such as Down's syndrome. Age related memory loss is
characterized by objective loss of memory in an older subject
compared to his or her younger years, but cognitive test
performance that is within normal limits for the subject's age. Age
related memory loss subjects score within a normal range on
standardized diagnostic tests for dementias, as set forth by the
DSM-IV. Moreover, the DSM-IV provides separate diagnostic criteria
for a condition termed Age-Related Cognitive Decline. In the
context of the present invention, as well as the terms
"Age-Associated Memory Impairment" and "Age-Consistent Memory
Decline" are understood to be synonymous with the age related
memory loss. Age-related memory loss may include decreased brain
weight, gyral atrophy, ventricular dilation, and selective loss of
neurons within different brain regions. For purposes of some
embodiments of the present invention, more progressive forms of
memory loss are also included under the definition of age-related
memory disorder. Thus persons having greater than age-normal memory
loss and cognitive impairment, yet scoring below the diagnostic
threshold for frank dementia, may be referred to as having a mild
neurocognitive disorder, mild cognitive impairment, late-life
forgetfulness, benign senescent forgetfulness, incipient dementia,
provisional dementia, and the like. Such subjects may be slightly
more susceptible to developing frank dementia in later life (See
also US patent application 2006/008517, which is incorporated by
reference). Symptoms associated with age-related memory loss
include but are not limited to alterations in biochemical markers
associated with the aging brain, such as IL-1beta, IFN-gamma,
p-JNK, p-ERK, reduction in synaptic activity or function, such as
synaptic plasticity, evidenced by reduction in long term
potentiation, diminution of memory and reduction of cognition.
[0045] As used herein "injury related memory loss" refers to damage
which occurs to the brain, and which may result in neurological
damage. Sources of brain injury include traumatic brain injury such
as concussive injuries or penetrating head wounds, brain tumors,
alcoholism, Alzheimer's disease, stroke, heart attack and other
conditions that deprive the brain of oxygen, meningitis, AIDS,
viral encephalitis, and hydrocephalus.
[0046] A subject shall mean a human or vertebrate animal or mammal
including but not limited to a dog, cat, horse, cow, pig, sheep,
goat, turkey, chicken, and primate, e.g., monkey. Subjects are
those which are not otherwise in need of an HDAC inhibitor.
Subjects specifically exclude subjects having Alzheimer's disease,
except in the instance where a subject having Alzheimer's disease
is explicitly recited.
[0047] The histone acetylation is preferably administered in an
amount effective to re-establish access to a memory. The term
re-establishing access as used herein refers to increasing
retrieval of a memory. Although Applicants are not bound by a
mechanism of action, it is believed that the compounds of the
invention are effective in increasing retrieval of memories by
re-establishing a synaptic network. The process of re-establishing
a synaptic network may include an increase in the number of active
brain synapses and or a reversal of neuronal loss. Data
demonstrating evidence of re-establishment of a synaptic network is
presented in the Examples below.
[0048] As used herein, the term re-establish access to long-term
memory when used with respect to a disorder comprising memory loss
or memory lapse refers to a treatment which increases the ability
of a subject to recall a memory. In some instances the therapeutic
of the invention also decreases the incidence and/or frequency with
which the memory is lost or cannot be retrieved.
[0049] The methods of the invention may also be directed to the
recapture of a memory in a subject having Alzheimer's disease.
Alzheimer's disease is a disorder in which the cognitive systems
that control memory have been damaged. Often long-term memory is
retained while short-term memory is lost; conversely, memories may
become confused, resulting in mistakes in recognizing people or
places that should be familiar.
[0050] Methods for recapturing a memory in a subject having
Alzheimer's disease by administering an HDAC inhibitor are also
provided according to the invention. Such methods involve
administering the inhibitor and monitoring the subject to identify
recapture of a memory that was previously lost. Subjects may be
monitored by routine tests known in the art. For instance some are
described in books such as DSM described above or in the medical
literature.
[0051] In some embodiments the method is accomplished by increasing
histone acetylation, by a method other than an HDAC inhibitor.
Methods for increasing histone acetylation, by a method other than
a classic HDAC inhibitor include but are not limited to nucleic
acid molecule inhibitors such as antisense and RNAi molecules which
reduce the expression of histone deacetylase and histone acetylase
activators. The invention embraces methods that regulate the
function of any protein involved with histone modification,
function and regulation.
[0052] A number of enzymes capable of chemical modification of
histones have been described. For example, histone acetyl
transferases include Gcn5p, p300/CBP-associated factor (P/CAF),
p300, CREB-binding protein (CBP), HAT1, TFIID-associated factor 250
(TAF.sub.II250), and steroid receptor coactivator-1 (SRC-1) (Wade
et al. (1997) Trends Biochem. Sci. 22:128-132; Kouzarides (1999)
Curr. Opin. Genet. Devel. 9:40-48; Sterner et al. (2000) Microbiol.
Mol. Biol. Rev. 64:435-459). The HDAC family of proteins have been
identified as histone deacetylases and include homologues to the
budding yeast histone deacetylase RPD3 (e.g., HDAC1, HDAC2, HDAC3
and HDAC8) and homologues to the budding yeast histone deacetylase
HDA1 (e.g., HDAC4, HDAC5, HDAC6 and HDAC7) (Ng et al. (2000) Trends
Biochem. Sci. 25:121-126). The Rsk-2 (RKS90) kinase has been
identified as a histone kinase. Sassone-Corsi et al. (1999) Science
285:886-891. A histone methyltransferase (CARM-1) has also been
identified. Chen et al. (1999) Science 284:2174-2177. Nucleosomes,
the primary scaffold of chromatin folding, are dynamic
macromolecular structures, influencing chromatin solution
conformations. The nucleosome core is made up of histone proteins,
H2A, H2B, H3 and H4. Histone acetylation causes nucleosomes and
nucleosomal arrangements to behave with altered biophysical
properties. The balance between activities of histone acetyl
transferases (HAT) and deacetylases (HDAC) determines the level of
histone acetylation. Acetylated histones cause relaxation of
chromatin and activation of gene transcription, whereas
deacetylated chromatin generally is transcriptionally inactive.
[0053] It has been demonstrated herein that general HDAC inhibitors
(e.g.; sodium butyrate, SAHA, TSA) enhance learning and memory.
Studies described in the Examples below were also undertaken to
determine which of the 11 histone deacetylases is responsible for
the observed function. It was discovered that while HDAC1 Tg mice
do not show any difference in learning behavior compared to the
control mice, HDAC2 Tg mice have impaired learning as evaluated by
Pavlovian fear conditioning and Morris water maze tests (FIGS. 10
and 11). Remarkably, HDAC2 neuron specific knockout mice (loss of
function) display enhanced learning (FIG. 12). Furthermore, it was
discovered that impaired learning exhibited by HDAC2 Tg mice is
accompanied by reduced neurogenesis in the adult dentate gyrus
(FIG. 13), and reduced CREB expression (FIG. 14). These
observations suggest that HDAC2 participates in learning and memory
and that it is likely to be the target of inhibition by the general
HDAC inhibitors. It was also discovered that novel inhibitors of
HDAC1, 2, 6, 8, and 10 also enhance memory (FIG. 21).
[0054] A histone deacetylase inhibitor as used herein is a compound
that inhibits the activity of histone deacetylase. One of ordinary
skill in the art can select suitable compounds on the basis of the
known structures of histone deacetylases. Examples of such
compounds are binding peptides such as antibodies, preferably
monoclonal antibodies, antibody fragments, scFv, etc that
specifically react with the histone deacetylase, small molecule
inhibitors referred to as HDAC inhibitors, and expression
inhibitors such as antisense and siRNA.
[0055] HDAC inhibitors include but are not limited to the following
compounds, functional analogs and salts thereof: trichostatin A
(TSA), trichostatin B, trichostatin C, trapoxin A, trapoxin B,
chlamydocin, sodium salts of butyrate, butyric acid, sodium salts
of phenylbutyrate, phenylbutyric acid, scriptaid, FR901228,
depudecin, oxamflatin, pyroxamide, apicidin B, apicidin C,
Helminthsporium carbonum toxin, 2-amino-8-oxo-9,10-epoxy-decanoyl,
3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide, suberoylanilide
hydroxamic acid (SAHA), valproic acid, FK228, or m-carboxycinnamic
acid bis-hydroxamide. In preferred embodiments the HDAC inhibitor
is an HDAC2 inhibitor such as sodium butyrate, SAHA or TSA.
Derivatives of the inhibitors showing increased pharmacological
half-life are also useful according to the invention (Brettman and
Chaturvedi, J. Cli. Pharmacol. 36 (1996), 617-622).
[0056] The HDAC inhibitors include SAHA. "SAHA" as used herein
refers to suberoylanilide hydroxamic acid, analogs, derivatives and
polymorphs. Polymorphs of SAHA are described in US Published Patent
Application No. 20040122101 which is incorporated by reference.
HDAC inhibitors that are SAHA include those described by the
following formula and having ability to inhibit HDAC2:
##STR00003##
[0057] wherein R1 is selected from an aryl ring system optionally
substituted; R2 is selected from either carbon or a heteroatom that
is optionally substituted with at least one hydrogen, alkyl,
alkenyl, aryl, halogen, and heteroatomic groups; R3 is absent or it
is a carbon or heteroatom that is optionally substituted with at
least one hydrogen, alkyl, alkenyl, aryl, halogen, and heteroatomic
groups; and L is 1 to 12 carbons optionally bonded to heteroatoms,
alkyl, alkenyl, or aryl groups.
[0058] "Alkyl" in general, refers to an aliphatic hydrocarbon group
which may be straight, branched or cyclic having from 1 to about 10
carbon atoms in the chain, and all combinations and sub
combinations of ranges therein. The term "alkyl" includes both
"unsubstituted alkyls" and "substituted alkyls," the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the backbone. In preferred
embodiments, a straight chain or branched chain alkyl has 12 or
fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.12 for
straight chain, C.sub.3-C.sub.12 for branched chain), and more
preferably 6 or fewer, and even more preferably 4 or fewer.
Likewise, preferred cycloalkyls have from 3-10 carbon atoms in
their ring structure, and more preferably have 5, 6 or 7 carbons in
the ring structure. Unless the number of carbons is otherwise
specified, "lower alkyl" as used herein means an alkyl group, as
defined above, but to having from one to ten carbons, more
preferably from one to six carbon atoms in its backbone structure,
and even more preferably from one to four carbon atoms in its
backbone structure. Likewise, "lower alkenyl" and "lower alkynyl"
have similar chain lengths. Preferred alkyl groups are lower
alkyls. In preferred embodiments, a substituent designated herein
as alkyl is a lower alkyl. Alkyl groups include, but are not
limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
t-butyl, n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl,
isohexyl, cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl,
2,2-dimethylbutyl, and 2,3-dimethylbutyl. Alkyl substituents can
include, for example, alkenyl, alkynyl, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic
moiety.
[0059] The term "alkenyl" refers to unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double bond.
[0060] As used herein, the term "halogen" designates --F, --Cl,
--Br or --I; the term "sulfhydryl" means --SH; and the term
"hydroxyl" means --OH.
[0061] The term "aryl," alone or in combination, means a
carbocyclic aromatic system containing one, two or three rings
wherein such rings may be attached together in a pendent manner or
may be fused. The term "aryl" embraces aromatic radicals such as
phenyl, naphthyl, tetrahydronaphthyl, indane and biphenyl, and
includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all
of which may be optionally substituted. The term "aryl" as used
herein includes 5-, 6- and 7-membered single-ring aromatic groups
that may include from zero to four heteroatoms, for example,
benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,
triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,
and the like. Those aryl groups having heteroatoms in the ring
structure may also be referred to as "aryl heterocycles" or
"heteroaromatics." The aromatic ring can be substituted at one or
more ring positions with such substituents as described above, for
example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,
cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino,
amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,
heterocyclyl, aromatic or heteroaromatic moieties, --CF.sub.3,
--CN, or the like. The term "aryl" also includes polycyclic ring
systems having two or more cyclic rings in which two or more
carbons are common to two adjoining rings (the rings are "fused
rings") wherein at least one of the rings is aromatic, e.g., the
other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls and/or heterocyclyls.
[0062] The term "biaryl" represents aryl groups which have 5-14
atoms containing more than one aromatic ring including both fused
ring systems and aryl groups substituted with other aryl groups.
Such groups may be optionally substituted. Suitable biaryl groups
include naphthyl and biphenyl. The term "carbocyclic" refers to a
cyclic compounds in which all of the ring members are carbon atoms.
Such rings may be optionally substituted. The compound can be a
single ring or a biaryl ring. The term "cycloalkyl" embraces
radicals having three to ten carbon atoms, such as cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and norboryl. Such
groups may be substituted.
[0063] "Heterocyclic" aryl or "heteroaryl" groups are groups which
have 5-14 ring atoms wherein 1 to 4 heteroatoms are ring atoms in
the aromatic ring and the remainder of the ring atoms being carbon
atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen.
Suitable heteroaryl groups include furanyl, thienyl, pyridyl,
pyrrolyl, N-lower alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl,
pyrazinyl, imidazolyl, indolyl and the like, all optionally
substituted. The term "heterocyclic" refers to cyclic compounds
having as ring members atoms of at least two different elements.
The compound can be a single ring or a biaryl. Heterocyclic groups
include, for example, thiophene, benzothiophene, thianthrene,
furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin,
pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine,
pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,
furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,
piperidine, piperazine, morpholine, lactones, lactams such as
azetidinones and pyrrolidinones, sultams, sultones, and the like.
The heterocyclic ring can be substituted at one or more positions
with such substituents as described above, as for example, halogen,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone,
aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic
moiety, --CF.sub.3, --CN, or the like.
[0064] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0065] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, the heteroatoms such as nitrogen may
have hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0066] Based upon the lead structure of the compounds described
herein such as SAHA (Compound 1, shown in FIG. 19), and the
"cap-linker-chelator" model for the design of HDAC inhibitors,
analogs of SAHA may be developed to improve potency and selectivity
for class II HDACs. The non-hydroxamate apicidin (Compound 2, FIG.
19) may be obtained from commercial sources (Fermentek). Compound 3
(FIG. 19), an electrophilic ketone with known submicromolar HDAC
inhibitory activity, may be synthesized as described by (Frey R R,
et al Trifluoromethyl ketones as inhibitors of histone deacetylase.
Bioorg Med Chem Lett 2002; 12:3443-3447). Compound 4 (FIG. 19),
also an electrophilic ketone may be synthesized based upon the
methods of (Wada C K, et al Alpha-keto amides as inhibitors of
histone deacetylase. Bioorg Med Chem Lett 2003; 13:3331-3335). The
purities of all compounds may be assessed by HPLC and the mass
spectra are confirmed relative to anticipated product structures.
Compounds may be profiled using a panel of all 11 human recombinant
HDACs (BPS Biosciences) to derive selectivity profiles and to
explore cofactor dependencies. In parallel, the cellular IC50 for
HDAC inhibition may be determined using a panel of antibody-based
assays (see e.g. Haggarty S J et al Multidimensional chemical
genetic analysis of diversity-oriented synthesis-derived
deacetylase inhibitors using cell-based assays. Chem Biol 2003a;
10:383-396).
[0067] Another HDAC inhibitor is Trapoxin
(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10--
epoxy-decanoyl)). Trapoxin is a microbially derived
epoxide-containing cyclotetrapeptide that inhibits histone
deacetylation in vivo and causes mammalian cells to arrest in the
cell cycle.
[0068] Trichostatin is another HDAC inhibitor. Trichostatins like
many of the HDAC inhibitors are commercially available from sources
such as Sigma, Vincibiochem (Italy) etc. Exemplary methods of
preparing trichostatin are described in U.S. Pat. No.
4,946,999.
[0069] Trichostatin A, SAHA, and trapoxin stimulate histone
acetylation by acting as direct inhibitors of HDAC enzyme activity.
Each of these compounds possess lysine-like side chains and act as
chemical analogs of lysine substrates. Molecular models based upon
the x-ray crystal structure of an HDAC-like protein indicate that
trichostatin A and SAHA can bind within the active site of the HDAC
enzyme and interact with a zinc metal ion within the catalytic
pocket that is critical for enzymatic activity.
[0070] HDAC inhibitors also include short chain fatty acids such as
Sodium Butyrate, Isovalerate, Valerate, 4 Phenylbutyrate (4-PBA),
Phenylbutyrate (PB), Propionate, Butyramide, Isobutyramide,
Phenylacetate, 3-Bromopropionate, Tributyrin, Valproic Acid and
Valproate and Pivanex.TM..
[0071] A DNA methylation inhibitor is an agent that directly or
indirectly causes a reduction in the level of methylation of a
nucleic acid molecule. DNA methylation inhibitors are well known
and routinely utilized in the art and include, but are not limited
to, inhibitors of methylating enzymes such as methylases and
methyltransferases. Non-limiting examples of DNA methylation
inhibitors include 5-azacytidine, 5-aza-2'deoxycytidine (also known
as Decitabine in Europe), 5,6-dihydro-5-azacytidine,
5,6-dihydro-5-aza-2'deoxycytidine, 5-fluorocytidine,
5-fluoro-2'deoxycytidine, and short oligonucleotides containing
5-aza-2'deoxycytosine, 5,6-dihydro-5-aza-2'deoxycytosine, and
5-fluoro-2'deoxycytosine, and procainamide, Zebularine, and
(-)-egallocatechin-3-gallate.
[0072] In addition to the traditional HDAC inhibitors described
above, histone deacetylase and DNA methylating enzymes can also be
inhibited by expression inhibitors such as antisense and RNAi
mechanisms. Thus, the invention embraces antisense oligonucleotides
that selectively bind to nucleic acid molecules encoding a histone
deacetylase or DNA methylating enzyme to decrease expression and
activity of this protein.
[0073] As used herein, the term "antisense oligonucleotide" or
"antisense" describes an oligonucleotide that is an
oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide, or modified oligodeoxyribonucleotide which
hybridizes under physiological conditions to DNA comprising a
particular gene or to an mRNA transcript of that gene and, thereby,
inhibits the transcription of that gene and/or the translation of
that mRNA. The antisense molecules are designed so as to interfere
with transcription or translation of a target gene upon
hybridization with the target gene or transcript. Antisense
oligonucleotides that selectively bind to a nucleic acid molecule
encoding a histone deacetylase are particularly preferred. Those
skilled in the art will recognize that the exact length of the
antisense oligonucleotide and its degree of complementarity with
its target will depend upon the specific target selected, including
the sequence of the target and the particular bases which comprise
that sequence.
[0074] It is preferred that the antisense oligonucleotide be
constructed and arranged so as to bind selectively with the target
under physiological conditions, i.e., to hybridize substantially
more to the target sequence than to any other sequence in the
target cell under physiological conditions. Based upon the
nucleotide sequences of nucleic acid molecules encoding histone
deacetylase, (e.g., GenBank Accession Nos NP.sub.--848512,
NP.sub.--848510, NP.sub.--478057, NP.sub.--478056, NP.sub.--055522)
or upon allelic or homologous genomic and/or cDNA sequences, one of
skill in the art can easily choose and synthesize any of a number
of appropriate antisense molecules for use in accordance with the
present invention. In order to be sufficiently selective and potent
for inhibition, such antisense oligonucleotides should comprise at
least about 10 and, more preferably, at least about 15 consecutive
bases which are complementary to the target, although in certain
cases modified oligonucleotides as short as 7 bases in length have
been used successfully as antisense oligonucleotides. See Wagner et
al., Nat. Med. 1(11):1116-1118, 1995. Most preferably, the
antisense oligonucleotides comprise a complementary sequence of
20-30 bases. Although oligonucleotides may be chosen which are
antisense to any region of the gene or mRNA transcripts, in
preferred embodiments the antisense oligonucleotides correspond to
N-terminal or 5' upstream sites such as translation initiation,
transcription initiation or promoter sites. In addition,
3'-untranslated regions may be targeted by antisense
oligonucleotides. Targeting to mRNA splicing sites has also been
used in the art but may be less preferred if alternative mRNA
splicing occurs. In addition, the antisense is targeted,
preferably, to sites in which mRNA secondary structure is not
expected (see, e.g., Sainio et al., Cell Mol. Neurobiol.
14(5):439-457, 1994) and at which proteins are not expected to
bind.
[0075] In one set of embodiments, the antisense oligonucleotides of
the invention may be composed of "natural" deoxyribonucleotides,
ribonucleotides, or any combination thereof. That is, the 5' end of
one native nucleotide and the 3' end of another native nucleotide
may be covalently linked, as in natural systems, via a
phosphodiester internucleoside linkage. These oligonucleotides may
be prepared by art recognized methods which may be carried out
manually or by an automated synthesizer. They also may be produced
recombinantly by vectors.
[0076] In preferred embodiments, however, the antisense
oligonucleotides of the invention also may include "modified"
oligonucleotides. That is, the oligonucleotides may be modified in
a number of ways which do not prevent them from hybridizing to
their target but which enhance their stability or targeting or
which otherwise enhance their therapeutic effectiveness.
[0077] The term "modified oligonucleotide" as used herein describes
an oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acid molecules
has been covalently attached to the oligonucleotide. Preferred
synthetic internucleoside linkages are phosphorothioates,
alkylphosphonates, phosphorodithioates, phosphate esters,
alkylphosphonothioates, phosphoramidates, carbamates, carbonates,
phosphate triesters, acetamidates, carboxymethyl esters and
peptides.
[0078] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose.
[0079] The present invention, thus, contemplates pharmaceutical
preparations containing modified antisense molecules that are
complementary to and hybridizable with, under physiological
conditions, nucleic acid molecules encoding a histone deacetylase,
together with pharmaceutically acceptable carriers. Antisense
oligonucleotides may be administered as part of a pharmaceutical
composition. In this latter embodiment, it may be preferable that a
slow intravenous administration be used. Such a pharmaceutical
composition may include the antisense oligonucleotides in
combination with any standard physiologically and/or
pharmaceutically acceptable carriers which are known in the art.
The compositions should be sterile and contain a therapeutically
effective amount of the antisense oligonucleotides in a unit of
weight or volume suitable for administration to a subject.
[0080] The methods of the invention also encompass use of isolated
short RNA that directs the sequence-specific degradation of a
histone deacetylase mRNA through a process known as RNA
interference (RNAi). The process is known to occur in a wide
variety of organisms, including embryos of mammals and other
vertebrates. It has been demonstrated that dsRNA is processed to
RNA segments 21-23 nucleotides (nt) in length, and furthermore,
that they mediate RNA interference in the absence of longer dsRNA.
Thus, these 21-23 nt fragments are sequence-specific mediators of
RNA degradation and are referred to herein as siRNA or RNAi.
Methods of the invention encompass the use of these fragments (or
recombinantly produced or chemically synthesized oligonucleotides
of the same or similar nature) to enable the targeting of histone
deacetylase mRNAs for degradation in mammalian cells useful in the
therapeutic to applications discussed herein.
[0081] The methods for design of the RNA's that mediate RNAi and
the methods for transfection of the RNAs into cells and animals is
well known in the art and are readily commercially available (Verma
N. K. et al, J. Clin. Pharm. Ther., 28(5):395-404 (2004), Mello C.
C. et al. Nature, 431(7006)338-42 (2004), Dykxhoorn D. M. et al.,
Nat. Rev. Mol. Cell. Biol. 4(6):457-67 (2003) Proligo (Hamburg,
Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce
Chemical (part of Perbio Science, Rockford, Ill., USA), Glen
Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and
Cruachem (Glasgow, UK)). The RNAs are preferably chemically
synthesized using appropriately protected ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. Most
conveniently, siRNAs are obtained from commercial RNA oligo
synthesis suppliers listed herein. In general, RNAs are not too
difficult to synthesize and are readily provided in a quality
suitable for RNAi. A typical 0.2 .mu.mol-scale RNA synthesis
provides about 1 milligram of RNA, which is sufficient for 1000
transfection experiments using a 24-well tissue culture plate
format.
[0082] The histone deacetylase cDNA specific siRNA is designed
preferably by selecting a sequence that is not within 50-100 bp of
the start codon and the termination codon, avoids intron regions,
avoids stretches of 4 or more bases such as AAAA, CCCC, avoids
regions with GC content <30% or >60%, avoids repeats and low
complex sequence, and it avoids single nucleotide polymorphism
sites. The histone deacetylase siRNA may be designed by a search
for a 23-nt sequence motif AA(N19). If no suitable sequence is
found, then a 23-nt sequence motif NA(N21) may be used with
conversion of the 3' end of the sense siRNA to TT. Alternatively,
the histone deacetylase siRNA can be designed by a search for
NAR(N17)YNN. The target sequence may have a GC content of around
50%. The siRNA targeted sequence may be further evaluated using a
BLAST homology search to avoid off target effects on other genes or
sequences. Negative controls are designed by scrambling targeted
siRNA sequences. The control RNA preferably has the same length and
nucleotide composition as the siRNA but has at least 4-5 bases
mismatched to the siRNA. The RNA molecules of the present invention
can comprise a 3' hydroxyl group. The RNA molecules can be
single-stranded or double stranded; such molecules can be blunt
ended or comprise overhanging ends (e.g., 5', 3') from about 1 to
about 6 nucleotides in length (e.g., pyrimidine nucleotides, purine
nucleotides). In order to further enhance the stability of the RNA
of the present invention, the 3' overhangs can be stabilized
against degradation. The RNA can be stabilized by including purine
nucleotides, such as adenosine or guanosine nucleotides.
Alternatively, substitution of pyrimidine nucleotides by modified
analogues, e.g., substitution of uridine 2 nucleotide 3' overhangs
by 2'-deoxythymidine is tolerated and does not affect the
efficiency of RNAi. The absence of a 2' hydroxyl significantly
enhances the nuclease resistance of the overhang in tissue culture
medium.
[0083] The RNA molecules used in the methods of the present
invention can be obtained using a number of techniques known to
those of skill in the art. For example, the RNA can be chemically
synthesized or recombinantly produced using methods known in the
art. Such methods are described in U.S. Published Patent
Application Nos. US2002-0086356A1 and US2003-0206884A1 that are
hereby incorporated by reference in their entirety.
[0084] The methods described herein are used to identify or obtain
RNA molecules that are useful as sequence-specific mediators of
histone deacetylase mRNA degradation and, thus, for inhibiting
histone deacetylase activity. Expression of histone deacetylase can
be inhibited in humans in order to prevent the protein from being
translated and thus contributing to the increased recapture of
memories.
[0085] The RNA molecules may also be isolated using a number of
techniques known to those of skill in the art. For example, gel
electrophoresis can be used to separate RNAs from the combination,
gel slices comprising the RNA sequences removed and RNAs eluted
from the gel slices. Alternatively, non-denaturing methods, such as
non-denaturing column chromatography, can be used to isolate the
RNA produced. In addition, chromatography (e.g., size exclusion
chromatography), glycerol gradient centrifugation, affinity
purification with antibody can be used to isolate RNAs.
[0086] Any RNA can be used in the methods of the present invention,
provided that it has sufficient homology to the histone deacetylase
gene to mediate RNAi. The RNA for use in the present invention can
correspond to the entire histone deacetylase gene or a portion
thereof. There is no upper limit on the length of the RNA that can
be used. For example, the RNA can range from about 21 base pairs
(bp) of the gene to the full length of the gene or more. In one
embodiment, the RNA used in the methods of the present invention is
about 1000 bp in length. In another embodiment, the RNA is about
500 bp in length. In yet another embodiment, the RNA is about 22 bp
in length. In certain embodiments the preferred length of the RNA
of the invention is 21 to 23 nucleotides.
[0087] Further, histone deacetylase DNA methylating enzymes can
also be inhibited by binding peptides such as antibodies. Numerous
histone deacetylase antibodies are commercially available from
sources such as Sigma, Vinci Biochem, Cell Signaling Technologies.
Such antibodies can be modified to produce antibody fragments or
humanized versions. Alternatively therapeutically useful antibodies
can be produced using techniques known to those of ordinary skill
in the art since HDACs are available.
[0088] The therapeutic compounds of the invention may be directly
administered to the subject or may be administered in conjunction
with a delivery device or vehicle. Delivery vehicles or delivery
devices for delivering therapeutic compounds to surfaces have been
described. The therapeutic compounds of the invention may be
administered alone (e.g., in saline or buffer) or using any
delivery vehicles known in the art. For instance the following
delivery vehicles have been described: Cochleates; Emulsomes,
ISCOMs; Liposomes; Live bacterial vectors (e.g., Salmonella,
Escherichia coli, Bacillus calmatte-guerin, Shigella,
Lactobacillus); Live viral vectors (e.g., Vaccinia, adenovirus,
Herpes Simplex); Microspheres; Nucleic acid vaccines; Polymers;
Polymer rings; Proteosomes; Sodium Fluoride; Transgenic plants;
Virosomes; Virus-like particles. Other delivery vehicles are known
in the art and some additional examples are provided below.
[0089] The term effective amount of a therapeutic compound of the
invention refers to the amount necessary or sufficient to realize a
desired biologic effect. For example, as discussed above, an
effective amount of a therapeutic compounds of the invention is
that amount sufficient to re-establish access to a memory. Combined
with the teachings provided herein, by choosing among the various
active compounds and weighing factors such as potency, relative
bioavailability, patient body weight, severity of adverse
side-effects and preferred mode of administration, an effective
prophylactic or therapeutic treatment regimen can be planned which
does not cause substantial toxicity and yet is entirely effective
to treat the particular subject. The effective amount for any
particular application can vary depending on such factors as the
disease or condition being treated, the particular therapeutic
compounds being administered the size of the subject, or the
severity of the disease or condition. One of ordinary skill in the
art can empirically determine the effective amount of a particular
therapeutic compounds of the invention without necessitating undue
experimentation. Compositions of the invention include compounds as
described herein, or a pharmaceutically acceptable salt or hydrate
thereof.
[0090] Subject doses of the compounds described herein for delivery
typically range from about 0.1 .mu.g to 10 mg per administration,
which depending on the application could be given daily, weekly, or
monthly and any other amount of time therebetween. The doses for
these purposes may range from about 10 .mu.g to 5 mg per
administration, and most typically from about 100 .mu.g to 1 mg,
with 2-4 administrations being spaced days or weeks apart. In some
embodiments, however, parenteral doses for these purposes may be
used in a range of 5 to 10,000 times higher than the typical doses
described above.
[0091] In one embodiment, the composition is administered once
daily at a dose of about 200-600 mg. In another embodiment, the
composition is administered twice daily at a dose of about 200-400
mg. In another embodiment, the composition is administered twice
daily at a dose of about 200-400 mg intermittently, for example
three, four, or five days per week. In another embodiment, the
composition is administered three times daily at a dose of about
100-250 mg. In one embodiment, the daily dose is 200 mg, which can
be administered once-daily, twice-daily, or three-times daily. In
one embodiment, the daily dose is 300 mg, which can be administered
once-daily or twice-daily. In one embodiment, the daily dose is 400
mg, which can be administered once-daily or twice-daily. The HDAC
inhibitor can be administered in a total daily dose of up to 800 mg
once, twice or three times daily, continuously (i.e., every day) or
intermittently (e.g., 3-5 days a week).
[0092] For any compound described herein the therapeutically
effective amount can be initially determined from animal models. A
therapeutically effective dose can also be determined from human
data for HDAC inhibitors which have been tested in humans (e.g. for
the treatment of cancer) and for compounds which are known to
exhibit similar pharmacological activities. Higher doses may be
required for parenteral administration. The applied dose can be
adjusted based on the relative bioavailability and potency of the
administered compound. Adjusting the dose to achieve maximal
efficacy based on the methods described above and other methods as
are well-known in the art is well within the capabilities of the
ordinarily skilled artisan.
[0093] The formulations of the invention are administered in
pharmaceutically acceptable solutions, which may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, and optionally other
therapeutic ingredients.
[0094] For use in therapy, an effective amount of the therapeutic
compounds of the invention can be administered to a subject by any
mode that delivers the therapeutic agent or compound to the desired
surface, e.g., mucosal, systemic. Administering the pharmaceutical
composition of the present invention may be accomplished by any
means known to the skilled artisan. Preferred routes of
administration include but are not limited to oral, parenteral,
intramuscular, intranasal, sublingual, intratracheal, inhalation,
ocular, vaginal, rectal and intracerebroventricular.
[0095] For oral administration, the therapeutic compounds of the
invention can be formulated readily by combining the active
compound(s) with pharmaceutically acceptable carriers well known in
the art. Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
subject to be treated. Pharmaceutical preparations for oral use can
be obtained as solid excipient, optionally grinding a resulting
mixture, and processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Optionally the oral formulations
may also be formulated in saline or buffers, i.e. EDTA for
neutralizing internal acid conditions or may be administered
without any carriers.
[0096] Also specifically contemplated are oral dosage forms of the
above component or components. The component or components may be
chemically modified so that oral delivery of the derivative is
efficacious. Generally, the chemical modification contemplated is
the attachment of at least one moiety to the component molecule
itself, where said moiety permits (a) inhibition of proteolysis;
and (b) uptake into the blood stream from the stomach or intestine.
Also desired is the increase in overall stability of the component
or components and increase in circulation time in the body.
Examples of such moieties include: polyethylene glycol, copolymers
of ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In:
Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience,
New York, N.Y., pp. 367-383; Newmark, et al., 1982, J. Appl.
Biochem. 4:185-189. Other polymers that could be used are
poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for
pharmaceutical usage, as indicated above, are polyethylene glycol
moieties.
[0097] The location of release may be the stomach, the small
intestine (the duodenum, the jejunum, or the ileum), or the large
intestine. One skilled in the art has available formulations which
will not dissolve in the stomach, yet will release the material in
the duodenum or elsewhere in the intestine. Preferably, the release
will avoid the deleterious effects of the stomach environment,
either by protection of the therapeutic agent or by release of the
biologically active material beyond the stomach environment, such
as in the intestine.
[0098] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is important. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0099] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic i.e. powder; for liquid
forms, a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0100] The therapeutic can be included in the formulation as fine
multi-particulates in the form of granules or pellets of particle
size about 1 mm. The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression.
[0101] Colorants and flavoring agents may all be included. For
example, the therapeutic agent may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0102] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, a-lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans and starch. Certain inorganic salts may
be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and
Avicell.
[0103] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates include but are not limited to starch, including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0104] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0105] An anti-frictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0106] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0107] To aid dissolution of the therapeutic into the aqueous
environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential non-ionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the therapeutic
agent either alone or as a mixture in different ratios.
[0108] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. Microspheres formulated for oral
administration may also be used. Such microspheres have been well
defined in the art. All formulations for oral administration should
be in dosages suitable for such administration.
[0109] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0110] For administration by inhalation, the compounds for use
according to the present invention may be conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0111] Also contemplated herein is pulmonary delivery of the
therapeutic compounds of the invention. The therapeutic agent is
delivered to the lungs of a mammal while inhaling and traverses
across the lung epithelial lining to the blood stream. Other
reports of inhaled molecules include Adjei et al., 1990,
Pharmaceutical Research, 7:565-569; Adjei et al., 1990,
International Journal of Pharmaceutics, 63:135-144 (leuprolide
acetate); Braquet et al., 1989, Journal of Cardiovascular
Pharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al.,
1989, Annals of Internal Medicine, Vol. III, pp. 206-212
(a1-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146
(a-1-proteinase); Oswein et al., 1990, "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery
II, Keystone, Colo., March, (recombinant human growth hormone);
Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-g and
tumor necrosis factor alpha) and Platz et al., U.S. Pat. No.
5,284,656 (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong
et al.
[0112] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0113] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, North Carolina; and the
Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford,
Mass.
[0114] All such devices require the use of formulations suitable
for the dispensing of therapeutic agent. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, and/or carriers useful in therapy. Also, the
use of liposomes, microcapsules or microspheres, inclusion
complexes, or other types of carriers is contemplated. Chemically
modified therapeutic agent may also be prepared in different
formulations depending on the type of chemical modification or the
type of device employed.
[0115] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise therapeutic agent dissolved
in water at a concentration of about 0.1 to 25 mg of biologically
active compound per mL of solution. The formulation may also
include a buffer and a simple sugar (e.g., for stabilization and
regulation of osmotic pressure). The nebulizer formulation may also
contain a surfactant, to reduce or prevent surface induced
aggregation of the compound caused by atomization of the solution
in forming the aerosol.
[0116] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the
therapeutic agent suspended in a propellant with the aid of a
surfactant. The propellant may be any conventional material
employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0117] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing therapeutic
agent and may also include a bulking agent, such as lactose,
sorbitol, sucrose, or mannitol in amounts which facilitate
dispersal of the powder from the device, e.g., 50 to 90% by weight
of the formulation. The therapeutic agent should most
advantageously be prepared in particulate form with an average
particle size of less than 10 mm (or microns), most preferably 0.5
to 5 mm, for most effective delivery to the distal lung.
[0118] Nasal delivery of a pharmaceutical composition of the
present invention is also contemplated. Nasal delivery allows the
passage of a pharmaceutical composition of the present invention to
the blood stream directly after administering the therapeutic
product to the nose, without the necessity for deposition of the
product in the lung. Formulations for nasal delivery include those
with dextran or cyclodextran.
[0119] For nasal administration, a useful device is a small, hard
bottle to which a metered dose sprayer is attached. In one
embodiment, the metered dose is delivered by drawing the
pharmaceutical composition of the present invention solution into a
chamber of defined volume, which chamber has an aperture
dimensioned to aerosolize and aerosol formulation by forming a
spray when a liquid in the chamber is compressed. The chamber is
compressed to administer the pharmaceutical composition of the
present to invention. In a specific embodiment, the chamber is a
piston arrangement. Such devices are commercially available.
[0120] Alternatively, a plastic squeeze bottle with an aperture or
opening dimensioned to aerosolize an aerosol formulation by forming
a spray when squeezed is used. The opening is usually found in the
top of the bottle, and the top is generally tapered to partially
fit in the nasal passages for efficient administration of the
aerosol formulation. Preferably, the nasal inhaler will provide a
metered amount of the aerosol formulation, for administration of a
measured dose of the drug.
[0121] The compounds, when it is desirable to deliver them
systemically, may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0122] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0123] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0124] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0125] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0126] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0127] Suitable liquid or solid pharmaceutical preparation forms
are, for example, aqueous or saline solutions for inhalation,
microencapsulated, encochleated, coated onto microscopic gold
particles, contained in liposomes, nebulized, aerosols, pellets for
implantation into the skin, or dried onto a sharp object to be
scratched into the skin. The pharmaceutical compositions also
include granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, drops or preparations with protracted release of active
compounds, in whose preparation excipients and additives and/or
auxiliaries such as disintegrants, binders, coating agents,
swelling agents, lubricants, flavorings, sweeteners or solubilizers
are customarily used as described above. The pharmaceutical
compositions are suitable for use in a variety of drug delivery
systems. For a brief review of methods for drug delivery, see
Langer, Science 249:1527-1533, 1990, which is incorporated herein
by reference.
[0128] The therapeutic compounds of the invention and optionally
other therapeutics may be administered per se (neat) or in the form
of a pharmaceutically acceptable salt. When used in medicine the
salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically acceptable salts thereof. Such salts
include, but are not limited to, those prepared from the following
acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric,
maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric,
methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0129] Suitable buffering agents include: acetic acid and a salt
(1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a
salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
Suitable preservatives include benzalkonium chloride (0.003-0.03%
w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and
thimerosal (0.004-0.02% w/v).
[0130] The pharmaceutical compositions of the invention contain an
effective amount of a therapeutic compound of the invention
optionally included in a pharmaceutically-acceptable carrier. The
term pharmaceutically-acceptable carrier means one or more
compatible solid or liquid filler, diluents or encapsulating
substances which are suitable for administration to a human or
other vertebrate animal. The term carrier denotes an organic or
inorganic ingredient, natural or synthetic, with which the active
ingredient is combined to facilitate the application. The
components of the pharmaceutical compositions also are capable of
being commingled with the compounds of the present invention, and
with each other, in a manner such that there is no interaction
which would substantially impair the desired pharmaceutical
efficiency.
[0131] The therapeutic agents may be delivered to the brain using a
formulation capable of delivering a therapeutic agent across the
blood brain barrier. One obstacle to delivering therapeutics to the
brain is the physiology and structure of the brain. The blood-brain
barrier is made up of specialized capillaries lined with a single
layer of endothelial cells. The region between cells are sealed
with a tight junction, so the only access to the brain from the
blood is through the endothelial cells. The barrier allows only
certain substances, such as lipophilic molecules through and keeps
other harmful compounds and pathogens out. Thus, lipophilic
carriers are useful for delivering non-lipohilic compounds to the
brain. For instance, DHA, a fatty acid naturally occurring in the
human brain has been found to be useful for delivering drugs
covalently attached thereto to the brain (Such as those described
in U.S. Pat. No. 6,407,137). U.S. Pat. No. 5,525,727 describes a
dihydropyridine pyridinium salt carrier redox system for the
specific and sustained delivery of drug species to the brain. U.S.
Pat. No. 5,618,803 describes targeted drug delivery with
phosphonate derivatives. U.S. Pat. No. 7,119,074 describes
amphiphilic prodrugs of a therapeutic compound conjugated to an
PEG-oligomer/polymer for delivering the compound across the blood
brain barrier. The compounds described herein may be modified by
covalent attachment to a lipophilic carrier or co-formulation with
a lipophilic carrier. Others are known to those of skill in the
art.
[0132] The therapeutic agents of the invention may be delivered
with other therapeutics for enhancing memory retrieval or treating
other symptoms or causes of disorders associated with the memory
loss. For instance, environmental enrichment (EE) has been used for
enhancing memories. EE involves creating a stimulating environment
around a subject. Other therapeutics may also be combined to treat
the underlying disorder or to enhance memory recall.
[0133] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting.
EXAMPLES
[0134] The following examples involve the use of a CK-p25 Tg mouse
model to evaluate therapeutic strategies aimed at restoring
learning and the access to long-term memory after synaptic and
neuronal loss had already occurred. We show that up-regulation of
the plasticity and function of the remaining neurons reinstates
learning and memory in a degenerated brain. CK-p25 Tg mice allow
for control of the onset and extent of neurodegeneration, thereby
accessing the determinants of consolidated long-term memories
experimentally. It was previously shown that a single fear
conditioning trial results in a stable long-term memory that is
initially encoded in the hippocampus but likely transferred to the
cortical network after 3-4 weeks (7). A similar time-window is
reported for human long-term memories (8).
[0135] The methods show a distinction between the learning and
memory aspects of neurodegenerative disease. The invention provides
methods both for the treatment of long-term memory loss and memory
lapse. In some embodiments these treatments are directed towards
subjects with neurodegenerative disease and synaptic loss.
Individuals suffering from neurodegenerative diseases can display
impairments that draw a distinction between learning & memory.
For example, while patients have difficulty learning new
information, they also suffer from inability to recognize close
relatives and other attributes of long-term memory. In some aspects
the invention provides methods for the recovery of impaired
learning and lost long-term memories after animals developed severe
neurodegeneration and synaptic loss.
[0136] Methods
[0137] Environmental enrichment: Up to four mice were continuously
housed in a cage that contained two wheels for voluntary running
and a variety of toys (obtained form from Petco) to create tunnels,
and climbing devices. Food and water was ad libitum. The food was
hidden within the bedding. Toys and running wheels were changed on
a daily basis.
[0138] Learning tests: All behavioral testing is described in
Fischer et al. (3).
[0139] Cannulation and injection: Microcannula were inserted into
the lateral brain ventricles as described by Fischer et al. (24).
Sodiumbutyrate (Sigma; St. Louis, Mo.) was dissolved in artificial
cerebrospinal fluid (aCSF). A stock solution of TSA (Sigma) was
dissolved in DMSO and diluted with aCSF before injection.
[0140] Immunoblotting and staining: Lysates for immunoblotting were
prepared as described by Fischer et al. (3). To isolate histones,
brain tissue was homogenized in TX-buffer (50 mM Tris HCL, 150 mM
NACL, 2 mM EDTA, 1% Triton-100) and incubated at 4.degree. C. for
15 min prior to centrifugation at 2000 rpm for 10 min. After a
wash-step in TX-buffer the pellet was dissolved in TX-buffer
containing 0.2M HCl and incubated on ice for 30 min, before a
second centrifugation at 10000 rpm for 10 min. The supernatant was
either dialysed or directly used for immunoblotting. Antibodies
were used in 1:1000 concentrations. All antibodies detecting
histones and anti-PSD-95 were form Upstate (Lake Placid, N.Y.).
Anti-synaptophysin (SVP38) was from Sigma. Anti-neuronal nuclei
(neuN) and anti-growth associated protein (Gap43) were from
Chemicon (Temecula, Calif.) and anti-N-cadherin,
anti-.beta.-catenin were from Santa Cruz (Santa Cruz, Calif.).
Immunostaining was performed as described by Fischer et al. (3).
Antibodies mentioned above were used in a 1:500 dilution.
Anti-MAP-2 antibody (Sigma) was used in a 1:200 dilution.
[0141] Statistical analysis: The data were analyzed by unpaired
student's t test and one-way ANOVA (ANalyis Of VAriance). One-way
ANOVA followed by post-hoc Scheffe's test was employed to compare
means from several groups. Error bars present S.E.M.
[0142] Results
Example 1
The Effect of EE on Learning Behavior after Neuronal Loss
[0143] To investigate the effect of EE on learning behavior after
neuronal loss had already occurred, p25 was induced in 11-month old
CK-p25 Tg mice for 6 weeks. Afterwards p25 expression was repressed
(3) and one group of CK-p25 Tg mice was subjected to EE, whereas
the other group was not enriched. Subsequently all mice, including
a control group that did not express p25, were subjected to fear
conditioning and water maze learning (n=8/group) (FIG. 1a). Despite
a comparable extent of brain atrophy (P=0.2435 enriched vs.
nonenriched CK-p25 Tg mice; P<0.0001 enriched/non-enriched
CK-p25 Tg mice vs. control), EE treated CK-p25 Tg mice showed
markedly increased associative and spatial learning when compared
to the non-enriched CK-p25 Tg mice (FIG. 1b, FIG. 7a). Non-enriched
CK-p25 Tg mice displayed impaired freezing behavior, when compared
to control mice (P=0.0337), indicating impaired associative
learning. Enriched CK-p25 Tg mice showed significantly more
freezing when compared to the non-enriched group (P<0.0001)
(FIG. 1c). In addition, the escape latency in the water maze test
was significantly impaired in non-enriched CK-p25 Tg mice when
compared to control mice (F.sub.1,568=154,814; P<0.001).
Enriched CK-p25 Tg mice performed significantly better than
non-enriched CK-p25 mice (F.sub.1,568=77.167; P<0.0001), but
still displayed impaired performance when compared to the control
group (F.sub.1,568=49.453; P<0.0001) (FIG. 1e). These data show
that EE can reinstate learning ability in mice with severe
neurodegeneration.
[0144] Plotting brain weight against the freezing behavior of
individual mice showed that improved learning but not brain weight
is associated with EE in CK-p25 Tg mice. No correlation between
brain weight and freezing behavior was found (P=0.9993) reflecting
that non-enriched and enriched CK-p25 Tg mice display brain atrophy
to the same degree but the freezing behavior of enriched CK-p25 Tg
mice is similar to the control group that shows no brain atrophy
(FIG. 1d). Consistently, the neuronal marker protein NeuN was
similarly reduced in EE treated and untreated CK-p25 Tg mice.
Notably, levels of synaptic markers proteins, synaptophysin and
MAP-2 immunoreativitiy (IR) were significantly higher in EE treated
CK-p25 Tg mice when compared to non-enriched CK-p25 Tg mice.
Although NeuN levels were decreased to similar levels, several
markers for synaptic plasticity and integrity were increased in
enriched when compared to non-enriched CK-p25 Tg mice (FIG. 1f, g;
see also FIG. 7) (*P<0.05 enriched vs. non enriched CK-p25 Tg
group, n=3. Quantification (*P<0.05 non enriched CK-p25 Tg
group, n=3, vs. control and enriched CK-p25 Tg mice). SVP (synaptic
vesicle protein) staining is commonly used to analyze synaptic
plasticity in animal models for neurodegeneration or post mortem
tissue from human AD patients. Confocal images (1 .mu.m) were
scanned and subjected to three-dimensional reconstruction. The
pictures are displayed as seen from the x-axis. LSMeta10 software
(Zeiss; Jena, Germany) was used to calculate the mean SVP
intensity. Brain sections with the strongest intensity were scanned
first. All other images included in the analysis where scanned
using the same microscope setting. Non-enriched CK-p25 Tg mice
showed significantly reduced hippocampal SVP intensity
(P<0.0001), when compared to control mice (no p25). Enriched
CK-p25 Tg mice displayed significantly increased hippocampal SVP
intensity when compared to non-enriched CK-p25 Tg mice (P=0.0304).
Scale bar: 20 .mu.m. py, pyramidal cell layer (FIG. 1g).
[0145] The data presented in this example indicate that EE promoted
growth of new dendrites and synapses in CK-p25 Tg mice. Thus,
despite the substantial loss of neurons, EE induced the refinement
of the synaptic network, which causes improved learning in the
CK-p25 Tg mice. Exposure of wild type mice to EE facilitated their
learning ability and caused elevated levels of marker proteins for
synaptic integrity and plasticity indicating dendritic branching
and synaptogenesis (See also FIG. 5 and FIG. 6).
Example 2
Experimental Model for the Investigation of Long-Term Memories
[0146] To establish an experimental model that allows investigation
of the fate of long-term memories, 11-month old CK-p25 Tg and
control mice were trained in the fear conditioning paradigm and
returned to their home cages for 4 weeks to allow the consolidation
of hippocampus-independent long-term memories. Subsequently p25 was
induced for either 3 (Exp. 1) or 6 weeks (Exp. 2) before the mice
were subjected to the memory test. These time points were chosen
because, in contrast to 6-week induction, after 3 weeks of p25
expression no overt pathology was observed (3) (FIG. 2a). In Exp.
1, CK-p25 Tg mice (n=8) showed similar freezing than control mice
(n=9; P=0.863) (FIG. 2b; Exp.1). In contrast, CK-p25 Tg mice
induced for 6 weeks (n=9) showed significant reduction in freezing
behavior during the memory test performed 10 weeks after the
training when compared to the control group (n=16; P<0.0001),
showing that the access to long-term memories has been lost. (FIG.
2b; Exp.2). The loss of consolidated long-term memory was also
evident in the water maze paradigm (FIG. 9a, b).
[0147] If memories became inaccessible due to synaptic and neuronal
loss it might be possible to re-establish the access to such
memories if sufficient refinement of the neuronal network can be
achieved by the remaining neurons. To investigate if memories to
became inaccessible due to synaptic and neuronal loss CK-p25 Tg and
control mice were trained in the fear conditioning paradigm and
returned to their home cages for 4 weeks followed by 6 weeks of p25
induction. Next, the mice were either subjected to EE or kept in
their home cages for an additional 4 weeks. The experiment was
performed as described under (a) but after 6 weeks of p25
induction, p25 expression was repressed and one group of CK-p25 Tg
mice was subjected to EE (n=13) whereas the other group was not
enriched (n=9). Control groups consisted of non-enriched mice that
were trained but did not express p25 (n=22) and an enriched group
that did not express p25 and were not subjected to the training
(n=6). (FIG. 2c). Afterwards all mice were subjected to the memory
test. Whereas non-enriched CK-p25 Tg mice showed significantly
impaired freezing behavior, indicating the loss of long-term
memories, enriched CK-p25 Tg mice displayed much improved freezing
behavior, indicating a marked recovery of long-term memories.
Whereas non-enriched CK-p25 Tg mice displayed decreased freezing
when compared to control mice (P<0.0001), enriched CK-p25 Tg
mice performed significantly better (P<0.0001 vs. non enriched
CK-p25 group) (FIG. 2d).
[0148] Enriched and non-enriched CK-p25 Tg mice have a similar
extent of neuronal loss and brain atrophy in the anterior
cingulated cortex (FIG. 2e; Right: Representative images showing
NeuN staining and brain atrophy; Left: Brain weight was plotted
against freezing behavior). No correlation between brain weight and
freezing was found. Evidence for the recovery of long-term memories
was also found by employing the water maze paradigm (FIG. 9a, c).
The fact that long-term memories can be recovered by EE supports
the idea that the apparent "memory loss" is really a reflection of
inaccessible memories. These findings are in line with the
phenomena known as "fluctuating memories" where demented patients
experience temporary time periods of apparent clarity (9, 10).
[0149] The anterior cingulate cortex (ACC) is implicated with the
consolidation and encoding of long-term memories (11). CK-p25 Tg
mice with no EE treatment displayed reduced synaptophysin-IR in the
ACC when compared to control mice (FIG. 2f). Non-enriched CK-p25
mice showed significantly reduced SVP intensity, when compared to
control mice. Enriched CK-p25 mice displayed significantly
increased hippocampal SVP intensity when compared to non-enriched
CK-p25 mice (P=0.0251). In contrast, cortical levels of synaptic
marker proteins, synaptophysin- and MAP-2 IR were increased in
enriched, relative to non-enriched CK-p25 Tg mice (FIG. 2f; FIG.
7c, d). These data show that EE leads to the recovery of long-term
memories by re-establishing the synaptic network.
Example 3
Mechanism Underlying EE
[0150] Other than a few genes involved in synaptic function,
relatively little is known about the mechanism underlying EE
(12-14). Since histone acetylation, an epigenetic mechanism
regulating gene expression via chromatin remodeling, has recently
been implicated in synaptic plasticity and learning behavior
(15-18), the induction of a transcriptional program by EE that
leads to activation of plasticity genes was evaluated. Wild type
mice were subjected to EE and hippocampal and cortical brain
lysates were obtained 3, 24 h or 2 weeks later (n=3/group) by acid
extraction and probed with antibodies detecting histone-tail
modification that indicate active chromatin. Increases in
hippocampal H3 (K-9, K14) and H4 (K-5) and in cortical H3 (K-9) and
H4 (K-5) acetylation were observed. In addition increased
methylation of H3 (K4) was observed in cortical lysates. No changes
in H4 acetylation were observed when the Ac-H4 (K5,8,12,16)
antibody was used, which is likely due to the fact that H4-K16 is a
marker of condensed chromatin. EE induced hippocampal and cortical
histone 3 (H3) and histone 4 (H4) acetylation and methylation as
soon as 3 hours after treatment (FIG. 3a, b). In addition,
intraperitoneal [ip] and intracerebroventricular [icv] injections
of histone deacetylases (HDACs) inhibitors sodiumbutyrate (SB) or
trichostatin (TSA) significantly facilitated associative learning
in wild type mice (FIG. 8a). To investigate whether inhibition of
HDACs mimics the effects of EE, SB was administered daily [ip] into
wild type mice for 4 weeks (FIG. 3c). This procedure resulted in a
robust increase in H3- and H4 acetylation in the hippocampus (FIG.
3d). Two days after the last injection mice were trained in the
fear conditioning paradigm. When tested 24 h later, SB injected
mice showed more freezing during the memory test (P<0.0001)
(FIG. 3e). Similarly, when trained in the water maze paradigm SB
injected mice displayed a shorter escape latency
(F.sub.1,138=24.119; P<0.0001) (FIG. 30 when compared to the
vehicle group. In addition increased hippocampal MAP-2 and
synaptophysin IR and elevated levels of synaptic and dendritic
marker proteins in SB-injected mice was observed (FIG. 8d-f).
Locomotor activity or basal anxiety was not altered in SB-injected
mice (FIG. 8c).
Example 4
Reinstatement of Learning Behavior Through Inhibition of HDAC
[0151] Whether sustained inhibition of HDACs would reinstate
learning behavior and recover access to long-term memories in
CK-p25 Tg mice that had developed synaptic and neuronal loss was
investigated. P25 was induced in 11 month old CK-p25 Tg mice for 6
weeks. Afterwards p25 expression was repressed and one group of
CK-p25 Tg mice was subjected to daily sodiumbutyrate (SB) injection
(1.2 g/kg) for 4 weeks (n=9), whereas the other group received
saline injection (n=9). Subsequently all mice, including a control
group that did not express p25 (n=9), were subjected to fear
conditioning and water maze learning. To this end p25 expression
was induced in 11-month old CK-p25 mice for 6 weeks, before one
group was injected daily for 4 weeks with SB whereas the control
group received saline injection (FIG. 4a). Compared to the vehicle
group, SB treated CK-p25 Tg mice showed significantly enhanced
associative (freezing behavior) when compared to saline (vehicle)
injected CK-p25 Tg mice (P=0.009) (FIG. 4b) and spatial learning,
as shown by significantly shorter escape latency, when compared to
the vehicle group (F.sub.1,538=87.484; P<0.0001) (FIG. 4c), and
increased levels of synaptic marker proteins (FIG. 4d, e; FIG. 7 f,
h). Also, SB and vehicle-injected CK-p25 Tg mice displayed a
similar extent of brain atrophy and hippocampal neuronal loss (FIG.
7e). These findings show that increased histone acetylation using
the HDAC inhibitor SB can reinstate learning ability in mice
exhibiting severe neurodegeneration.
[0152] The effect of HDAC inhibition on the recovery of
inaccessible long-term memories was evaluated. Eleven month old
CK-p25 Tg mice were trained in the fear conditioning paradigm and
returned to their home cages for 4 weeks. Subsequently p25 was
induced for 6 weeks before the mice were injected with either
saline (vehicle, n=12) or SB (1.2 g/kg, n=14) for 4 weeks (daily).
Afterwards all mice, including a vehicle injected control groups
that did not express p25 (n=15) and a group of SB-injected control
mice that were not trained (n=8), were subjected the memory test.
(FIG. 4f). Vehicle injected CK-p25 Tg mice showed impaired access
to long-term memory as revealed by the markedly reduced freezing
behavior compared to the control mice that did not express p25.
When compared to the trained control group, saline-injected CK-p25
Tg mice showed a significant reduction in freezing behavior during
the memory test (P<0.0001) test, suggesting the loss of
consolidated long-term memories. SB-injected CK-p25 Tg mice
performed significantly better than vehicle injected CK-p25 Tg mice
(P=0.0496) (FIG. 4g). Despite a similar degree of brain atrophy and
neuronal loss (FIG. 7g), SB-injected CK-p25 Tg mice showed
significantly increased freezing and elevated levels of synaptic
marker proteins when compared to the vehicle CK-p25 Tg group (FIG.
4g-i; FIG. 7g, h). Similarly, SB administration also leads to the
recovery of long-term spatial memories (FIG. 9a, d). Thus chronic
injection of SB led to the recovery of memories in CK-p25 Tg mice
that had developed severe neuronal loss.
Example 5
Exposure to EE Facilitates Learning Ability in WT Mice
[0153] Exposure of WT mice to EE facilitated their learning ability
and caused elevated levels of marker proteins for synaptic
integrity and plasticity indicating dendritic branching and
synaptogenesis (6). Images showing a home cage and a cage used for
environmental enrichment (EE) are depicted in FIG. 5A. For EE
stimulus up to 4 mice were constantly housed in a large cage that
contained various toys and running wheels (1 wheel/2 mice). All
toys were changes on daily basis. C57BL6J mice (n=8/group) were
subjected to EE for 2 weeks and trained in the fear conditioning
paradigm. Enriched mice showed significantly more freezing behavior
during the memory test performed 24 h later, when compared to the
home cage group (P<0.0001), indicating facilitated associative
learning (FIG. 5b). Similar results were obtained in the Morris
water maze paradigm (P<0.0001 control vs. enrichment) (FIG.
5b).
[0154] To investigate the relationship between EE and synaptic
plasticity, C57BL6J mice (n=3/group) were subjected to EE for 2
weeks and subsequently used for analysis. Representative images
showing increased MAP-2 immunoreativitiy (IR) in the hippocampus
and anterior cingulate cortex of enriched mice are shown in FIG. 6a
and FIG. 6b. Representative images of the hippocampal and anterior
cingulate cortex region of enriched and non-enriched mice showing
staining of the synaptic marker protein synaptophysin (SVP) are
shown in FIG. 6c and FIG. 6d. Confocal images (1 .mu.m) were
scanned and subjected to three-dimensional reconstruction. Pictures
are displayed as seen from the x-axis. LSMeta10 software was used
to calculate the mean SVP intensity. Brain sections with the
strongest intensity were scanned first. All other images included
in the analysis where scanned using the same microscope setting.
Enriched mice displayed significantly increased hippocampal SVP
(IR) when compared to non-enriched mice. Representative immunoblots
from the hippocampus and cortex of enriched and non-enriched mice
show that several markers for synaptic plasticity and integrity
were increased in enriched mice when compared to non-enriched mice,
indicating facilitated synaptic plasticity (P<0.0001) (FIG. 6e).
**P<0.001. py, pyramidal cell layer; oriens; rad, stratum
radiatum; Lmol, molecular layer; mol, lower molecular layer; GrDG,
granula cell layer of the dentate gyrus, DG, dentate gyrus; CA1,
hippocampal regions CA 1; cg, cingulate cortex.
Example 6
Effects of Enrichment or Sodiumbutyrate Treatment on Plasticity
Factors in CK-p25 Tg Mice that Developed Severe
Neurodegeneration
[0155] For EE treatment p25 was induced in 11 month old CK-p25 Tg
mice for 6 weeks. Afterwards p25 expression was repressed as
described in (3) and one group of CK-p25 Tg mice was subjected to
EE. Representative pictures showing immunostaining for NeuN in the
hippocampal region confirm similar degree of hippocampal neuronal
loss in enriched and non-enriched CK-p25 Tg mice are shown in FIG.
7a. Brain sections from the same mice used in the experiment
described under of FIG. 7a were immunostained for the dendritic
marker protein MAP-2. MAP-2 immunoreativitiy of enriched CK-p25 Tg
mice increased when compared to the non-enriched group (FIG. 7b).
Similar results were observed in other brain regions such as the
anterior cingulated cortex (FIG. 7c). Representative immunoblots
from cortical lysates of control mice, enriched and non-enriched
CK-p25 Tg mice are shown in FIG. 7d. Although NeuN levels were
decreased to similar levels in enriched and non-enriched CK-p25 Tg
mice, several markers for synaptic plasticity and integrity were
increased in enriched when compared to non-enriched CK-p25 Tg mice.
*P<0.05 vs. non enriched group, n=3.
[0156] For SB treatment, p25 was induced in 11 month old CK-p25 Tg
mice for 6 weeks. Afterwards p25 expression was repressed and one
group of CK-p25 Tg mice was subjected to daily sodiumbutyrate (SB)
injection (1.2 g/kg) for 4 weeks, whereas the other group received
saline injection. Representative images showing hippocampal NeuN
staining confirming that SB and vehicle injected CK-p25 mice
displayed hippocampal neuronal loss to the same degree are shown in
FIG. 7e. Representative images showing hippocampal MAP-2 staining
are depicted in FIG. 7f. MAP-2 immunoreativitiy of CK-p25 Tg mice
treated with SB is increased when compared to the vehicle group.
Representative images showing NeuN staining of the ACC. While SB
and vehicle-injected CK-p25 Tg mice displayed similar levels of
neuronal loss in the ACC (light spots, NeuN staining) MAP-IR was
increased in SB treated when compared to vehicle treated CK-p25 Tg
mice are shown in FIG. 7g. Representative immunoblots from the
hippocampus and cortex of all groups are shown in FIG. 7h. Although
NeuN levels were decreased to similar levels in SB and vehicle
injected CK-p25 Tg mice, several markers for synaptic plasticity
and integrity were increased in SB-injected when compared to
vehicle injected CK-p25 Tg mice. *P<0.05 vs. vehicle group, n=3.
py, pyramidal cell layer; rad, stratum radiatum; Lmol, molecular
layer; mol, lower molecular layer; GrDG, granula cell layer of the
dentate gyms, DG, dentate gyrus; CA1, hippocampal regions CA1; cg,
cingulate cortex.
Example 7
The Effect of Sodiumbutyrate Injection on Learning, Basal Anxiety,
Explorative Behavior and Brain Plasticity
[0157] C57BL/6J mice were subjected to fear conditioning and
injected intraperitoneally [ip] with 1.2 g/kg sodiumbutyrate (SB,
n=8) or saline (vehicle; n=8) immediately afterwards. SB injected
mice displayed significantly more freezing behavior during the
memory test performed 24 h later (P<0.0001) (FIG. 8b). C57BL/6J
mice were implanted with microcannulae into the lateral brain
ventricles (icy) and injected with either 100 ng SB, 50 ng TSA or
vehicle immediately after fear conditioning (n=8/group). SB or TSA
injected mice displayed more freezing behavior than vehicle
injected mice during the memory test performed 24 h later
(P<0.0001) (FIG. 8b). C57BL/6J were daily injected [ip] with SB
or saline (n=8/group) for 4 weeks before all mice were subjected to
the elevated plus maze and open field test. No significant group
difference was observed indicating that SB-treatment did not affect
basal anxiety or explorative behavior (FIG. 8c). FIG. 8d shows
representative images showing increased MAP-2 immunoreativitiy (IR)
in the hippocampus of wild type mice injected with SB for 4 weeks,
when compared to vehicle injected mice. FIG. 8e shows
representative immunoblots (n=3/group) from hippocampal lysates of
wild type mice injected with SB or vehicle for 4 weeks. Immunoblots
were quantified relative to the optical density of the
vehicle-group (set to 1). Several markers for brain plasticity were
increased in SB injected mice. FIG. 8f shows representative images
with increased SVP IR in the hippocampus of mice injected with SB
for 4 weeks, when compared to vehicle injected mice (P=0.0243). The
analysis was performed as described under FIG. 1g.
Example 8
Recovery of Spatial Memories Upon Environmental Enrichment and
Sodiumbutyrate Injection
[0158] CK-p25 Tg mice in which p25 expression was repressed and
control mice were trained in the water maze paradigm until all mice
reliably found the hidden platform. Afterwards all mice were
returned to their home cages for 4 weeks to to allow the
consolidation of hippocampus independent long-term memories.
Afterwards p25 expression was induced for 6 weeks, followed by p25
repression. Pilot studies showed that a probe test, commonly used
to analyze memory retrieval in the water maze paradigm, was not a
reliable read out to analyze long-term memory retrieval. In fact,
without extensive re-training even wild type mice showed no
significant preference for the target quadrant when tested 10 weeks
after the training in a probe test (data not shown). To measure the
retrieval of long-term memory mice were instead exposed to only 2
reminder-training sessions on a single day. The mean escape latency
during the reminder-training sessions was compared to control mice
that did not receive the initial training. Using this approach
control mice that did not receive the initial training (n=7) and
showed a significantly longer escape latency than trained control
mice, suggesting that this difference reflects spatial long-term
memory in trained control mice. Notably, 6-week induced trained
CK-p25 Tg mice (n=8) showed significantly longer escape latency
than trained control mice (n=8) during the reminder-training
session. These data indicate that 6 weeks of p25 expression causes
loss of spatial memory (FIG. 9b). The recovery of spatial memories
upon environmental enrichment was evaluated by a similar
experiment, except that after 6 weeks of p25 expression mice were
either subjected to EE or returned to their home cages for 4 weeks.
When the memory test was performed as described above non-enriched
CK-p25 Tg mice (n=8) showed significantly reduced escape latency
when compared to the trained control group (n=8). However, the
escape latency of enriched CK-p25 Tg mice was significantly shorter
when compared to non-enriched CK-p25 Tg mice. Enriched control mice
that did not receive the initial training served as a control to
delineate learning from long-term memory retrieval. These data
provide evidence that spatial long-term memories can recover upon
EE (FIG. 9c). To evaluate the recovery of spatial memories upon SB
injection, a similar experiment was performed as described above,
but instead of EE, one group of CK-p25 Tg mice (n=10) received
daily injection of SB, whereas the other group was injected with
vehicle (n=10). Similar to the data described above, the results
provide evidence that spatial long-term memories recover after
neuronal loss upon injection of SB. *P<0.001 vs. CK-p25 vehicle
or "no p25 no training" groups (FIG. 9d).
Example 9
The Over Expression of HDAC2, but not HDAC1, Impairs Fear
Conditioning without Affecting Sensory-Motor Functions
[0159] A fear conditioning experiment is depicted in FIG. 10a was
performed as previously described (24). Mice were trained in
chamber for 3 minutes, following by a 30-second tone cue
immediately before a 0.8 mA electoral stimulation. Mice were
represented in the same chamber without tone cue after 24 hrs and
the time freezing behavior appeared was recorded. Tone-induced
freezing was recorded in another chamber with different decoration
24 hrs later. The freezing behavior was monitored during the
context- and tone-dependent memory test of HDAC1 transgenic mice
(Tg), HDAC2 transgenic mice, and control mice (FIG. 10b; HDAC1 Tg
n=17; HDA 2 Tg n=14; control, n=19). HDAC2 Tg mice showed impaired
fear conditioning. During the initial 3 min exposure to the
training box, no significant difference between distances traveled
could be detected between HDAC1 Tg, HDAC2 Tg, and control mice
(FIG. 10c). Average activity during the training procedure, and an
increased activity during electoral stimulation, reflected an
escape response to the electric foot shock that was applied during
the training procedure. No difference could be detected between
HDAC1 Tg, HDAC2 Tg, and control mice (FIG. 10d).
Example 10
The Over Expression of HDAC2 Impairs Spatial Learning
[0160] The water maze paradigm (Morris, R. G. M., Garrud, P.,
Rawlins, J. N. P., O'Keefe, J. 1982. Place navigation impaired in
rats with hippocampal lesions. Nature, 297, 681-683) was performed
in a circular tank (diameter 2 m) filled with opaque water. A
platform (11.times.11 cm) was submerged below the water's surface
in the center of the target quadrant. The swimming path of the mice
was recorded by a video camera and analyzed by the Videomot 2
software (TSE). For each training session, the mice were placed
into the maze subsequently from four random points of the tank.
Mice were allowed to search for the platform for 60 seconds. If the
mice did not find the platform within 60 seconds, they were gently
guided to it. Mice were allowed to remain on the platform for 15
seconds. During the memory test (probe test), the platform was
removed from the tank, and the mice were allowed to swim in the
maze for 60 s. HDAC2 transgenic mice (n=8) and control mice (n=8)
were subjected to the water maze paradigm for nine consecutive
days, with 2 trials everyday (FIG. 11a). The hidden platform was
located in the target quadrant (T). Escape latencies of control
mice improved significantly faster than control HDAC2 Tg mice
throughout the training (p=0.0044). In the probe test, performed
after 5 days of training trials, control mice (p=0.0145) spent
significantly more time in the target quadrant (T) than HDAC2 Tg
mice (FIG. 11b).
Example 11
The HDAC 2 Gene Knockout Enhances Associative Learning
[0161] The freezing behavior of HDAC2 knockout (KO) mice and
control mice (HDAC2 KO n=10; control, n=10) during the contextual
dependent memory test is shown in FIG. 12a. HDAC2 KO mice showed
enhanced fear conditioning. The distance traveled during the
initial 3 minutes exposure to the training box (FIG. 12b). No
significant difference could be detected between HDAC2 KO and
control mice. Average activity during the training procedure and an
increased activity during electoral stimulation reflected an escape
response to the electric foot shock that was applied during the
training procedure (FIG. 12c). No difference could be detected
between HDAC2 KO and control mice.
Example 12
The Over Expression of HDAC 2 Decreases Newly Generated Neurons in
Dentate Gyrus of Adult Mice
[0162] Representative images are shown of hippocampal brain
sections from two month-old--mice group (n=3/group) immunostained
for the newborn neuron marker Doublecortin (DCX). Representative
pictures show DCX immunoreativitiy (light regions) in the
hippocampus dentate gyrus, scale bar, 40 um (FIG. 13a). Note the
significant decrease in HDAC2Tg group (p=0.0193). Representative
pictures show BrdU immunoreativitiy (light regions) in the
hippocampus dentate gyrus (FIG. 13b). BrdU was i.p. injected into
two-month-old mice groups (n=3) 30 days before analysis. Note the
significant decrease in HDAC2Tg group (p=0.0001).
Example 13
HDAC 2 Tgs Show Reduced CREB Expression in Hippocampus
[0163] Representative images of hippocampal brain sections from
two-month-old mice group (n=3/group) immunostained for the CREB in
CA1 and CA3 regions (FIG. 14a). Representative pictures show CREB
immunoreativitiy (light regions) in the hippocampus dentate gyrus,
DAPI, blue, scale bar, 150 um. Protein levels are analyzed in
forebrain lysates of all experimental groups (n=3/group) by
immunoblotting (FIG. 14b).
Example 14
HDAC2 Directly Binds to the Promoter Region of Specific Genes and
Regulates Gene Expression
[0164] Whole brains from two-month-old experimental groups were
fixed and homogenized. Lysates of all experimental groups were
immune-precipitated with HDAC2 antibody and subjected to
semi-quantitative PCR analysis (FIG. 15). DNA samples taken
directly from lysates were prepared in 100 ng/ul (WBL). Equal
volumes of ChIP samples from each group were used in PCR analysis.
Primers were designed to target the promoter region of specific
genes. Note that WT, but not the HDAC2 KO group, showed a strong
signal in PCR amplification for the promoter region of CREB and
BDNF, indicating that HDAC2 directly binds to those regions. Globin
and Tubulin showed equal amounts of amplification in WT and LO
group.
Example 15
The Effects of SAHA and MS-275, Relative to Sodium Butarate (SB),
on the Hippocampus Dependent Associative Learning in Wildtype
Mice
[0165] After chronic treatment for 10-21 days, we found that SAHA
is more potent than SB in facilitating associative learning using
the contextual fear conditioning paradigm (FIG. 16a). Conversely,
MS-275, a class 1 HDAC inhibitor, does not facilitate associative
learning in mice (FIG. 16b). Brain lysates from SAHA and MS-275
treated mice exhibit increased acetylation of lys9lys14 on histone
3 and lys5 on histone 4 compared to saline treated mice (FIG. 16c)
demonstrating that both drugs affect HDAC activity in the
brain.
Example 16
HDAC Inhibitors Regulate Neuronal Activity and Neurogenesis in
Dentate Gyrus
[0166] In agreement with its beneficial effect on associative
learning, SAHA treatment increased BrdU positive cells in the
subgranule zone of the dentate gyrus (FIG. 17a). In addition, DCX,
a marker for newly generated neurons, labeled more cells in SAHA
treated but not MS-275 treated dentate gyrus (FIG. 17b). These
observations suggest that SAHA upregulates adult neurogenesis in
the dentate gyrus. Furthermore, SAHA treated mice showed higher
number of c-fos positive cells after fear conditioning training but
MS-275 treated mice showed lower number of c-fos positive cells
compared to saline treated group (FIG. 17c). Thus, activity induced
c-fos expression may also be used to evaluate the effect of HDAC
inhibitors on hippocampus dependent learning. FIG. 17d is a bar
graph depicting the significant enhancement by SAHA.
Example 17
Cell-Based and Biochemical Assays for Optimizing HDAC Inhibitor
Potency and Selectivity
[0167] To assess the effects of compounds on histone acetylation as
part of the proposed medicinal chemistry we have implemented an
automated microscopy system that enables the rapid and quantitative
assessment of chromatin modifications over a wide dynamic range and
the simultaneous measurement of the effect of compounds on cell
viability, morphology, and proliferation (FIGS. 18a-c). To measure
the in vitro biochemical selectivity of novel HDAC inhibitors, we
have optimized a 384-well plate based fluorimetric deacetylase for
class I and class II HDACs (FIG. 18d) and discovered that apicidin,
an ethyl ketone containing, non-hydroxamic acid, is a potent HDAC5
(class 2) inhibitor (FIG. 18e). Recombinant HDAC1-11 preparations
will allow testing of the selectivity and potency of inhibitors as
part of the medicinal chemistry efforts proposed here. Using
additional biochemical assays of a diverse collection of over 1,500
compounds, we discovered two new biasing elements for HDAC
inhibition (FIG. 180 that show selectivity for class II HDACs (FIG.
18g).
Example 18
UW_WT-I.sub.--06 Significantly Enhances Associative Learning
[0168] The structure of UW_WT-I.sub.--06 and UW_WT-I.sub.--05 are
shown in FIG. 20. Mice were injected 10 days with either 5 mg/kg of
MS275 or 25 mg/kg of UW_WT-I.sub.--06 or UW_WT-I.sub.--05 before
training and memory tests as described above. UW_WT-I.sub.--06 was
discovered to significantly enhance associative learning. These
compounds show strong inhibitory activity toward HDAC 1, 2, and 10
with UW_WT-I.sub.--06 also inhibiting HDAC 6 and 8. The results are
shown in FIG. 21. [0169] 1. Andorfer, C. et al. Cell-cycle reentry
and cell death in transgenic mice expressing nonmutant human tau
isoforms. J neurosci 25, 5446-5454 (2005). [0170] 2. Santacruz, K.
et al. Tau suppression in a neurodegenerative mouse model improves
memory function. Science 309, 476-481 (2005). [0171] 3. Fischer,
A., Sananbenesi, F., Pang, P. T., Lu, B. & Tsai, L. H. Opposing
roles of transient and prolonged expression of p25 in synaptic
plasticity and hippocampus-dependent memory. Neuron 48, 825-838
(2005). [0172] 4. Cruz, J. C. & Tsai, L. H. Jekyll and Hyde
kinase: roles for Cdk5 in brain development and disease. Curr Opin
Neurobiol. 14, 390-394 (2004). [0173] 5. Cruz, J. C., Tseng, H. C.,
Goldman, J. A., Shih, H. & Tsai, L. H. Aberrant Cdk5 activation
by p25 triggers pathological events leading to neurodegeneration
and neurofibrillary tangles. Neuron 40, 471-83 (2003). [0174] 6.
Nithianantharajah, J. & Hannan, A. J. Enriched environments,
experience-dependent plasticity and disorders of the nervous
system. Nat Rev Neurosci 7, 697-709 (2006). [0175] 7. Kim, J. J.
& Fanselow, M. S. Modality-specific retrograde amnesia of fear.
Science 256, 675-7 (1992). [0176] 8. Gilmore, E. C. & Herrup,
K. Neocortical cell migration: GABAergic neurons and cells in
layers I and VI move in a cyclin-dependent kinase 5-independent
manner. J Neurosci 21, 9690-700 (2001). [0177] 9. Bradshaw, J.,
Saling, M., Hopwood, M., Anderson, V. & Brodtmann, A.
Fluctuating cognition in dementia with Lewy bodies and Alzheimer's
disease is qualitatively distinct. J Neurol Neurosurg Psychiatry
75, 382-387 (2004). [0178] 10. Palop, J. J., Chin, J. & Mucke,
L. A network dysfunction perspective on neurodegenerative diseases.
Nature 443, 768-773 (2006). [0179] 11. Frankland, P. W., Bontempi,
B., Talton, L. E., Kaczmarek, L. & Silva, A. J. The involvement
of the anterior cingulate cortex in remote contextual fear memory.
Science 304, 881-883 (2004). [0180] 12. Need, A. C. & Giese, K.
P. Handling and environmental enrichment do not rescue learning and
memory impairments in alphaCamKII(T286A) mutant mice. Genes Brain
Behav. 2, 132-139 (2003). [0181] 13. Tang, Y. P., Wang, H. S.,
Feng, M., Kyin, Y. Z. & Tsien, J. Z. Differential effects of
enrichment on learning and memory function in NR2B transgenic mice.
Neuropharmacology, 779-790 (2001). [0182] 14. Rampon, C. et al.
Effects of environmental enrichment on gene expression in the
brain. Proc Natl Acad Sci USA 97, 12880-12884 (2000). [0183] 15.
Levenson, J. M. et al. Regulation of histone acetylation during
memory formation in the hippocampus. J Biol Chem. 279, 40545-40559
(2004). [0184] 16. Kumar, A. et al. Chromatin remodeling is a key
mechanism underlying cocaine-induced plasticity in striatum. Neuron
48, 303-314 (2005). [0185] 17. Alarcon, J. M. et al. Chromatin
acetylation, memory, and LTP are impaired in CBP+/- mice: a model
for the cognitive deficit in Rubinstein-Taybi syndrome and its
amelioration. Neuron 42, 947-959 (2004). [0186] 18. Korzus, E.,
Rosenfeld, M. G. & Mayford, M. CBP histone acetyltransferase
activity is a critical component of memory consolidation. neuron
42, 961-972 (2004). [0187] 19. Voss, H. U. et al. Possible axonal
regrowth in late recovery from the minimally conscious state. J
Clin Invest. 116, 2005-2011 (2006). [0188] 20. van Praag, H.,
Kempermann, G. & Gage, F. H. Neuronal consequences of
environmental enrichment. Nat Rev Neurosci 1, 191-198 (2000).
[0189] 21. Horn, D., Ruppin, E., Usher, M. & Hermann, M. Neural
network modeling of Alzheimer's Disease. Neural Computation 5,
736-749 (1993). [0190] 22. Ruppin, E., Reggia, J. A. & Horn, D.
Pathogenesis of schizophrenic delusions and hallucinations: a
neural model. Schizophr Bull 22, 105-123 (1996). [0191] 23. Horn,
D., Levy, N. & Ruppin, E. Neuronal-based synaptic compensation:
a computational study in Alzheimer's disease. Neuroal comput 8,
1227-1243 (1996). [0192] 24. Fischer, A., Sananbenesi, F., Schrick,
C., Spiess, J. & Radulovic, J. Cyclin-dependent kinase 5 is
required for associative learning. J Neurosci 22, 3700-7. (2002).
[0193] 25. Frey R R, Wada C K, Garland R B, Curtin M L, Michaelides
M R, Li J, Pease L J, Glaser K B, Marcotte P A, Bouska J J, Murphy
S S, Davidsen S K. Trifluoromethyl ketones as inhibitors of histone
deacetylase. Bioorg Med Chem Lett 2002; 12:3443-3447. [0194] 26.
Wada C K, Frey R R, Ji Z, Curtin M L, Garland R B, Holms J H, Li J,
Pease L J, Guo J, Glaser K B, Marcotte P A, Richardson P L, Murphy
S S, Bouska J J, Tapang P, Magoc T J, Albert D H, Davidsen S K,
Michaelides M R. Alpha-keto amides as inhibitors of histone
deacetylase. Bioorg Med Chem Lett 2003; 13:3331-3335. [0195] 27.
Flaggarty S J, Wong J C, Koeller K M, Butcher R A, Schreiber S L.
Multidimensional chemical genetic analysis of diversity-oriented
synthesis-derived deacetylase inhibitors using cell-based assays.
Chem Biol 2003a; 10:383-396.
[0196] All references, patents and patent publications that are
recited in this application are incorporated in their entirety
herein by reference.
[0197] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
* * * * *