Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF INHIBITING CALCINEURIN WITH APOE ANALOGS
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of U.S. Provisional Application
Serial No.
60/953,043, filed July 31, 2007, which is herein incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[002] The invention relates to methods of inhibiting calcineurin activity with
ApoE analogs.
The invention also relates to methods of treating demyelinating disorders and
spinal cord injury
by modulating calcineurin activity.
BACKGROUND OF THE INVENTION
[003] Calcineurin, also known as PP2B, is a calcium-dependent serine/threonine
phosphatase
that plays a pivotal role in the activation of T cells during the immune
response. Calcineurin
dephosphorylates the NFAT (nuclear factor of activated T cells) transcription
factor, which
causes a conformational change that reveals a nuclear localization signal.
NFAT is then
translocated to the nucleus where it induces transcription of a number of
cytokines, such as
interleukin-2, that orchestrate the immune response. Calcineurin is targeted
by the
immunosuppressant drugs, cyclosporin A and FK506. Thus, one approach to the
design of
immunosuppressant therapies for preventing transplant rejection and treating
autoimmune
disorders is the development of novel calcineurin inhibitors.
[004] In addition to its role in activation of the immune response,
calcineurin signaling has
been implicated in a number of other functions, such as angiogenesis, learning
and memory,
schizophrenia, myocardial hypertrophy, skeletal muscle differentiation,
apoptosis, intimal
hyperplasia, and heart disease (Aramburu et al. (2004) EMBO reports, Vol. 5:
343-348; Bueno et
al. (2002) Cardiovascular Research, Vol. 53: 806-82 1). In recent years,
calcineurin has been
identified as a mediator of calcium-dependent axon repulsion in the central
nervous system
induced by myelin-associated proteins. Such axon repulsion limits the ability
of neuronal axons
to regenerate after an injury to the spinal cord and thus prevents functional
recovery.
Furthermore, oligodendrocytes, which are the cells that myelinate the axons of
the central
nervous system, are particularly vulnerable to apoptosis following injury.
Loss of these cells
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leads to demyelination and further loss of neuronal function (Liu et al.
(1997) J. Neurosci., Vol.
17: 5395-5406). Calcineurin has been reported to promote apoptosis by
activating the pro-
apoptotic protein Bad through dephosphorylation (Wang et al. (1999) Science,
Vol. 284:339-
343). Inhibition of calcineurin after spinal cord injury in rats has been
shown to result in a fewer
number of apoptotic oligodendrocytes, suggesting that calcineurin is a
mediator of apoptosis in
these cells (Nottingham et al. (2002) Exp. Neurol., Vol. 177: 242-25 1).
Therefore, calcineurin
activation exacerbates neuronal damage caused by spinal cord injury by
promoting loss of
oligodendrocytes and hindering regeneration of damaged axons by mediating axon
repulsion
mechanisms. Thus, there is a need for the development of novel calcineurin
inhibitors for
treating spinal cord injury.
[005] Other conditions, such as ischemia and multiple sclerosis, are
associated with apoptosis
of oligodendrocytes and demyelination of nerve fibers. Inhibition of
calcineurin would be an
effective therapeutic approach for treating these diseases as well. In the
case of multiple
sclerosis, inhibition of calcineurin would not only enhance the survival of
oligodendrocytes and
reduce demyelination, but also suppress the inflammatory response, which is
dysfunctional in
this autoimmune disorder. Given that aberrant calcineurin signaling appears to
be associated
with abnormal cell function and various disease states, the development of
agents that regulate
this phosphatase could be effective therapeutics in treating a number of
conditions.
SUMMARY OF THE INVENTION
[006] The present invention is based, in part, on the surprising discovery
that ApoE analogs
directly interact with and inhibit calcineurin activity. Accordingly, the
invention provides a
method of modulating calcineurin activity in a subject comprising
administering at least one
ApoE analog to the subject.
[007] In one embodiment, the invention provides a method of modulating
calcineurin activity
in a cell comprising contacting the cell with at least one ApoE analog. In
another embodiment,
calcineurin activity is inhibited following contact with the at least one ApoE
analog. In another
embodiment, NFAT-mediated transcription is reduced in the cell following
contact with the at
least one ApoE analog. The cell may be in vitro or in vivo.
[008] In another embodiment, the cell is in a subject. In some embodiments,
the subject suffers
from an inflammatory condition, heart disease, a renal condition, a fungal
infection, a pulmonary
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disorder or muscular dystrophy. In other embodiments, the subject has a
transplanted organ or
tissue. In another embodiment, the subject is at risk for restenosis.
[009] The present invention also provides a method of promoting remyelination
in a subject in
need thereof. In one embodiment, the method comprises administering an
effective amount of at
least one ApoE analog to the subject, wherein myelination is enhanced in the
subject following
administration of the at least one ApoE analog. In another embodiment, the
subject is suffering
from a demyelinating disorder or condition. In another embodiment, the subject
has a spinal
cord or nerve injury. In some embodiments, the number of oligodendrocytes is
increased in the
subject following administration of the at least one ApoE analog. In other
embodiments, the
amount of neuronal inflammation is decreased in the subject following
administration of at least
one ApoE analog. Preferably, one or more symptoms of demyelination is reduced
in the subject
following administration of the at least one ApoE analog.
[010] The present invention also encompasses a method of treating multiple
sclerosis in a
subject in need thereof comprising administering an effective amount of at
least one ApoE
analog to the subject. In one embodiment, the amount of myelination is
increased in the subject
following administration of the at least one ApoE analog. In another
embodiment, the number of
oligodendrocytes is increased in the subject following administration of the
at least one ApoE
analog. In still another embodiment, the amount of neuronal inflammation is
decreased in the
subject following administration of the at least one ApoE analog. One or more
symptoms of
multiple sclerosis is preferably reduced in the subject following
administration of the least one
ApoE analog.
[011] The present invention provides a method of treating spinal cord injury
or nerve injury in a
subject in need thereof. In one embodiment, the method comprises administering
an effective
amount of at least one ApoE analog to the subject. The spinal cord injury may
be a contusive
injury or a compressive injury. In another embodiment, the nerve injury is a
peripheral nerve
crush injury. In another embodiment, axonal degeneration is decreased in the
subject following
administration of the at least one ApoE analog. In still another embodiment,
inflammation at the
injured site is decreased following administration of the at least one ApoE
analog. Inflammation
may include activation of microglia or macrophages as well as secretion of
inflammatory
cytokines.
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[012] The present invention also contemplates a method of promoting the
survival and/or
proliferation of oligodendrocyte precursor cells. In one embodiment, the
method comprises
treating the oligodendrocyte precursor cells with at least one ApoE analog,
wherein the number
of oligodendrocyte precursor cells is increased following treatment with the
at least one ApoE
analog. The treatment with the at least one ApoE analog may be in vitro or in
vivo. In another
embodiment, calcineurin activity is inhibited in the oligodendrocyte precursor
cells following
treatment with the at least one ApoE analog.
BRIEF DESCRIPTION OF THE FIGURES
[013] Figure 1. COG 133 and COG 112 promote the recovery of animals from
clinical
disability of EAE when applied after the onset of disease. (A) Changes in the
mean daily
clinical score (CS) after treatment with COG 133 (1 mg/kg,i.p.) or COG 112 (1
mg/kg, i.p.). (B)
Percentage of animals in remission. Statistical analysis was conducted by
ANOVA comparing
the CS after day 18, which was defined as the recovery phase.
[014] Figure 2. Representative histopathology of EAE in mice treated with COG
112 or
saline control. 5 m thick sections were stained with Luxol fast blue and
eosin to reveal
demyelination and peripheral infiltrates. The left panels show the
pathological changes in spinal
cord from cervical (A), thoracic (B) and lumbar (C) segments of a saline-
treated control animal.
Demyelination is shown as loss of blue staining in white matter and peripheral
infiltrates are
stained with purple particles. Panels D, E and F show the corresponding
segments of a COG
112-treated animal where no obvious demyelination is observed. The boxed-
insets of panel A
and D are magnified l Ox in panel G and H, respectively.
[015] Figure 3. Profiling and quantification of T cell subset infiltrates in
the spinal cords
of animals on 21 days post-immunization that were treated with or without COG
112.
Single-cell suspensions of spinal cord were stained with anti-CD3, CD4, and
CD8 mAbs.
Frequency of CD3+CD45+ cytometry (T cell) is represented in control (A) and
COG112-treated
(C) animals. Frequency of CD4+ and CD8+ T cells is represented in saline
control (B) and COG
112-treated (D) mice. Total cell number for each of the specific cell surface
markers is calculated
and expressed as number per spinal cord (E).
[016] Figure 4. In vivo exposure to COG 133 suppressed in vitro production of
inflammatory effectors by peritoneal macrophages. TNF-a (A) and IL-6 (B) were
measured
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by ELISA, and nitrite in culture medium was measured by a Sievers NO Analyzer
(C). Statistical
analysis was performed by two-tailed student's t test and significance was
shown as ** (p<0.01)
and *** (p<0.001).
[017] Figure 5. In vitro effects of COG compounds on LPS and IFN-y-induced
production
of inflammatory mediators in cultured peritoneal macrophages isolated from EAE
mice.
Conditioned medium with or without COG 133 or COG 112 was collected 45 hours
after
immunogen exposure for analysis of TNF-a (A) and IL-6 (B), or 72 hours after
inflammatory
stimulation for measurement of nitrite (C). Level of NOS2 expression was
assessed by
quantitative real-time PCR (D). NOS2 expression was first normalized by the
endogenous
control (18S rRNA) and then expressed as fold change compared to control
group. Statistical
analysis was performed by ANOVA and significance was shown as *(p<0.05) and **
(p<0.001).
[018] Figure 6. COG 112 promotes remyelination of the corpus callosum in a
cuprizone
model of demyelination. C57BL/6J male mice were fed with 0.2% cuprizone-
containing diet for
weeks to induce complete demyelination in the corpus callosum. Starting from
week 6,
cuprizone was removed and the animals were treated with either vehicle or COG
112 (1mg/kg)
by i.p. injection three times a week for 4 weeks. Myelination in corpus
callosum was evaluated
on a scale from 0 to 3 after staining brain sections with Luxol Fast Blue
(LFB). The data were
collected from 4 animals of each group and 2 sections from each animal. A non-
parametric
Mann-Whitney test was used to compare differences between groups. Panels A and
B(100x) are
representative pictures of LFB-stained sections from vehicle- and COG 112-
treated animals,
respectively. Panels C and D (400x) are representative sections labeled with
anti-GST-n-FITC
antibody (marker for mature oligodendrocytes) from vehicle- and COG 112-
treated animals,
respectively.
[019] Figure 7. COG 112 promotes remyelination in a lysolecithin-induced
demyelination
model in cerebellum slice culture. Cerebellum slices from postnatal day 10 rat
pups were
cultured for 7 days. Lysolecithin was added to a final concentration of 0.5
mg/ml and then
removed after incubation overnight (15-17 hr). 1 M COG 112 was added to the
slice and
cultured for 5 days. The cerebellar slices were either immunohistologically
stained with MBP; or
collected and lysed for western blotting with myelin marker MBP. (A) naive
slice; (B) 2 days
after lysolecithin treatment; (C) day 7 after lysolecithin treatment; (D) day
7 after lysolecithin
treatment with COG 112 added on day 3; (E) Western blot of cerebellar lysates
probed for MBP;
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(F) Densitometry analysis of blots probed for MBP. The density of each MBP
band was
normalized to density of (3-actin band. One-way ANOVA was used for statistical
analysis. n=4
[020] Figure 8. COG 112 prevents lysolecithin-induced cell death of
oligodendrocytes in
cerebellum slice culture. Cerebellum slices from postnatal day 10 rat pups
were cultured for 7
days. Lysolecithin was added to a final concentration of 0.5 mg/ml with and
without COG 112 (1
M). After incubation overnight (16 hr), medium was replaced with fresh
lysolecithin-free
medium, but still containing COG 112 for the COG 112 treatment group. After
two days, the
cerebellar slices were collected and lysed for western blotting with myelin
marker MBP (left
panel) and oligodendrocyte marker CNPase (right panel). The density of each
MBP or CNPase
band was normalized to the density of the (3-actin band. One-way ANOVA was
used for
statistical analysis. n=4, * p< 0.01.
[021] Figure 9. COG 112 protects OPCs from LPS-induced cell death in
OPC/microglia
mixed culture. LPS (final conc. 10 ng/ml) was added to an OPC/microglia mixed
culture with or
without 1 M COG 112. The prefix peptide of COG 112, antennapedia, was used as
a negative
control. Cells were fixed with 4% formaldehyde and stained with the OPC
marker, NG2, and
counterstained with hoescht. (A) LPS-treated cultures; (B) LPS + antennapedia
(Antp)-treated
cultures; (C) LPS + COG 112-treated cultures; (D) Bar graph summarizing the
number of NG2+
cells per field in each condition. ** p<0.01.
[022] Figure 10. ApoE analog promotes proliferation of OPCs in culture.
Cultures
enriched in OPCs were grown in proliferation media containing BrdU in the
presence and
absence of 1 M COG 112 or a peptide control. Cells were subsequently labeled
with Hoescht
stain to detect nuclei, a BrdU antibody to detect proliferating cells, and an
A2B5 antibody to
identify OPCs (left panel). The number of cells per field were counted for
each condition (right
panel). In graph, the Hoescht bars correspond to the total number of
A2B5+/Hoescht+ cells,
while the BrdU bars correspond to A2B5+/BrdU+ cells.
[023] Figure 11. ApoE analogs increase the number of mature oligodendrocytes
in
culture. ApoE analogs (COG 133, COG 112, and COG 68) were added to enriched
OPC culture
in proliferation media for four days. Cultures were then differentiated in the
absence of the ApoE
analogs. Cells were labeled with a GST-n antibody to detect mature
oligodendrocytes.
[024] Figure 12. COG 112 reduces the size of the lesion in white matter after
spinal cord
injury by inhibition of microglia activation. Rats received 175 kdyn contusive
spinal cord
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injury and subsequent treatment with saline or COG 112 starting immediately
after injury for 1
week. The injured area and spared white matter from the injured spinal cord
were evaluated and
quantified by luxol fast blue (LFB) staining area (top left panel). Activation
of microglia was
examined by staining adjacent sections with an ED1 antibody (bottom left
panel). Compared to
animals treated with saline (A-E), the lesion was significantly decreased in
COG 112-treated
animals (A"-E"). The percentage of spared white matter area to the total
spinal cord was
significantly increased in the COG 112-treated group (top right panel). COG
112 dramatically
suppressed the activation of microglia after injury as shown by the
significant decrease in area of
ED 1 immunoreactivity in the injured spinal cord (bottom right panel). Scale
bar =1000 m.
[025] Figure 13. COG 112 attenuates axonal neurodegeneration and promotes
functional
recovery after sciatic nerve crush. C57BL mice were subjected to sciatic nerve
crush on left
hindlimb using a number 5 jeweler's forceps. The animals received lactated
Ringer's buffer or
COG 112 (1mg/kg) by i.p. 2 hr after crush followed by daily dose for 14 days.
(A) To assess
functional deficits after nerve crush, footprints were recorded and the
Sciatic Functional Index
(SFI) was calculated. COG 112 treatment for 14 days significantly improved
functional recovery.
*** P<0.001. Treatment with COG 112 also robustly augmented the mRNA
expression of
peripheral myelin marker P0 (B) and axonal regeneration marker GAP43 (C)
quantified by real-
time PCR. **P<0.01; P<0.001. (D) On day 4 after injury, the sciatic nerves
were dissected and
stained with FD NeuroSilver kit to reveal degenerating axons manifested as
dotted lines as
depicted in the schematic above the micrographs.
[026] Figure 14. ApoE analogs bind directly to calcineurin and inhibit its
activity. (A)
Dose-dependent inhibition of calcineurin activity by different ApoE analogs.
Inactive peptide
COG 125 and antennapedia (antp) were used as negative controls. (B)
Immunoprecipitation of
human brain lysate with a biotin-COG 133 conjugate. Lane 1= markers; lane 2 =
clear bead;
lane 3 = biotin-COG 133 bead conjugate; and lane 4 = 10 g lysate. (C)
Immunoprecipitation of
different glial cultures with a biotin-COG 133 conjugate. Lane 1= markers;
lane 2 = astrocyte;
lane 3 = microglia=, and lane 4 = oligodendrocyte precursor cells (OPC). Blots
in B and C were
probed for calcineurin (PP2B).
[027] Figure 15. ApoE analog inhibits LPS-induced NFAT translocation. Murine
microglial cell line, BV2 cells were stimulated with LPS in the presence or
absence of COG 112.
NFAT (green signal) and nuclear label (red signal). (A) Untreated cultures;
(B) LPS-treated
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cultures; (C) LPS + COG 112-treated cultures; (D) NFAT translocation was
quantified by the
fluorescence intensity of green fluorescence in the nucleus defined by yellow
area.
[028] Figure 16. ApoE analogs suppress the release of nitric oxide (A) and TNF-
a (B) in LPS-
treated BV2 microglia cells. Series concentrations of ApoE analogs were added
to culture
medium together with LPS (10 ng/ml). 24 hours later, culture medium was
collected for
measurement of NO by Griess assay or TNF-a by ELISA. (C) ApoE analogs inhibit
superoxide
formation in PMA + LPS-treated human macrophage cell line U937. ApoE analogs
(10 M) were
added to the cell medium of human macrophage U937 5 minutes prior to PMA+LPS
treatment.
The release of superoxide in culture medium was measured by Luminol assay kit
(Calbiochem).
[029] Figure 17. ApoE analogs suppress LPS-induced TNF-a production in plasma
of
C57BL mice. Mice received i.p. injection of LPS (1 mg/kg). Five minutes later,
they were given
COG 133 (4 mg/kg, i.v.) or molar equivalent doses of the indicated ApoE
analogs. Whole blood
was collected 1 hour after LPS injection, and the level of TNF-a in plasma was
quantified by
ELISA. The level of plasma TNF-a is expressed as percent of TNF-a production
in plasma vs.
vehicle control (LR buffer). ANOVA was used for statistical analysis followed
by Newman-
Keuls Multiple Comparison Test. a= p<0.001 compared to LR group; b=p<0.05
compared to
COG 133 group.
[030] Figure 18. Comparison of anti-excitotoxicity activity of ApoE analogs in
primary
neuron-glia mixed culture. Primary neurons growing on an astrocyte layer for
14 days were
treated with the indicated concentrations of the ApoE analogs for 15 min. NMDA
was then
added to the medium to a final concentration of 200 M. After incubation for 5
min, NMDA-
containing medium was replaced with fresh medium with the same ApoE peptides
at the
designated concentration. After 24 hours, LDH in medium was measured and used
as index of
cell death. The wells treated with NMDA for 24 hours were considered 100% cell
death and used
for normalization with the other treatments.
[031] Figure 19. COG 345 promotes remyelination in lysolecithin-induced
demyelination
model of cerebellum slices. Significant demyelination was induced by
lysolecithin (0.5 mg/ml)
in cerebellar slices. COG 112 or COG 345 (1 M) was added on day 3 when full
demyelination
was achieved. 4 days later, the slices were fixed and double stained with
myelin marker MBP
(green) and Purkinje cell marker calbindin (red). (A) Untreated-slices; (B)
lysolecithin-treated
slices; (C) COG 112-treated slices after lysolecithin exposure; (D) COG 345-
treated slices after
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lysolecithin exposure. E-F. ApoE analogs protect OPCs from LPS-induced cell
death in
OPC/microglia mixed culture. OPCs and microglia derived from brain culture of
P2 rat pups
were plated in 96-well plates in 1:1 ratio. LPS (10 ng/ml) together with 1 M
of the indicated
ApoE analogs was added to the cultures. After 24 hours, OPC cell death was
quantified by LDH
assay (E) and MTT assay (F). Statistical analysis was conducted by one-way
ANOVA followed
by Dunnett comparion vs. LPS group. * P<0.01, ** P<0.001, n=3.
DETAILED DESCRIPTION OF THE INVENTION
[032] Aberrant calcineurin signaling has been implicated in a number of
disorders ranging from
nerve injury to heart disease. The inventors have surprisingly discovered that
ApoE analogs can
bind directly to calcineurin and inhibit its activity. Therefore, ApoE analogs
represent a novel
therapeutic for treating a myriad of conditions associated with over-
activation of calcineurin.
[033] The present invention provides a method of modulating calcineurin
activity in a cell. In
one embodiment, the method comprises contacting the cell with at least one
ApoE analog. As
used herein, "modulating" refers to changes in calcineurin activity observed
in vitro or in vivo,
including an increase or decrease in calcineurin activity. As previously
discussed, activation of
calcineurin is reported to be involved in multiple biological processes
associated with disease.
For example, calcineurin is known to play a vital role in activation of the
immune response and
is a target for current therapies used to prevent transplant rejection.
Calcineurin is also associated
with other inflammatory conditions, such as autoimmune disorders, inflammatory
skin
conditions, and asthma. In addition, calcineurin signaling also appears to be
important in
neurological conditions, such as demyelinating disorders and the repair of the
nervous system
after injury.
[034] Activation of calcineurin by any number of stimuli results in the
dephosphorylation and
activation of the nuclear factor of activated T cell (NFAT) family of
transcription factors, which
in turn stimulate transcription of interleukin-2 (IL2) and other cytokines. In
one embodiment of
the present invention, modulation of calcineurin in a cell by at least one
ApoE analog results in
inhibition of calcineurin activity. In another embodiment, NFAT-mediated
transcription is
reduced in the cell. In another embodiment, interleukin-2 expression is
decreased in the cell.
[035] Activities of calcineurin can be either direct activities or indirect
activities. A direct
activity of calcineurin occurs when calcineurin interacts directly with a
substrate and
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dephosphorylates it, thereby causing an activation or inactivation of the
substrate protein. An
indirect activity of calcineurin occurs when a protein is affected by
calcineurin activity although
the protein is not a direct substrate for calcineurin. For instance,
downstream molecules in
signaling cascades can be indirectly affected by calcineurin as the result of
activation or
inactivation of upstream proteins in the cascade.
[036] In one embodiment, the at least one ApoE analog for modulating
calcineurin activity is a
peptide derivative of COG 133 (LRVRLASHLRKLRKRLL; SEQ ID NO: 1). COG 133 is a
truncated peptide comprised of residues 133-149 of ApoE and has previously
proved useful in
treating or reducing cerebral ischemia or cerebral inflammation. See U.S.
Application No.
10/252,120, filed September 23, 2002, incorporated herein by reference in its
entirety. In
another embodiment of the invention, the ApoE analog may be a peptide
derivative containing a
sequence selected from the group consisting of:
Ac-As-Aib-LRKL-Aib-KRLL-NH2 (SEQ ID NO: 2)
Ac-LRVRLAS-Aib-LRKLRK(nitro-Arg)LL-NH2 (SEQ ID NO: 3),
Ac-LRVRLAS-Aib-LRKLRK(acetyl-Arg)LL-NH2 (SEQ ID NO: 4),
Ac-RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-NH2 (SEQ ID NO: 5),
Ac-Aib-LRKL-Aib-(n acetyl K)RLL-NH2 (SEQ ID NO: 6), and
Ac-RRLSYSRRRFLRVRLASHLRKLRKRLL-NH2 (SEQ ID NO: 7),
wherein Aib is amino iso-butyric acid, (nitro-Arg) is nitro arginine, (acetyl-
Arg) is acetyl
arginine, (n acetyl K) is n-acetyl lysine, and Ac is an acetlyated amino
terminus. The ApoE
analog may be derived from a peptide containing residues 130-150 of the ApoE
holoprotein. As
used herein, "derived" means that the derivative contains the basic structure
of the peptide from
which it is derived, but has either one or more insertions, deletions, or
substitutions in its amino
acid sequence. Substitutions encompass non-natural amino acids as well as both
conservative
and non-conservative amino acid substitutions. The peptide derivative may
retain the alpha-
helical character of residues 130-150 of ApoE. Other suitable ApoE analogs
that may be used in
the methods of the invention include those described in WO 2006/029028, filed
September 2,
2005, which is herein incorporated by reference in its entirety. See Table 1.
[037] In one embodiment of the invention, the efficacy of the ApoE peptide
derivative can be
improved by conjugation to a protein transduction domain (PTD). PTDs are short
basic peptides
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that promote the intracellular delivery of cargo that would otherwise fail to,
or only minimally,
traverse the cell membrane. PTDs can be used to enhance CNS penetration of
compounds. For
instance, empirical testing of PTDs can be performed to identify PTDs that are
capable of
transporting cargo across the blood brain barrier. PTDs can be antimicrobial
peptides, such as
protegrin 1, Bactenecin 7, Buforin, and Maginin; a host of arginine-rich RNA-
and DNA-binding
peptides (e.g., HIV-1 transactivating protein (TAT) and Drosophila homeodomain
transcription
factor Antennapedia (a.k.a. Penetratin); chimeric PTDs such as Transportan;
lysine- and
arginine-rich peptides derived from phage-display libaries; polyarginine; and
(3-homolysine
oligomers (See, Fisher et al. (2001) Bioconjugate Chemistry, Vol. 12: 825-841;
Lindsay (2002)
Current Opinions in Pharmacology, Vol. 2: 587-594; Tung and Weissleder (2003)
Advanced
Drug Delivery Reviews, Vol. 55: 281-294; Liefert and Whitton (2003) Molecular
Therapy, Vol.
8: 13-19; Bogoyevitch et al. (2002) DNA and Cell Biology, Vol. 21: 879-894;
and Garcia-
Echeverria and Ruetz (2003) Bioorganic and Medicinal Chemistry Letters, Vol.
13: 247-251, all
of which are incorporated by reference in their entireties). In some
embodiments, the ApoE
peptide derivative is conjugated to a protein transduction domain selected
from the group
consisting of peptides derived from antennapedia, TAT, SynBl, SynB3, SynB5,
and
polyarginine. For example, COG 112 (SEQ ID NO: 5) and COG 68 (SEQ ID NO: 7)
are ApoE
peptides linked to PTDs. COG 112 is linked to antennapedia, while COG 68 is
linked to SynB3.
Such PTD peptides may comprise a sequence selected from the group consisting
of:
GRKKRRQRRRPPQ (SEQ ID NO: 9)
RQIKIWFQNRRMKWKK (SEQ ID NO: 10)
RRMKWKK (SEQ ID NO: 11)
RGGRLSYSRRRFSTSTGR (SEQ ID NO: 12)
RRLSYSRRRF (SEQ ID NO: 13)
RGGRLAYLRRRWAVLGR (SEQ ID NO: 14)
RRRRRRRR (SEQ ID NO: 15).
[038] ApoE analogs suitable for use in the methods of the present invention
can be produced by
standard techniques as are known in the art. In some embodiments, the ApoE
analogs may be
peptide derivatives. Modification of the peptide derivatives disclosed herein
to enhance the
functional activities associated with these peptides could be readily
accomplished by those of
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skill in the art. For instance, the peptides used in the methods of the
present invention can be
chemically modified or conjugated to other molecules in order to enhance
parameters like
solubility, serum stability, etc, while retaining functional activity. In
particular, the peptides of
the invention may be acetylated at the N-terminus and/or amidated at the C-
terminus, or
conjugated, complexed or fused to molecules that enhance serum stability,
including but not
limited to albumin, immunoglobulins and fragments thereof, transferrin,
lipoproteins, liposomes,
a-2-macroglobulin and a-1 -glycoprotein, PEG, lipids, and dextran. Such
molecules are
described in detail in US 6,762,169, which is herein incorporated by reference
in its entirety.
Small molecules that target the conjugate to specific cells or tissues may
also be used. It is
known that presence of a biotin-avidin complex increases uptake of such
modified peptides
across endothelial cells. Linkage of peptides to carbohydrate moieties, for
example to a(3-
glycoside through a serine residue on the peptide to form a(3-0-linked
glycoside, enhances
transport of the glycoside derivative via glucose transporters (PoIt, R. et
al. Proc. Natl. Acad. Sci.
USA 91 : 7144-7118 (1994); Oh et al. Drug Transport and targeting, In Membrane
Transporters
as Drug Targets, Amidon, G. L. and Sadee, W. eds., pg 59-88, Plenum Press, New
York, 1999).
The peptides may have attached various label moieties such as radioactive
labels, heavy atom
labels, and fluorescent labels for detection and tracing. Fluorescent labels
include, but are not
limited to, luciferin, fluorescein, eosin, Alexa Fluor, Oregon Green,
rhodamine Green,
tetramethylrhodamine, rhodamine Red, Texas Red, coumarin and NBD fluorophores,
the QSY 7,
dabcyl and dabsyl chromophores, BODIPY, Cy5, etc.
[039] Another variation of the ApoE analogs of the present invention is the
linking of from one
to fifteen amino acids or analogs to the N-terminal or C-terminal amino acid
of the peptide
derivatives disclosed herein. Analogs of the peptide derivatives can also be
prepared by adding
from one to fifteen additional amino acids to the N-terminal, C- terminal, or
both N- and C-
terminals, of an active peptide, where such amino acid additions do not
adversely affect the
ability of the peptide to bind to calcineurin and modulate its activity. For
example, COG 1410
(SEQ ID NO: 2), COG 248 (SEQ ID NO: 3), COG 345 (SEQ ID NO: 4), COG 112 (SEQ
ID
NO: 5), COG 241 (SEQ ID NO: 6), and COG 68 (SEQ ID NO: 7) variants can be
created by
adding from one to fifteen additional amino acids to the N-terminal, C-
terminal, or both N- and
C-terminals, of the active peptide. An active peptide is any peptide capable
of binding to
calcineurin and modulating calcineurin activity.
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[040] The ApoE analogs of the present invention further include conservative
variants of the
peptides herein described. As used herein, a conservative variant refers to
alterations in the
amino acid sequence that do not adversely affect the biological functions of
the peptide. A
substitution, insertion or deletion is said to adversely affect the peptide
when the altered
sequence prevents or disrupts a biological function associated with the
peptide. For example, the
overall charge, structure or hydrophobic/hydrophilic properties of the peptide
may be altered
without adversely affecting a biological activity. Accordingly, the amino acid
sequence can be
altered, for example to render the peptide more hydrophobic or hydrophilic,
without adversely
affecting the biological activities of the peptide. Ordinarily, the
conservative substitution
variants, analogs, and derivatives of the peptides, will have an amino acid
sequence identity to
the disclosed sequences SEQ ID NOs: 1-7 of at least about 55%, at least about
65%, at least
about 75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or at
least about 96% to 99%. Identity or homology with respect to such sequences is
defined herein
as the percentage of amino acid residues in the candidate sequence that are
identical with the
known peptides, after aligning the sequences and introducing gaps, if
necessary, to achieve the
maximum percent homology, and not considering any conservative substitutions
as part of the
sequence identity. N-terminal, C-terminal or internal extensions, deletions,
or insertions into the
peptide sequence shall not be construed as affecting homology. Thus, the ApoE
analogs of the
present invention include peptide derivatives having the amino acid sequence
disclosed in SEQ
ID NOs: 1-7; fragments thereof having a consecutive sequence of at least about
3, 4, 5, 6, 10, 15,
or more amino acid residues of the peptide; amino acid sequence variants of
such peptides
wherein an amino acid residue has been inserted N- or C-terminal to, or
within, the disclosed
sequence; and amino acid sequence variants of the disclosed sequence, or their
fragments as
defined above, that have been substituted by another residue. Peptide
compounds comprising the
peptide sequences of the invention may be 15, 20, 25, 30, 35, 40, 45, 50 or
more amino acids.
Contemplated variants further include those containing predetermined mutations
by, e.g.,
homologous recombination, site-directed or PCR mutagenesis, and the
corresponding peptides of
other animal species, including but not limited to rabbit, rat, porcine,
bovine, ovine, equine and
non-human primate species, and derivatives wherein the peptide has been
covalently modified by
substitution, chemical, enzymatic, or other appropriate means with a moiety
other than a
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naturally occurring amino acid (for example a detectable moiety such as an
enzyme or
radioisotope).
[041] ApoE analogs capable of modulating calcineurin activity, including but
not limited to
peptide derivatives of COG 133 (SEQ ID NO: 1), can be in free form or the form
of a salt, where
the salt is pharmaceutically acceptable. These include inorganic salts of
sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and the
like. Various
organic salts of the peptide derivatives may also be made with, including, but
not limited to,
acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid,
tartaric acid, citric acid,
benozic acid, cinnamic acid, salicylic acid, etc.
[042] The present invention contemplates a method of modulating calcineurin
activity in a cell
by contacting the cell with at least one ApoE analog in vivo. In one
embodiment, the cell is in a
subject. The subject may suffer from an inflammatory condition, heart disease,
a renal condition,
a fungal infection, a pulmonary disorder, or muscular dystrophy. Accordingly,
the present
invention provides methods of treating these disorders in a subject in need
thereof by
administering at least one ApoE analog to the subject. In preferred
embodiments, the ApoE
analog is a peptide derivative having the sequence of SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID
NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
[043] As discussed above, calcineurin signaling is instrumental in the
activation of the immune
response. As such, calcineurin is an attractive therapeutic target for
designing treatments for
inflammatory conditions. Several calcineurin inhibitors are currently
available as
immunosuppressants for preventing organ transplant rejection and treating
autoimmune
disorders. In addition, inhibition of calcineurin has previously shown to be
an effective
treatment for inflammatory skin conditions including atopic dermatitis
(eczema), facial and
intertriginous psoriasis, hand dermatitis, lichen planus, and vitiligo
(leukoderma) among others.
The inventive methods of modulating calcineurin in a cell by contacting the
cell with at least one
ApoE analog can be used to ameliorate one or more symptoms associated with an
inflammatory
condition. In one embodiment, the inflammatory condition is an inflammatory
skin condition,
such as those described above. In another embodiment, the inflammatory
condition is asthma.
In another embodiment, the inflammatory condition is an autoimmune disorder.
Non-limiting
examples of autoimmune disorders include rheumatoid arthritis, myasthenia
gravis, systemic
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lupus erythematosus, psoriasis, multiple sclerosis, ulcerative colitis,
Crohn's disease, and
diabetes (type 1).
[044] In another embodiment, the methods of the invention may be used to
prevent rejection of
transplanted organs or tissues in a subject. Rejection of transplanted tissues
occurs because the
immune system of the recipient attacks the donor organ or tissue. Transplant
recipients are
typically administered an immunosuppressant, such as a calcineurin inhibitor,
to reduce the
immune response to the foreign tissue. Thus, at least one ApoE analog may be
administered to
the subject to suppress calcineurin activation and prevent transplant
rejection. ApoE analogs,
such as COG 1410 (SEQ ID NO: 2), COG 248 (SEQ ID NO: 3), COG 345 (SEQ ID NO:
4),
COG 112 (SEQ ID NO: 5), COG 241 (SEQ ID NO: 6), and COG 68 (SEQ ID NO: 7) may
be
administered to subjects receiving any type of organ or tissue transplant,
such as heart, lung,
liver, kidney, pancreas, intestine, skin graft, cornea, bone marrow, heart
valve, blood vessels, and
bone.
[045] Calcineurin activation has also been linked to various forms of heart
disease, such as
myocardial infarction, dilated cardiomyopathy, pathologic cardiac hypertrophy,
and heart failure
(see, for example, Bueno et al. (2002) Cardiovascular Research, Vol. 53: 806-
821 and
Mitsuhashi et al. (2003) J. Biol. Chem., Vol. 134: 269-276). Administration of
at least one
ApoE analog to a subject can reduce or prevent one or more symptoms of heart
disease. In one
embodiment, the heart disease is pathologic cardiac hypertrophy, dilated
cardiomyopathy, or
heart failure.
[046] In addition to its involvement in diseases of cardiac muscle,
calcineurin is known to
regulate gene expression in skeletal muscle, such as genes related to fiber-
type switching.
Genetic deletion of calcineurin in skeletal muscles of scgd -/- mice, a mouse
model of limb-
girdle muscular dystrophy, reduced skeletal muscle degeneration and pathology,
suggesting
calcineurin signaling may mediate, in part, the skeletal muscle pathogenesis
observed in the
disease (Parsons et al. (2007) J. Biol. Chem., Vol. 282: 10068-10078). ApoE
analogs may be an
effective treatment for muscular dystrophy by inhibiting calcineurin activity,
and thus
ameliorating skeletal muscle degeneration.
[047] Modulation of calcineurin activity by ApoE analogs may also be effective
in treating
several renal conditions. Inhibition of calcineurin activity was shown to
decrease renal
hypertrophy in diabetic rats (Gooch et al. (2003) Am. J. Physiol. Renal
Physiol., Vol. 284: F144-
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F154). Renal hypertrophy is often a harbinger of several pathological
conditions. As such,
reduction of renal hypertrophy may prevent the development of more serious
renal
complications. The present invention contemplates a method of treating a renal
condition in a
subject in need thereof comprising administering at least one ApoE analog to
the subject. In one
embodiment, the renal condition is diabetic nephropathy or renal hypertrophy.
[048] Disruption of the endothelial barrier leading to tissue edema underlies
many acute
inflammatory diseases of the lung. Calcineurin has been implicated in the
control of endothelial
barrier function and the subsequent development of pulmonary edema. Regulation
of calcineurin
activity by ApoE analogs according to the methods of the present invention
provide a therapeutic
approach to treating pulmonary disorders, such as pulmonary edema and
respiratory failure, in a
subject.
[049] In another embodiment, the present invention provides a method for
treating a fungal
infection in a subject comprising administering at least one ApoE analog
capable of modulating
calcineurin activity in a cell of the subject. Candida albicans, a yeast-like
fungi, is one of the
most frequent causes of keratitis, which can progress to endophthalmitis
posing a risk for loss of
vision. Calcineurin activity appears to be required for survival of C.
albicans, especially in the
presence of standard anti-fungal azole drugs. Thus, ApoE analogs administered
alone or in
combination with other anti-fungal treatments, may alleviate fungal infections
caused by drug-
resistant strains.
[050] In yet another embodiment, the present invention provides a method of
preventing
restenosis in a subject comprising administering at least one ApoE analog
capable of modulating
calcineurin activity in a cell of the subject. Restenosis is the re-narrowing
of a blood vessel,
which is a common occurrence after vascular surgery or angioplasty to open a
blocked artery or
vessel. One cause of restenosis is intimal hyperplasia induced by inflammatory
cytokines
stimulated by injury to the vessel as a result of the vascular procedure.
Inhibition of calcineurin
in combination with an anti-proliferative therapy has been shown to produce a
synergistic
reduction in intimal thickening after angioplasty in rats. In addition,
tacrolimus, a calcineurin
inhibitor, was reported to inhibit the proliferation of vascular smooth muscle
cells and prevent
restenosis in clinical trials. In one embodiment, ApoE analogs, such as COG
1410 (SEQ ID NO:
2), COG 248 (SEQ ID NO: 3), COG 345 (SEQ ID NO: 4), COG 112 (SEQ ID NO: 5),
COG 241
(SEQ ID NO: 6), and COG 68 (SEQ ID NO: 7) may be used to coat stents that can
be placed in
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vessels after angioplasty or other vascular surgical procedure to prevent the
development of
intimal hyperplasia and subsequent restenosis. Alternatively or additionally,
ApoE analogs may
be administered alone or with anti-proliferative drugs, such as sirolimus, to
the subject after a
vascular procedure to prevent restenosis of the unblocked vessel.
[051] Recently, calcineurin has been identified to play a role in
demyelination of axons.
Oligodendrocytes, which myelinate the axons of the central nervous system, are
susceptible to
apoptosis following neuronal injury. Loss of oligodendrocytes causes
demyelination and
neuronal dysfunction (Liu et al. (1997) J. Neurosci., Vol. 17: 5395-5406).
Inhibition of
calcineurin following spinal cord injury was shown to result in fewer
apoptotic oligodendrocytes
indicating that calcineurin activation promotes apoptosis in these cells
(Nottingham et al. (2002)
Exp. Neurol., Vol. 177: 242-25 1). Thus, modulation of calcineurin activity by
contacting
oligodendrocytes with at least one ApoE analog would promote the survival of
oligodendrocytes
and prevent demyelination following injury. In fact, the inventors have
demonstrated that ApoE
analogs enhance the survival of both oligodendrocyte precursor cells as well
as mature
oligodendrocytes in vivo and in vitro (See Examples 3-5). Furthermore, ApoE
holoprotein has
been implicated in remyelination after nerve injury due to its role in lipid
transport. Thus, the
present invention provides a method of promoting remyelination in a subject in
need thereof. In
one embodiment, the method comprises administering an effective amount of at
least one ApoE
analog to the subject, wherein myelination is enhanced in the subject
following administration of
the at least one ApoE analog.
[052] An effective amount of at least one ApoE analog is an amount that
increases myelination
in a subject as compared to that which would occur in the absence of the
analog. In one
embodiment, the effective amount of an ApoE analog increases the number of
oligodendrocyte
precursor cells or mature oligodendrocytes compared to that which would occur
in the absence of
the analog. In another embodiment, the effective amount of an ApoE analog is
an amount that
modulates calcineurin activity in a subject. The effective amount (and the
manner of
administration) will be determined on an individual basis and will be based on
the specific agent
being used and a consideration of the subject (size, age, general health), the
condition being
treated, the severity of the symptoms to be treated, the result sought, the
specific carrier or
pharmaceutical formulation being used, the route of administration, and other
factors as would
be apparent to those skilled in the art. The effective amount can be
determined by one of
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ordinary skill in the art using techniques as are known in the art.
Therapeutically effective
amounts of the ApoE analogs described herein can be determined using in vitro
tests, animal
models or other dose-response studies, as are known in the art.
[053] In another embodiment, the method comprises administering an effective
amount of at
least one ApoE analog to promote remyelination in a subject suffering from a
demyelinating
disorder or condition. Demyelinating disorders or conditions include, but are
not limited to,
optic neuritis, devic disease, transverse myelitis, acute disseminated
encephalomyelitis,
adrenoleukodystrophy, adrenomyeloneuropathy, multiple sclerosis, Guillain-
Barre syndrome,
Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), and diabetic
peripheral
neuropathy. In another embodiment, the subject in need of remyelination has a
spinal cord or
nerve injury.
[054] Any of the ApoE analogs described herein are suitable for use in the
method of
promoting remyelination in a subject. In one embodiment, the at least one ApoE
analog is a
peptide derivative of COG 133 (SEQ ID NO: 1). In preferred embodiments, the
peptide
derivative contains a sequence selected from the group consisting of COG 1410
(SEQ ID NO: 2),
COG 248 (SEQ ID NO: 3), COG 345 (SEQ ID NO: 4), COG 112 (SEQ ID NO: 5), COG
241
(SEQ ID NO: 6), and COG 68 (SEQ ID NO: 7). In another embodiment, the number
of
oligodendrocytes is increased in the subject following administration of the
at least one ApoE
analog. In still another embodiment, the amount of neuronal inflammation is
decreased in the
subject following administration of the at least one ApoE analog. In yet
another embodiment of
the invention, one or more symptoms of demyelination is reduced in the subject
following
administration of the at least one ApoE analog. Symptoms of demyelination
include, but are not
limited to, conduction block, conduction slowing, numbing, tingling, pain,
progressive muscle
weakness, loss of deep tendon reflexes (areflexia), fatigue, abnormal
sensations, and paralysis.
[055] The present invention also encompasses a method of treating spinal cord
injury or nerve
injury in a subject in need thereof comprising administering an effective
amount of at least one
ApoE analog to the subject. In one embodiment, the spinal cord injury is a
contusive injury or a
compressive injury. In another embodiment, the nerve injury is a peripheral
nerve crush injury.
Preferably, the ApoE analog is administered shortly after the occurrence of
the spinal cord or
nerve injury, such as within 1 hour, 2 hours, 3, 4, 5, 6, 7, 8, 9, 10 hours,
18 hours, 24, hours, 36
hours, or 48 hours after the injury.
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[056] Axonal degeneration typically occurs after spinal cord or nerve injury.
When a nerve
fiber is cut or crushed, the part distal to the injury (i.e. the part of the
axon separated from the
neuron's nucleus) will degenerate. This process typically commences within 24
hours of a
lesion. Axonal degeneration is followed by loss of the myelin sheath (i.e.
demyelination) and
infiltration by macrophages, which scavenge the cellular debris left by the
degenerating axons.
The loss of axons is the cause of the loss of neuronal function (e.g. motor
and sensory function)
below the level of the injury. In one embodiment, axonal degeneration is
decreased in the
subject following administration of the at least one ApoE analog. In another
embodiment,
inflammation at the injured site is decreased following administration of the
at least one ApoE
analog. Inflammation may include the activation of microglia or macrophages as
well as the
release of inflammatory cytokines. Any of the ApoE analogs described herein
may be used in
treating spinal cord or nerve injury in a subject.
[057] The present invention also provides a method of promoting the survival
and/or
proliferation of oligodendrocyte precursor cells. In one embodiment, the
method comprises
treating the oligodendrocyte precursor cells with at least one ApoE analog,
wherein the number
of oligodendrocyte precursor cells is increased following treatment with the
at least one ApoE
analog. The treatment with the at least one ApoE analog may be in vitro or in
vivo. Methods of
detection and quantification of oligodendrocyte precursor cells are known in
the art. Such
methods include, but are not limited to, immunohistochemistry, in situ
hybridization, cell sorting
(e.g. FACS), and enzymatic assays. Oligodendrocye precursor cells as well as
mature
oligodendrocytes can be identified by one or more proteins that are
specifically expressed in
these cell types (i.e. markers). Some non-limiting examples of oligodendrocyte
markers include
the ganglioside GD3, the NG2 chondroitin sulfate proteoglycan, platelet-
derived growth factor-
alpha receptor subunit (PDGF-alphaR), glutathione S-transferase, and 2',3'-
Cyclic Nucleotide 3'-
Phosphodiesterase (CNPase).
[058] In another embodiment, calcineurin activity is inhibited in the
oligodendrocyte precursor
cells following treatment with the at least one ApoE analog. Methods of
measuring calcineurin
phosphatase activity are known in the art and include, but are not limited to,
in vitro
phosphorylation assays that monitor release of phosphate from a peptide
substrate, and western
blot methods that detect phosphorylation status of endogenous substrates in
cellular lysates. In
some embodiments, the at least one ApoE analog is a peptide derivative of COG
133 (SEQ ID
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NO: 1). The peptide derivative may contain a sequence of COG 1410 (SEQ ID NO:
2), COG
248 (SEQ ID NO: 3), COG 345 (SEQ ID NO: 4), COG 112 (SEQ ID NO: 5), COG 241
(SEQ ID
NO: 6), or COG 68 (SEQ ID NO: 7).
[059] Multiple sclerosis is an autoimmune disease that is one of the most
common neurological
diseases in young adults and affects more than 2 million people worldwide.
Multiple sclerosis is
characterized by inflammation and demyelination of the brain and spinal cord
accompanied by
extensive depletion of oligodendrocytes and axonal degeneration. Current
therapies for multiple
sclerosis target the inflammation associated with the disease rather than
restoring myelination.
Increased expression of ApoE protein, known for its role in lipid metabolism,
is reported to
occur at sites of active remyelination. Furthermore, levels of ApoE protein in
the cerebrospinal
fluid of multiple sclerosis patients proved to be a reliable marker of
patients in remission (Rifai
et al. (1987) Clin. Chem., Vol. 33: 1155-1157). Given that ApoE analogs
promote the survival
of oligodendrocyte precursor cells and oligodendrocytes as well as enhance
remyelination of
demyelinated axons (see Examples 3-5), ApoE analogs may be an effective
treatment for
multiple sclerosis. In fact, the inventors demonstrated that ApoE analogs
could promote
remyelination in an experimental autoimmune encephalomyelitis (EAE) model of
human
multiple sclerosis (MS) and this remyelination correlated with recovery from
clinical symptoms
of the disease (see Example 1).
[060] The present invention contemplates a method of treating multiple
sclerosis in a subject in
need thereof comprising administering an effective amount of at least one ApoE
analog to the
subject. ApoE analogs suitable in the inventive method include those ApoE
analogs described
herein. In some embodiments, the ApoE analog is a peptide derivative
containing a sequence
selected from the group consisting of COG 248 (SEQ ID NO: 3), COG 345 (SEQ ID
NO: 4),
COG 241 (SEQ ID NO: 6), and COG 68 (SEQ ID NO: 7). In a preferred embodiment,
the ApoE
peptide derivative contains the sequence of COG 345 (SEQ ID NO: 4). The ApoE
peptide
derivatives may be conjugated to a protein transduction domain as described
herein to facilitate
transport across the blood brain barrier and access to the central nervous
system. In another
embodiment, the amount of myelination is increased in the subject following
administration of
the at least one ApoE analog. In another embodiment, the number of
oligodendrocytes is
increased in the subject following administration of the at least one ApoE
analog. In still another
embodiment, the amount of neuronal inflammation is decreased in the subject
following
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administration of the at least one ApoE analog. Neuronal inflammation may be
measured by the
number of inflammatory cells infiltrating central nervous system tissue as
well as by activation
of microglia and macrophages.
[061] In another embodiment of the invention, one or more symptoms of multiple
sclerosis is
reduced in the subject following administration of the at least one ApoE
analog. Symptoms of
multiple sclerosis include, but are not limited to, changes in sensation
(hypoesthesia), muscle
weakness, abnormal muscle spasms, difficulty in moving, difficulties with
coordination and
balance (ataxia), problems in speech (dysarthria) or swallowing (dysphagia),
visual problems
(nystagmus, optic neuritis, or diplopia), fatigue and acute or chronic pain
syndromes, and bladder
and bowel difficulties. Cognitive impairment of varying degrees, or emotional
symptoms in the
form of depression or pseudobulbar affect are also common. Neuropathic pain,
described as
constant, boring, burning or tingling intensely, is typical and usually occurs
in the legs. Other
symptoms of multiple sclerosis include paraesthesias, such as pins and
needles, tingling,
shivering, burning pains, feelings of pressure, and areas of skin with
heightened sensitivity to
touch.
[062] In some embodiments, the ApoE analogs of the present invention are used
in combination
with a pharmaceutically acceptable carrier. The present invention thus also
provides
pharmaceutical compositions suitable for administration to a subject. Such
compositions
comprise an effective amount of an ApoE analog as described herein in
combination with a
pharmaceutically acceptable carrier. The carrier can be a liquid, so that the
composition is
adapted for parenteral administration, or can be solid, i.e., a tablet or pill
formulated for oral
administration. Further, the carrier can be in the form of a nebulizable
liquid or solid so that the
composition is adapted for inhalation. When administered parenterally, the
composition should
be pyrogen free and in an acceptable parenteral carrier. ApoE analogs can
alternatively be
formulated for encapsulation in liposomes, using known methods. Additionally,
the intranasal
administration of peptides to treat CNS conditions is known in the art (see,
e.g., U.S. Patent No.
5,567,682, incorporated herein by reference to Pert, regarding intranasal
administration of
peptide T to treat Alzheimer's Disease). Preparation of an ApoE analog of the
present invention
for intranasal administration can be carried out using techniques as are known
in the art.
Pharmaceutical preparations of the agents of the present invention can
optionally include a
pharmaceutically acceptable diluent or excipient.
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[063] An alternative method of administering ApoE analogs, such as peptide
derivatives, of the
present invention is carried out by administering to the subject a vector
carrying a nucleic acid
sequence encoding the peptide derivative, where the vector is capable of
entering cells of the
body, such as cells of the central nervous system, heart, kidney, and lung, so
that the peptide
derivative is expressed and secreted. In particular, expression of peptide
derivatives in the brain
and spinal cord make the ApoE peptide derivatives available to microglial
cells and
oligodendrocytes. Suitable vectors are typically viral vectors, including DNA
viruses, RNA
viruses, and retroviruses. Techniques for utilizing vector delivery systems
and carrying out gene
therapy are known in the art. Herpesvirus vectors, adenovirus vectors, adeno-
associated virus
vectors and lentiviral vectors are particular types of vectors that can be
employed in
administering compounds of the present invention.
[064] In another embodiment, an ApoE analog of the invention may be formulated
for topical
administration, particularly in the treatment of inflammatory skin conditions,
such as atopic
dermatitis (eczema), psoriasis, hand dermatitis, lichen planus, and vitiligo
(leukoderma). The
topical formulation may be a cream, gel, ointment, lotion, paste, spray, or
powder.
Pharmaceutical carriers for topical formulations include aqueous, powder or
oily bases,
thickeners, emulsifers, semi-solid preparations of hydrocarbons (petrolatum,
mineral oil,
paraffins, synthetic hydrocarbons), surfactants, emollients and the like.
Other suitable carriers
and ingredients for topical formulations are discernable to one skilled in the
art.
[065] In another embodiment, an ApoE analog as described herein may be
formulated as a
coating for a medical device, such as a stent or catheter. Particularly useful
in methods of
treating restenosis in a subject, the ApoE analog may be used to coat a metal
stent to produce a
drug-eluting stent. A drug-eluting stent is a scaffold that holds open
narrowed or diseased
arteries and releases a compound to prevent cellular proliferation and/or
inflammation. ApoE
analogs may be applied to a metal stent imbedded in a thin polymer for release
of the ApoE
analog over time. Methods of coating stents with therapeutic compounds are
known in the art.
See, e.g., U.S. Patent No. 7, 144, 422; U.S. Patent No. 7, 055, 237; and WO
2004/004602, which
are here incorporated by reference in their entireties. In some embodiments,
the ApoE analog
may be used in combination with other anti-restenosis compounds to produce a
formulation for
incorporation into drug-eluting stents. Suitable compounds for use in
combination with the
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ApoE analogs disclosed herein include, but are not limited to, paclitaxel,
rapamycin (sirolimus),
tacrolimus, zotarolimus, everolimus, docetaxel, pimecrolimus, and derivatives
thereof.
[066] The ApoE analogs described herein may be used alone to modulate
calcineurin activity
and promote remyelination or in combination with other standard therapeutic
agents prescribed
to treat the indicated conditions. In one embodiment, the ApoE analogs may be
administered in
combination with other immunosuppressants, including sirolimus (rapamycin),
other calcineurin
inhibitors, glucocorticoids, cytostatics, antibodies, opioids, tumor necrosis
factor-alpha binding
proteins, such as infliximab (Remicade), etanercept (Embrel), and adalimumab
(Humira), and
small molecules, such as FTY720. Other calcineurin inhibitors include
tacrolimus (FK506),
cyclosporin A, and pimecrolimus (Elidel). In another embodiment, the ApoE
analogs may be
co-administered with other anti-inflammatory agents, such as corticosteroids,
hydrocortisone,
prednisone and the like as well as anti-inflammatory cytokines, growth
factors, or leukocyte
migration inhibitory compounds. Useful cytokines include, but are not limited
to, IL-4, IL-10,
IL-11, and IL- 13, particularly IL-4 and IL- 10, which are known to suppress
production of
inflammatory cytokines and to be involved in restoring the immune system.
Growth factors
include GM-CSF among others. These cytokines and growth factors may be
administered as
purified proteins--obtained naturally or from recombinant sources--or
administered in the form
of nucleic acids that express these peptides, particularly as fusion proteins.
[067] The ApoE analogs may also be used in combination with anti-diarrheal
agents such as
loperamide and the like, antibacterial agents such as penicillin,
cephalosporins, bacitracin and the
like; antiparasitic agents such as quinacrine, chloroquine and the like;
antifungal agents such as
nystatin, gentamicin, and the like; antiviral agents such as acyclovir,
gancyclovir, ribavirin,
interferons and the like; analgesic agents such as salicylic acid,
acetaminophen, ibuprofen,
flurbiprofen, morphine and the like; local anesthetics such as lidocaine,
bupivacaine, benzocaine
and the like; growth factors such as colony stimulating factor, granulocyte-
macrophage colony
stimulating factor, and the like; antihistamines such as diphenhydramine,
chlorphencramine and
the like; anti-nausea medications, nutritional additives such as leukovorin,
standard multiple
sclerosis therapies, such as beta interferon la (AvoneX and Rebif ) and lb
(Betaserori ),
glatiramer acetate (Copaxone(x), mitoxantrone (Novantrone(x), and Tysabri
(Biogen-Idec and
Elan), and other like substances. Combination therapies (e.g. ApoE analogs and
another
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therapeutic agent) can be co-administered or formulated together in a single
pharmaceutical
composition.
[068] The ApoE analogs of the present invention can be administered acutely
(i.e., during the
onset or shortly after events leading to an inflammatory, neurological, heart,
renal, or pulmonary
condition), or can be administered prophylactically (e.g., before scheduled
surgery, or before the
appearance of inflammatory, neurological, cardiac, renal, and/or pulmonary
signs or symptoms),
or administered during the course of a degenerative disease (e.g. multiple
sclerosis) to reduce or
ameliorate the progression of symptoms that would otherwise occur. The timing
and interval of
administration is varied according to the subject's symptoms, and can be
administered at an
interval of several hours to several days, over a time course of hours, days,
weeks or longer, as
would be determined by one skilled in the art.
[069] The typical daily regime can be from about .01 g/kg body weight per
day, from about 1
mg/kg body weight per day, from about 10 mg/kg body weight per day, from about
100 mg/kg
body weight per day, from about 1,000 mg/kg body weight per day. Dosages can
be between
about 0.01 g/kg and about 10 mg/kg body weight per day, depending on the ApoE
analog, or
between about 1 mg/kg and about 10 mg/kg body weight per day.
[070] As used herein, the term "administering to the central nervous system of
a subject" refers
to the use of routes of administration, as are known in the art, that provide
the compound to the
central nervous system (CNS) tissues, and in particular the brain and spinal
cord, of a subject
being treated.
[071] The blood-brain barrier presents a barrier to the passive diffusion of
substances from the
bloodstream into various regions of the CNS. However, active transport of
certain agents is
known to occur in either direction across the blood-brain barrier. Substances
that can have
limited access to the brain from the bloodstream can be injected directly into
the cerebrospinal
fluid. Cerebral ischemia and inflammation are also known to modify the blood-
brain barrier and
result in increased access to substances in the bloodstream.
[072] Administration of an ApoE analog directly to the central nervous system
is known in the
art. Intrathecal injection administers agents directly to the brain ventricles
and the spinal fluid.
Surgically-implantable infusion pumps are available to provide sustained
administration of
agents directly into the spinal fluid. Lumbar puncture with injection of a
pharmaceutical agent
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into the cerebrospinal fluid ("spinal injection") is known in the art, and is
suited for
administration of the present ApoE analogs.
[073] Pharmacologic-based procedures are also known in the art for
circumventing the blood
brain barrier, including the conversion of hydrophilic compounds into lipid-
soluble drugs. The
ApoE anlaog can be encapsulated in a lipid vesicle or liposome.
[074] The intra-arterial infusion of hypertonic substances to transiently open
the blood-brain
barrier and allow passage of hydrophilic drugs into the central nervous system
is also known in
the art. US Patent No. 5,686,416 to Kozarich et al. discloses the co-
administration of receptor
mediated permeabilizer (RMP) peptides with compounds to be delivered to the
interstitial fluid
compartment of the brain, to cause an increase in the permeability of the
blood-brain barrier and
effect increased delivery of the compounds to the brain.
[075] In particular methods of the invention, it is desirable to administer an
ApoE analog to a
subject such that the ApoE analog can access cells of the central nervous
system. To do so, the
ApoE analog is required to cross the blood brain barrier. One method of
transporting an active
agent across the blood-brain barrier is to couple or conjugate the ApoE analog
to a second
molecule (a "carrier"), which is a peptide or non-proteinaceous moiety
selected for its ability to
penetrate the blood-brain barrier and transport the ApoE analog across the
blood-brain barrier.
In addition to the protein transduction domains described herein, which can
facilitate transport
across the blood brain barrier, other suitable carriers include pyridinium,
fatty acids, lipids,
inositol, cholesterol, and glucose derivatives. The carrier can be a compound
which enters the
brain through a specific transport system in brain endothelial cells. Chimeric
peptides adapted
for delivering neuropharmaceutical agents into the brain by receptor-mediated
transcytosis
through the blood-brain barrier are disclosed in U.S. Patent No. 4,902,505
(Pardridge et al).
These chimeric peptides comprise a pharmaceutical agent conjugated with a
transportable
peptide capable of crossing the blood-brain barrier by transcytosis. Specific
transportable
peptides disclosed by Pardridge et al. include histone, insulin, transferrin,
and others.
Conjugates of a compound with a carrier molecule, to cross the blood-brain
barrier, are also
disclosed in U.S. Patent No. 5,604,198. Specific carrier molecules disclosed
include
hemoglobin, lysozyme, cytochrome c, ceruloplasmin, calmodulin, ubiquitin and
substance P.
See also U.S. Patent No. 5,017,566, which is herein incorporated by reference
in its entirety.
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[076] The examples which follow are set forth to illustrate the present
invention, and are not to
be construed as limiting thereof.
EXAMPLES
Example 1. COG peptides reduce spinal cord demyelination and inflammation in a
mouse
model of multiple sclerosis
[077] A myelin oligodendrocyte glycoprotein (MOG)-induced experimental
autoimmune
encephalomyelitis (EAE) model of human multiple sclerosis (MS) was used to
test whether COG
peptides had a therapeutic effect on animals with established disease.
C57BL/6J mice were first
immunized with MOG to create an EAE model. On the day that an animal first
showed a clinical
score (CS) > 2, it was randomly assigned to a group treated daily with 1
mg/kg, i.p. of COG 133
(SEQ ID NO: 1), 1 mg/kg, i.p. of COG 112 (SEQ ID NO: 5) or normal saline.
Treatment with
COG 112 and COG 133 significantly slowed the deleterious progress of the
disease and
promoted recovery to a normal clinical behavior (Figure 1A and B). Even though
the peptide
treatment started much later (from about day 14 post-immunization (dpi) on
average) than that of
a pre-treatment paradigm (dpi 2), COG 112 and COG 133 still significantly
reduced the maximal
severity of disability. The most profound improvement was found with post-
treatment of COG
112 where significantly more animals displayed complete recovery or remission
from the disease
when compared to saline controls. On dpi 35, 71% of COG 112-treated and 50% of
COG 133-
treated mice exhibited complete remission, however, only 22% of mice in the
saline control
group completely recovered (Figure 1B). In addition, both COG 112 and COG 133
significantly
reduced the burden of disability as measured by area under curve of Figure 1A
(data not shown).
[078] One of the pathological hallmarks of MS is extensive demyelination of
the myelin sheath
surrounding neurons accompanied by massive infiltration of peripheral
leukocytes into the brain
and the spinal cord, all of which contribute to the clinical manifestations of
neurological
disability. We further examined the effects of COG peptides on demyelination
and leukocyte-
influx into the spinal cords by Luxol Fast Blue (LFB) and Eosin staining.
Saline treated mice
displayed severe demyelination (shown as loss of blue staining in white
matter) in all segments
of the spinal cord as depicted in Figure 2A, B, and C, corresponding to the
cervical, thoracic and
lumbar segments, respectively. Panel G is a magnified view of the boxed area
in A. Interestingly,
COG 112 and COG 133 treatments greatly reduced demyelination and infiltration
of peripheral
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mononuclear cells in these regions as shown in a representative section from a
COG 112-treated
mouse in Figure 2D, E, F, and H (magnified view of the boxed area in D). These
histopathological findings are consistent with the decreased clinical severity
of EAE observed in
COG 112- and COG 133-treated mice.
[079] In accord with the histopathological demonstration of inflammatory
infiltrates shown
above, we also quantified and profiled T lymphocyte infiltrates in the spinal
cord to evaluate the
effect of COG peptides on T cell migration. Since COG 112 displayed a more
potent therapeutic
effect than COG 133, we examined the frequency of CD3+, CD4+, and CD8+ T cells
in the
spinal cords of COG 112-treated mice compared to vehicle controls on dpi 21.
Single-cell
suspensions of spinal cord were stained with anti-CD3, CD4, and CD8 mAbs. As
shown in
Figure 3A & C, the frequency of CD3+ T cells in the spinal cords of mice
treated with COG 112
was reduced as compared to vehicle controls on dpi 21. COG 112 treatment also
significantly
reduced CD4+ T cell infiltration into the spinal cord (Figure 3E). However, no
significant
difference was observed in the number of CD8+ T cells with peptide treatment
(Figure B & D).
[080] The results of these experiments show that COG peptides (i.e. ApoE
analogs) improve
the clinical symptoms of EAE as well as reduce the associated demyelination
and inflammation
of the spinal cord.
Example 2. COG peptides inhibit macrophage activation in EAE mice
[081] To examine the effect of COG 133 (SEQ ID NO: 1) treatment on activation
of
macrophages in EAE mice, macrophages were collected from a subset of saline
and COG 133-
treated mice on dpi 35 and then challenged in vitro with the immune
stimulators
lipopolysaccharide and interferon-y (LPS + IFN-y). Nitrite in culture medium
was measured by a
Sievers NO Analyzer and TNF-a and IL-6 were measured by ELISA. Upon treatment
with LPS
and IFN-y, macrophages derived from saline-treated mice responded with a
robust release of
TNF-a and IL-6 compared to the significantly lower response of macrophages
derived from
COG 133-treated mice (p < 0.001; Fig. 4A and B). Similarly, macrophages
derived from saline-
treated mice exhibited a robust release of NO when treated in vitro with LPS +
IFN-7 compared
to the significantly lower levels of NO released from macrophages derived from
COG 133-
treated animals (p < 0.001; Fig. 4C).
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[082] We then examined the ability of COG 112 (SEQ ID NO: 5) and COG 133 (SEQ
ID NO:
1) to inhibit the in vitro release of NO, TNF-a and IL-6 using macrophages
obtained from saline-
treated EAE mice on dpi 35. Conditioned medium with or without COG 133 or COG
112 was
collected 45 hours after immunogen (LPS+IFN-y) exposure for analysis of TNF-a
and IL-6, or
72 hours after stimulation for measurement of nitrite. COG 112 treatment
resulted in a dose-
dependent suppression of TNF-a and IL-6 levels, with complete inhibition
occurring at about 5
M (Figure 5A and B), while 5 M of COG 133 exhibited only moderate, yet
statistically
significant, suppression of both TNF-a and IL-6. In LPS+IFN-y treated
macrophages, 5 M
COG 112 completely inhibited NO release (p < 0.00 1 versus control), while the
same
concentration of COG 133 inhibited NO release by about 37% (p < 0.05 versus
control) (Figure
5C). Levels of NOS2 expression were quantified by real-time PCR. NOS2
expression was first
normalized by the endogenous control (18S rRNA) and then expressed as fold
change compared
to control group. As shown in Figure 5D, COG 112 and COG 133 affect NOS2
expression as
well as NO production, with COG 112 exhibiting a more potent effect than COG
133.
[083] In summary, macrophage activation in response to stimulation with an
immuogen is
suppressed by exposure to COG peptides either in vivo or in vitro. Thus, COG
peptides are able
to modulate the activation status of macrophages, which play a pivotal role in
the initiation and
progress of inflammatory disease, such as EAE and MS.
Example 3. COG 112 promotes remyelination in an in vivo model of
demyelination.
[084] In an EAE model, treatment with COG 112 reduced demyelination in the
spinal cord
(Example 1). Therefore, we tested the effect of COG peptides on remyelination
in a cuprizone-
induced demyelination model. C57BL/6J male mice were fed with 0.2% cuprizone-
containing
mouse chow for 5 weeks to cause demyelination. At the end of week 5, cuprizone-
containing
chow was replaced with regular chow and the animals started receiving either
vehicle or 1
mg/kg, i.p. COG 112 (SEQ ID NO: 5) three times a week (Monday, Wednesday and
Friday
paradigm) for 4 weeks. At the end of the treatment period, animals were
sacrificed for evaluation
of demyelination. 5 m thick coronal sections containing corpus callosum were
stained with
Luxol Fast blue (LFB) and eosin. Adjacent sections were labeled with
glutathione S-transferase-
n (GST-n) antibody to identify mature oligodendrocytes.
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[085] As shown by the loss of blue staining in the corpus callosum, cuprizone
administration
for 5 weeks caused extensive demyelination (Figure 6A). The removal of
cuprizone is expected
to allow remyelination to occur to some extent. However, no significant
remyelination was
observed in vehicle-treated animals, in which cuprizone had been absent for 4
weeks (Figure
6E). In contrast, 1 mg/kg COG 112, administered by i.p. injection three times
a week,
significantly increased LFB staining of myelin within corpus callosum (Figure
6B and E). Given
that the treatment was initiated after full demyelination at 5 weeks, these
data suggest that COG
112 promotes remyelination. Significantly more GST-7u+ cells within the corpus
callosum ribbon
were observed in COG 112-treated animals compared to vehicle-treated control
animals (Figure
6C and D). This finding is consistent with the enhanced remyelination within
the same area after
treatment with COG 112, suggesting COG 112 enhances the migration and/or
maturation of
oligodendrocyte precursor cells in vivo. Taken together, the results of these
experiments suggest
that ApoE analogs (i.e. COG peptides) act as remyelinating agents as well as
immunomodulatory
agents. Thus, ApoE analogs represent a novel therapy for demyelinating
disorders.
Example 4. COG 112 promotes remyelination in an in vitro demyelination model
[086] The effect of COG peptides (i.e. ApoE analogs) on remyelination in a
second model of
demyelination was examined. For this series of experiments, a lysolecithin-
induced in vitro
demyelination model in an organotypic cerebellar slice culture was used.
Parasagittal slices of
postnatal Day 10 (P10) rat cerebellum were cut at 400 m using a Tissue Slicer
(SD Instruments,
Grants Pass, OR). After culture in conditioned medium for 7 days in vitro
(DIV), fresh medium
with lysolecithin (0.5 mg/ml) was added to the slice and incubated overnight
(15-17 hours) at
37 C. The lysolecithin-containing medium was then replaced with fresh medium
without
lysolecithin. On day 3, COG 112 (1 M) was added to the slice with fresh
medium. After
incubation for 5 days, the cerebellar slices were either immunohistologically
stained for myelin
basic protein (MBP), a myelin marker, (Figure 7A-D) or lysed for western
blotting and
subsequent probing for MBP (Figure 7E and F). The glial specific toxin,
lysolecithin, induced
significant demyelination 5 days later (Figure 7C), while post-treatment with
COG 112 robustly
elevated the level of MBP protein (Figure 7D), suggesting COG 112 treatment
enhanced
remyelination. Similar results were obtained from the Western blot analysis of
lysates of the
cerebellar slices (Figure 7E and F). Lysolecithin-treated slices exhibited a
reduced level of
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MBP, while post-treatment with COG 112 increased MBP levels. In addition, the
blots of
cerebellar slice lysate were also probed for oligodendrocyte (OL) marker 2',3'-
Cyclic Nucleotide
3'-Phosphodiesterase (CNPase). COG 112 significantly increased the level of
CNPase (data not
shown), suggesting that COG 112 increased the number of OLs. Because COG 112
was added
after demyelination was established, the increased number of OLs is likely to
be the result of
maturation of oligodendrocyte precursor cells (OPCs).
[087] We next investigated whether COG 112 could prevent lysolecithin-induced
demyelination. Cerebellar slices from postnatal day 10 rat pups were cultured
for 7 days.
Lysolecithin was added to a final concentration of 0.5 mg/ml in the presence
or absence of COG
112 (1 M). After incubation overnight (16 hours), medium was replaced with
fresh lysolecithin-
free medium, but the medium in the COG 112-treatment group still contained COG
112. After 2
days, the cerebellar slices were collected and lysed for western blotting with
myelin marker MBP
and oligodendrocyte marker CNPase. The density of each MBP or CNPase band was
normalized
to the density of the (3-actin band. As shown in Figure 8, COG 112 did not
prevent lysolecithin-
induced demyelination (left panel), but did prevent oligodendrocyte cell death
as evidenced by
the higher level of CNPase in the COG 112-treated group (right panel).
Example 5. COG peptides enhance the survival and proliferation of
oligodendrocyte
precursor cells
[088] A shortage of oligodendrocyte precursor cells/oligodendrocytes
(OPCs/OLs) in the locus
of demyelination could be responsible for the failure to remyelinate. The
insufficient number of
OPCs may be largely due to progressive cell death by repeated or chronic
autoimmune insults. It
is known that OPCs are very vulnerable to the inflammatory microenvironment
during the
autoimmune attack, which is mostly dependent on microglia activation (Lehnardt
et al. (2002) J
Neurosci., Vol. 22(7):2478-2486). Therefore, inhibition of microglia
activation and subsequent
inflammatory factors may preserve the OPCs and benefit subsequent
remyelination by surviving
oligodendrocytes.
[089] Given that COG peptides can robustly inhibit microglia and macrophage
activation (Li et
al. (2006) J Pharmacol Exp Ther., Vol. 318(3):956-965), we tested whether COG
112 could
protect OPCs from inflammation-mediated cell death in an OPC-microglia mixed
culture. A
primary culture prepared from the brains of P2 Sprague-Dawley rat pups was
grown for seven
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days. Microglia and OPCs were shaken off from the primary culture with an
orbital shaker at
150-200 rpm. The OPCs and microglia were plated in 24-well plates at a 1:1
ratio with 5x104
cells per well of each cell type. After 24 hours, LPS (final conc. 10 ng/ml)
was added with or
without 1 M COG 112. The prefix peptide of COG 112, antennapedia, was used as
a negative
control. After incubation for 72 hours, the medium was removed and cells were
fixed with 4%
formaldehyde and stained with the OPC marker, NG2, and counterstained with
hoescht. Five
fields from each of the wells were randomly chosen to count the number of NG2-
positive cells.
As shown in Figure 9, COG 112 significantly reduced LPS-induced cell death of
OPCs, while its
prefix peptide (antennapedia) did not have any effect.
[090] We next determined whether ApoE analogs (e.g. COG peptides) had an
effect on OPC
proliferation in addition to their ability to enhance OPC survival. Enriched
OPCs were collected
by shaking a primary culture of P2 rat pup brain. Proliferation medium
containing COG 112 (1
M), BrdU (20 M), 10 ng/mL FGF and PDGF was added to the enriched OPC cultures.
After
three days in culture, cells were stained with Hoescht stain to show nuclei, a
BrdU antibody to
identify proliferating cells, and an A2B5 antibody to identify OPCs (Figure
10). Antennapedia,
the prefix peptide of COG 112, was used as a control. COG 112 significantly
increased the
number of A2B5+/BrdU+ cells, suggesting that ApoE analogs promote OPC
proliferation (Figure
10, right panel).
[091] Since COG 112 increased the proliferation of OPCs, we next examined
whether the
ApoE analogs could also increase the number of mature oligodendrocytes.
Enriched OPC
cultures were prepared as described for the previous experiment. 2 M COG 133
(SEQ ID NO:
1), 1 M COG 112 (SEQ ID NO: 5), or 1 M COG 68 (Ac-
RRLSYSRRRFLRVRLASHLRKLRKRLL-NH2; SEQ ID NO: 7) were added to the enriched
OPC cultures in proliferation media for four days. The media was then replaced
with
differentiation medium without the ApoE analogs for an additional six days.
Cells were labeled
with a GST-n antibody to detect mature oligodendrocytes. All three ApoE
analogs significantly
increased the number of mature oligodendrocytes in culture (Figure 11).
[092] The findings of this series of experiments demonstrate that ApoE analogs
enhance the
proliferation of OPCs as well as their survival in an inflammatory
environment. ApoE analogs
also increase the number of mature oligodendrocytes (OLs). The ability of ApoE
analogs to
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preserve the number of OPCs/OLs and their function may be one mechanism by
which ApoE
analogs promote remyelination.
Example 6. Effect of COG peptides on spinal cord injury
[093] Similar to MS, spinal cord injury (SCI) is another devastating
neurological disorder
manifested as massive demyelination in the white matter of the spinal cord and
extensive
microglial activation surrounding the injured site. To examine the therapeutic
effect of ApoE
analogs on spinal cord injury, COG 112 (SEQ ID NO: 5) was administered to rats
immediately
after a contusive spinal cord injury. Female Sprague-Dawley rats were
subjected to 175 kdyn
contusive impact with the Infinite Horizon Spinal Cord Impactor (Infinite
Horizon, L.L.C.,
Lexington, KY) at the T9 level of the exposed spinal cord. Immediately after
injury, the animal
received the first dose of COG 112 (1mg/kg) or vehicle by tail vein injection.
The second dose of
COG 112 or vehicle was given i.v. 5 hours later followed by daily i.p. doses
for 7 days. The
animals were sacrificed on day 7 and the spinal cord spanning the injured site
was subjected to
histological examination. The injured area and spared white matter of the
spinal cord were
evaluated by Luxol Fast blue (LFB) staining. Activation of microglia was
examined by staining
adjacent sections with an antibody to ED 1. The percentage of spared white
matter (WM) to total
spinal cord area at epicenter, 0.6 mm, 1.2mm, 1.8mm or 2.4 mm rostral (R) or
caudal (C) from
epicenter was significantly higher in COG 112-treated animals than animals
treated with saline
(Figure 12, top panels). These results show that COG 112 significantly reduced
the size of the
lesion and preserved the white matter from contusive injury to the spinal
cord. Most
impressively, about 50% of spared tissue remained at the epicenter of COG 112-
treated animals,
while only 15% remained in control animals. Similar protection was also
observed with COG
112 in a compression model of spinal cord injury in mice (data not shown).
[094] The microglia surrounding the injured spinal cord were robustly
activated as shown by
ED1-immunoreactivity 7 days after contusion (Figure 12, bottom panels).
Treatment with COG
112 dramatically decreased the ED 1 immunoreactive area in the injured spinal
cord. Decrease of
ED1+ area in caudal or rostral spinal cord was more obvious than in the
injured epicenter (Figure
12, bottom right panel), suggesting that COG 112 suppresses the activation of
microglia and the
inflammatory reaction after spinal cord injury.
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[095] Taken together, these findings support the use of ApoE analogs for
treatment of spinal
cord injury as well as other demyelinating disorders. ApoE analogs would
prevent
inflammation-mediated demyelination thus preserving nerve tissue, which would
benefit
functional recovery.
Example 7. COG 112 promotes the functional recovery from sciatic nerve injury
[096] We used a sciatic nerve crush model to investigate whether ApoE analogs
can prevent
functional loss and/or promote functional recovery after nerve injury.
C57BL/6J male mice were
anesthetized and the sciatic nerve on left hindlimb was crushed with Number 5
Jeweler's forceps
at mid-thigh level for 30 seconds. The animals were then randomly assigned to
receive either
COG 112 (1mg/kg, i.p.) or lactated Ringer's buffer two hours after crush
followed by daily dose
for 14 days. On day 4, two animals from each group were sacrificed and crushed
sciatic nerves
were dissected and silver stained using a FD NeuroSilver staining kit to
reveal degenerating
axons. As shown in Figure 13D, treatment with COG 112 significantly reduced
axonal
degeneration, indicating that COG 112 could protect nerves from degenerative
damage after
injury.
[097] On day 7 and 14, footprints for each animal were taken for quantitative
assessment of
hindlimb motor function. A Sciatic Functional Index (SFI), which takes into
account the
relationship between toes and feet of an animal's hindlimb, was calculated for
each animal as
previously described (Bain et al. (1989) Plast. Reconstr. Surg., Vol.
83(1):129-38). SFI values
of zero and 100 indicate normal and complete dysfunction, respectively. As
shown in Fig. 13A,
animals developed serious hindlimb dysfunction one week after sciatic nerve
crush.
Administration of COG 112 significantly improved functional recovery after
treatment for 2
weeks compared to the vehicle control group (P<0.001).
[098] In order to examine the effect of COG 112 on remyelination and
regeneration of axons,
segments of sciatic nerves spanning the crushed sites were dissected and
collected on day 3, 7
and 14 for examination of P0, a marker of peripheral myelinaton, and growth
associated protein
43 (GAP43), a marker of axonal regeneration, mRNA by real time PCR. COG 112
robustly
increased the expression of both P0 (Figure 13B) and GAP43 (Figure 13C) after
treatment for 2
weeks. These data collectively suggest that COG compounds can promote
functional recovery
33
CA 02692847 2010-01-07
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by inhibiting Wallerian degeneration after sciatic nerve crush and promoting
axonal
remyelination and regeneration.
Example 8. ApoE analogs inhibit calcineurin activity
[099] Calcineurin (also referred to as protein phosphatase 2B or PP2B) is a
calcium-dependent
serine/threonine phosphatase that is activated in response to inflammatory
stimuli. Calcineurin
can dephosphorylate a number of targets, such as the NFAT family of
transcription factors, that
lead to the expression of proteins involved in the immune response. To further
elucidate the
mechanism by which ApoE analogs inhibit inflammation-induced demyelination,
the effect of
COG peptides on calcineurin activity was examined.
[0100] Calcineurin activity was measured in a cell-free in vitro assay in the
presence of various
concentrations of COG 133 (SEQ ID NO: 1), COG 112 (SEQ ID NO: 5), COG 68 (SEQ
ID NO:
7), COG 125 (Ac-AS(Aib)LRKL(Aib)KR-COOH; SEQ ID NO: 8), or antennapedia
(antp), the
prefix peptide for COG 112. Inactive COG 125 peptide and antennapedia were
used as negative
controls. Using a BioMol Calcineurin Phosphatase Activity Kit (Cat.# AK-804),
the assay was
conducted according to manufacturer's instructions. All three active COG
peptides produced a
dose-dependent inhibition of calcineurin activity (Figure 14A). This
inhibition of calcineurin
activity by ApoE analogs is mediated by a direct interaction of the ApoE
analog with the
phosphatase. As shown in Figure 14B and C, immunoprecipitates from human brain
or primary
culture revealed that calcineurin (PP2B) bound to COG 133.
[0101] Calcineurin dephosphorylates the transcription factor NFAT, which leads
to its activation
and translocation to the nucleus. Activation of calcineurin and subsequent
activation of NFAT is
induced by inflammatory stimuli. To further examine the effect of ApoE analogs
on calcineurin
signaling, murine microglial cell line, BV2 cells were stimulated with LPS (10
ng/mL) in the
absence and presence of COG 112 (2 M). After incubation for 15 min at 37 C,
the cells were
washed and fixed with 4% formaldehyde. After staining with 1/100 anti-NFAT
antibody
coupled with Alexaflour and then counterstained with hoescht nuclear dye, NFAT
translocation
was quantified by the fluorescence intensity of Alexaflour in the nucleus. As
shown in Figure
15, COG 112 prevented NFAT translocation in response to stimulation with LPS.
These data
demonstrate that ApoE analogs can inhibit calcineurin signaling, and may be
one mechanism by
which ApoE analogs modulate the inflammatory response.
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CA 02692847 2010-01-07
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Example 9. ApoE analogs with enhanced efficacy in vitro
[0102] We have identified several novel ApoE analogs that exhibit improved
characteristics
compared to COG 133, such as enhanced potency and efficacy in both in vitro
and in vivo
models of inflammation and/or neuroprotection, enhanced remyelination-
promoting activity,
increased stability and increased blood brain barrier (BBB) penetration, as
well as decreased
toxicity. The following ApoE analogs, COG 112, COG 1410, COG 241, COG 248, COG
68,
and COG 345, have been identified as having improved properties as compared to
the ApoE
peptide, COG 133. COG 125 (a truncated version of COG 1410) did not exhibit
any anti-
inflammatory properties in vitro or in vivo and is used routinely as a
negative control.
Table 1. ApoE Analog Sequences
COG 133 SEQ ID NO: 1 acetyl-LRVRLASHLRKLRKRLL-amide
COG 1410 SEQ ID NO: 2 acetyl-AS-Aib-LRKL-Aib-KRLL-amide
COG 248 SEQ ID NO: 3 acetyl-LRVRLAS-Aib-LRKLRK(nitro-Arg)LL-amide
COG 345 SEQ ID NO: 4 acetyl-LRVRLAS-Aib-LRKLRK(acetyl-Arg)LL-amide
COG 112 SEQ ID NO: 5 acetyl-
RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-
amide
COG 241 SEQ ID NO: 6 acetyl-Aib-LRKL-Aib-(n acetyl K)RLL-amide
COG 68 SEQ ID NO: 7 acetyl-RRLSYSRRRFLRVRLASHLRKLRKRLL-amide
COG 125 SEQ ID NO: 8 acetyl-AS-Aib-LRKL-Aib-KR-amide
[0103] The murine microglia cell line BV2 was used as the first screening
platform to compare
the potency of ApoE analogs in anti-inflammatory activity. In response to LPS,
BV2 produces
amounts of reactive radical NO and inflammatory cytokines, such as TNF-a and
IL-6. We
stimulated the cells with LPS (10 ng/ml) in the presence of several
concentrations of each of the
ApoE analogs. We measured NO in medium by Griess assay and TNF-a by ELISA 24
hours
after LPS stimulation of the cells. The IC50 value was calculated for each of
the ApoE analogs.
As shown in Figure 16A and B, the activity of COG 1410, COG 68, COG 112, COG
345, and
COG 248 was clearly superior to that of COG 133 in suppression of microglia
activation.
CA 02692847 2010-01-07
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[0104] To determine the anti-oxidant effect of the different ApoE analogs, the
human
macrophage cell line U937 was differentiated with IFN-7 (40 U/ml) for 4 days
and treated with
phorbol-12-myristate-l3-acetate (PMA) and LPS with or without ApoE analogs (10
M). The
release of superoxide in the medium was measured by Luminol Superoxide Assay
kit
(Calbiochem, CA). Both COG 112 and COG 345 significantly inhibited superoxide
release
during inflammation (Figure 16C).
[0105] The activity of four of the analogs was compared to that of COG 133
using an in vivo
model of inflammation. Groups of five mice received i.p. injection of LPS (1
mg/kg). Within 5
min, they were given COG 133 (4 mg/kg, i.v.) or molar equivalent dose of COG
112, COG
1410, COG 248, COG 345, or COG 125 (negative control). Whole blood was
collected 1 hour
after LPS injection. The level of TNF-a in plasma was quantified with ELISA
and expressed as
percent of TNF-a production in plasma vs. vehicle control (LR buffer). All of
the ApoE analogs
demonstrated enhanced suppression of TNF-a release as compared to COG 133
(Figure 17).
[0106] In addition to testing their anti-inflammatory activities, we compared
the anti-
excitotoxicity activity of the novel ApoE analogs. Primary neurons growing on
an astrocyte layer
for 14 days were treated with COG 133, COG 68, COG 112, or COG 345 for 15 min.
NMDA
was subsequently added to the medium to a final concentration of 200 M. After
incubation for 5
minutes, the NMDA-containing medium was replaced with fresh medium with the
same ApoE
analogs at the designated concentration. After 24 hours, LDH in the medium was
measured and
used as an index of cell death (Figure 18). The wells treated with NMDA alone
for 24 hours
were considered 100% cell death and used for normalization with the other
treatments. MK801 is
a NMDA-receptor antagonist and was used as a negative control.
[0107] Example 10. Novel ApoE analogs promote remyelination and protect
oligodendrocyte precursor cells from cell death
[0108] We tested the remyelinating effect of COG 345 on the lysolecithin-
induced
demyelination model of cerebellum slice culture. As described in Example 4,
cerebellar slices
obtained from P10 rat pups were first incubated with lysolecithin (0.5 mg/ml)
for 16 hours to
induce demyelination. Three days after lysolecithin exposure, 1 M COG 345
(SEQ ID NO: 4)
or COG 112 (SEQ ID NO: 5) was added to fresh medium and the slice was
incubated for 4 days.
Slices were fixed and double stained with myelin marker MBP (green) and
Purkinje cell marker
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CA 02692847 2010-01-07
WO 2009/018477 PCT/US2008/071815
calbindin (red). As shown in Figure 19A-D, COG 345 treatment dramatically
restored
myelination similar to COG 112, suggesting COG 345 is capable of promoting
remyelination.
[0109] To further assess the ability of the novel ApoE analogs to promote
remyelination, the
effect of the analogs on OPC survival was assessed in an OPC/microglia mixed
culture. OPC and
microglia derived from primary culture of P2 rat pup brain were plated on 96-
well plates in 1:1
ratio. LPS (10 ng/ml) was added together with 1 M COG 112, COG 345, COG 248,
COG 1410,
COG 133, COG 125 (negative control) or FTY720. FTY720 is an immunosuppressive
agonist
for sphingosine-l-phosphate (S1P) receptors and is currently in clinical
trials for treating
multiple sclerosis. After 24 hours, cell death of OPC was quantified by both
LDH and MTT
assays. All ApoE analogs significantly reduced OPC cell death mediated by
microglia after
stimulation with LPS, with COG 112 and COG 345 being the most potent (Figure
19E and F). In
contrast, FTY720 did not show a clear protective effect.
[0110] To further compare the activity of the various ApoE analogs, the
efficacy of the analogs
was assessed in a PLP-induced relapsing-remitting experimental autoimmune
encephalomyelitis
(EAE) model. The first dose of the ApoE analog (4 mg/kg, s.c.) or vehicle (LR)
was started on
the day when the animals exhibited a clinical score (CS) > 1.5. The second
dose was
administered the next day followed by three times a week (M, W, F). The
results are
summarized in Table 2. Relapse rate represents the number of animals
experiencing a relapse,
while the CSmax indicates the maximal clinical score during relapse. Area
under curve
represents the area under the curve in a plot of clinical score vs. days post-
inoculation (out to 60
days). In particular, COG 248 and COG 345 exhibited a significant improvement
in relapse rate
and maximum clinical score during relapse. The improvement in relapse rate and
clinical score
in the EAE model by ApoE analog treatment may be due, in part, to the
remyelination-promoting
effect of the ApoE analogs.
Table 2. Efficacy of ApoE Analogs on PLP EAE Model
Ta=eaiine,-it Rt-laps:e .Raie CS,;..,, du!rli g relv,p:e Ai e:,i under
,me.:an,{SE.'N[?
LR s4 3,~t=~ T 3 4-.1 '? 9
t Of:il_3_i 14 i 2 i_} 7
=
t OGI12 5s4 ~ -=-~t~.;~
t ~ ~~~i24 1 8.. 151 ~ ~~. S 1 n -2 ~':
C'0 G ?4S, 4;141_6'-t0_2 .I.402t2_ 2
{ 'O G~-1, 4:" 5 1 z4"+o __ 1_
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CA 02692847 2010-01-07
WO 2009/018477 PCT/US2008/071815
[0111] To fully evaluate the use of ApoE analogs as therapeutic compounds, the
margin of
safety should also be determined. Towards that end, we determined the maximum
tolerated dose
of COG 112, COG 248, and COG 345 when administered by intravenous and
subcutaneous
injection. The results of this analysis is given in Table 3. For all compounds
except COG 133,
efficacy in an animal model of human diseases has been demonstrated and a
safety window of
greater than 20 (the maximum tolerated dose/minimum effective dose) by i.v.
injection and of >
100 for s.c. has been established. In the case of COG 345, this analog
demonstrated a superior
safety profile by intravenous injection compared to the other analogs when
compared on a molar
basis.
Table 3. Maximum Tolerated Dose and Minimum Effective Dose of ApoE Analogs
axiinuin TG-lera.t:ed Dose -kli:irimiitTi Efl'ective.
C'ompiiuud ln~tra-veno-us: Subc:utaneou.s Dos:e (~nodel)
e_"1 4:==. ~ ~ J' '7 {x -.._ . = .-~x i I \
~ ~~:1 1'~ ~ > 10' 1, ~~{7' I
~.L ~:
C0~.~1 ~ 2 2 " , 2 1 3 1 g v y ! h 100 1 l lg kg ~ .AE TBT;
t 0 (--214 ~' ~ InT>'k~ SQ: n :ka 1 mg Iig (F AEi
C0~.~34: 20 2 150 ~~=õ"lcg 1~~~ge1g :;~AE )
1-:AE. - Ea:l.~~n-artlita1 Atl~omuinma TBI t:on>; ut.ed Conlir al
zrqpactTBI
[0112] It is understood that the disclosed invention is not limited to the
particular methodology,
protocols and reagents described as these may vary. It is also understood that
the terminology
used herein is for the purposes of describing particular embodiments only and
is not intended to
limit the scope of the present invention which will be limited only by the
appended claims.
[0113] It must be noted that as used herein and in the appended claims, the
singular forms "a",
"an", and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such host cells,
reference to "the
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
[0114] Unless defined otherwise, all technical and scientific terms used
herein have the same
meanings as commonly understood by one of skill in the art to which the
disclosed invention
belongs. Although any methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, the exemplary
methods, devices, and
materials are as described. All patents, patent applications and other
publications cited herein
38
CA 02692847 2010-01-07
WO 2009/018477 PCT/US2008/071815
and the materials for which they are cited are specifically incorporated by
reference in their
entireties.
[0115] Those skilled in the art will recognize, or be able to ascertain using
no more than routine
experimentation, many equivalents to the specific embodiments of the invention
described
herein. Such equivalents are intended to be encompassed by the following
claims.
39
CA 02692847 2010-01-07
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TABLE 1. Other Suitable ApoE Analogs
LFt.I'RI.,s4S H Ki:<l:.-\H:>
.t _A :=r :`3Ri=F R K.E:-A 3:i?-'KRI L,(,N Lc),-NRz
._..
~,..~i~.i k~:.~>I.:s1.~~f T;.F`.,Fta ti:-=:~4-.r~y~ -Ã:~.F ~:-.1~39~ I ~i ~
~at). ~: ~Iiz
A ,>D S .,As3=-I R.KL"lb3k I,Y.,, M[.
ai::-:~~ j13-El(K t.>A at,
&c-A8M.Ex.M -.=23i;-KRLI,1v.E1z Ac-s~~z-r~31?-:Ã:~.~A:-.ai1y F tt~,~3Z
={~~.;o;~ ~1T.~{.
Ac-:g i-A-.AI a1'1A Ac;=A4-,aiE - E<RK~.=.~Yi~. \H2
t=vr:DR ,13b :o-4.SFf -LRl>dL.~,'~x1~~5,:~.,~3~3:
Ni1];
:^~t: TA~ ~:i}r 1 kt~.T::"t~=z s~i:~~[: E`~:Fi tu-:t~-,SiF_r=~1a~;i..,a'tF R
S:zRYii{. F1{.z, . l~r;
:~c;-:~t;~ .~,ik,-1;t~A,:a.-,='~i~ .~ti~z~,~} {4s..~r .L :~:E;:?
~at 3~k s~~.-~ti-~t~-~ 1:k~i:~ IC~~:~: \~~=; ~sC--A~~ _~ilr~.~c~r1;;sF ~; a~?
1~~:~ \f~:
c~t;-s'4;~ ,Ri~ :..~~rTt'=~~.~,: 3'~~l, :lt._~{L {o-~;T_~:;..;~~-Ã:r
AC-13 * :Zab-i RiF,-A a4>- KP,UL-N.T:i~
.ri_:-AS-r'i~b-1R.K'E.-A s`.:.K3~.L3"-\H at;...rIS- Az.,*-.r:Rarrr
A aAx ~-:u5 r k;1F .SL'-K#+y ~3 r L<,)-(N tti)NET;
:is::,w ,-',..A<ka-LRKE,-- Aif =K?~T_;=NH,,
Ac:..A:-S HIRKLAKIrLI N 10:#
Air-AS-:S:Er
~i:=,"t~_~~.;1~Iu~~~;:.~ `s-~:(t yt~ ~:.~.,r ~3~~ ~c<..;~_~~Ã~~ttit
Ib+~s'`.~.~<~ i{~~;
~~c :~~ ~:h ~ ~#~-~;.-.~õx-~ iF ar~`=~,~ s ~1#$a r~.^..,;~tiF-~~,:~ ~:
~:~F:i,`.F ;~i~~r
