U.S. patent application number 13/520623 was filed with the patent office on 2013-01-03 for apoe peptide dimers and uses thereof.
Invention is credited to Dale J. Christensen, Michael P. VITEK.
Application Number | 20130005645 13/520623 |
Document ID | / |
Family ID | 44305786 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130005645 |
Kind Code |
A1 |
VITEK; Michael P. ; et
al. |
January 3, 2013 |
APOE PEPTIDE DIMERS AND USES THEREOF
Abstract
The present invention provides novel pharmaceutical compositions
comprising ApoE-derived peptide dimers. In particular, the ApoE
peptide dimers of the invention comprise at least two ApoE mimetic
domains and can comprise one or more protein transduction domains.
Methods of treating various conditions, such as cancer,
inflammatory conditions, and neurodegenerative diseases, by
administering the pharmaceutical compositions of the invention are
also disclosed.
Inventors: |
VITEK; Michael P.; (Rescarch
Triangle, NC) ; Christensen; Dale J.; (Research
Triangle, NC) |
Family ID: |
44305786 |
Appl. No.: |
13/520623 |
Filed: |
January 6, 2011 |
PCT Filed: |
January 6, 2011 |
PCT NO: |
PCT/US11/20393 |
371 Date: |
September 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61292668 |
Jan 6, 2010 |
|
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Current U.S.
Class: |
514/1.4 ;
514/1.9; 514/13.2; 514/16.6; 514/17.5; 514/17.7; 514/17.8;
514/17.9; 514/18.2; 514/18.6; 514/19.3; 514/19.4; 514/19.6;
514/21.3; 530/324 |
Current CPC
Class: |
A61K 47/64 20170801;
A61P 25/16 20180101; A61P 1/00 20180101; A61P 9/10 20180101; A61P
29/00 20180101; A61P 35/00 20180101; A61K 38/1709 20130101; A61K
38/00 20130101; A61P 25/28 20180101; A61P 25/00 20180101; A61P
35/02 20180101; C07K 14/775 20130101; A61P 7/10 20180101; A61P
25/18 20180101; A61P 1/04 20180101; A61P 17/06 20180101 |
Class at
Publication: |
514/1.4 ;
530/324; 514/21.3; 514/19.3; 514/17.7; 514/17.5; 514/1.9; 514/17.9;
514/18.6; 514/18.2; 514/16.6; 514/13.2; 514/17.8; 514/19.4;
514/19.6 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 35/00 20060101 A61P035/00; A61P 25/00 20060101
A61P025/00; A61P 25/28 20060101 A61P025/28; A61P 9/10 20060101
A61P009/10; A61P 35/02 20060101 A61P035/02; A61P 25/16 20060101
A61P025/16; A61P 25/18 20060101 A61P025/18; A61P 29/00 20060101
A61P029/00; A61P 17/06 20060101 A61P017/06; A61P 1/00 20060101
A61P001/00; A61P 1/04 20060101 A61P001/04; C07K 14/775 20060101
C07K014/775; A61P 7/10 20060101 A61P007/10 |
Claims
1. A peptide dimer comprising a first ApoE peptide and a second
ApoE peptide, wherein said first and second ApoE peptides are
covalently linked by a linking moiety, and wherein the first and
second ApoE peptides contain a sequence derived from amino acids
133-149 of ApoE protein.
2. The peptide dimer of claim 1, wherein said linking moiety is
selected from the group consisting of a disulfide bridge, a
bismaleimide, a 1,4-disubstituted triazole, and
N,N-dipropargylamine.
3. The peptide dimer of claim 2, wherein said bismaleimide is
bismaleimido-ethane or bismaleimido-hexane.
4. The peptide dimer of claim 1, wherein said first and second ApoE
peptides are the same.
5. The peptide dimer of claim 4, wherein said first and second ApoE
peptides are peptides having a sequence selected from the group
consisting of LRVRLASHLRKLRKRLL (SEQ ID NO: 3),
AS(Aib)LRKL(Aib)KRLL (SEQ ID NO: 5),
LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4), and
LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35).
6. The peptide dimer of claim 1, wherein said first and second ApoE
peptides are different.
7. The peptide dimer of claim 1, wherein said first ApoE peptide is
conjugated to a first protein transduction domain through one or
more first linking residues.
8. The peptide dimer of claim 7, wherein said first protein
transduction domain is selected from the group consisting of
peptides derived from antennapedia, TAT, SynB1, SynB3, SynB5, and
polyarginine.
9. The peptide dimer of claim 8, wherein said first protein
transduction domain has a sequence of RQIKIWFQNRRMKWKK (SEQ ID NO:
8), YGRKKRRQRRR (SEQ ID NO: 9), or WKK.
10. The peptide dimer of claim 7, wherein said one or more first
linking residues is cysteine, azidohomoalanine, or
propargylglycine.
11. The peptide dimer of claim 7, wherein said first and second
ApoE peptides are peptides having a sequence selected from the
group consisting of LRVRLASHLRLRKRLL (SEQ ID NO: 3),
AS(Aib)LRKL(Aib)KRLL (SEQ ID NO: 5),
LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4), and
LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35).
12. The peptide dimer of claim 7, wherein said second ApoE peptide
is conjugated to a second protein transduction domain through one
or more second linking residues.
13. The peptide dimer of claim 12, wherein said second protein
transduction domain is selected from the group consisting of
peptides derived from antennapedia, TAT, SynB1, SynB3, SynB5, and
polyarginine.
14. The peptide dimer of claim 13, wherein said second protein
transduction domain has a sequence of RQIKIWFQNRRMKWKK (SEQ ID NO:
8), YGRKKRRQRRR (SEQ ID NO: 9), or WKK.
15. The peptide dimer of claim 12, wherein said one or more second
linking residues is cysteine, azidohomoalanine, or
propargylglycine.
16. The peptide dimer of claim 12, wherein said first and second
ApoE peptides are peptides having a sequence selected from the
group consisting of LRVRLASHLRKLRKRLL (SEQ ID NO: 3), AS
(Aib)LRKL(Aib)KRLL (SEQ ID NO: 5), LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL
(SEQ ID NO: 4), and LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35).
17. A pharmaceutical composition comprising an effective amount of
a peptide dimer of claim 1 and a pharmaceutically acceptable
carrier.
18. A method of treating cancer, CNS inflammation, traumatic brain
injury, cerebral ischemia, cerebral edema, neurolathyrism,
amyotrophic lateral sclerosis (ALS), Huntington's disease,
Parkinson's disease, schizophrenia, atherosclerosis, bacterial
sepsis, multiple sclerosis, rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, polyarticular-course juvenile
rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's
disease, ulcerative colitis, or Alzheimer's disease in a subject in
need thereof comprising administering to the subject a composition
of claim 17.
19. The method of claim 18, wherein said cancer is leukemia,
lymphoma, or breast cancer.
20. (canceled)
21. The method of claim 19, wherein said leukemia is chronic
lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), or
acute lymphocytic leukemia (ALL).
22-43. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/292,668, filed Jan. 6, 2010, which is herein
incorporated by reference in its entirety.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
COGO.sub.--022.sub.--01WO_SeqList_ST25.txt, date recorded: Jan. 6,
2011, file size 40 kilobytes).
FIELD OF THE INVENTION
[0003] The present invention relates to pharmaceutical compositions
comprising peptide dimers derived from apolipoprotein E (ApoE). The
present invention also relates to methods of treating various
disease states, such as cancer and neurodegenerative diseases, with
the novel compositions.
BACKGROUND OF THE INVENTION
[0004] Cancer is a class of diseases in which a group of cells
exhibit uncontrolled growth, invasion and destruction of adjacent
tissues, and metastasis (spread of aberrant cells spread to other
locations in the body), or in which cells fail to undergo
programmed cell death (e.g. apoptosis) at the appropriate time.
Cancer causes about 13% of all deaths worldwide and according to
the American Cancer Society, 7.6 million people died from cancer in
the world during 2007. Current treatment for cancer depends upon
the specific type of cancer and tissue involved, but includes
surgery, chemotherapy, radiation therapy, immunotherapy, and
monoclonal antibody therapy among other methods. Although these
treatment methods have been successful in some cases, they are
hindered by adverse side effects or limited efficacy. For example,
the efficacy of eliminating cancerous tissue by surgical removal of
tumors is often limited by the tendency of cancers to invade
adjacent tissue and metastasize to other sites in the body.
Chemotherapy, as well as radiation treatment, is often limited by
toxicity or damage to other tissues in the body. Thus, cancer
remains a major health concern and there is a need for improved
methods of treating cancer.
[0005] Inflammation is strongly correlated to cancer initiation,
progression and metastasis (Mantovani et al. (2008) Nature, Vol.
454: 436-444). Pro-inflammatory mediators such as prostaglandins,
cytokines, reactive oxygen/nitrogen species, and growth factors,
activate PI3K/Akt signaling that increases pro-survival,
proliferative, and metastatic processes (Dillon et al. (2007)
Oncogene, Vol. 26: 1338-1345; Qiao et al. (2008) Cell Cycle, Vol.
7: 2991-2996; Prueitt et al. (2007) International Journal of
Cancer, Vol. 120: 796-805; Wang and DuBois (2006) Gut, Vol. 55:
115-122). Mutations in the PI3K/Akt pathway are common in human
tumors, which result in unregulated PI3K/Akt signaling (Carnero et
al. (2008) Curr Cancer Drug Targets, Vol. 8: 187-98; Dillon et al.,
2007; Yuan and Cantley (2008) Oncogene, Vol. 27: 5497-5510). Thus,
pharmacological control of the PI3K/Akt signaling axis is an aim
for cancer therapeutics.
[0006] Akt kinase activity is directly regulated by the tumor
suppressor protein phosphatase 2A (PP2A), which functions to
dephosphorylate Akt at threonine 308 and serine 473 (Andjelkovic et
al. (1996) Proc. Natl. Acad. Sci., Vol. 93: 5699-5704; Resjo et al.
(2002) Cellular Signalling, Vol. 14: 231-238). However, PP2A
activity is commonly decreased in human cancers (Chen et al. (2004)
Cancer Cell, Vol. 5: 127-136). One mechanism by which PP2A activity
is suppressed in cancer is by the formation of complexes with
endogenous protein inhibitors such as CIP2A and I.sub.2PP2A
(Junttila et al. (2007) Cell, Vol. 130: 51-62; Li et al. (1996) J.
Biol. Chem., Vol. 271: 11059-11062). I.sub.2PP2A, which is also
known as SET, is a potent inhibitor of PP2A and has been implicated
in AML and blast crisis CML (Li et al., 1996; Neviani et al. (2005)
Cancer Cell, Vol. 8: 355-368). Despite the endogenous inhibition in
many human cancers, PP2A activity can be pharmacologically
increased and is a potential molecular target for cancer
therapeutics (Guichard et al. (2006) Carcinogenesis, Vol. 27:
1812-1827; Perrotti and Neviani (2008) Cancer and Metastasis
Reviews, Vol. 27: 159-168; Switzer et al. (2009) Oncogene, Vol. 28:
3837-3846).
[0007] ApoE-derived peptides have shown promising effects in
abating injury in inflammation-associated neuropathologies, such as
Alzheimer's disease, multiple sclerosis and traumatic brain injury
(Hoane et al. (2009) Journal of Neurotrauma, Vol. 26: 121-129; Li
et al. (2006) J Pharmacol Exp Ther, Vol. 318: 956-965; Wang et al.
(2007) Neuroscience, Vol. 144: 1324-33; WO 2006/029028; WO
2003/026479). Inflammation is a common feature of both neurological
diseases and cancer, and PI3K/Akt signaling is also unregulated in
neurodegenerative diseases such as Alzheimer's (Griffin et al.
(2005) J Neurochem, Vol. 93: 105-17; Pei et al. (2003) Acta
Neuropathol, Vol. 105: 381-92). Also, the expression of PP2A
subunits is decreased in Alzheimer's patients, which is consistent
with increased tau hyper-phosphorylation observed in this pathology
(Vogelsberg-Ragaglia et al. (2001) Experimental Neurology, Vol.
168: 402-412). ApoE peptides have also been reported to increase
PP2A activity by relieving inhibition by SET (see, e.g., WO
2008/080082). Thus, ApoE peptides represent a viable therapeutic
approach for treating various conditions, including cancer,
inflammatory conditions, and neurodegenerative diseases.
[0008] Although several ApoE peptides have proven to be effective
in treating specific conditions, there is a need in the art to
develop new ApoE-derived peptides with increased potency and
greater safety windows. In particular, it is desirable to develop
new ApoE-based peptide therapeutics that can effectively treat
multiple conditions.
SUMMARY OF THE INVENTION
[0009] The present invention is based, in part, on the discovery
that dimerization of ApoE peptides increases their biological
activity. Thus, the present invention provides novel ApoE peptide
therapeutics with increased potency as compared to monomeric ApoE
peptides. For instance, in one embodiment, a peptide dimer of the
invention comprises a first ApoE peptide and a second ApoE peptide,
wherein said first and second ApoE peptides are covalently linked
by a linking moiety. In certain embodiments, the first and second
ApoE peptides contain a sequence derived from the LDL receptor
binding region of the native ApoE holoprotein. The first and second
ApoE peptides may be identical or may be different.
[0010] In some embodiments, at least one of the ApoE peptides in
the dimer is conjugated to a protein transduction domain through,
optionally, one or more linking residues. In other embodiments,
both the first and second ApoE peptides in the dimer are each
conjugated to a protein transduction domain through, optionally,
one or more linking residues. The one or more linking residues can
include cysteine residues, or modified amino acids, such as
azidohomoalanine or propargylglycine. The protein transduction
domain can be a peptide derived from antennapedia, TAT, SynB1,
SynB3, SynB5, and polyarginine.
[0011] The first and second ApoE peptides in the peptide dimers of
the invention may be covalently linked by a linking moiety. The
linking moiety can include a disulfide bridge, a bismaleimide
(e.g., bismaleimido-ethane or bismaleimido-hexane), a
1,4-disubstituted triazole, and N,N-dipropargylamine.
[0012] The present invention also includes pharmaceutical
compositions of the ApoE peptide dimers of the invention. In one
embodiment, the pharmaceutical composition comprises an effective
amount of an ApoE peptide dimer as described herein and a
pharmaceutically acceptable carrier. In some embodiments, the
pharmaceutical compositions may further comprise additional
therapeutic compounds depending on the particular condition to be
treated.
[0013] The present invention also provides methods of treating,
preventing, or ameliorating various conditions or diseases,
including cancer, neurodegenerative disorders (e.g., ALS,
Alzheimer's disease, Parkinson's disease, and Huntington's
disease), and inflammatory conditions (e.g., multiple sclerosis,
inflammatory bowel disease, Crohn's disease, and rheumatoid
arthritis) by administering an effective amount of at least one
ApoE peptide dimer as described herein.
[0014] The present invention also includes a method for predicting
or evaluating the efficacy of a therapeutic intervention for
treating cancer in a patient. In one embodiment, the method
comprises measuring the expression level of SET protein in a
biological sample from a patient, and comparing the measured level
to the expression level of SET protein in a control sample, wherein
the measured expression level of SET protein is indicative of the
therapeutic efficacy of the therapeutic intervention. In certain
embodiments, the therapeutic intervention is an ApoE peptide or
peptide dimer described herein. The biological sample can be, for
example, a tumor biopsy from a solid tumor or mononuclear cells
isolated from a blood sample. In one embodiment, the biological
sample is CD19+/CD5+ leukemia cells.
[0015] The present invention also encompasses a kit for predicting
the therapeutic efficacy of ApoE peptides or peptide dimers for
treating cancer in a patient. In one embodiment, the kit comprises
a reagent for measuring SET protein expression in a biological
sample and instructions for measuring SET protein expression for
predicting or evaluating the efficacy of an ApoE peptide or peptide
dimer for treating cancer in a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. COG 112 inhibits LPS-induced production of
inflammatory cytokines in BV2 microglia cells. Inhibition curves
for COG112 in the production of NO (green squares), TNF.alpha. (red
diamonds), and IL-6 (blue triangles) by BV2 microglia. Compounds
were added to the final concentrations indicated on the graph along
with 100 ng/mL of LPS. After 24 hours the media was removed and
assayed by two-site ELISA (BioSource) and quantitated relative to a
standard curve on the same plate.
[0017] FIG. 2. Mass spectra of the original lot #313, reduced, and
chemically oxidized COG112. Mass spectra were obtained by LC/MS
with electrospray ionization in the positive mode for mass
detection. A. Mass spectra of original lot #313 of COG112. B. Mass
spectra of COG112 from lot #313 reduced with dithiothreitol. C.
Mass spectra of COG112 exposed to oxidizing conditions to form a
dimer. Red arrows indicate peaks that are characteristic of a
COG112 disulfide bridged dimer.
[0018] FIG. 3. NO Inhibition curve for COG449. BV2 microglia were
treated with COG449 at the final concentrations indicated on the
graph, along with 100 ng/mL of LPS. After 24 hours the media was
removed and assayed by two-site ELISA (BioSource) and quantitated
relative to a standard curve on the same plate.
[0019] FIG. 4. The BMOE-linked dimer peptide COG449 activates PP2A
in CML cells. A. 32D-BCR/Abl chronic myelogenous leukemia cells
were treated with COG449 (1 .mu.M), COG445 (1
.mu.M)(disulfide-linked COG112 dimer) or no treatment, and the PP2A
activity was measured. B. 32D:BCR/Abl cell cultures were treated
with no compound, 1 .mu.M COG449, or 5 .mu.M FTY720 for 30 minutes
followed by lysis in an NP40 lysis buffer. PP2A was
immunoprecipitated and assayed with the PP2A Immunoprecipitation
assay kit (Upstate) according to the manufacturer's directions.
[0020] FIG. 5. A. Dose response curves for COG449 cytotoxicity on
CLL cells from 4 leukemia patients. Human CLL cells were isolated
from blood samples and assayed for COG449 cytotoxicity. COG449 was
applied to B-CLL cells (0.25.times.10.sup.6 cells/well in a 96 well
plate) and after 72 hours, viable cells were assessed using the MTS
assay (Pharmacia) to determine the concentration of COG449 that was
effective in killing 50% of the input CLL cells (EC50). B. Dose
response curves for COG445 on CLL cells from 7 patients or normal
B-cells from 5 patients. Human CLL cells and PBMC were isolated and
assayed for cytotoxicity of COG445.
[0021] FIG. 6. A. Dose response curves for the indicated COG
peptides for LPS-induced nitric oxide production in BV2 microglia
cells. B. Dose response curves for the indicated COG peptides for
inhibition of MDA-MB-231 breast cancer cell growth. C. Dose
response curves for the indicated COG peptides for inhibition of
U87MG glioblastoma cell growth.
[0022] FIG. 7. Schematic representation of the approach to create
an ApoE peptide dimer library. PTD=protein transduction domain;
ApoE=apoE-mimetic domain; "X" and "Y" represent different linking
moieties.
[0023] FIG. 8. Bismaleimide coupling. A. The chemical
transformation involved in the bismaleimide coupling reaction.
Cysteine residues incorporated during peptide synthesis are coupled
together through a bismaleimide compound such as
bismaleimido-ethane (BMOE) or bismaleimido-hexane (BMH). B.
Chemical structures of BMOE and BMH.
[0024] FIG. 9. The chemical transformation involved in the "Click
Chemistry" reaction. Copper catalyzed 3+2 condensation results in
coupling through formation of a stable 1,4-disubstituted triazole.
A. Heterocoupling of a propargylglycine peptide and an
azidohomoalanine peptide results in a heterodimeric peptide. B.
Homodimeric peptides can be synthesized by coupling two
azidohomoalanine-containing monomer peptides with
N,N-dipropargylamine.
[0025] FIG. 10. COG445 inhibits EGF-induced Akt activation in
breast cancer and glioblastoma cell lines. A. Western blot analysis
of U87 glioblastoma cells exposed to the indicated concentrations
of COG445 peptide in the presence of EGF. The blot is probed with
antibodies for the activated EGF receptor (P-EGFR), total EGF
receptor, activated PDK1 (P-PDK1), and total PDK1. B. Western blot
and densitometry analysis of Akt activation induced by EGF in U87
cells in the presence of increasing concentrations of COG445.
[0026] FIG. 11. The effect of COG445 on Akt activation is mediated
through PP2A. A. Western blot analysis of MDA-MB-231 cells treated
with the indicated concentrations of COG445 and EGF in the presence
or absence of okadaic acid. The blot is probed with an antibody for
activated Akt (P-Akt) and total Akt. B. Densitometry analysis of
Akt activation induced by EGF in MDA-MB-231 cells in the presence
of increasing concentrations of COG445 with and without okadaic
acid treatment. The ratio of phosphorylated Akt to total Akt is
normalized to that of the EGF alone control. C. Nonlinear
regression analysis of the data depicted in panel B.
[0027] FIG. 12. A. Total PP2A activity immunoprecipitated from
MDA-MB-231 cells treated with EGF and increasing concentrations of
COG445. B. Western blot and densitometry analysis of total c-myc
protein levels in U87 cells treated with the indicated
concentrations of COG445 in the presence or absence of okadaic
acid. C. Immunoprecipitation of the catalytic subunit of PP2A from
MDA-MB-231 cells exposed to EGF and two different concentrations of
COG445. The blot is probed for I.sub.2PP2A (SET) and PP2A catalytic
subunit.
[0028] FIG. 13. A. Western blot and densitometry analysis of
phosphorylated m-TOR levels in MDA-MB-231 cells treated with EGF
and the indicated concentrations of COG445 in the presence or
absence of okadaic acid. B. Western blot analysis of phosphorylated
GSK-3.beta. levels in MDA-MB-231 cells treated with EGF and the
indicated concentrations of COG445 in the presence or absence of
okadaic acid.
[0029] FIG. 14. A. Dose response curve of COG445 on proliferation
of U87 (open circles) and MDA-MB-231 (filled circles) cells as
measured by MTT reduction. B. Dose response curve of COG445 on
proliferation of MDA-MB-231 cells as measured by cell count.
[0030] FIG. 15. SET antagonism reduces c-Myc phosphorylation at
S62. Raji cells were treated with COG449 or a vehicle control for
20 hrs and lysed. Lysates were analyzed by Western blotting with
anti-Phoshpo-S62 and total c-Myc antibodies. * indicates
p<0.05.
[0031] FIG. 16. SET is overexpressed in CLL. A. Scatter plot of the
SET/.beta.-Actin ratio measured for 16 CLL patients and 6 normal
B-cell samples showing a significant increase in expression of SET
in B-CLL cells relative to normal B-cells. Representative Western
blots are shown. B. mRNA was isolated from the same patient and
volunteer samples and SET mRNA was quantified by qPCR.
[0032] FIG. 17. SET is overexpressed in B-cell lymphoma lines. A.
mRNA was isolated from Raji and Ramos cells and normal B-cells and
SET mRNA was quantified by qPCR. B. Western blots showing SET and
GAPDH from the same cells in panel A showing a significant increase
in expression of SET in B-cell lymphoma lines relative to normal
B-cells (N004 and N007).
[0033] FIG. 18. Silencing of SET inhibits growth of Raji cells.
Growth of Raji cells monitored by MTT 72 hr after shRNA for a
control or SET was introduced by lentiviral transduction. Western
blots show that SET was reduced by about half relative to
.beta.-Actin loading controls.
[0034] FIG. 19. SET levels may be predictive of CLL disease
progression. The time from diagnosis to first needed treatment (the
"time-to-treatment") was assessed relative to CLL cell SET level
determined by immunoblot. Patients with high levels were compared
to those with lower levels (determined by receiver operating
characteristics) and had a statistically significantly shorter
time-to-treatment (n=226; p<0.002).
[0035] FIG. 20. Proposed regulatory mechanisms of Mcl-1 stability.
A. Sequence homology of the c-myc regulatory sites and the Mcl-1
sequence from 159-164 showing conservation of the S/T-X-S-S-S/T-P
(SEQ ID NO: 89) motif. B. A schematic representation of the
proposed regulatory complex for Mcl-1 (adapted from figure provided
by R. Sears).
[0036] FIG. 21. Co-Immunoprecipitation of Mcl-1 associated proteins
that may regulate Mcl-1 stability. Immunoblots of proteins (SET
(A), PP2A (B), Pin-1 (C), and Axin 1 (D)) that co-immunoprecipitate
with Mcl-1. "I" indicates lanes with input loading control and "IP"
indicates immunoprecipitation lanes.
[0037] FIG. 22. SET Antagonism reduces cellular Mcl-1
concentrations. Primary human CLL cells were plated and incubated
with the indicated concentrations of COG449 for 24 hrs. Cells were
lysed, subjected to PAGE and immunoblotted to quantify the Mcl-1
and .beta.-Actin ratio (* indicates p<0.01).
[0038] FIG. 23. Inhibition of the Ramos cell line of c-myc
dependent Burkitt's lymphoma growth in vivo by treatment with
COG449. Tumor growth in SCID mice treated with vehicle or COG449
peptide 19 days after injection with 10.sup.7 cells from the Ramos
cell line of Burkitt's lymphoma.
[0039] FIG. 24. A. A plot of tumor volume of Ramos cell tumor
xenografts in SCID mice with COG449 treatment (open squares) or
lactated Ringer's solution control (filled squares) being initiated
on day 11 once tumors reached a palpable size of 150-200 mm.sup.3.
B. Final tumor mass for treated and untreated Ramos tumors
harvested on day 19 after implantation. ***=p<0.001 by
T-test.
[0040] FIG. 25. SET and CIP2A are overexpressed in human primary
triple negative breast cancer (TNBC). SET (panel A) and CIP2A
(panel B) expression by qRT-PCR in 13 TNBC patient samples relative
to normal tissue (N).
[0041] FIG. 26. SET is overexpressed in human breast cancer cell
lines. SET protein expression along with actin in TNBC cell lines
by western blotting.
[0042] FIG. 27. COG449 reduces phosphorylation of eIF4E. U87MG
glioblastoma cells were treated with COG449 (1 .mu.M) or a vehicle
control for 20 hrs and phosphorylation of eIF4E was determined by
Western blotting with a phospho-specific antibody and a total eIF4E
antibody. Treatment with COG449 reduced the ratio of the phospho-
to total-eIF4E protein (n=3). * indicates p<0.01 compared to the
vehicle control.
[0043] FIG. 28. Cytotoxic effects of COG449 in breast cancer cells.
Cell lines were grown in serum-free media and COG449 as indicated
for 24 hrs. Cellular proliferation was measured by cell counting.
Cell number is represented relative to control, untreated
cells.
[0044] FIG. 29. Combination treatment with COG449 and sorafenib or
gefitinb on triple negative breast cancer (TNBC) cell line growth.
MDA-231 cells were grown in the presence of COG449, sorafenib, or
gefitinb at sub-lethal doses as indicated. After 48 hrs, live cells
were quantified using the MTT assay. *** indicates p<0.001.
[0045] FIG. 30. Inhibition of triple negative breast cancer (TNBC)
tumor growth in xenografts with COG449 treatment. 4.times.10.sup.6
MDA-MB-231 cells were injected with Matrigel into the 4th mammary
glands of immune compromised mice and treated daily by subcutaneous
injection of 10 mg/kg COG449 starting at day 10 (panel A), or mice
were treated by intravenous tail vein injection 3.times. week at 1
mg/kg starting at day 27 (panel B) post injection. Tumor volume was
determined by caliper measurement.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The inventors previously discovered that ApoE synthetic
peptides were useful in treating various types of cancer. See WO
2010/002982, filed Jul. 1, 2009, which is herein incorporated by
reference in its entirety. Here, the inventors have expanded upon
their earlier work finding surprisingly that dimers of the
synthetic ApoE peptides exhibit increased biological activity as
compared to their monomeric counterparts. The inventors discovered
that one particular lot of ApoE peptide, which was particularly
potent in activity assays, had been oxidized to form peptide
dimers. Additional experiments demonstrated that ApoE peptide
dimers formed through irreversible linkages were even more potent
than the reversibly-linked dimers. Accordingly, the present
invention provides novel peptide dimers derived from the receptor
binding region of ApoE. In one embodiment, the peptide dimer
comprises a first ApoE peptide and a second ApoE peptide, wherein
said first and second ApoE peptides are covalently linked by a
linking moiety.
[0047] ApoE peptides, also referred to as COG peptides, are
peptides derived from the native ApoE holoprotein. The peptide
dimers of the present invention comprise at least two ApoE peptides
or ApoE mimetic domains. The ApoE peptides or mimetic domains may
be derived from the LDL receptor binding region of the ApoE
holoprotein, namely amino acids 130-150 of full-length ApoE
protein. In certain embodiments, the ApoE peptides or mimetic
domains of the invention may be derived from at least amino acids
133-140 of ApoE. In one embodiment of the invention, the ApoE
peptide is derived from amino acids 130-149 of ApoE. In another
embodiment, the ApoE peptide is derived from amino acids 133-149 of
ApoE. In still another embodiment, the ApoE peptide is derived from
amino acids 138-149 of ApoE. As used herein, the phrase "derived
from" refers to a peptide that contains at least 80% identity to a
particular amino acid sequence from the ApoE protein or a peptide
that has at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 contiguous amino acid residues from a receptor
binding region of the ApoE protein (e.g. amino acids 130-150). By
way of example, a peptide having a sequence corresponding to amino
acids 133-149 with one, two, or three point mutations or amino acid
modifications would be considered to be derived from amino acids
133-149 of the ApoE protein. ApoE peptides or mimetic domains can
be derivatives of a peptide containing five or more, ten or more
residues, or 15 or more residues from amino acids 133-149 of native
ApoE protein, including derivatives having non-natural amino acid
substitutions, such as amino isobutyric acid and acetyl lysine, and
other modifications that enhance the alpha-helical content of the
peptide.
[0048] In one embodiment of the invention, the first and/or second
ApoE peptide of the peptide dimer has a sequence of
LRVRLASHLRKLRKRLL (SEQ ID NO: 3 (COG133)). The COG133 monomer has
previously proven useful in treating or reducing cerebral ischemia
or cerebral inflammation. See U.S. Application Publication No.
2003/0077641 A1, filed Sep. 23, 2002, incorporated herein by
reference in its entirety. In another embodiment, the first and/or
second ApoE peptide is an analog or derivative of COG133. For
instance, the first and/or second ApoE peptide has a sequence
selected from the group consisting of AS(Aib)LRKL(Aib)KRLL (SEQ ID
NO: 5 (COG1410)), LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL (SEQ ID NO: 4
(COG248)), and LRVRLAS(Aib)LRKLR(K-Ac)RLL (SEQ ID NO: 35 (COG345)).
Other ApoE analogs or derivatives can be incorporated into the ApoE
peptide dimers of the invention. For instance, a large number of
analogs of the ApoE 130-150 peptide were previously created and
their activity tested in a cell-based assay for suppression of
release of inflammatory cytokines and free radicals and in receptor
binding assays. Lynch et al. (2003) J. Biol. Chem., Vol. 278(4):
48529-33 and U.S. Application Publication Serial No. 2003/0077641
A1, filed Sep. 23, 2002; U.S. Pat. No. 7,205,280, issued Apr. 17,
2007; and U.S. application Ser. No. 09/260,430, filed Mar. 1, 1999,
the contents of each of which are incorporated herein by reference
in their entireties. In particular, the improved ApoE analogs
described in WO 2006/029028, which is herein incorporated by
reference in its entirety, are suitable first and/or second ApoE
peptides or mimetic domains of the peptide dimers of the invention.
For instance, ApoE peptides or mimetic domains can include, but are
not limited to:
TABLE-US-00001 (SEQ ID NO: 42) LRVRLASH-(NMe)-LRKLRKRLL-NH.sub.2
(SEQ ID NO: 43) Ac-ASH-Aib-RKLRKRLL-NH.sub.2 (SEQ ID NO: 44)
Ac-AS-Aib-LRKLRKRLL-NH.sub.2 (SEQ ID NO: 45) Ac-DS-Aib-LRKLRKRLL-
NH.sub.2 (SEQ ID NO: 46) Ac-ASHLRKL-Aib-KRLL-NH.sub.2 (SEQ ID NO:
47) Ac-DR-Aib-ASHLRKLRKR-Aib-L-NH.sub.2 (SEQ ID NO: 48)
Ac-DS-Aib-LRKLRKR-Aib-L-NH.sub.2 (SEQ ID NO: 49)
Ac-DR-Aib-ASHLRKL-Aib-KRLL-NH.sub.2 (SEQ ID NO: 50)
Ac-DS-Aib-LRKL-Aib-KRLL-NH.sub.2 (SEQ ID NO: 51)
Ac-DR-Aib-AS-Aib-LRKLRKRLL-NH.sub.2 (SEQ ID NO: 52)
Ac-DR-Aib-ASHLRKLRKRLL-NH.sub.2 (SEQ ID NO: 53)
Ac-CAS-Aib-LRKL-Aib-KRLL-NH.sub.2 (SEQ ID NO: 54)
Ac-DS-Aib-LRKL-Aib-KRLL-NH.sub.2 (SEQ ID NO: 55)
Ac-AS-Aib-LRKL-Aib-KRLV-NH.sub.2 (SEQ ID NO: 56)
Ac-AS-Aib-LRKL-Aib-KRLM-NH.sub.2 (SEQ ID NO: 57)
Ac-AS-Aib-LRKL-Aib-KRLI-NH.sub.2 (SEQ ID NO: 58)
Ac-AS-Aib-LRKL-Aib-KRLA-NH.sub.2 (SEQ ID NO: 59)
Ac-AS-Aib-LRKL-Aib-KALL-NH.sub.2 (SEQ ID NO: 60)
Ac-AS-Aib-LRKL-Aib-K(orn)LL-NH.sub.2 (SEQ ID NO: 61)
Ac-AS-Aib-LRKL-Aib-K(narg)LL-NH.sub.2 (SEQ ID NO: 62)
Ac-AS-Aib-LRKL-Aib-K(harg)LL-NH.sub.2 (SEQ ID NO: 63)
Ac-AS-Aib-LRKL-Aib-K(dmarg)LL-NH.sub.2 (SEQ ID NO: 64)
Ac-AS-Aib-LRKL-Aib-ARLL-NH.sub.2 (SEQ ID NO: 65)
Ac-AS-Aib-LRKL-Aib-(aclys)RLL-NH.sub.2 (SEQ ID NO: 66)
Ac-AS-Aib-LRKL-Aib-(azlys)RLL-NH.sub.2 (SEQ ID NO: 67)
Ac-ASH-Aib-RKL-Aib-KRLL-NH.sub.2 (SEQ ID NO: 68)
Ac-AS-Aib-LRKL-Aib-KRL-(NLe)-NH.sub.2 (SEQ ID NO: 69)
Ac-AS-Aib-LRKL-Aib-KR-(NLe)-L-NH.sub.2 (SEQ ID NO: 70)
Ac-AS-Aib-LRKL-Aib-KR-(NLe)-(NLe)-NH.sub.2 (SEQ ID NO: 71)
Ac-AS-Aib-LRKL-Aib-K(orn)L-(NLe)-NH.sub.2 (SEQ ID NO: 72)
Ac-AS-Aib-LRKL-Aib-K(orn)-(NLe)-L-NH.sub.2 (SEQ ID NO: 73)
Ac-AS-Aib-LRKL-Aib-K(orn)-(NLe)-(NLe)-NH.sub.2 (SEQ ID NO: 74)
Ac-AS-Aib-LRKL-Aib-K(harg)L-(NLe)-NH.sub.2 (SEQ ID NO: 75)
Ac-AS-Aib-LRKL-Aib-K(harg)-(NLe)-L-NH.sub.2 (SEQ ID NO: 76)
Ac-AS-Aib-LRKL-Aib-K(harg)-(NLe)-(NLe)-NH.sub.2 (SEQ ID NO: 77)
Ac-AS-Aib-L(orn)KL-Aib-KRLL-NH.sub.2 (SEQ ID NO: 78)
Ac-AS-Aib-L(orn)KL-Aib-K(orn)LL-NH.sub.2 (SEQ ID NO: 79)
Ac-AS-Aib-L(orn)KL-Aib-KRL-(NLe)-NH.sub.2 (SEQ ID NO: 80)
Ac-AS-Aib-L(orn)KL-Aib-KRL-(NLe)-(NLe)-NH.sub.2 (SEQ ID NO: 81)
Ac-AS-Aib-L(orn)KL-Aib-K(orn)L-(NLe)-NH.sub.2 (SEQ ID NO: 82)
Ac-AS-Aib-L(orn)KL-Aib-K(orn)-(NLe)-(NLe)-NH.sub.2 (SEQ ID NO: 83)
Ac-ASHLRKLRKRLL-NH.sub.2 (apoE138-149) (SEQ ID NO: 84)
Ac-ASHCRKLCKRLL-NH.sub.2 (SEQ ID NO: 85) Ac-ASCLRKLCKRLL-NH.sub.2
(SEQ ID NO: 86) Ac-CSHLRKLCKRLL-NH.sub.2 (SEQ ID NO: 87)
Ac-ASHLRKCRKRCL-NH.sub.2 (SEQ ID NO: 88)
Ac-ASHCRKLRKRCL-NH.sub.2
wherein (NMe)-L is an N-methylated Leucine, Aib is amino
iso-butyric acid, (orn) is ornithine, (narg) is nitroarginine,
(NLe) is norleucine, (harg) is homoarginine, (dmarg) is dimethyl
arginine, (aclys) is acetyl lysine, (azlys) is azalysine and Ac is
an acetylated amino terminus. Other ApoE peptides or mimetic
domains derived from the receptor binding region of ApoE protein
are also contemplated. For instance, ApoE peptides or mimetic
domains that comprise a sequence corresponding to amino acids
133-149 of ApoE protein and retain (i.e. not substituted) one or
more key residues selected from the group consisting of S139, R142,
K143, L144, K146, R147 and L149, but have one or more amino acid
substitutions at other positions are suitable first and/or second
ApoE peptides or mimetic domains of the peptide dimers of the
invention.
[0049] In certain embodiments of the invention, the first and
second ApoE peptides of the peptide dimer are the same. For
example, in one embodiment, the peptide dimer comprises a first
ApoE peptide and a second ApoE peptide, wherein the first and
second peptide have a sequence of SEQ ID NO: 3 (COG133). In other
embodiments, the first and second ApoE peptides of the peptide
dimer are different. By way of example, the peptide dimer can
comprise a first ApoE peptide and a second ApoE peptide, wherein
the first ApoE peptide has a sequence of SEQ ID NO: 3 (COG133) and
the second ApoE peptide has a sequence of SEQ ID NO: 5 (COG1410).
Peptide dimers including all possible permutations of the different
ApoE peptides described herein are encompassed by the present
invention.
[0050] In another embodiment of the invention, the first and/or
second ApoE peptide of the peptide dimer is conjugated to a protein
transduction domain (PTD). PTDs are short basic peptides that
enhance the intracellular delivery of cargo. Some non-limiting
examples of PTDs that may be conjugated to the ApoE peptides
include peptides derived from antennapedia, SynB1, SynB3, SynB5,
TAT, and polyarginine. For instance, exemplary PTD sequences that
can be conjugated to the first and/or second ApoE peptides
include:
TABLE-US-00002 (SEQ ID NO: 8) RQIKIWFQNRRMKWKK (SEQ ID NO: 9)
YGRKKRRQRRR (SEQ ID NO: 36) GRKKRRQRRRPPQ (SEQ ID NO: 37) RRMKWKK
(SEQ ID NO: 38) RGGRLSYSRRRFSTSTGR (SEQ ID NO: 39) RRLSYSRRRF (SEQ
ID NO: 40) RGGRLAYLRRRWAVLGR (SEQ ID NO: 41) RRRRRRRR WKK
[0051] In certain embodiments, the first and/or second ApoE peptide
is conjugated to a PTD having a sequence of RQIKIWFQNRRMKWKK (SEQ
ID NO: 8); YGRKKRRQRRR (SEQ ID NO: 9), or WKK. In one embodiment,
the first ApoE peptide is conjugated to a first PTD through one or
more first linking residues. Thus, the peptide dimers of the
invention can comprise a first ApoE peptide conjugated to a first
PTD and a second ApoE peptide that is not conjugated to a PTD. In
such embodiments, the peptide dimers comprise two ApoE peptides or
mimetic domains and a single PTD. In another embodiment, the first
ApoE peptide is conjugated to a first PTD through one or more first
linking residues and the second ApoE peptide is conjugated to a
second PTD through one or more second linking residues. In such
embodiments, the peptide dimers comprise two ApoE peptides or
mimetic domains and two PTDs. Thus, the peptide dimers of the
invention can comprise two ApoE peptide domains with zero, one, or
two PTDs. See Example 3 and FIG. 7.
[0052] The ApoE peptides and PTDs of the dimers can be any
combination of the ApoE peptides and PTDs described herein. In
particular embodiments, the PTDs are selected from peptides derived
from antennapedia or TAT (e.g., SEQ ID NO: 8, SEQ ID NO: 9, or the
WKK sequence) and the ApoE peptides are selected from COG133 (SEQ
ID NO: 3), COG248 (SEQ ID NO: 4), COG1410 (SEQ ID NO: 5), or COG345
(SEQ ID NO: 35) as described in Tables II and IV of Example 3. In a
certain embodiment, the ApoE peptide is COG133 (SEQ ID NO: 3) and
the PTD has a sequence of SEQ ID NO: 8. In another embodiment, the
ApoE peptide is COG133 (SEQ ID NO: 3) and the PTD has a sequence of
WKK. In another embodiment, the peptide dimer comprises a first
peptide and a second peptide, wherein said first peptide and said
second peptide are covalently linked by a bismaleimido-ethane, and
wherein the first and second peptide have a sequence of SEQ ID NO:
1, SEQ ID NO: 15, or SEQ ID NO: 90. For example, in one particular
embodiment, the peptide dimer comprises a first peptide and a
second peptide, wherein the first and second peptide have a
sequence of SEQ ID NO: 1, and wherein said first peptide and said
second peptide are covalently linked by a bismaleimido-ethane
linker between the cysteine residues at position 17 in SEQ ID NO: 1
(e.g., the peptide dimer is COG449; see Table I). In another
particular embodiment, the peptide dimer comprises a first peptide
and a second peptide, wherein the first and second peptide have a
sequence of SEQ ID NO: 15, and wherein said first peptide and said
second peptide are covalently linked by a bismaleimido-ethane
linker between the cysteine residues at the amino terminus of each
peptide (i.e. at position 1 in SEQ ID NO: 15) (e.g., the peptide
dimer is COG492; see Table I). In still another particular
embodiment, the peptide dimer comprises a first peptide and a
second peptide, wherein the first and second peptide have a
sequence of SEQ ID NO: 90, and wherein said first peptide and said
second peptide are covalently linked by a bismaleimido-ethane
linker between the cysteine residues at position 4 in SEQ ID NO: 90
(e.g., the peptide dimer is COG493; see Table I).
[0053] The first and/or second ApoE peptide may optionally be
conjugated to the PTD through one or more linking residues. As used
herein, a "linking residue" refers to at least one amino acid or
modified amino acid that is capable of undergoing a reaction to
cross-link ApoE peptide monomers to form stable dimers. In some
embodiments, the linking residues are amenable to cross-linking
using maleimide groups, such as those described in FIG. 8, or
cross-linking through the formation of stable 1,4-disubstituted
triazoles as described in FIG. 9. Exemplary linking residues
include cysteine, azidohomoalanine, and propargylglycine. Other
suitable linking residues can be ascertained by those of skill in
the art.
[0054] The peptide dimers of the invention comprise a first ApoE
peptide and a second ApoE peptide, wherein said first and second
ApoE peptides are covalently linked by a linking moiety. As used
herein, a "linking moiety" may be a compound or molecule that
cross-links peptide monomers such that the peptide chains are
separated by at least four atoms. The linking moiety can be
selected to create various lengths of the linker between the
peptide monomers. For instance, the linking moiety may be selected
such that the peptide chains are separated by at least 6 atoms, at
least 8 atoms, at least 10 atoms, or at least 12 atoms. Linking
moieties can be heterologous amino acids not found in the native
ApoE sequence, such as additional cysteine residues or modified
amino acids, such as azidohomoalanine or propargylglycine. In some
embodiments, linking moieties can include molecules or compounds
that are produced from a cross-linking reaction with amino acids in
the peptide chains. For instance, in certain embodiments, the
linking moiety is selected from the group consisting of a disulfide
bridge, a bismaleimide, a 1,4-disubstituted triazole, and
N,N-dipropargylamine. The bismaleimide can include, but is not
limited to, bismaleimido-ethane or bismaleimido-hexane. In some
embodiments, the linking moiety is not a peptide bond.
[0055] In embodiments in which the peptide dimer comprises ApoE
peptides that are not conjugated to PTDs, the two ApoE peptides can
be linked such that the carboxy terminus of the first ApoE peptide
is linked to the amino terminus of the second ApoE peptide (e.g.,
direct linkage). Alternatively, the two ApoE peptides can be linked
such that the two ApoE peptides are in reverse orientation relative
to each other. For example, the carboxy terminus of the first ApoE
peptide can be linked to the carboxy terminus of the second ApoE
peptide or the amino terminus of the first ApoE peptide can be
linked to the amino terminus of the second ApoE peptide. Such
linkages may be accomplished by adding one or more amino acid
residues capable of undergoing cross-linking reactions (e.g.
cysteine, azidohomoalanine, or propargylglycine residues) to the
appropriate terminus of the first and second ApoE peptides. By way
of example, cysteine residues added to the amino terminus of both
the first and second ApoE peptides will generate a dimer in which
the first and second ApoE peptides are linked at their amino
termini (see, e.g., COG492 in Example 2).
[0056] In embodiments, in which the peptide dimer comprises at
least one ApoE peptide conjugated to a PTD, the dimer can be formed
by cross-linking at least one of the linking residues in the
ApoE-PTD conjugate and an amino acid at either the carboxy or amino
terminus of the second, unconjugated ApoE peptide. If both ApoE
peptides are conjugated to PTDs, the dimer is preferably formed by
cross-linking at least one of the linking residues in each ApoE-PTD
conjugate such that the two peptide chains are linked through
internal amino acid residues.
[0057] ApoE peptides or mimetic domains may be incorporated into
multimers such that an ApoE multimer contains three or more ApoE
peptides or mimetic domains. One or more of the ApoE peptides in
the multimer can be conjugated to a PTD as described herein. In one
embodiment, the present invention provides an ApoE trimer
comprising a first ApoE peptide, a second ApoE peptide, and a third
ApoE peptide, wherein the first, second, and third ApoE peptides
are covalently linked by a linking moiety. Other ApoE multimers are
contemplated within the scope of the invention.
[0058] Peptides of the present invention can be produced by
standard techniques as are known in the art. The peptides of the
invention 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.
[0059] Modification of the peptides disclosed herein to enhance the
functional activities associated with these peptides could be
readily accomplished by those of skill in the art. For instance,
the peptide dimers of the present invention can be chemically
modified or conjugated to other molecules in order to enhance
parameters such as solubility, serum stability, etc., while
retaining functional activity. In particular, the first and/or
second ApoE peptide of the dimer may be acetylated at its
N-terminus and/or amidated at its C-terminus, or the dimers can be
further conjugated, complexed or fused to molecules that enhance
serum stability, including but not limited to albumin,
immunoglobulins and fragments thereof, transferrin, lipoproteins,
liposomes, .alpha.-2-macroglobulin and .alpha.-1-glycoprotein, PEG
and dextran. Such molecules are described in detail in U.S. Pat.
No. 6,762,169, which is herein incorporated by reference in its
entirety.
[0060] The ApoE peptides of the inventive peptide dimers can
further include conservative variants of the peptides described
herein. 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: 3, 4, 5, and 35,
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.
[0061] Thus, the first and/or second ApoE peptides of the peptide
dimers of the present invention include molecules having the amino
acid sequence disclosed in SEQ ID NOs: 3, 4, 5, and 35; fragments
thereof having a consecutive sequence of at least about 3, 4, 5, 6,
10, 15, or more amino acid residues of the therapeutic 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. 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 naturally
occurring amino acid (for example, a detectable moiety such as an
enzyme or radioisotope).
[0062] Methods of cross-linking peptides to form peptide dimers are
known to those in the art and can include, but are not limited to,
coupling via maleimide groups and coupling using "click chemistry"
(see Example 3 and FIGS. 8-9). The skilled artisan can ascertain
other suitable methods for covalently linking the ApoE peptides
described herein to form peptide dimers without undue
experimentation.
[0063] The ApoE peptide dimers of the invention 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 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.
[0064] In one embodiment, the peptide dimers of the present
invention are used in combination with a pharmaceutically
acceptable carrier. Thus, the present invention also provides
pharmaceutical compositions suitable for administration to a
subject. Such compositions comprise an effective amount of an ApoE
peptide dimer of the present invention 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. Active
agents can alternatively be formulated encapsulated in liposomes,
using known methods. Preparation of a peptide dimer of the present
invention for intranasal administration can be carried out using
techniques as are known in the art. The inventive peptide dimers
may also be formulated for topical administration, for example in
the form of creams or gels. Topical formulations are particularly
useful for treating skin cancers or inflammatory skin conditions.
In other embodiments, the ApoE peptide dimers may be formulated for
rectal administration, such as in the form of suppositories. In
some embodiments, rectal administration of the ApoE peptide dimers
may be preferred for treatment of colorectal cancer, inflammatory
bowel disease, or Crohn's disease.
[0065] Pharmaceutical preparations of the peptide dimers of the
present invention can optionally include a pharmaceutically
acceptable diluent or excipient.
[0066] The ApoE peptide dimers of the invention may contain further
modifications or be formulated to specifically target specific
tissues, such as inflamed tissues or cancerous tumors. For
instance, the ApoE peptide dimers may be conjugated to other
peptides that localize to tumor cells, such as those described in
U.S. Pat. No. 6,380,161, U.S. Publication No. 2003/0232013, WO
2009/155556, and WO 2009/143023. Additionally or alternatively, the
ApoE peptide dimers may be encapsulated into liposomes. The
liposomes may contain a targeting ligand to localize the liposomes
to particular tissues or tumor sites.
[0067] An effective amount of an ApoE peptide dimer of the present
invention is an amount that decreases at least one symptom or
pathology associated with cancer, such as tumor size, tumor growth,
spread of cancer cells, number of cancer cells, and survival,
compared to that which would occur in the absence of the peptide.
An effective amount of an ApoE peptide dimer can also be an amount
that decreases microglial activation (i.e., an amount that
decreases the production of neurotoxic and neuromodulatory
compounds by microglia) as compared to that which would occur in
the absence of the compound. The effective amount (and the manner
of administration) will be determined on an individual basis and
will be based on the specific composition of the peptide dimer
being used and a consideration of the subject (size, age, general
health), the specific condition being treated (e.g. cancer,
neurodegenerative disorder, inflammatory condition), 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 ordinary skill in the art using techniques as are known in the
art. Therapeutically effective amounts of the peptide dimers
described herein can be determined using in vitro tests, animal
models or other dose-response studies, as are known in the art.
[0068] The peptide dimers of the present invention can be
administered acutely (i.e., during the onset or shortly after
events leading to a diagnosis of a particular condition), or can be
administered prophylactically (e.g., before scheduled surgery, or
before the appearance of signs or symptoms of a particular
condition), or administered during the course of a particular
disease or condition 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.
[0069] The typical daily regime can be from about 0.01 .mu.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.
Depending on the particular ApoE peptide dimer to be administered,
dosages can be between about 1 mg/kg and about 500 mg/kg body
weight per day, preferably between about 25 mg/kg and about 400
mg/kg body weight per day, or more preferably between about 50
mg/kg and about 250 mg/kg body weight per day.
[0070] The present invention provides methods of treating cancer in
a subject in need thereof by administering an effective amount at
least one ApoE peptide dimer as described herein. In certain
embodiments, said at least one ApoE peptide dimer comprises a
sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4,
5, 6, 7, 10, 11, 12, 13, 14, 15, 35, 90, and effective fragments
and variants thereof. ApoE peptide dimers can reduce one or more
symptoms associated with cancer, including but not limited to tumor
formation, tumor growth, number of cancerous cells, spread of
cancerous cells to healthy tissue, and decreased survival. Cancers
that may be treated with the peptide dimers and methods of the
invention include, but are not limited to, various forms of
leukemia (CLL, CML, ALL, AML), breast cancer, ovarian cancer,
cervical cancer, prostate cancer, colorectal cancer, lung cancer,
pancreatic cancer, brain cancer (e.g., gliomas), skin cancer
(melanoma and nonmelanoma), head and neck cancers, bladder cancer,
endometrial cancer, renal cell cancer, thyroid cancer, stomach
cancer, esophageal cancer, gall bladder cancer, liver cancer,
lymphoma (e.g. non-Hodgkin's lymphoma), and sarcoma. In one
embodiment, the ApoE peptide dimers can reduce activation of
signaling pathways, such as the Akt pathway, that are aberrantly
activated in various forms of cancer (see Example 5). ApoE peptides
can also activate PP2A (Examples 2, 5, and 10). PP2A has been
reported to negatively regulate endothelial cell motility, which is
required for angiogenesis and tumor metastasis in cancers (Gabel et
al., 1999, Otolaryngol Head Neck Surg. 121: 463-468; Young, MR.,
1997, Adv Exp Med Biol. 407: 311-318). Inhibition of PP2A by
okadaic acid increased cell motility by disrupting the cytoskeletal
network thereby enhancing the invasive properties of the tumor
cells. Thus, peptide dimers of the present invention would reduce
tumor cell metastasis and cancer-associated angiogenesis by
activating PP2A. In one embodiment of the invention, administration
of the ApoE peptide dimer increases PP2A activity in a cancer cell
of the subject. In another embodiment, administration of the ApoE
peptide dimer decreases Akt kinase activity in a cancer cell of the
subject. In yet another embodiment, administration of the ApoE
peptide dimer induces cytotoxicity in cancer cells in the
subject.
[0071] The present invention also provides a method for the
treatment of leukemia comprising administering at least one ApoE
peptide dimer in an amount that would reduce symptoms of the
disease as compared to that which would occur in the absence of the
peptide dimer. In one embodiment, the leukemia is chronic
myelogenous leukemia (CML). SET (i.e., I.sub.2PP2A), an endogenous
negative regulator of PP2A, is overexpressed in CML and inhibits
PP2A, thus maintaining activation of the oncogenic BCR/ABL kinase
pathway (Neviani et al. (2005) Cancer Cell. 8: 355-368). Therefore,
administration of an ApoE peptide dimer of the invention would
activate PP2A, which would then be free to dephosphorylate
regulators of cell proliferation and survival as well as suppress
the oncogenic activity of the BCR/ABL kinase thus reducing
leukemogenesis. In another embodiment, the leukemia is chronic
lymphocytic leukemia (CLL). In preferred embodiments,
administration of the ApoE peptide dimer decreases the number of
CD5+ B cells in the subject. In another embodiment, the leukemia is
acute lymphocytic leukemia (ALL).
[0072] The present invention also encompasses methods of treating
breast cancer in a subject by administering an effective amount of
at least one ApoE peptide dimer to the subject. In one embodiment,
the breast cancer is characterized by Her2 expression. In another
embodiment, the breast cancer is characterized by estrogen receptor
expression. In another embodiment, the breast cancer is
characterized by progesterone receptor expression. The ApoE peptide
dimers of the invention can be used to treat any of the three main
subtypes of breast cancer: luminal tumors (ER+/HER2-), HER2
amplified tumors (HER2+), and triple negative breast cancer (TNBC,
ER-/PR-/HER2-). In certain embodiments, the breast cancer to be
treated with an ApoE peptide dimer of the invention is triple
negative breast cancer characterized by lack of expression of the
estrogen receptor, progesterone receptor, and HER2 receptor.
Administration of ApoE peptide dimers preferably reduce tumor
growth following their administration.
[0073] The ApoE peptide dimers of the present invention may be used
alone to treat cancer or in combination with other therapeutic
agents commonly used to treat cancer, such as, e.g. chemotherapy
agents (chlorambucil, cyclophosphamide), corticosteroids
(prednisone, prednisolone), fludarabine, pentostatin, cladribine,
imatinib (Gleevec), dasatinib (Sprycel), hormonal therapy
(tamoxifen, aromatase inhibitors), sorafenib, gefitinib, and
radiation. In some embodiments, the ApoE peptide dimers are
administered in combination with sorafenib or gefitinib to treat
cancer. As used herein, "in combination" means that the ApoE
peptide dimer and other therapeutic agents are administered such
that their effects overlap in time. Thus, the ApoE peptide dimer
can be administered simultaneously with the other therapeutic agent
or before or after the other therapeutic agent.
[0074] The present invention provides a method for predicting or
evaluating the efficacy of a therapeutic intervention for treating
cancer in a patient. In one embodiment, the method comprises
measuring the expression level of SET protein in a biological
sample from a patient, and comparing the measured level to the
expression level of SET protein in a control sample, wherein the
measured expression level of SET protein is indicative of the
therapeutic efficacy of the therapeutic intervention. In certain
embodiments, the therapeutic intervention is an ApoE peptide or
peptide dimer of the invention. The present inventors have
discovered that ApoE mimetic peptides and peptide dimers bind to
SET (i.e., I.sub.2PP2A) and relieve its inhibition of endogenous
PP2A, thereby increasing PP2A activity in the cell. Without being
bound by any particular theory, it is believed that this increase
in PP2A activity induced by ApoE peptides or peptide dimers
triggers apoptosis leading to cytotoxicity of cancer cells.
Therefore, cancer cells that overexpress SET protein are
particularly susceptible to ApoE peptide-induced cytotoxicity.
Accordingly, the present invention includes a method for predicting
therapeutic efficacy of an ApoE peptide or peptide dimer for
treating cancer in a patient by measuring the expression level of
SET protein in a biological sample from the patient and comparing
the measured level to the expression level of SET protein in a
control sample, wherein the measured expression level of SET
protein is predictive of the therapeutic efficacy of the ApoE
peptide or peptide dimer.
[0075] In one embodiment, a measured SET expression level of at
least 2-fold relative to the control sample is predictive of
therapeutic efficacy of an ApoE peptide or peptide dimer for
treating cancer in the patient. In some embodiments, a measured SET
expression level of at least 4-fold, at least 5-fold, at least
8-fold, at least 10-fold, at least 12-fold, at least 15-fold, or at
least 20-fold relative to the control sample is predictive of
therapeutic efficacy of an ApoE peptide or peptide dimer for
treating cancer in the patient. In one embodiment, the method is
predictive of therapeutic efficacy of an ApoE peptide or peptide
dimer for treating breast cancer in the patient. In another
embodiment, the method is predictive of therapeutic efficacy of an
ApoE peptide or peptide dimer for treating triple negative breast
cancer (estrogen receptor negative, progesterone receptor negative,
and HER2 receptor negative) in the patient. In another embodiment,
the method is predictive of therapeutic efficacy of an ApoE peptide
or peptide dimer for treating B-cell lymphoma (e.g. non-Hodgkin's
lymphoma) in the patient. In still another embodiment, the method
is predictive of therapeutic efficacy of an ApoE peptide or peptide
dimer for treating leukemia (e.g. CML or CLL) in the patient.
[0076] SET expression can be measured by assessing the level of SET
protein or SET transcript. SET expression can be measured by
methods known in the art including, but not limited to, Northern
Blot, PCR, RT-PCR, Western Blot, immunoassay (e.g. ELISA or
multiplexed assays), 2D gel electrophoresis, and hybridization. In
one embodiment, SET protein expression is measured.
[0077] In certain embodiments, the method for predicting the
efficacy of ApoE peptide therapy for treating cancer in a patient
further comprises administering at least one ApoE peptide or
peptide dimer to the patient following assessment of SET expression
in the patient's biological sample. Any ApoE peptide or dimer
thereof described herein is suitable for use in the method. For
instance, in some embodiments, an ApoE peptide or dimer thereof
having a sequence selected from the group consisting of SEQ ID NOs:
1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 35, 90 and effective
fragments and variants thereof is administered to the patient. In
another embodiment, the method further comprises adjusting the
particular type of ApoE peptide or peptide dimer or dosage of the
ApoE peptide or peptide dimer based on the expression level of the
SET protein in the patient's biological sample. SET expression
levels can be measured multiple times over a particular period of
time or treatment period in a patient.
[0078] The biological sample can be any tissue sample that contains
cancerous cells. For instance, the biological sample can include,
but is not limited to, a biopsy from a solid tumor (e.g. breast
cancer, lymphoma, sarcoma, etc.) or peripheral blood mononuclear
cells (PBMCs) isolated from blood. In one embodiment, the
biological sample is CD19+/CD5+ leukemia cells. The control sample
can be any tissue sample that contains normal or non-cancerous
cells such as PBMCs isolated from a normal, age-matched patient or
non-cancerous tissue (e.g. breast, lymph, skin, etc.) isolated from
the patient to be treated or from a normal, age-matched control
patient.
[0079] The present invention also encompasses a kit for predicting
or evaluating the efficacy of an ApoE peptide or peptide dimer for
treating cancer in a patient. In one embodiment, the kit comprises
a reagent for measuring SET protein expression in a biological
sample and instructions for measuring SET protein expression for
predicting or evaluating the efficacy of an ApoE peptide or peptide
dimer for treating cancer in a patient. In some embodiments, the
reagent for measuring SET expression can include SET-specific
antibodies, ELISA reagents, and primers and probes for amplifying
and detecting SET mRNA. In other embodiments, the kit may further
comprise one or more normalization controls. For example, the
normalization control may be an exogenously added RNA or protein
that is not naturally present in the sample or it may be a protein
or RNA known to be expressed constitutively in a particular
biological sample or cell, such as beta-actin. In such embodiments,
the kit may further provide reagents (antibodies, primers, probes,
etc.) for detecting and quantitating the normalization control. In
some embodiments, the kit can further comprise a set of reference
values to which the measured SET expression levels can be
compared.
[0080] The present invention provides a method of reducing glial
activation or microglial activation in a subject in need thereof by
administering to the subject at least one of the ApoE peptide
dimers of the invention. In one embodiment, the microglial
activation is associated with central nervous system (CNS)
inflammation, traumatic brain injury, cerebral ischemia or cerebral
edema. Thus, the present methods and compositions are useful in
preventing, suppressing or reducing the activation of glia in the
CNS that occurs as a part of acute or chronic CNS disease. The
effect of the present methods and peptide dimers can be assessed at
the cellular or tissue level (e.g., histologically or
morphometrically), or by assessing a subject's neurological status.
The suppression or reduction of glial activation can be assessed by
various methods as would be apparent to those in the art; one such
method is to measure the production or presence of compounds that
are known to be produced by activated glia, and compare such
measurements to levels of the same compounds in control situations.
Alternatively, the effects of the present methods and peptide
dimers in suppressing, reducing or preventing microglial activation
can be assessed by comparing the signs and/or symptoms of CNS
disease in treated and control subjects, where such signs and/or
symptoms are associated with or secondary to activation of
microglia.
[0081] The present methods and peptide dimers are useful in
preventing, treating, or ameliorating neurological signs and
symptoms associated with acute CNS injury. As used herein, acute
CNS injury includes but is not limited to stroke (caused by
thrombosis, embolism or vasoconstriction), closed head injury,
global cerebral ischemia (e.g., ischemia due to systemic
hypotension of any cause, including cardiac infarction, cardiac
arrhythmia, hemorrhagic shock, and post coronary artery bypass
graft brain injury), focal ischemia and intracranial hemorrhage.
Ischemic damage to the central nervous system can result from
either global or focal ischemic conditions. Global ischemia occurs
where blood flow to the entire brain ceases for a period of time,
such as during cardiac arrest. Focal ischemia occurs when a portion
of the brain is deprived of normal blood flow, such as during
thromboembolytic occlusion of a cerebral vessel, traumatic head
injury, edema and brain tumors. Much of the CNS damage due to
cerebral ischemia occurs during the hours or even days following
the ischemic condition, and is secondary to the release of
cytotoxic products by damaged tissue.
[0082] The present methods and peptide dimers are also useful in
preventing, treating, or ameliorating the neurological signs and
symptoms associated with inflammatory conditions affecting the
nervous system including the CNS, including but not limited to
multiple sclerosis, vasculitis, acute disseminated
encephalomyelitis and Guillain-Barre syndrome. In this regard, the
ApoE peptide dimers of the invention can be used alone or in
combination with other known anti-inflammatory drugs or cytokines
to formulate pharmaceutical compositions for the treatment of CNS
inflammatory conditions.
[0083] In another embodiment, the present invention provides a
method of reducing neuronal cell death in a subject in need thereof
comprising administering to the subject an effective amount of at
least one ApoE peptide dimer described herein. In some embodiments,
the neuronal cell death is associated with glutamate
excitotoxicity. It was previously found that the COG 133 monomer
peptide significantly suppressed neuronal cell death and calcium
influx associated with N-methyl-D-aspartate exposure (see, e.g.,
U.S. Application Publication No. 2003/0077641 A1, herein
incorporated by reference in its entirety). Thus, the peptide
dimers of the present invention provide the basis for improved
therapeutic compositions for treating diseases associated with
glutamate excitotoxicity mediated by overstimulation of the NMDA
receptor. For instance, glutamate excitotoxicity has been
associated with neurolathyrism, amyotrophic lateral sclerosis (ALS)
(Doble (1999) Pharmacol. Ther., Vol. 81:163-221), schizophrenia
(Nguimfack (2002) Encephale, Vol. 28: 147-153), Huntington's
chorea, Parkinson's (Nguimfack, 2002; Mytilineou et al. (1997) J.
Neurochem., Vol. 68: 33-39; Klopman and Sedykh (2002) BMC
Pharmacol., Vol. 2: 8; Le and Lipton (2001) Drugs Aging, Vol. 18:
717-724), bipolar disorder (Farber et al. (2002) Mol. Psychiatry,
Vol. 7: 726-733), multiple sclerosis in humans and experimental
autoimmune encephalitis (EAE) in animals (Paul and Bolton (2002) J.
Pharmacol. Exp. Ther., Vol. 302: 50-57), depression, stroke (Le and
Lipton, 2001), epilepsy and the inherited neurometabolic disease
d-2-hydroxyglutaric aciduria (Kolker et al. (2002) Eur. J.
Neurosci., Vol. 16: 21-28), in addition to Alzheimer's Disease (Bi
et al. (2002) Neuroscience, Vol. 112: 827-840; Bi et al. (2002) J.
Neurol. Sci., Vol. 200: 11-18) and traumatic brain injury (Rao et
al. (2001) Brain Res., Vol. 911: 96-100; Regner et al. (2001) J.
Neurotrauma, Vol. 18: 783-792; Xu and Luo (2001) Chin. J.
Traumatol., Vol. 4: 135-138).
[0084] Thus, the present invention includes the use of the
disclosed peptide dimers in methods and pharmaceutical formulations
for the treatment of any of the above diseases or disorders, and in
combined therapeutic compositions containing other known compounds
useful for treating the various disorders. For instance, in some
embodiments, the peptide dimers of the invention can be used to
treat neurolathyrism, amyotrophic lateral sclerosis (ALS),
Huntington's disease, Parkinson's disease, or schizophrenia in a
subject in need thereof.
[0085] Riluzole (RILUTEK.RTM., Rhone-Poulenc) is a substance with
glutamate antagonistic properties that is used for neuroprotective
treatment in amyotrophic lateral sclerosis and which has been
tested in clinical trials for treatment of Huntington's disease and
Parkinson's disease (Schiefer et al. (2002) Mov. Disord., Vol. 17:
748-757; Doble, 1999). Schiefer and colleagues recently
demonstrated that riluzole prolongs survival time and alters
nuclear inclusion formation in a transgenic mouse model of
Huntington's disease. Thus, given the probable NMDA antagonistic
role of the peptide dimers of the invention, these peptide dimers
could be used in pharmaceutical formulations for the treatment of
ALS, Huntington's and Parkinson's, alone or in combination with
other glutamate antagonists, such as riluzole.
[0086] L-deprenyl is an inhibitor of monoamine oxidase (MAO)-B that
delays the emergence of disability and the progression of signs and
symptoms of Parkinson's disease, and is predicted to exert a
protective effect from events occurring downstream from activation
of glutamate receptors (Mytilineou et al., 1997). MAO-B inhibitors,
dopamine receptor antagonists, such as levodopa, and NMDA receptor
antagonists have all been shown to have an antiparkinson effect,
and multidrug combinations have been shown to synergistically
enhance the antiparkinson effects of the drugs (Klopman and Sedykh,
2002). Thus, given the probable NMDA antagonistic role of the
peptide dimers of the invention, these peptide dimers could be used
in pharmaceutical formulations for the treatment of Parkinson's,
alone or in combination with other NMDA receptor antagonists, MAO-B
inhibitors, such as L-deprenyl, and dopamine receptor agonists,
such as levodopa.
[0087] The production of free radicals as a result of glutamate
excitotoxicity has been implicated in the pathogenesis of
schizophrenia (Nguimfack, 2002). Thus, researchers have begun to
examine treatment of schizophrenia with antioxidizing substances
used in other neurological diseases such as ALS, Parkinson's and
Huntington's disease. Given that the peptide dimers of the
invention likely have NMDA receptor antagonistic properties and can
be used to inhibit the production of free radicals as a result of
glutamate excitotoxicity, these peptide dimers can be used in
pharmaceutical formulations for the treatment of schizophrenia,
alone or in combination with other antioxidizing substances.
[0088] The present invention also includes a method of treating,
preventing or ameliorating the symptoms of multiple sclerosis in a
subject in need thereof by administering to the subject an
effective amount of at least one ApoE peptide dimer of the
invention. Multiple sclerosis (MS) is an immunologically mediated
disease, as determined by observation of the response to
immunotherapy and the existence of an animal model, experimental
autoimmune encephalitis (EAE). See, for example, Mix et al. (2004)
J. Neuroimmunol., Vol. 151(1-2): 158-70, Anderson, et al. (2004),
Ann. Neurol., Vol. 55(5):654-9, and Ni et al. (2004) Mult. Scler.,
Vol. 10(2): 158-64. Interferon (IFN) beta-1b, IFN beta-1a, and
glatiramer acetate (COPAXONE.RTM., Teva), current therapies used
for relapsing or remitting MS, have mechanisms of action that
address the immunologic pathophysiology of MS (Dhib-Jalbut (2002)
Neurology, Vol. 58: S3-S9). For instance, the interferons bind to
cell surface-specific receptors, initiating a cascade of signaling
pathways that end with the secretion of antiviral,
antiproliferative, and immunomodulatory gene products. Glatiramer
acetate, a synthetic molecule, inhibits the activation of myelin
basic protein-reactive T cells and induces a T-cell repertoire
characterized by anti-inflammatory effects. Several currently
marketed treatments, including IV immunoglobulin (GAMAGARD.RTM.,
Baxter), methotrexate (RHEUMATREX.COPYRGT., American Cyanamid), and
azathioprine (IMURAN.RTM., GlaxoSmithKline), have been evaluated as
treatments for relapsing-remitting multiple sclerosis in
combination with the approved therapies (Calabresi (2002)
Neurology, Vol. 58: S10-S22). Given that the NMDA receptor
antagonist memantine (NAMENDA.RTM., Merz) has been shown to prevent
the breakdown of and restore the blood-brain barrier and reduce
symptoms associated with pathogenesis of EAE in vivo (Paul and
Bolton, 2002), the peptide dimers of the present invention can be
used alone or in combination with other NMDA receptor antagonists
or in addition to interferons or glatiramer acetate for the
treatment of MS in humans.
[0089] The present invention encompasses a method of treating,
preventing or ameliorating the symptoms of rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis or polyarticular-course
juvenile rheumatoid arthritis in a subject in need thereof by
administering to the subject at least one ApoE peptide dimer as
described herein. Current therapies for arthritis include peptides
and proteins that bind with tumor necrosis factor. Etanercept
(ENBREL.RTM., Amgen) is a dimeric fusion protein consisting of the
extracellular ligand binding portion of the human 75 kd tumor
necrosis factor receptor linked to the Fc portion of human IgG1.
Adalimumab (HUMIRA.RTM., Abbott) is a recombinant human IgG1
monoclonal antibody. Tumor necrosis factor binding proteins have
shown outstanding results in slowing the progression and lessening
the symptoms of rheumatoid arthritis and other rheumatic diseases.
Thus, the ApoE peptide dimers of the present invention can be used
alone or in combination with other drug for the treatment of
rheumatic diseases, including for example, rheumatoid arthritis,
ankylosing spondylitis, polyarticular-course juvenile rheumatoid
arthritis, and psoriatic arthritis.
[0090] The present methods and ApoE peptide dimers are also useful
in treating, preventing, or ameliorating neurological signs and
symptoms associated with chronic neurological disease, including
but not limited to Alzheimer's disease (AD) and HIV-associated
encephalopathy. The finding by the present inventors that ApoE
peptide dimers are particularly potent in suppressing microglial
activation provides a role for the peptide dimers of the invention
in the treatment of any neurological disease involving microglial
activation. For example, microglia express markers of activation in
AD, suggesting that crucial inflammatory events in AD involve
microglia. Such activated microglia cluster near amyloid plaques
(Griffin et al. (1995) J. Neuropath. Exp. Neurol., Vol. 54: 276).
Microglia are also activated in epilepsy (Sheng et al. (1994) J.
Neurochem, Vol. 63: 1872).
[0091] It has been shown that uptake and pathogenic effects of
amyloid beta peptide are blocked by NMDA receptor antagonists (Bi
et al., 2002). Other studies indicate that anti-inflammatory drugs
can delay the onset or progression of AD (Breitner et al. (1995)
Neurobiol. Aging, Vol. 16: 523; Rogers et al. (1993) Neurology,
Vol. 43: 1609). Thus, the peptide dimers of the present invention
can be used alone or in combination with other NMDA receptor
antagonists or other known pharmaceuticals and especially
anti-inflammatory drugs used for the treatment of AD in
compositions and methods for the treatment of AD in humans.
[0092] The present invention includes a method of treating,
preventing or ameliorating the symptoms of bacterial sepsis in a
subject in need thereof by administering to the subject an
effective amount of an ApoE peptide dimer of the invention.
Monomeric ApoE receptor binding peptides have been shown to protect
against LPS-induced production of cytokines in the periphery in an
in vivo animal model of sepsis. See U.S. Application Publication
No. 2003/0077641 A1, which is herein incorporated by reference in
its entirety. Thus, the peptide dimers of the present invention can
be used alone or in combination with other known anti-inflammatory
cytokines and antibodies in compositions and methods for the
treatment of sepsis.
[0093] It is known that the inflammatory process mediates an aspect
of the atherosclerotic process. See, e.g., Hansson (1994) Basic
Res. Cardiol., Vol. 89: 41; Berliner et al. (1995) Circulation,
Vol. 91: 2488; Watanabe et al. (1997) Int. J. Cardiol., Vol. 54:
551. ApoE is known to be secreted by macrophages locally at blood
vessel walls (although the amount secreted by macrophages in an
individual is trivial compared to the amount of ApoE produced by
the liver). In the classic model of atherosclerosis, ApoE functions
to remove cholesterol from the blood stream and deliver it to
macrophages or to the liver. However, it has become apparent that
ApoE secreted by macrophages at the blood vessel wall decreases
atherosclerotic plaque formation, independent of any lipid
metabolism effects. For instance, ApoE-deficient mice are accepted
as a model of hypercholesteremia and atherosclerotic disease.
Providing ApoE-secreting macrophages to such mice dramatically
decreases atherosclerotic plaque formation. Linton et al. (1995)
Science, Vol. 267: 1034. Conversely, replacing a wild-type mouse's
macrophages with ApoE-deficient macrophages accelerates
atherosclerotic changes, even though the animal continues to
produce ApoE by the liver. Fazio et al. (1997) Proc. Natl. Acad.
Sci., Vol. 94: 4647.
[0094] In atherosclerosis, it is hypothesized that ApoE, via a
receptor-mediated event, downregulates macrophage activation in the
vicinity of blood vessel walls. Such down-regulation of macrophage
activation interrupts or interferes with the cascade of events
associated with atherosclerotic plaque formation, to thereby reduce
or slow the formation of atherosclerotic lesions. The cascade of
events known to be associated with atherosclerosis includes smooth
muscle cell and endothelial cell proliferation, and foam cell
formation. Evidence exists that ApoE downregulates each of these
processes. ApoE thus affects the presence and progression of
atherosclerosis in vivo, independent of its effects on lipids. The
progression of atherosclerosis can be assessed by measuring the
amount or size of atherosclerotic plaques, or the percentage of the
blood vessel blocked by an atherosclerotic lesion, or the rate of
growth of such plaques.
[0095] Atherosclerosis refers to the thickening of the arterial
intima and accumulation of lipid in artherosclerotic plaques. The
present invention provides a method of treating atherosclerosis or
of reducing the formation of atherosclerotic plaques in a subject
in need thereof by administering one or more peptide dimers of the
present invention. Conditions that can be treated by the present
method include atherosclerosis of the coronary arteries; arteries
supplying the CNS, such as carotid arteries; arteries of the
peripheral circulation or the splanchnic circulation; and renal
artery disease. Administration, such as parenteral administration,
can be site-specific or into the general blood stream. In some
embodiments, the peptide dimers can be combined with an additional
anti-atherosclerotic drug, including HMG-CoA reductase inhibitors,
also termed statins. Suitable statins for use in the methods of the
invention include, for example, lovastatin (MEV ACOR.RTM., Merck),
simvastatin (ZOCOR.RTM., Merck), pravastatin (PRAVACHOL.RTM.,
Bristol Myers Squibb), rosuvastatin (CRESTOR.RTM., AstraZeneca),
fluvastatin (LESCOL.RTM., Novartis) and atorvastatin (LIPITOR.RTM.,
Warner-Lambert).
[0096] The present invention further provides a method of treating,
preventing or ameliorating the symptoms of inflammatory bowel
disease (IBD), Crohn's disease, or ulcerative colitis in a subject
in need thereof by administering an effective amount of at least
one ApoE peptide dimer of the invention. In practicing the methods
of this invention, the therapeutic peptides and/or derivatives
thereof may be used alone or in combination with other active
ingredients. If desired, one or more agents typically used to treat
inflammatory bowel disease may be used as a substitute for or in
addition to the therapeutic peptides in the methods and
compositions of the invention. Such agents include biologics (e.g.,
inflixamab, adelimumab, and CDP-870), small molecule
immunomodulators (e.g., VX 702, SCIO 469, doramapimod, RO 30201
195, SCIO 323, DPC 333, pranalcasan, mycophenolate, and
merimepodib), non-steroidal immunophilin-dependent
immunosuppressants (e.g., cyclosporine, tacrolimus, pimecrolimus,
and ISAtx247), 5-amino salicylic acid (e.g., mesalamine,
sulfasalazine, balsalazide disodium, and olsalazine sodium), DMARDs
(e.g., methotrexate and azathioprine) and alosetron.
[0097] Suitable subjects benefiting from the compositions and
methods of the present invention include male and female mammalian
subjects, including humans, non-human primates, and non-primate
mammals. Subjects include veterinary (companion animal) subjects,
as well as livestock and exotic species.
[0098] The examples which follow are set forth to illustrate the
present invention, and are not to be construed as limiting
thereof.
EXAMPLES
Example 1
The Cytotoxic Activity of ApoE Peptides is Enhanced by Formation of
Disulfide Dimers
[0099] We have previously shown that the addition of a protein
transduction domain (PTD), such as an antennapedia peptide, to the
apoE-mimetic COG133 peptide (LRVRLASHLRKLRKRLL (SEQ ID NO: 3))
enhances its anti-inflammatory activity. A series of fusion
peptides with COG133 conjugated to a PTD were prepared by chemical
synthesis. Notably, we found that COG112 with the sequence
RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL (SEQ ID NO: 1), was effective in
suppressing production of NO, TNF.alpha. and IL-6 with IC50s of 21
nM, 58 nM, and 12 nM, respectively, in BV2 cells following
stimulation with LPS (FIG. 1). These results demonstrate a
significant safety window for COG112 where effective suppression
occurs at concentrations of 12-58 nM while the LD50 is >120-fold
higher at 7 .mu.M.
[0100] During the course of testing various compounds for
cytotoxicity against CLL cells, we found that COG112 had an ED50 of
220 nM. These data were generated using lot #313 of the COG112
peptide. Upon depletion of the stock of lot #313, we began using a
new synthesis of COG112 (lot #411) and discovered that the ED50 was
reduced to 1.2 .mu.M. While still being more potent than the
apoE-mimetic COG133 lacking the antennapedia PTD, this lot was less
active than lot #313.
[0101] To determine any possible structural differences in the two
lots of COG112, we assayed COG112 from the two different lots using
liquid chromatography/mass spectrometry (LC/MS) techniques with
electrospray ionization in the positive detection mode. For COG112
from lot #313, a predominant peak with a mass to charge ratio (M/Z)
of 1126.3 and peaks at M/Z=1800.9, 1286.6, and 1001.5 were observed
(red arrows in FIG. 2A). Upon analysis, the peak at M/Z of 1800.9
arises from the Mass+5 proton form of a dimerized peptide with 5
positive charges (represented as [M+5H].sup.5+/5) and the 1286.6
peak arises from the [M+7H].sup.7+/7 species. Indeed, the dimer
peptide would be expected to have peaks at M/Z=1801.3, 1501.2,
1286.9, 1126.2, 1001.1, 901.1, while the monomer peptide would be
expected to give peaks at M/Z=1501.6, 1126.4, 901.3, 751.3, and
644.1.
[0102] To confirm this finding, we prepared the reduced COG112 by
treatment of COG112 from lot #313 with dithiothreitol to reduce the
disulfides to the free thiol and repeated the LC/MS analysis (FIG.
2B). In the reduced peptide, peaks of M/Z=1501.0, 1126.4. 901.3,
751.3 and 644.2 were observed in good agreement with the peaks
expected from a monomeric peptide. Confirmation that the dimer was
the active form of COG112 was accomplished by forcing the formation
of the disulfide by stirring the monomer in oxidative conditions
and purifying the dimer (also known as COG445 to discern the dimer
form from the monomer form of COG112). Analysis of COG445 by LC/MS
gave MS peaks of M/Z=1800.5, 1501.3, 1286.6, 1126.1, 1001.0, and
901.2 (FIG. 2C) with the peaks at 1800.5, 1286.6 and 1001.0 being
unique to the dimer form of the peptide, thereby confirming the
disulfide bridge of this compound.
[0103] Having confirmed the dimer structure of COG445, we then
evaluated this peptide in both the BV2 cell assay for NO release
and the CLL cytotoxicity cell assay. In the BV2 assay, we confirmed
an IC50 of 20 nM for NO release and an ED50 of 110 nM for
cytotoxicity of CLL cells. In the case of COG445, it is important
to note that the previous ED50 values (e.g., 220 nM) were reported
using the molecular weight of the monomer of 4502 rather than the
actual molecular weight of the dimer of 9004. Adjusting for the
correct molecular weight of COG445, the ED50 value for CLL
cytotoxicity is reduced to 110 nM.
Example 2
Non-Reducible COG112 Dimer Peptides Activate PP2A and are Cytotoxic
to Cancer Cells
[0104] After discovery that COG112 was active as a disulfide-linked
dimer, we sought a method to stabilize the dimer state of COG112.
We initially treated the reduced COG112 peptide with a 5-fold molar
excess of bismaleimidoethane (BMOE) in dilute solution. The peptide
was precipitated by addition of ether, collected by filtration, and
the unreacted BMOE removed by washing prior to drying under vacuum.
The BMOE-linked peptide was dissolved in buffer and mixed with a
1.5-2.0 molar excess of freshly reduced COG112. Coupling was
monitored by HPLC until the reaction was complete and the resultant
peptide-BMOE-linker-peptide dimer was precipitated with ether,
collected, washed, and purified by reverse phase HPLC to a purity
of 98%. The identity of this peptide (known as COG449) was
confirmed by MS and was assayed in the BV2 NO release assay. As
shown in FIG. 3, we observed an IC50 of 9.4 nM for nitric oxide
release from BV2 microglia with COG449, an approximate 2-fold
improvement in activity over COG445 (disulfide-linked COG112
dimer).
[0105] To further evaluate the effect of COG449, we measured the
ability of the stable dimerized COG449 compound to activate PP2A in
32D:p210.sup.BCR/Abl chronic myelogenous leukemia cells. Treatment
with either COG445 or COG449 resulted in increased phosphate
release due to activation of PP2A relative to untreated control
cells (FIG. 4A). However, COG449 treatment increased the rate to a
greater extent than COG445, which suggests that COG449 and other
stable dimer peptides may be found to have improved potency for
killing CLL cells. COG449 also exhibited enhanced PP2A activation
compared to FTY720, an agent previously shown to activate PP2A
(Neviani et al. (2007) J Clin Invest, Vol. 117: 2408-2421).
32D:p210.sup.BCR/Abl chronic myelogenous leukemia cells were
treated with no compound, 1 .mu.M COG449, or 5 .mu.M FTY720. We
observed a robust increase of approximately 45% relative specific
activity (phosphate release/minute/unit protein) of PP2A upon
treatment with COG449 alone when compared to the untreated control,
and about 20% activation compared with FTY720 (FIG. 4B).
[0106] Based on the activation of PP2A in serum containing media
and potent suppression of NO in the BV2 assay, we tested COG 445
and COG449 for cytotoxicity against patient-derived CLL cells and
normal B cells (FIG. 5). Blood from CLL patients was collected and
CD5+/CD19+ CLL cells were isolated using the RosetteSep.TM. Human
B-Cell Enrichment Cocktail, according to the manufacturer's
instructions, and treated with COG compounds. Compounds were
applied to B-CLL cells (2.5.times.10.sup.5 cells/well in a 96 well
plate), after which the cells were treated for 72 hours. After the
treatment period, viable cells were assessed using the MTS assay
(Pharmacia) to determine the concentration of COG compound that was
effective in killing 50% of the input CLL cells (EC50). Like the
values for PP2A activation and NO release, COG449 showed increased
potency compared to COG445 as listed in Table I below. The EC50
values for cytotoxicity of normal B-cells from volunteers treated
with COG445 and COG449 were nearly 200 fold higher (greater than 10
.mu.M).
[0107] In order to more fully understand the role that the PTD
domain and the apoE domain of the COG peptides play in anti-CLL
cytotoxic activity, we tested additional compounds with an HIV-TAT
PTD (COG226) and peptides with altered apoE sequences (COG1410, and
COG248) as shown in Table I. It is notable that either the
antennapedia (ANTP) or TAT PTD increases potency of COG1410 from
5.7 .mu.M to 1.0 .mu.M and 1.4 .mu.M, respectively. It is also
interesting that COG1410 attached to ANTP (COG 120) is more potent
as a monomer than the COG112 monomer, with EC50 values of 1.0 .mu.M
and 1.4 .mu.M for COG120 and COG112, respectively. This result
suggests that creation of dimeric peptides containing altered apoE
domains plus the ANTP or TAT PTD domains may further improve the
potency of the peptides. These data demonstrate that apoE-mimetic
compounds display potent and selective cytotoxic activity for
freshly isolated human B-CLL cells with a wide safety margin.
[0108] We also evaluated the various peptides for efficacy in
suppressing nitric oxide production induced by LPS stimulation of
BV2 microglia cells as a measure of anti-inflammatory activity as
well as the maximum dose tolerated in mice (Table I and FIG. 6A).
For the LPS assay, EC50 is the concentration of compound that
caused a 50% suppression of nitric oxide release from BV2 cells
following LPS stimulation. For mouse toxicity, MTD is the maximum
dose of the compound that can be given by intravenous injection
without causing deaths after 24 hrs. The ApoE peptide dimers, in
particular, exhibited significant potency in the anti-inflammatory
assay (FIG. 6A). Coupling the ApoE domain to a protein transduction
domain further enhanced the potency of the dimers.
[0109] Next, we examined the potency of an ApoE peptide dimer
versus the monomeric form on proliferation of the MDA-MB-231 breast
cancer cell line. MDA-MB-231 cells were treated with various
concentrations of either COG435 (monomer; SEQ ID NO: 90) or COG493
(a BMOE-linked dimer of COG 435) peptides for 48 hours. Following
peptide treatment, cells were quantified using a MTT assay. The
results, shown in FIG. 6B, show that the dimeric form of the ApoE
mimetic peptide was significantly more cytotoxic to breast cancer
cells than the monomeric form.
[0110] To determine whether ApoE peptide dimers were cytotoxic to
other types of cancer, we evaluated the effect of three different
ApoE BMOE-linked peptide dimers (COG449, COG492, COG493; see Table
I) on the growth characteristics of U87MG glioblastoma cells.
Various concentrations of COG449, COG492, COG493 or Sorafenib were
used to treat U87MG glioblastoma cells and viable cells were
quantitated using MTT. Sorafenib, which has previously been
reported to be cytotoxic to glioblastoma cells (Yang et al. (2010)
Mol Cancer Ther., Vol. 9(4):953-962), was used as a positive
control. The dose response curve shown in FIG. 6C show that each of
the dimeric peptides were cytotoxic to U87MG cells.
[0111] This series of experiments demonstrate that ApoE peptides
are cytotoxic to three different types of cancer cells.
Interestingly, the dimeric form of the ApoE peptides is
considerably more potent in inducing cytotoxicity of cancer cells
than the monomeric form.
TABLE-US-00003 TABLE I Activity of COG compounds on cancerous B-CLL
cells, inflammation, and mouse toxicity LPS Mouse CLL Normal Assay
MTD EC50 Fold EC50 EC50 (mg/ Compound Sequence Form (.mu.M) Change
(.mu.M) (.mu.M) kg) COG056 LLRKRLKRLHSALRVRL Monomer 12.9 .+-. 1.0
>20 >20 n.d. (rev133) (SEQ ID NO: 2) 4.6 COG133
LRVRLASHLRKLRKRLL Monomer 4.4 .+-. 2.9 >20 8.8 16 (SEQ ID NO: 3)
1.5 COG248 LRVRLAS(Aib)LKRLRK(nitroR)LL Monomer 2.3 .+-. 5.5 >20
0.9 n.d. (SEQ ID NO: 4) 1.3 [Aib is amino isobutyric acid and
nitroR is a nitroarginine] COG1410 AS(Aib)LRKL(Aib)KRLL Monomer 5.7
.+-. 2.3 >20 4.5 15 (SEQ ID NO: 5) 3.0 [Aib is amino isobutyric
acid] COG226 YGRKKRRQRRR-C-AS(Aib)LRKL Monomer 1.4 .+-. 9.2 >20
<1.0 n.d. (TAT- (Aib)KRLL 0.2 COG1410) (SEQ ID NO: 6) COG120
RQIKIWFQNRRMKWKK-C- Monomer 1.0 .+-. 12.6 >20 <1.0 n.d.
(ANTP- AS(Aib)LRKL(Aib)KRLL 0.2 COG1410) (SEQ ID NO: 7) COG112
RQIKIWFQNRRMKWKK-C- Monomer 1.4 .+-. 9.2 >20 <1.0 28 (ANTP-
LRVRLASHLRKLRKRLL 0.7 COG133) (SEQ ID NO: 1) COG445
COG112-C-C-COG112 Dimer 0.11 .+-. 117.3 >10 <1.0 28
(disulfide- [C--C is a disulfide 0.08 linked bridge] COG112) COG449
COG112-C-BMOE-C-COG112 Dimer 0.077 .+-. 167.5 >10 0.22 12 (BMOE-
[BMOE is a bismalei- 0.011 linked midoethane linker] COG112) COG492
C-LRVRLASHLRKLRKRLL Dimer n.d. n.d. n.d. 0.75 25 (BMOE- (SEQ ID NO:
15) linked <BMOE> COG133) C-LRVRLASHLRKLRKRLL (SEQ ID NO: 15)
[BMOE is a bismalei- midoethane linker] COG435
WKK-C-LRVRLASHLRKLRKRLL Monomer n.d. n.d. n.d. n.d. n.d. (SEQ ID
NO: 90) COG493 WKK-C-LRVRLASHLRKLRKRLL Dimer n.d. n.d. n.d. 0.17 20
(BMOE- (SEQ ID NO: 90) linked <BMOE> COG435)
WKK-C-LRVRLASHLRKLRKRLL (SEQ ID NO: 90) [BMOE is a bismalei-
midoethane linker]
Example 3
Creation of an ApoE Peptide Dimer Library
[0112] Based on the results demonstrated in Examples 1 and 2 that
dimers of ApoE peptides are more potent in inducing cytotoxicity of
cancer cells and activating PP2A, twenty eight different monomer
ApoE peptides are synthesized that can be coupled with two
different coupling chemistries to create a dimer library of sixty
four unique compounds. The goal of this Example is to establish a
library of chemically stable peptide dimers designed to explore the
structure activity relationship between apoE-mimetic peptides and
cytotoxicity for CLL cells. Our initial screens with COG peptides
were limited to a single dimer peptide, COG112, which has the
sequence Ac-RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-amide (SEQ ID NO: 1)
that contained a disulfide bridge through the cysteine at position
17. This peptide has an antennapedia-derived PTD domain at the
N-terminal end and an apoE-mimetic domain in the C-terminal portion
such that the dimerized peptide contained two PTD domains and two
apoE-mimetic domains. While this dimer of COG112 demonstrated
superior potency, it is not possible to determine whether the PTD
domain is essential for improved potency or whether a dimer
composed of two COG peptides will be sufficient for high potency in
cytotoxicity assays. It appears that a PTD does improve cytotoxic
potency based on the observation that monomeric COG112 had an ED50
of 1.4.+-.0.7 .mu.M while COG133 that lacks a PTD was three fold
less potent with an ED50 of 4.4.+-.1.5 .mu.M (Table I). Based on
these results, our strategy for making dimer peptides relies on the
combinatorial mixing of peptides that contain a PTD and an
apoE-mimetic domain. FIG. 7 illustrates this approach and all
peptides in the dimer library contain two apoE-mimetic domains. A
series of monomer peptides with reactive groups that can be
chemically coupled to create stable dimer peptides is created and
combined to make dimer peptides that contain zero, one, or two PTD
domains. In order to complete the chemical coupling, we have
identified and validated two unique approaches to perform the
coupling reactions, namely bismaleimide coupling and click
chemistry coupling.
[0113] The first method used to create apoE-mimetic dimer peptides
utilizes the reaction of a maleimide group with the sulfhydryl
group of cysteine. Monomer peptides are created with a single
cysteine residue in the peptide monomers and coupled to form a
dimer using a bismaleimide linker to create the stable dimer (FIG.
8). Both bismaleimido-ethane (BMOE) and bismaleimido-hexane (BMH)
are utilized in the creation of dimers, which allows for bridges of
12 or 16 atoms between the two peptide chains in the dimer
peptides. Use of two different length bridging groups allows us to
determine the effect of bridging group length on anti-CLL activity.
In order to create the bismaleimide linked library, twelve unique
monomers are synthesized as listed in Table II. We have selected
four unique sequences for the apoE-mimetic domains (COG133,
COG1410, COG248, and COG345). COG1410, COG248, and COG345 have
previously been shown to exhibit improved anti-inflammatory
activity relative to COG133 in NO release assays. An antennapedia
PTD (RQIKIWFQNRRMKWKK (SEQ ID NO: 8)) and an HIV TAT PTD
(YGRKKRRQRRR (SEQ ID NO: 9)) are used for the PTD domain in the
library peptides.
TABLE-US-00004 TABLE II Monomer Peptide Sequences for Bismaleimide
Library Construction SEQ ID apoE- Designation Sequence NO
PTD-domain domain A1 RQIKIWFQNRRMKWKK-C- 1 Antennapedia COG133
LRVRLASHLRKLRKRLL A2 RQIKIWFQNRRMKWKK-C- 7 Antennapedia COG1410
AS(Aib)LRKL(Aib)KRLL A3 RQIKIWFQNRRMKWKK-C- 10 Antennapedia COG248
LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL A4 RQIKIWFQNRRMKWKK-C- 11
Antennapedia COG345 LRVRLAS(Aib)LRKLR(K-Ac)RLL A5
YGRKKRRQRRR-C-LRVRLASHLRKLRKRLL 12 HIV-TAT COG133 A6
YGRKKRRQRRR-C-AS(Aib)LRKL(Aib)KRLL 6 HIV-TAT COG1410 A7
YGRKKRRQRRR-C-LRVRLAS(Aib)LKRLRK 13 HIV-TAT COG248 (Nitro-Arg)LL A8
YGRKKRRQRRR-C-LRVRLAS(Aib)LRKLR 14 HIV-TAT COG345 (K-Ac)RLL B1
C-LRVRLASHLRKLRKRLL 15 -- COG133 B2 C-AS(Aib)LRKL(Aib)KRLL 16 --
COG1410 B3 C-LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL 17 -- COG248 B4
C-LRVRLAS(Aib)LRKLR(K-Ac)RLL 18 -- COG345 Aib = aminoisobutyric
acid, K-Ac = N.epsilon.-Acetyl-lysine, and Nitro-Arg =
Nitroguanidinoarginine
[0114] Following synthesis of the peptides listed in Table II, the
peptides are coupled to form dimers using either BMOE or BMH as
listed in Table III. In Table III, an X indicates which monomer
peptides are coupled together, thereby creating a focused library
containing 40 unique members. The initial library is not strictly
combinatorial in that only matched apoE sequences are coupled,
resulting in dimer peptides that contain two COG133, two COG1410,
two COG248, or two COG345 apoE domains. Final compounds that
contain two PTD domains and two apoE domains are highlighted in the
blue area of the Table, compounds that contain one PTD domain and
two apoE domains are highlighted in the yellow area of the Table,
and compounds lacking a PTD domain but containing two apoE domains
are found in the green area of the Table.
TABLE-US-00005 TABLE III Combinatorial plan for construction of
dimer peptides using bismaleimides ##STR00001##
[0115] The second method for coupling of the peptides utilizes
"Click" chemistry for coupling the monomers. This coupling method
relies on the copper catalyzed 3+2 Huisgen cycloaddition of an
azide and a primary alkyne to form a stable 1,4-disubstituted
triazole as shown in FIG. 9. The reactive azido and primary alkyne
groups are incorporated into peptides during synthesis through
commercially available L-azidohomoalanine and L-propargylglycine,
respectively. After synthesis of the peptide monomers containing
either L-azidohomoalanine or L-propargylglycine, two methods can be
used to create dimerized peptides. The first approach is the simple
coupling of one monomer containing azidohomoalanine with a
propargylglycine-containing monomer using standard reaction
conditions (FIG. 9A). This reaction generates a dimer with a short
6 atom bridge between the peptide chains. The second approach
utilizes two azidohomoalanine containing monomers that are coupled
together through N,N-dipropargylamine (FIG. 9B) to create a 13 atom
bridge between the peptide chains. In order to complete the "Click"
chemistry library construction, 16 unique monomer peptides are
synthesized as listed in Table IV.
TABLE-US-00006 TABLE IV Monomer Peptide Sequences for Click
Coupling Library Construction SEQ ID apoE- Designation Sequence NO
PTD-domain domain A1 RQIKIWFQNRRMKWKK-Azh- 19 Antennapedia COG133
LRVRLASHLRKLRKRLL A2 RQIKIWFQNRRMKWKK-Azh- 20 Antennapedia COG1410
AS(Aib)LRKL(Aib)KRLL A3 RQIKIWFQNRRMKWKK-Azh- 21 Antennapedia
COG248 LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL A4 RQIKIWFQNRRMKWKK-Azh- 22
Antennapedia COG345 LRVRLAS(Aib)LRKLR(K-Ac)RLL A5
YGRKKRRQRRR-Azh-LRVRLASHLRKLRKRLL 23 HIV-TAT COG133 A6
YGRKKRRQRRR-Azh-AS(Aib)LRKL(Aib)KRLL 24 HIV-TAT COG1410 A7
YGRKKRRQRRR-Azh-LRVRLAS(Aib)LKRLRK 25 HIV-TAT COG248 (Nitro-Arg)LL
A8 YGRKKRRQRRR-Azh-LRVRLAS(Aib)LRKLR 26 HIV-TAT COG345 (K-Ac)RLL B1
Pgy-LRVRLASHLRKLRKRLL 27 -- COG133 B2 Pgy-AS(Aib)LRKL(Aib)KRLL 28
-- COG1410 B3 Pgy-LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL 29 -- COG248 B4
Pgy-LRVRLAS(Aib)LRKLR(K-Ac)RLL 30 -- COG345 C1
Azh-LRVRLASHLRKLRKRLL 31 -- COG133 C2 Azh-AS(Aib)LRKL(Aib)KRLL 32
-- COG1410 C3 Azh-LRVRLAS(Aib)LKRLRK(Nitro-Arg)LL 33 -- COG248 C4
Azh-LRVRLAS(Aib)LRKLR(K-Ac)RLL 34 -- COG345 Azh = azidohomoalanine,
Pgy = propargylglycine, Aib = aminoisobutyric acid, K-Ac =
N.epsilon.-Acetyl-lysine, and Nitro-Arg =
Nitroguanidinoarginine
[0116] Following synthesis of the peptides listed in Table IV, the
peptides are coupled to form dimers using either the direct Click
coupling (FIG. 9A) or via N,N-dipropargylamine coupling (FIG. 9B)
using the combinatorial plan shown in Table V. In Table V, an X
indicates which monomer peptides are coupled together while the
designation Xa or Xb indicates the use of direct Click coupling or
via N,N-dipropargylamine Click coupling, respectively, to create
the dimerized peptides. This approach generates a focused library
containing 24 unique members. Final compounds that contain two PTD
domains and two apoE domains are highlighted in the blue area of
the Table, compounds that contain one PTD domain and two apoE
domains are highlighted in the yellow area of the Table, and
compounds lacking a PTD domain but containing two apoE domains are
found in the green area of the Table. In the case of the dimers
that contain two apoE domains and lacking PTD domains (green area
of Table), there are two lengths of linkers between the
apoE-mimetic domains. Heterodimerization through direct Click
coupling (B1 to C1, B2 to C2, etc.) leads to a shorter 6 atom
bridge between the peptide chains and homodimerization using
N,N-dipropargylamine results in a longer 13 atom bridge between the
peptide chains.
TABLE-US-00007 TABLE V Combinatorial plan for construction of Click
dimer peptides ##STR00002## Xa = direct Click coupling, Xb =
coupling through N,N-dipropargylamine
Methods
[0117] Bismaleimide Coupling.
[0118] Coupling of the cysteine containing peptides with
bismaleimide compounds is performed as a two step process where a
3-5 fold molar excess BMOE or BMH are initially reacted with the
reduced monomer peptide in dilute solution. The peptide is
precipitated by addition of ether, collected by filtration, and
unreacted BMOE/BMH is removed by washing prior to drying under
vacuum. The BMOE/BMH-linked peptide monomer peptide is dissolved in
buffer and mixed with a 1.5-2.0 molar excess of the second monomer
peptide. Coupling is monitored by HPLC until the reaction is
complete. The resultant peptide is precipitated with ether,
collected, washed, purified by reverse phase HPLC to a purity of
>90%, and analyzed by LC/MS to determine the molecular mass of
the product. Any peptides that do not match the expected mass are
rejected and the coupling is repeated.
[0119] Click Coupling.
[0120] Coupling of the azidohomoalanine and propargylglycine
containing peptides is performed using previously described
protocols (Chan et al. (2004) Org Lett, Vol. 6(17): 2853-2855).
Briefly, equimolar amounts of each monomer peptide are mixed
together with copper iodide and ascorbic acid or
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). Initially
coupling is accomplished with ascorbic acid (Rostovtsev et al.
(2002) Angew Chem Int Ed Engl, Vol. 41(14): 2596-2599), but TBTA is
used if the ascorbic acid coupling results in poor yield or if
damage to the peptide scaffolds is observed. Following coupling,
peptides are precipitated with ether, collected and purified by
reverse phase HPLC to a purity of >90%. Peptides are analyzed by
LC/MS to determine the molecular mass of the product and any
peptides that do not match the expected mass are rejected and the
coupling is repeated.
Example 4
Evaluation of ApoE Peptide Dimers
[0121] This example outlines experiments designed to evaluate the
potency of the peptide dimers from the library described in Example
3. A two step screening cascade is employed. It has previously been
shown that high cytotoxicity and leukemia cell apoptosis occur when
CLL cells are cultured with nitric oxide synthase (NOS) inhibitors,
thereby reducing the concentration of nitric oxide (NO)(Thomas et
al. (2008) Free Radic Biol Med, Vol. 45: 18-31). This phenomenon
occurs because low to moderate levels of NO are required for
maintenance of the anti-apoptotic state of CLL cells (Zhao et al.
(1998) Blood, Vol. 92(3): 1031-1043; Levesque et al. (2008) Leuk
Res, Vol. 32(7): 1061-70; Kolb et al. (2003) Cardiovasc Haematol
Disord, Vol. 3(4): 261-86). Within cells, NO is produced from
L-arginine by three NOS isoforms in humans that are encoded by
separate genes. NOS1 ("neuronal" NOS) and NOS3 ("endothelial" NOS)
generally produce low levels of NO and are constitutively
expressed, while inducible NOS (NOS2) is induced by cytokines and
microbial factors through activation of NF.kappa.B. Like BV2 cells,
human cells express NOS2 and produce NO in response to several
stimuli including IFN-.alpha., IFN-.gamma., IL-1, TNF.alpha., IL-6,
and LPS (Weinberg (1998) Molecular Med, Vol. 4: 577-591). In human
CLL cells, it has been reported that high levels of NOS2 mRNA and
protein are constitutively expressed and the cells have high NOS
enzyme activity (Zhao et al. (1998) Blood, Vol. 92(3): 1031-1043).
Based on these data and the limitations on use of human CLL cells,
compounds are initially screened for suppression of NO production
in lipopolysaccharide stimulated BV2 microglial cells. This assay
is used for the initial screen because BV2 cells grow rapidly and
readily express inducible NO synthase (NOS) in response to LPS
treatment, leading to measurable amounts of NO production.
Therefore, the first screening assay entails treating BV2 cells in
an 8 point dose titration curve followed by stimulation with LPS
and measuring the ability of the peptide dimers to suppress NO
production.
[0122] Following determination of the IC50 for NO production in BV2
cells, the twenty peptide dimers with the greatest potency in the
NO suppression assay are screened for cytotoxic activity against
purified CD19+/CD5+ leukemia cells from CLL patients. Whole blood
from CLL patients is obtained and the CD19+/CD5+ CLL cells are
isolated by using RosetteSep.TM. Human B Cell Enrichment Cocktail
as described in the methods section below. Unlike homogeneous BV2
cells from culture, it is difficult to obtain enough cells from one
patient to screen many peptide dimers with cells from a single
patient. Furthermore, due to the diverse nature of CLL with many
documented chromosomal abnormalities and phenotypes, it is
necessary to screen a single peptide dimer against CLL cells from
multiple patients. Therefore, to ensure that pharmacogenomic
effects are minimized in this screening step, ED50 values for
cytotoxicity of each peptide dimer is determined for CLL cells from
not less than six randomly selected patients and the ED50 curves
from each individual patient are averaged. Following completion of
this screening step, the safety window of each of the 10 most
potent peptide dimers from the CLL cell cytotoxicity assays is
determined by isolating CD19+ B-cells from normal, age-matched
volunteers and determining the ED50 for cytotoxicity of normal
B-cells. Similar to the analysis with the CLL cells, the ED50 in
B-cell samples is determined from not less than 4 volunteers. A
safety window for each peptide dimer is calculated by dividing the
ED50 value for cytotoxicity on CLL cells by the ED50 value for
cytotoxicity on normal B-cells. The five peptide dimers with the
greatest safety window is selected for use in pharmacokinetic
profiling.
Pharmacokinetic Profiling of Peptide Dimers
[0123] Male C57Bl/6 mice (20-24 g, Charles River, Raleigh, N.C.)
are injected with a peptide dimer subcutaneously at a volume of 5
mL/kg in the scruff of the neck. Ten minutes before the desired
timepoint, mice are anesthetized and at the desired timepoint blood
is drawn by cardiac puncture. Blood from at least 4 mice is used
for each timepoint with the samples collected at 5, 10, 15, 30, 45,
60, 90, 120, 180 and 240 minutes for the initial analysis. The
samples are processed and the pharmacokinetic analysis is
completed. These timepoints were selected based on previous
experience with subcutaneously injected COG1410 that shows a dose
dependent Tmax of 20-30 minutes and a half life for clearance of
60-90 minutes. In the event that the half life has not been
reached, a repeat of the experiment is performed that uses one
timepoint before the observed Tmax, a timepoint at the observed
Tmax and timepoints extending long enough to adequately determine
the half life based on extrapolation of the previous observations
of clearance from the plasma. Analysis of three concentrations of
the peptide dimer is also performed to determine if there is a dose
dependent effect on the pharmacokinetic parameters.
[0124] Noncompartmental model analysis is used to estimate
pharmacokinetic parameters (Gibaldi and Perrier, 1982) including
area under the plasma-concentration time curve from time 0 to time
infinity (AUC, 0-.infin.), peak plasma concentration (C.sub.max),
systemic clearance (CL, which is calculated based on the ratio
between the dose and AUC, 0-.infin.), volume of distribution at
steady state (Vss), terminal half-life (t1/2), which is calculated
using a minimum of the last three concentration-time data, and mean
residence time in the body (MRT). Data analysis is conducted using
Win-Nonlin professional version 3.1 (Pharsight Corporation, Cary,
N.C., USA).
Peptide Dimer Treatment of E.mu.-TCL1 Transgenic Mice
[0125] Following pharmacokinetic profiling, select peptide dimers
are tested in a transgenic mouse model of CLL. Blood is drawn by
retro-orbital bleeding from transgenic E.mu.-TCL1 mice aged to 9
months for initial analysis to total white cell counts and leukemia
cell burden. Animals that show leukemia signs are randomly assigned
to treatment groups. At the initiation of treatment, blood is drawn
to determine baseline CD5+/CD19+ CLL cell counts and groups of
animals (n=20) are subcutaneously injected with a vehicle control
(lactated Ringer's solution) or one of the selected peptide dimers
at doses and a dose frequency schedule determined by the
pharmacokinetic profile data for a total treatment time of 35
days.
[0126] Blood is collected from each mouse on a weekly basis by
retro-orbital bleeds. This blood is used for determination of total
blood leukocyte and lymphocyte counts as well as CD19+/CD5+ cell
counts to determine the leukemia burden. After 35 days of
treatment, mice are euthanized and the post treatment leukemia
burden is measured by cell counting, spleen weight, and
histological analysis of bone marrow, spleen, liver, and lymph
nodes. All mice dying before 35 days are analyzed in a comparable
fashion.
Methods
[0127] BV2 Cell Growth, LPS Stimulation and NO Quantitation.
[0128] Low passage BV2 microglial cell cultures are maintained in
10% HI (Heat Inactivated) FBS DMEM media (supplemented with MEM
NEAA (Non-Essential Amino Acids), sodium pyruvate and Pen-Strep)
and continuously cultured until needed. To determine the IC50
values for NO production, peptides are added to BV2 cultures in 1%
HI FBS DMEM media (supplemented with MEM NEAA and sodium pyruvate)
at a range of final assay concentrations from 1.0 .mu.M in 2 fold
dilutions to 7.8 nM followed immediately by addition of LPS (100
ng/mL final concentration) as previously described (Laskowitz et
al. (2001) Exp Neurol, Vol. 167(1): 74-85). After incubating for
18.+-.1 hours, conditioned media is removed for analysis of nitrite
(the stable oxidation product of released nitric oxide) by the
Griess colorimetric assay (Promega). Remaining cells are assayed
for viability in an MTT assay (Promega) and the nitrite assay
values are normalized for each concentration using the percent
viability of cells in the MTT assay. IC50 values for NO inhibition
are calculated under the assumption that LPS-only (no peptide
added) cultures exhibit a 100% response and no-LPS (no peptide
added) cultures exhibit a 0% response. Typically, the absence of
LPS exposure to BV2 cells results in levels below the limit of
detection in our assays. Similarly, addition of any of the peptides
up to 25 .mu.M without LPS results in undetectable NO levels.
[0129] CLL Cell Isolation.
[0130] Normal B-cells from volunteers and B-CLL cells from patients
are isolated using the RosetteSep.TM. Human B Cell Enrichment
Cocktail according to the manufacturer's instructions. This method
depletes whole blood of T cells, monocytes, and NK cells using a
proprietary antibody cocktail that cross-links unwanted cells in
human whole blood to multiple red blood cells (RBCs) forming
immunorosettes. This increases the density of the unwanted
(rosetted) cells, such that they pellet along with the free RBCs
when centrifuged over a buoyant density medium such as
Ficoll-Paque.RTM.. This leaves the highly enriched B-cell or B-CLL
cells at the interface between Ficoll and the plasma. The
antibodies in the cocktail contain anti-CD14, anti-CD2 and
anti-CD16 antibodies to remove T cells, monocytes, and NK cells,
respectively. The purity of these B-cell and B-CLL preparations is
then determined by flow cytometry. Preparations typically average
less than 2% CD3+ T-cells and less than 0.5% CD14+ monocytes. In
the case of B-CLL cell isolation, we routinely obtain preparations
that contain less than 1.5% normal CD19+/CD5- B-cells using this
method.
[0131] CLL Cell Culture/Cytotoxicity Assays.
[0132] For cytotoxicity assays, 3.times.10.sup.6 CLL cells/well are
cultured in 24 well tissue culture plates in 1.5 mL of Hybridoma
SFM.TM. (Gibco, Long Island, N.Y.) as described by Levesque et al.
(2001, 2003). All cultures are incubated at 37.degree. C., 5% CO2
in air. Peptide dimers are applied to B-CLL cells
(0.25.times.10.sup.6 cells/well in a 96 well plate) and after 72
hours, viable cells are assessed using the MTS assay (Pharmacia) to
determine the concentration of peptide dimer that is effective in
killing 50% of the input CLL cells (ED50) (Levesque et al.,
2003).
Example 5
An ApoE Peptide Dimer Inhibits Akt Signaling in Cancer Cells
[0133] Akt signaling is often dysregulated in cancers thereby
promoting cellular survival and proliferation. ApoE-based peptides
have shown anti-inflammatory effects in various neuropathologies
associated with increased Akt signaling. Given the effect of ApoE
peptides on other inflammatory pathologies, we investigated the
effect of an ApoE peptide dimer on Akt signaling in breast and
brain cancer cells. A disulfide dimer of COG112 (SEQ ID NO: 1) was
prepared as described in Example 1 (COG445). Adherent breast cancer
cells, MDA-MB-231 (MB231), or glioblastoma cells, U87-MG (U87),
were serum starved overnight and then exposed to COG445 for 2 hours
before stimulating with epidermal growth factor (EGF) for 5
minutes. Western blot analysis showed that COG445 at concentrations
up to 1 .mu.M did not alter EGFR activation as determined by EGFR
tyrosine 1045 phosphorylation in both U87 (FIG. 10A) and MB231
(data not shown) cell lines. EGFR activation results in the
activation of the PI3K/Akt signaling pathway, which is mediated by
PDK1 activation. COG445 treatment did not alter PDK1 activation as
measured by serine 241 phosphorylation levels in U87 (FIG. 10A) and
MB231 (data not shown) cell lines. However, COG445 exposure did
result in a dose-dependent decrease of Akt activation in both MB231
and U87 cells (FIG. 10B). Akt serine 473 phosphorylation was
decreased in both cell lines at 100 nM and reaches statistical
significance at 1 .mu.M COG445.
[0134] Inhibition of Akt phosphorylation by COG445 appears to be
downstream of EGFR/PI3K signaling as EGFR and PDK1 phosphorylation
were not altered (FIG. 10A). An endogenous negative regulator of
Akt is protein phosphatase 2A (PP2A). To address the potential role
of PP2A on COG445 mediated inhibition of Akt, MB231 and U87 cells
were exposed to COG445 for two hours in the presence of a PP2A
inhibitor, okadaic acid (OA), and then stimulated with EGF. Western
blot analysis of MB231 cells shows that COG445 did not inhibit Akt
phosphorylation in the presence of OA (FIG. 11A). The difference in
Akt phosphorylation between MB231 cells treated with and without
OA, in terms of percent of EGF control and with respect to COG445
concentration, is shown in FIG. 11B. At COG445 concentrations
.gtoreq.100 nM there was a significant difference in Akt
phosphorylation, indicating that the inhibition of Akt by COG445 is
sensitive to OA. Nonlinear regression analysis further illustrates
that COG445 inhibits Akt activation in an okadaic acid sensitive
mechanism, consistent with protein phosphatase mediated mechanism
of action (FIG. 11C).
[0135] To further characterize the effect of COG445 on PP2A
activity, MB231 cells were treated as above and the phosphatase
activity of immunoprecipitated PP2A was measured. EGF stimulation
caused a significant decrease in total PP2A activity compared to
untreated serum starved cells (FIG. 12A). Cells treated with COG445
exhibited a significant (P<0.01) increase in PP2A activity at
concentrations .gtoreq.100 nM. However, the level of PP2A activity
did not return to unstimulated control levels. To further explore
the extent of PP2A activation in response to COG445, total c-myc
protein levels were determined by western blot analysis. C-myc is a
substrate for PP2A and upon dephosphorylation is metabolized by
ubiquitination and proteasomal degradation. In both U87 (FIG. 12B)
and MB231 (data not shown) cell lines, c-myc levels decreased after
a two-hour exposure to COG445 in a dose-dependent manner, reaching
statistical significance at 1000 nM. Furthermore, c-myc levels are
unaltered in response to COG445 in the presence of OA, indicating
that PP2A may mediate the decrease in c-myc levels induced by
COG445.
[0136] To examine the mechanism of COG445 on PP2A activity,
recombinant human PP2A catalytic subunit was incubated with COG445
and activity was measured. COG445 did not have any effect on the
rate of phosphatase activity up to 10 .mu.M (data not shown). These
data indicate that COG445 affects PP2A activity on a biochemical
level. To further explore the changes of PP2A protein complexes,
co-immunoprecipitation experiments were performed on MB231 cells.
The potent endogenous inhibitor of PP2A, I.sub.2PP2A (also known as
SET), was strongly associated to PP2A in EGF stimulated cells,
corresponding to the low PP2A activity, while the association was
significantly decreased in unstimulated cells (FIG. 12C).
Pre-treatment of the cells with either 0.1 or 1 .mu.M COG445 also
strongly diminished the association of I.sub.2PP2A to the catalytic
subunit of PP2A (data not shown).
[0137] Akt exerts its proliferative signal by phosphorylating
protein substrates such as mTOR and GSK-3.beta.. To examine the
effects of COG445 on downstream Akt signaling in MB231 cells, mTOR
activation and GSK-3.beta. inhibition was measured by western blot
analysis. COG445 caused a dose-dependent decrease in mTOR and
GSK-3.beta. phosphorylation upon EGF stimulation (FIGS. 13A and B).
The phosphorylation of Akt substrates was markedly reduced at
COG445 concentration .gtoreq.100 nM, corresponding to Akt
activation levels. The reduction in mTOR and GSK-3.beta.
phosphorylation was eliminated in the presence of okadaic acid,
further evidence that PP2A mediates the effects of COG445 on Akt
signaling.
[0138] Because COG445 inhibited Akt activation and downstream
signaling, we examined the effects of COG445 on cellular
proliferation. Adherent MB231 and U87 cells were grown for
.about.24 hours in the presence of COG445 and cellular
proliferation was measured by MTT reduction. COG445 caused a
dose-dependent decrease in proliferation for both cancer cell lines
tested (FIG. 14A). Furthermore, COG445 inhibited MB231 cellular
proliferation as determined by cell count (FIG. 14B). Approximately
6.times.10.sup.4 cells were plated in growth media (RPMI+10% FBS)
for 24-hours with increasing concentrations of COG445. Incubation
of MB231 cells with 0.1 .mu.M COG445 resulted in a slight decrease
in cellular proliferation, consistent with the MTT assay data.
However at concentrations .gtoreq.1 .mu.M COG445, there appears to
be a loss of cells. This is partially explained by decreased
proliferation, although it is possible that the rate of cellular
degradation was increased in the presence of COG445. However, there
was no indication of apoptosis after 24 hours in response to 1-1000
nM COG445 administration (data not shown).
[0139] The results of these experiments described in this example
suggest that ApoE-based peptides may have beneficial effects in
cancer chemotherapy by activating the tumor suppressor PP2A.
Example 6
An ApoE Peptide Dimer Reduces c-myc Phosphorylation in a Burkitt's
Lymphoma Cell Line
[0140] Aberrant c-myc expression has been implicated in various
forms of cancer. It has been reported that phosphorylation at
serine 62 of c-myc stabilizes the c-myc protein, while
dephosphorylation at this serine residue by PP2A directs
ubiquitin-mediated degradation of c-myc (see, e.g., Sears et al.
(2004) Cell Cycle, Vol. 3: 1133-1137). Because an ApoE peptide
dimer increases PP2A activity and promotes c-myc degradation in
cancer cell lines (see Examples 2 and 5), we further examined the
effect of a stabilized ApoE peptide dimer on c-myc phosphorylation
in a c-myc dependent human Raji cell line of Burkitt's lymphoma. We
treated Raji cells with COG449, a BMOE-linked dimer of COG112 (see
Example 2), or a vehicle control for 20 hr and probed the extracts
by Western blotting with an antibody for P-S62 and a total c-myc
antibody, which showed a significant reduction of phosphorylation
at S62 (FIG. 15).
[0141] These results are consistent with those obtained in the
experiments described in Examples 2 and 5 and suggest that ApoE
peptide dimers can modulate c-myc protein levels in cancer cells
perhaps by antagonizing SET and relieving the inhibition of PP2A.
Thus, ApoE peptide dimers may represent a new approach to cancer
treatment, especially in cancers where SET is overexpressed.
Example 7
SET is Overexpressed in CLL and B-Cell Lymphoma Cells
[0142] In seeking to study the dysregulation of PP2A in chronic
lymphocytic leukemia (CLL), we chose to focus on the endogenous
physiological inhibitory proteins of PP2A. Recently, Neviani et al.
(Cancer Cell, Vol. 8: 355-68, 2005) reported that the SET
oncoprotein (also known as Inhibitor-2 of PP2A, I.sub.2PP2A) was
overexpressed in patient-derived chronic myelocytic leukemia (CML)
cells and that the SET concentration increased as the patient
developed blast crisis. This reference also demonstrated that PP2A
activity decreased during blast crisis, resulting in reduced
ability of cells to regulate the Akt signaling pathway following
BCR/Abl stimulated Akt phosphorylation. Given the convergence of
dysregulated Akt signaling in both CML and CLL, we sought to
determine whether SET might also be overexpressed in fresh,
patient-derived CLL cells. Using primary B-CLL samples from 16
patients and normal B-cells from volunteers, we prepared cell
lysates and 40 .mu.g of each lysate was loaded on to a SDS PAGE,
transferred to nitrocellulose, and immunoblotting was performed.
The bands detected using an anti-SET antibody were quantitated and
normalized using .beta.-Actin as a loading control on a LiCor
Odyssey fluorescence scanner. We discovered that SET was
significantly overexpressed (p<0.05) in the CLL patient samples
relative to normal B-cells from volunteers (FIG. 16A). This result
was corroborated by determining the expression levels of SET mRNA
in patient CLL cells by quantitative PCR (qPCR). The results of
this analysis showed statistically significant (p<0.05) higher
SET mRNA levels relative to normal B-cells (FIG. 16B). This SET
overexpression was independent of apoE genotype or cytogenetic
abnormalities of the patients. Documenting SET overexpression in
CLL indicates that SET is an important factor in multiple cancers:
it is overexpressed in CML (Neviani et al. (2005) Cancer Cell, Vol.
8: 355-68) and diffuse large B-cell lymphoma (Nenasheva et al.
(2004) Mol Biol (Mosk), Vol. 38: 265-75). Also in microarray
studies of CLL cells, SET upregulation was noted in unmutated IgVH
cells (Rosenwald et al. (2001) J Exp Med, Vol. 194:1639-47).
[0143] To expand this work, we also evaluated SET overexpression in
the Raji and Ramos cell lines of Burkitt's lymphoma, a B-cell
Non-Hodgkin's lymphoma (NHL), which unlike CLL cells, is
proliferating and can be genetically manipulated. We grew these
cells using conditions recommended by ATCC. Cell lysis for Western
blotting and isolation of mRNA was performed using standard
protocols from 3 separate cultures. Following synthesis of cDNA
from the isolated mRNA, qPCR was performed analyzing SET mRNA and
18S primers and the fold change of the SET expression in Raji and
Ramos cells were normalized to that of normal B-cell cDNA
expression level (standardized to 1). SET expression levels were
10.5.+-.0.7 fold higher than normal B-cells in Raji cells and
8.2.+-.0.4 for the Ramos cells (p<0.001) (FIG. 17A). Western
blotting revealed elevated levels of SET protein as well in Raji
and Ramos cells relative to normal B-cell extracts (FIG. 17B).
Taken together, these results indicate that overexpression of SET
in B-CLL cells and lymphoma cells would decrease PP2A activity and
inhibit the ability of PP2A to regulate numerous signaling
pathways, such as the Akt-NF.kappa.B pathway, the c-Myc oncogene,
and the anti-apoptotic Mcl-1 protein, thereby allowing for a pro
growth, anti-apoptotic cancerous state to develop in these cells.
Thus, SET overexpression may be a key to the maintenance of the
cancerous anti-apoptotic state in these cells, and it suggests that
antagonism of SET may be an innovative method to treat B-cell
malignancies.
[0144] To analyze the effect of reducing the SET activity in
cancerous B-cells, we used lentivirus to introduce a shRNA
construct to silence SET production in the Raji cell line. When we
transduced cells with a SET-specific shRNA construct, the growth
rate measured using the MTT assay was significantly decreased
relative to a noncoding control shRNA construct (FIG. 18). The
SET-specific shRNA construct produced a reduction of the cellular
SET levels by approximately 50% relative to the .beta.-Actin
loading control protein in Western blots.
[0145] To determine if SET levels are indicative of more rapid CLL
disease progression, we used Western blotting to quantify SET
levels in cell extracts from 226 of the 435 patients in our
repository. We created a receiver operator curve to determine a
cutoff for high and not-high SET levels. Analysis of the time to
first treatment for each of these two groups showed a significant
difference--the group with highest SET levels had a reduced time to
first treatment relative to patients with lower SET levels (FIG.
19). This preliminary result supports our hypothesis that high CLL
SET levels render CLL more aggressive.
[0146] Overall, our data demonstrate that SET is overexpressed in
CLL and NHL relative to normal B-cells and that antagonism of SET
function by reducing its level in cells inhibits growth.
Furthermore, our results indicate that measurement of the
overexpression of SET from CLL cells or biopsied NHL tissue might
be a useful biomarker to predict which patients may require therapy
sooner and which patients may benefit from anti-SET therapy, such
as ApoE peptide dimers described herein.
Example 8
ApoE Peptide Dimers Reduce Cellular Concentrations of the
Anti-Apoptotic Mcl-1 Protein
[0147] The Myeloid Cell Leukemia-1 (Mcl-1) protein is a member of
the Bcl-2 family that regulates apoptosis. Members of this family
include the anti-apoptotic members Bcl-2, Bcl-XL and Mcl-1 while
pro-apoptotic members include BAD, BID, and BAX (Buggins and Pepper
(2010) Leuk Res, Vol. 34: 837-842). The anti-apoptotic Bcl-2 family
members associate with pro-apoptotic family members to inhibit
mitochondrial outer membrane permeabilization that releases
cytochrome-C and initiates the intrinsic apoptotic pathways. CLL
cells have been shown to overexpress both Bcl-2 and Mcl-1 (Buggins
and Pepper (2010) Leuk Res, Vol. 34: 837-842) and high levels of
Bcl-2 and Mcl-1 correlate with poor response to fludaribine therapy
in patients (Kitada et al. (1998) Blood, Vol. 91: 3379-8947). Mcl-1
overexpression was demonstrated to arise from B-cell receptor (BCR)
engagement and that stimulation of BCR may promote selection of
neoplastic B-cell clones (Stevenson and Caligaris-Cappio (2004)
Blood, Vol. 103: 4389-9548).
[0148] Recently, Peppers et al. measured Mcl-1, Bcl-2, and BAX
levels from 185 CLL patients and found that patients with high
Mcl-1 levels and low BAX levels, giving rise to a high Mcl-1/BAX
ratio had significantly shorter time-to-first-treatment and lower
overall survival than patients with lower Mcl-1 levels and ratios
between Mcl-1 and BAX (Pepper et al. (2008) Blood, Vol. 112:
3807-3817). In addition to CLL, Mcl-1 overexpression had been
reported in B-cell non-Hodgkin's lymphoma (NHL) patients and the
expression level correlated with tumor grade where higher
expression levels were found in high grade lymphomas (Cho-Vega et
al. (2004) Hum Pathol, Vol. 35: 1095-1100). Taken together, these
data have been used to suggest that Mcl-1 is the most significant
anti-apoptotic protein associated with B-cell malignancies (Gandhi
et al. (2008) Blood, Vol. 112: 3538-4051).
[0149] Given our results with destabilization of c-myc (see
Examples 5 and 6), we began our evaluation of the Mcl-1 protein by
analyzing the sequence of c-myc near the T58 and S62 sites that
regulates the ubiquitination and proteosomal degradation process
and compared this sequence to the Mcl-1 sequence. We observed that
there is distinct homology between the c-myc and Mcl-1 motifs with
S/T residues at 159 and 163 in Mcl-1 that correspond to the T58 and
S62 residues of c-myc as indicated by red arrows in FIG. 20A.
Furthermore, there is a proline residue at position 163 in Mcl-1
that corresponds to the proline-63 in c-myc (represented by the
purple chevron in FIG. 20A). Based on this overlap in sequences
between the regulatory region of c-myc and the sequence of Mcl-1,
we hypothesized that a similar regulatory mechanism exists for
degradation of Mcl-1. This mechanism would rely upon
phosphorylation of T163 followed by GSK3.beta.-mediated
phosphorylation of S159 prior to Pin1-mediated proline
isomerization at P164. After proline isomerization, T163 would be
dephosphorylated by PP2A and the pS159-Mcl-1 protein would then be
ubiquitinated and degraded by the proteosome (FIG. 20B).
Furthermore, we propose that the regulatory complex would utilize
Axin as a scaffolding protein in the same manner as c-myc (FIG.
20B).
[0150] To test this hypothesis, we performed immunoprecipitation of
Mcl-1 from human CLL cells and checked for co-immunoprecipitation
of Pin1, PP2A, Axin, and SET (FIG. 21). Pin1, Axin, and PP2A have
all been reported to co-immunoprecipitate with c-myc (Arnold et al.
(2009) EMBO J, Vol. 28: 500-512) and each of these proteins were
observed to co-immunoprecipitate with Mcl-1. It was also notable
that we observed SET in the Mcl-1 immunoprecipitated protein (FIG.
21). It was previously been reported that GSK3.beta.
co-immunoprecipitates with Mcl-1 (Ding et al., (2007) Cancer Res,
Vol. 67: 4564-4571). Based on the report from Ding et al., and our
observations, we have shown that all six of the proteins in the
regulatory complex proposed in FIG. 20 have been
co-immunoprecipitated with Mcl-1. These data also suggested that
antagonism of SET in this complex would increase PP2A activity
allowing for dephosphorylation of GSK3.beta., which was shown to be
inversely correlated with Mcl-1 stability, leading to
phosphorylation of S159, P164 isomerization, and pT163
dephosphorylation. Following T163 dephosphorylation, ubiquitination
and proteosomal degradation of Mcl-1 would reduce the Mcl-1 levels
in the cell and allow for activation of apoptosis.
[0151] We next sought to determine whether SET antagonism would
destabilize Mcl-1. Destabilization of Mcl-1 by treatment with SET
antagonists was evaluated by treating primary human CLL cells for
24 hr with the ApoE peptide dimer COG449 (a BMOE-linked dimer of
COG112; see Example 2) and evaluating the level of Mcl-1 in the
cells. We observed a significant dose-dependent decrease in the
Mcl-1 concentration relative to .beta.-Actin as a loading control
(FIG. 22). This effect indicates that treatment with COG449 should
induce apoptosis in CLL cells and we observed a dose dependent
increase in Annexin-V staining with an EC50 of approximately 110 nM
(data not shown). These results are consistent with the cytotoxic
effects of COG449 and other ApoE mimetic peptides on primary human
CLL cells observed in Example 2.
Example 9
ApoE Peptide Dimers Inhibit Growth of Tumor Cells In Vitro and In
Vivo
[0152] To evaluate the effect of COG449, an ApoE peptide dimer (see
Example 2), on cancer cell growth in vivo, we analyzed the effects
of COG449 treatment of the Ramos cell line of Burkitt's lymphoma, a
B-cell non-Hodgkin's lymphoma. Ramos cells are B-cells that
overexpress c-myc and form tumors throughout the body. After
determining that COG449 inhibited growth with an EC50 of 125 nM in
culture, 10.sup.7 Ramos cells were subcutaneously injected into the
left flank of female SCID mice (Schliemann et al. (2009) Blood,
Vol. 113: 2275-2283). Tumor growth was monitored daily by
palpitation and caliper measurement until tumors reached
approximately 150 mm.sup.3. At day 11, mice were assigned to two
groups so that initial tumor size was approximately equal between
groups.
[0153] Tumor measurements and treatment with COG449 (5 mg/kg,
subcutaneous injection into the right shoulder area) or a vehicle
control were performed by a technician who was blinded to the
treatment solutions. At day 19, the tumor volume reached the
predetermined size for termination of the experiment and animals
were photographed (FIG. 23) and euthanized. Tumors were dissected,
weighed, and segmented for pathological examination.
[0154] The measured tumor volumes and final tumor weights are
plotted in FIG. 24. Statistical analysis by one way ANOVA indicated
that tumor growth was significantly inhibited by COG449 and final
tumor mass was significantly lower in COG449 treated animals
(p<0.001). Disaggregated cells from one portion of the tumors
were analyzed by flow cytometry. This analysis showed that the
tumor cells were indeed B-cells. Importantly, the significant
reduction in tumor growth in the xenograft model demonstrates that
COG449 possesses acceptable pharmacological properties for in vivo
treatment of cancer.
Example 10
SET and CIP2A are Overexpressed in Triple-Negative Breast
Cancer
[0155] Breast cancer, the most common cancer affecting women, is a
heterogeneous disease comprised of several molecular subtypes (Tang
et al. (2009) Diagn Mol Pathol, Vol. 18(3): 125-132). Three main
subgroups have been defined based on the pattern of expression of
the hormone receptors estrogen (ER) and/or progesterone (PR), and
the HER2 receptor status: luminal tumors (ER+/HER2-), HER2
amplified tumors (HER2+), and triple negative breast cancer (TNBC,
ER-/PR-/HER2-). The identification of subgroups of breast cancer
based on biologic differences has allowed the development of
targeted therapeutic agents (Di Cosimo and Baselga (2010) Nat Rev
Clin Oncol, Vol. 7(3): 139-147). For example, hormone therapies are
effective in the treatment of hormone-receptor positive breast
cancers while HER2-targeted therapies are useful in the treatment
of HER2-positive tumors. TNBC, which lack expression of hormone
receptors and HER2, is therefore insensitive to such targeted
therapies. TNBC, and the molecularly related Basal-type breast
cancer accounts for 15-20% of all invasive breast cancer cases and
is characterized by its aggressive clinical behavior, high rates of
relapse following chemotherapy, and poor patient survival (Di
Cosimo and Baselga, 2010; Ray and Polite (2010) Cancer J, Vol.
16(1): 17-22). In addition, TNBC/Basal-like BC disproportionately
afflicts African-American women with incidence as high as 39%
(Carey et al. (2006) JAMA, Vol. 295(21): 2492-2502).
[0156] The most promising approach to treating women with this
devastating disease is the use of molecularly targeted drugs that
are specific for activated oncogenic pathways in the disease and
thus generally present less toxicity. Thus, understanding the
unique molecular changes associated with the development of TNBC is
necessary in order to develop new targeted therapies that will be
effective against this aggressive tumor type. However, little is
actually known about the underlying genetic changes associated with
the development of TNBC. Recent work has described aberrant
activation of various receptor tyrosine kinase signaling pathways
in TNBC, including the EGF, HGF, FAK, FGF, VEGF, and IGF-1
pathways, which can upregulate kinase cascades including
Raf/MEK/ERK and PI3K/AKT (Di Cosimo and Baselga, 2010; Turner et
al. (2009) Oncogene, Vol. 29(14): 2013-2023; Kurebayashi (2009)
Breast Cancer, Vol. 16(4): 275-280). Additionally, defects in
apoptotic pathways, including p53, Bcl2, and Mcl-1, are also common
in TNBC.
[0157] Several naturally occurring inhibitors of PP2A have been
identified, including SET (also known as I.sub.2PP2A) and Cellular
Inhibitor of PP2A (CIP2A). CIP2A has recently been described as an
important PP2A inhibitor in multiple cancer types (Khanna et al.
(2009) Cancer Inst, Vol. 101(11): 793-805). It is overexpressed in
39% of breast cancers and this is associated with clinical
aggressiveness (Come et al. (2009) Clin Cancer Res, Vol. 15(16):
5092-5100). CIP2A overexpression cooperates with Ras and c-Myc for
cell transformation, while its suppression inhibits tumor growth
(Sablina et al. (2008) Cancer Metastasis Rev, Vol. 27(2): 137-146).
CIP2A has been shown to interact with c-Myc and PP2A and display
c-Myc stabilization activity (Junttila et al. (2007) Cell, Vol.
130(1): 51-62). CIP2A appears to selectively inhibit PP2A targeted
to c-Myc (Westermarck and Hahn (2008) Trends Mol Med, Vol. 14(4):
152-160). The phosphoprotein SET is reported to have general PP2A
inhibitory activity (Li et al. (1995) Biochemistry, Vol. 34(6):
1988-1996). SET was originally identified as a fusion protein in
acute myelogenous leukemia and it is upregulated in multiple cancer
types (Westermarck and Hahn, 2008).
[0158] To determine whether these endogenous PP2A inhibitors are
expressed in triple negative breast cancers as well, we evaluated
SET and CIP2A expression by qRT-PCR in 13 cDNA samples from TNBC
tumors and found overexpression in 7 of 13 for SET and 12 of 13 for
CIP2A (FIGS. 25A and B). We also examined SET protein levels in
human triple negative breast cancer cell lines relative to an actin
control and found that SET is overexpressed in several breast
cancer cell lines (FIG. 26).
[0159] ApoE mimetic peptides bind to SET and activate PP2A (see
Example 5). We previously found that a peptide derived from amino
acids 133-149 (known as COG133) inhibited inflammation and the
production of cytokines and nitric oxide through reduced activation
of the NF-.kappa.B pathway (Singh et al. (2008) J Biol Chem, Vol.
283(24):16752-16761). In order to study the underlying mechanism of
this effect, COG133 was biotin labeled and used to affinity purify
protein binding partners. Interestingly, the primary binding
partner was the SET oncoprotein. The identification of SET, a
potent PP2A inhibitor (Li et al. (1996) Journal of Biological
Chemistry, Vol. 271(19): 11059-11062, as the binding partner of the
apoE-mimetic peptide suggested that the peptides would bind SET and
prevent it from binding to and inhibiting PP2A. COG449, a dimer
derivative of COG133 (see Example 2) with improved potency and
bioavailability, was also found to bind SET (data not shown).
[0160] Together, these studies emphasize a critical role for PP2A
suppression in breast cancer and support an innovative approach for
re-activating the PP2A tumor suppressor through therapies
antagonizing its inhibitors.
Example 11
SET Antagonism with an ApoE Peptide Dimer Reduces Phosphorylation
of a Cancer Related PP2A Target
[0161] To determine if antagonism of SET with COG449, a BMOE-linked
dimer of COG112 (see Example 2), reduced the phosphorylation of a
known PP2A target that is implicated in breast cancer, we evaluated
the status of eIF4E.
[0162] We previously demonstrated that antagonism of SET using
related COG peptides reduced the phosphorylation of Akt (see
Example 5), and the activity of NF.kappa.B (Singh et al., 2008). To
analyze the effect of PP2A activation on the mTOR pathway, we
analyzed the phosphorylation status of eIF4E and found that SET
antagonism by COG449 treatment resulted in decreased
phosphorylation of eIF4E (FIG. 27). Taken together, these data
suggest that targeting a single protein, SET, with an ApoE peptide
dimer results in downregulation of signaling pathways that are
implicated in proliferation and maintenance of an anti-apoptotic
state that is required for tumorigenic growth of triple negative
breast cancer.
Example 12
The ApoE Peptide Dimer COG449 Inhibits Growth of Breast Cancer
Tumor Cells In Vitro and In Vivo
[0163] Following demonstration of the activation of PP2A by the
BMOE-linked COG112 peptide dimer, COG449 (see Example 2), and the
inhibitory effects of COG449 on several PP2A targets, we explored
whether this peptide had any anti-tumor activity. To determine
whether COG449 might be effective in the treatment of human breast
cancers, we treated several breast cancer cell lines with COG449
and found that COG449 was cytotoxic to all cell lines, including
several triple negative breast cancer (TNBC) lines (MDA-231,
MDA-468, and HCC38) (FIG. 28).
[0164] To begin assessment of the potential of combination
therapies with COG449, we analyzed the effect of treating MDA-231
cells with sub-lethal doses of COG449 and the multi-kinase
inhibitor Sorafenib or the EGFR inhibitor Gefitinib at
concentrations below their ED50 doses. The combination of COG449
and Sorafenib or Gefitinib produced a robust cytotoxic effect that
was greater than the effect of either compound alone (FIG. 29). We
also evaluated the effects of COG449 in vivo using xenograft
experiments. To determine whether COG449 might be effective against
TNBC tumors in xenografts, immune compromised NOD/SCID gamma-chain
null (NSG) mice were injected with MDA-231 cells into their 4th
mammary glands. Once tumors became palpable at around 10 days,
tumors were treated by twice weekly subcutaneous injection of
COG449 at 10 mg/kg. At 28 days post-xenograft, daily intra-tumor
injection of COG449 was initiated and continued until sacrifice
(FIG. 30A). In order to use a more clinically relevant treatment
paradigm, MDA-231 xenografted mice were treated 3-times a week with
1 mg/kg COG449 by intravenous injection (FIG. 30B). No cytotoxic
effects were observed in mice in either of these studies. The
effective inhibition of tumor growth in these xenograft models
indicate that COG449 has suitable pharmacological properties for
the treatment of cancer. Furthermore, we have administered COG449
by intravenous infusion at doses of 10-15 mg/kg without any
observed adverse effects. When COG449 doses were increased to 20
mg/kg we observed mild edema in the front paws and lethargy as the
first adverse events associated with the administration of COG449.
Together, these data suggest that a wide safety window exists
between tumor suppressive doses and doses that elicit toxic
effects.
[0165] 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.
[0166] 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 and the materials for which they are cited are
specifically incorporated by reference in their entireties.
[0167] 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.
* * * * *