U.S. patent application number 15/328593 was filed with the patent office on 2018-03-29 for bipartite molecules and uses thereof in treating diseases associated with abnormal protein aggregates.
This patent application is currently assigned to Academia Sinica. The applicant listed for this patent is Academia Sinica. Invention is credited to Rita PY Chen, Yijuang Chern, Joseph Jen-Tse Huang, Yu-Song Jang, Te-Hsien Kung, Xiang-Me Lai, Tai-Yan Liao, Benjamin Pang-hsien Tu.
Application Number | 20180086801 15/328593 |
Document ID | / |
Family ID | 55163818 |
Filed Date | 2018-03-29 |
United States Patent
Application |
20180086801 |
Kind Code |
A9 |
Tu; Benjamin Pang-hsien ; et
al. |
March 29, 2018 |
BIPARTITE MOLECULES AND USES THEREOF IN TREATING DISEASES
ASSOCIATED WITH ABNORMAL PROTEIN AGGREGATES
Abstract
Bipartite molecules comprising a peptide affinity moiety and at
least one charged moiety and uses thereof in reducing formation of
abnormal protein aggregate and treating diseases associated with
such abnormal protein aggregate, including neurodegenerative
disease characterized by formation of protein aggregates.
Inventors: |
Tu; Benjamin Pang-hsien;
(Taipei, TW) ; Chen; Rita PY; (Taipei, TW)
; Huang; Joseph Jen-Tse; (Taipei City, TW) ;
Chern; Yijuang; (Taipei, TW) ; Jang; Yu-Song;
(Kaohsiung City, TW) ; Lai; Xiang-Me; (Chiayi
County, TW) ; Liao; Tai-Yan; (Tainan City, TW)
; Kung; Te-Hsien; (New Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Academia Sinica |
Taipei |
|
TW |
|
|
Assignee: |
Academia Sinica
Taipei
TW
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170226169 A1 |
August 10, 2017 |
|
|
Family ID: |
55163818 |
Appl. No.: |
15/328593 |
Filed: |
July 24, 2015 |
PCT Filed: |
July 24, 2015 |
PCT NO: |
PCT/US2015/041921 PCKC 00 |
371 Date: |
January 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62029030 |
Jul 25, 2014 |
|
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 38/00 20130101; A61P 25/28 20180101; C07K 14/4703 20130101;
C07K 14/4711 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Claims
1. A bipartite molecule comprising; (i) a peptide that binds an
abnormal protein aggregate associated with a disease or a component
of the abnormal protein aggregate; and (ii) at least one charged
moiety; wherein the peptide and the at least one charged moiety are
conjugated covalently.
2. The bipartite molecule of claim 1, wherein the abnormal protein
aggregate is associated with a neurodegenerative disease.
3. The bipartite molecule of claim 1, wherein the neurodegenerative
disease is Alzheimer's disease (AD), Huntington's disease (HD),
Parkinson's disease (PD), Dementia with Lewy body (DLB),
amyotrophic lateral sclerosis (ALS), Frontotemporal lobar
degeneration-TDP-43 proteinopathy, Frontotemporal lobar
degeneration-Tauopathy, Frontotemporal lobar degeneration with
ubiquitinated inclusions (FTLD-U), Pick's disease, Cortical basal
degeneration, Progressive supranuclear palsy, FTDP-17, or
Creutzfeldt-Jacob disease (CJD).
4. The bipartite molecule of claim 1, wherein the peptide comprises
a fragment of amyloid .beta. or TDP-43, which interferes with
protein aggregation of amyloid .beta. or TDP.
5. The bipartite molecule of claim 4, wherein the peptide comprises
the amino acid sequence GSNKGAIIGLM (SEQ ID NO: 1); or
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV (SEQ ID NO: 2).
6. The bipartite molecule of claim 1, wherein the peptide is a
polyglutamine (PolyQ) fragment.
7. The bipartite molecule of claim 6, wherein the PolyQ fragment
consists of about 5-20 glutamine residues.
8. The bipartite molecule of claim 1, wherein the charged moiety is
a polyarginine (PolyR) fragment or polyethylenimine (PEI).
9. The bipartite molecule of claim 1, wherein the molecule
comprises two charged moieties, one of which is a polyR fragment
and the other one is polyethylenimine (PEI).
10. The bipartite molecule of claim 1, wherein the molecule is
selected from the group consisting of: TABLE-US-00006 (SEQ ID NO:
3) RRRRRRRRGSNKGAIIGLM, (SEQ ID NO: 4) RRRRRRRRGSNKGAIIGLM-PEI,
(SEQ ID NO: 5) YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV-PEI, (SEQ ID
NO: 6) RRRRRRRRWDQQQQQQQQQQ, and (SEQ ID NO: 7)
RRRRRRRRWDQQQQQQQQQQQQQQQ.
11. A pharmaceutical composition, comprising a bipartite molecule
of claim 1 and a pharmaceutically acceptable carrier.
12-15. (canceled)
16. A method for treating a disease associated with an abnormal
protein aggregate, the method comprising administering to a subject
in need thereof an effective amount of the pharmaceutical
composition of claim 11.
17. The method of claim 16, wherein the subject is a human patient
having, suspected of having, or at risk for a neurodegenerative
disease.
18. The method of claim 17, wherein the neurodegenerative disease
is selected from the group consisting of Alzheimer's disease (AD),
Huntington's disease (HD), Parkinson's disease (PD), Dementia with
Lewy body (DLB), amyotrophic lateral sclerosis (ALS),
Frontotemporal lobar degeneration-TDP-43 proteinopathy,
Frontotemporal lobar degeneration-Tauopathy, Frontotemporal lobar
degeneration with ubiquitinated inclusions (FTLD-U), Pick's
disease, Cortical basal degeneration, Progressive supranuclear
palsy, FTDP-17, and Creutzfeldt-Jacob disease (CJD).
19. The method of claim 18, wherein the neurodegenerative disease
is Alzheimer's disease (AD) and the bipartite molecule comprises a
peptide that comprises the amino acid sequence of GSNKGAIIGLM (SEQ
ID NO: 1) or YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV (SEQ ID NO:
2).
20. The method of claim 16, wherein the bipartite molecule is:
TABLE-US-00007 (SEQ ID NO: 3) RRRRRRRRGSNKGAIIGLM, (SEQ ID NO: 4)
RRRRRRRRGSNKGAIIGLM-PEI, or (SEQ ID NO: 5)
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV-PEI..
21. The method of claim 16, wherein the neurodegenerative disease
is Huntington's disease and the bipartite molecule comprises a
peptide that comprises a polyQ fragment.
22. The method of claim 20, wherein the bipartite molecule is:
TABLE-US-00008 (SEQ ID NO: 6) RRRRRRRRWDQQQQQQQQQQ, or (SEQ ID NO:
7) RRRRRRRRWDQQQQQQQQQQQQQQQ.
23. The method of claim 16, wherein the bipartite molecule is
administered by an intranasal route.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This PCT application claims the benefit of U.S. Provisional
Application No. 62/029,030, filed Jul. 25, 2014, under 35 U. S. C.
.sctn.119. The entire content of the prior application is herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Neurodegenerative diseases, such as Alzheimer's disease
(AD), Huntington's disease (HD), synucleinopathy (e.g., Parkinson's
disease (PD) and dementia with Lewy bodies (DLB)), tauopathy (e.g.,
Pick's disease, progressive supranuclear palsy, corticobasal
degeneration, and frontotemporal dementia with Parkinsonism linked
to chromosome 17), TDP-43 proteinopathy (e.g., amyotrophic lateral
sclerosis (ALS) and frontotemporal lobar degeneration with
ubiquitinated inclusions (FTLD-U)), and Creutzfeldt-Jacob disease
(CJD), are characterized by an abnormal aggregation of pathogenic
proteins, leading to the formation of inclusion bodies (IBs).
Recently, the prion-like behavior of abnormal protein aggregates
has been established, showing that these IBs not only serve as a
diagnostic pathological marker, but also play an important role in
the pathogenesis of these diseases.
[0003] Because neurodegenerative diseases are usually age-related,
they primarily affect patients in mid- to late-life. It is expected
that their incidence will increase as the population ages. Since
the processes of many neurodegenerative diseases are not
well-understood, there is currently no cure for these diseases. It
is therefore of great importance to develop effective therapies for
neurodegenerative diseases.
SUMMARY OF THE INVENTION
[0004] The present disclosure is based, at least in part, on the
unexpected discovery that a number of bipartite molecules, e.g.,
polyR-A.beta.40-(25-35), PEI-V24P (10-40), and polyR-polyQ,
successfully decreased abnormal protein aggregation and
demonstrated beneficial therapeutic effects in murine models of
Huntington's disease and Alzheimer's disease (APP/PS1). More
specifically, polyR-polyQ was found to bind mutant huntingtin
(mHtt) protein aggregates, decrease mHtt-mediated toxicity, and
delay the onset and progress of neurologic dysfunctions observed in
the R6/2 murine model of Huntington's disease. Furthermore,
polyR-A.beta.40 (25-35) and PEI-V24P (10-40) were found to
ameliorate A.beta..sub.40 cytotoxicity in mouse neuroblastoma
cells, prevent memory deterioration, and decrease the level of
A.beta. plaque in the brains of the APP/PS1 transgenic murine model
of Alzheimer's disease. The bipartite molecules described herein
all contain an affinity moiety (e.g., the polyQ portion, the
A.beta.40 (25-35) portion, and the V24P (10-40) portion) capable of
binding to an abnormal protein aggregate or a component thereof
(e.g., a monomer of the aggregate) and a charged moiety (e.g., the
polyR portion or the PEI portion).
[0005] Accordingly, one aspect of the present disclosure relates to
a bipartite molecule comprising (i) a peptide affinity moiety that
binds to a disease-associated abnormal protein aggregate, or
component thereof; and (ii) at least one charged moiety. The
affinity moiety is linked (e.g., covalently) to the at least one
charged moiety. In some examples, the bipartite molecule described
herein may contain one charged moiety (e.g., a charged peptide
fragment), which may be conjugated to either the N-terminus or the
C-terminus of the peptide affinity moiety. In other examples, the
bipartite molecule described herein may contain two charged
moieties (e.g., the same or different), one being conjugated to the
N-terminus of the peptide affinity moiety and the other being
conjugated to the C-terminus of the peptide affinity moiety.
[0006] In some embodiments, the peptide affinity moiety binds an
abnormal protein aggregate or a component thereof that is
associated with a neurodegenerative disease (e.g., Alzheimer's
disease, Huntington's disease, synucleinopathy (e.g., Parkinson's
disease, and dementia with Lewy bodies), tauopathy (e.g., Pick's
disease, progressive supranuclear palsy, corticobasal degeneration,
and frontotemporal dementia with Parkinsonism linked to chromosome
17), TDP-43 proteinopathy (e.g., amyotrophic lateral sclerosis
(ALS), frontotemporal lobar degeneration-TDP-43 proteinopathy,
frontotemporal lobar degeneration-tauopathy, Pick's disease,
cortical basal degeneration, progressive supranuclear palsy,
FTDP-17 with ubiquitinated inclusions (FTLD-U)), or
Creutzfeldt-Jacob disease.
[0007] In some examples, the peptide moiety of the bipartite
molecule is a fragment of amyloid .beta. or TDP-43, which can
interfere with amyloid .beta. or TDP protein aggregation. The
fragment of amyloid .beta. may comprise the amino acid sequence of
GSNKGAIIGLM (SEQ ID NO: 1) or YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV
(SEQ ID NO: 2) (.sup.DP refers to the D-form of proline), which is
capable of interfering with amyloid .beta. protein aggregation.
[0008] In other examples, the peptide affinity moiety of the
bipartite molecule described herein can be a polyglutamine (PolyQ)
fragment (containing, e.g., 5-20 Q residues such as 10 Q or 15 Q
residues), which is capable of binding to the polyQ stretch of
huntingtin protein, thereby preventing the formation of abnormal
protein aggregates.
[0009] In any of the bipartite molecules described herein, the at
least one charged moiety of the bipartite molecule can be a
polyarginine (PolyR) fragment (containing, e.g., at least 5, 8, 10,
or 12 R residues) or polyethylenimine (PEI). In some embodiments,
the bipartite molecules may contain more than 2 charged moieties
(e.g., 2, 3, or more). Examples of the bipartite molecules as
described herein include, but are not limited to:
TABLE-US-00001 (SEQ ID NO: 3) RRRRRRRRGSNKGAIIGLM, (SEQ ID NO: 5)
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV-PEI, (SEQ ID NO: 6)
RRRRRRRRWDQQQQQQQQQQ, (SEQ ID NO: 7) RRRRRRRRWDQQQQQQQQQQQQQQQ, or
(SEQ ID NO: 4) RRRRRRRRGSNKGAIIGLM-PEI.
[0010] In another aspect, the present disclosure provides a
pharmaceutical composition comprising one or more bipartite
molecules as described herein and a pharmaceutically acceptable
carrier.
[0011] In yet another aspect, the present disclosure provides a
method for reducing the formation of an abnormal protein aggregate
associated with a disease (e.g., a neurodegenerative disease) or
treating such a disease, the method comprising administering (e.g.,
via an intranasal route) to a subject in need of the treatment an
effective amount of one or more bipartite molecules as described
herein. In some examples, the subject is a human patient having,
suspected of having, or at risk for, a neurodegenerative disease,
e.g., Alzheimer's disease, Huntington's disease, Parkinson's
disease, dementia with Lewy bodies, amyotrophic lateral sclerosis,
frontotemporal lobar degeneration-TDP-43 proteinopathy,
frontotemporal lobar degeneration-tauopathy, Pick's disease,
cortical basal degeneration, progressive supranuclear palsy,
FTDP-17, and/or Creutzfeldt-Jacob disease.
[0012] In some examples, the neurodegenerative disease is
Alzheimer's disease (AD) and the bipartite molecule for use in
treating AD comprises an affinity moiety having the amino acid
sequence GSNKGAIIGLM (SEQ ID NO: 1) or
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV (SEQ ID NO: 2). Such a
bipartite molecule can be RRRRRRRRGSNKGAIIGLM (SEQ ID NO: 3),
RRRRRRRRGSNKGAIIGLM-PEI (SEQ ID NO: 4), or
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV-PEI (SEQ ID NO: 5).
[0013] In another example, the neurodegenerative disease is
Huntington's disease and the bipartite molecule for use in treating
this disease comprise an affinity moiety having a PolyQ fragment.
Such a bipartite molecule can be RRRRRRRRWDQQQQQQQQQQ (SEQ ID NO:
6), or RRRRRRRRWDQQQQQQQQQQQQQQQ (SEQ ID NO: 7).
[0014] Also within the scope of the present disclosure are (a)
pharmaceutical compositions for use in interfering with abnormal
protein aggregation associated with a disease (e.g., preventing the
formation of, or disrupting existing, aggregates) or treating such
a disease, the pharmaceutical composition comprising a
pharmaceutically acceptable carrier and one or more bipartite
molecule as described herein; and (b) uses of any of the
pharmaceutical compositions or bipartite molecules for
manufacturing a medicament for treating a disease associated with
abnormal protein aggregation. Such a disease can be a
neurodegenerative diseases characterized by abnormal protein,
including, but not limited to, Alzheimer's disease, Huntington's
disease, Parkinson's disease, dementia with Lewy bodies,
amyotrophic lateral sclerosis, frontotemporal lobar
degeneration-TDP-43 proteinopathy, frontotemporal lobar
degeneration-tauopathy, Pick's disease, cortical basal
degeneration, progressive supranuclear palsy, FTDP-17, and/or
Creutzfeldt-Jacob disease.
[0015] The details of one or more embodiments of the disclosure are
set forth in the description below. Other features or advantages of
the present disclosure will be apparent from the following drawings
and detailed description of several embodiments, and also from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. Schematic illustration of the modular design of the
therapeutic peptide for HD.
[0017] FIG. 2. Stability of the therapeutic peptides determined by
HPLC. A: diagrams showing the retention time of the tested peptides
from day 0 to day 28. B: charts showing the amount of the tested
peptides at day 0 and day 28. Note the 8R5Q and 8R10Q remained
stable in water for 28 days, but the soluble 8R15Q and 8R20Q
gradually decreased with time.
[0018] FIG. 3. Diagrams showing the co-localization of tested
bipartite peptides with the 109QmHtt aggregates. Panel A: Structure
of TAMRA-labeled 8R10Q used in this study. Panel B: Epifluorescence
micrographs of Neuro2a cells expressing 109QmHtt treated with
TAMRA-8R10Q (8R10Q) at various time points. Note the
co-localization of 8R10Q and 109QmHttGFP aggregates (GFP) from 8-24
hours (arrows). Scale bar: 10 .mu.m. Panel C: TIRF micrographs of
the Neuro2a expressing 109QmHtt treated with water or peptides as
indicated at various time points. Note the decrease in aggregate
size in 8R10Q and 8R15Q-treated cells. Panel D: Quantitation of the
size of individual aggregates in Neuro2a at 8 hours (left) and 24
hours (right) after peptide treatment. The size was significantly
decreased by 8R10Q or 8R15Q peptide at both time points. Panel E:
Quantitation of the number of aggregates in 20 Neuro2a cells at 12
hours (left) and 24 hours (right) after peptide treatment. Both the
8R10Q and 8R15Q increased the number of aggregates. Statistics
performed with one way ANOVA. **p<0.01; ***p<0.001.
[0019] FIG. 4. Testing the ability of therapeutic peptides to
decrease mHtt aggregation in Neuro2a cells overexpressing mHtt.
Panel A: Filter trap assays and corresponding quantitation of cells
(panel B) treated as indicated at 8 hours (T1), 24 hours (T2), or
both (T1+2) after transfection with the 109QmHtt construct. Both
8R10Q and 8R15Q significantly decreased mHtt aggregates when
treated at T1 or both time points, but not at T2 alone. Panel C:
Western blots and quantitation (panel D) of RIPA-soluble (sol)
and--insoluble (ins) fractions of Neuro2a cells overexpressing
25QHtt or 109QmHtt and treated as indicated. The levels of 109QmHtt
in both the soluble and insoluble fractions were significantly
decreased by 8R10Q. Panel E: Western blot of the 109QmHtt in cells
treated as indicated. Note that MG132 blocked the effect of 8R10Q
peptide with respect to decreasing insoluble 109QmHtt. Panel F:
Quantitation of ratio of 109QmHtt in cells treated MG132 (+) over
DMSO (-) in relationship to peptides. Statistical analysis
conducted with one way ANOVA, *<0.05, **<0.01, ***<0.001,
ns: not significant.
[0020] FIG. 5. Therapeutic effect of 8R10Q and 8R15Q in
109QmHtt-expressing cells. Panel A: Cell viability assay by MTT in
Neuro2a cells expressing 25QHtt (left) or 109QmHtt (right) treated
with H.sub.2O.sub.2 at 50 .mu.M and the indicated peptide. Panel B:
Cell growth curve of Neuro2a cells expressing 25QHtt or 109QmHtt
treated with indicated peptide. The cells in right subpanel were
further treated with H.sub.2O.sub.2 at 12.51. .mu.M 16 hours after
transfection. 109QmHtt cells growth was slower than that of the
25QHtt cells. 8R10Q treatment rescued the slow growth of Neuro2a
cells by 109QmHtt with or without H.sub.2O.sub.2. Panel C:
Micrographs of the phase contrast and fluorescent images of
retinoic acid-differentiated Neu2a cells expressing GFP-25QHtt or
GFP-109QmHtt (GFP) treated with the indicated peptide. Note the
neurites of the differentiated cells. Panel D: Quantitation of the
percentage of differentiated cells with neurites. Fewer
109QmHtt-expressing cells had neurites vs. 25QHtt-expressing cells.
8R10Q treatment could significantly rescue the defect in neurite
outgrowth. The experiments were conducted in triplicate and
repeated twice. Statistical analysis was performed with one way
ANOVA, *p<0.05; **p<0.01; ***p<0.001, ns: not
significant.
[0021] FIG. 6. 8R10Q ameliorated functional deterioration of R6/2
transgenic mice. Panel A: Longitudinal rotarod performance of wild
type (WT) and R6/2 mice treated with PBS or 8R10Q peptide. Note the
significant delay in motor deterioration of R6/2 mice treated with
8R10Q from 11 weeks of age. Panel B: T maze test of WT and R6/2
mice at 13 weeks of age. The 8R10Q significantly rescued the memory
deficit in R6/2 mice. Panel C: The curves of blood sugar in serum
of WT and R6/2 mice treated as indicated. Note 8R10Q treatment
significantly decreased the rise in blood sugar in R6/2 mice. Panel
D: Lifespan of WT and R6/2 mice treated with PBS vs. 8R10Q. 100% of
R6/2 mice died before 16 weeks of age. 8R10Q significantly extended
the lifespan of R6/2 mice. WT mice N=6/group; R6/2 mice N=10/group;
statistical analysis performed with two way ANOVA (panels A and C),
one way ANOVA (panel B), *p<0.05; **p<0.01; ***p<0.001,
ns: not significant, and log-rank (Mantel-Cox) Test (D),
***p<0.0001.
[0022] FIG. 7. Amelioration of neuronal damage of 13 week-old R6/2
mice by 8R10Q peptide. Panel A: Micrographs of the Nissl stain
sections of cortex (CTX) of WT and R6/2 mice. The cortex and its
thickness are highlighted by the dashed lines and red solid lines,
respectively. Panel B: Quantitation of the cortical thickness. Note
the decrease in cortical thickness in R6/2 mice which was reversed
by the 8R10Q. Panels C and E: Micrographs (panel D) and (panel F)
show the corresponding quantitation of the Nissl stained sections
of the cortex and striatum, respectively. Note the decrease in the
number of neurons, which was reversed by 8R10Q. N=3/group, each bar
represents the average of 5 sections. Statistical analysis
performed with one way ANOVA, *p<0.05; ***p<0.001, ns: not
significant.
[0023] FIG. 8. Decrease in mHtt aggregates and glial pathology in
13-week-old R6/2 mice by 8R10Q peptide treatment. Panel A:
Micrographs and corresponding quantitation (panel B) of the
immunostained sections of cortex (CTX) and striatum with EM48
antibody. The brown dots show the mHtt aggregates. The left
subpanels of panel B show the quantitative data with respect to the
total area occupied by the aggregates, and the right, the intensity
of individual aggregates. Panel C: Photographs of the
immunofluorescent-stained sections of the cortex of R6/2 mice with
anti-GFAP antibody. Panel D: Quantitation of the GFAP intensity
showed a significant decrease in 8R10Q treated mice. Panel E:
Micrographs of the immunostained sections of the cortex (CTX) and
striatum of WT and R6/2 mice with anti-Ibal antibody. Panel F:
Quantitation of lbal intensity revealed an obvious increase in lbal
immunoreactivity in R6/2 mice which was reversed by the 8R10Q.
N=3/group. Each bar represented an average of 15 sections/mouse
group. Statistics were performed with Student's t test for panels B
and D, but with one way ANOVA, *p<0.05; **p<0.01;
***p<0.001, ns: not significant.
[0024] FIG. 9. Changes in the levels of Htt mRNA by quantitative
RT-PCR in Neuro2a transfected with 25QHtt or 109QmHtt and treated
with the indicated peptide. Statistical analysis was performed with
one way ANOVA, ***p<0.001, ns: not significant
[0025] FIG. 10. Effect of the designed bipartite peptides on
inhibition of fibrillization. The peptides were dissolved in 20 mM
sodium phosphate buffer with 150 mM KCl (pH 7) and incubated at
25.degree. C. CD spectra and TEM images were taken for A.beta.40
(panels A and D), R.sub.8A.beta.(25-35) (panels B and E),
.sup.DR.sub.8-A.beta. (25-35) (panels C and F), and the 1:1 mixture
of A.beta.40 with R.sub.8-A.beta. (25-35) (panels G and J) or
.sup.DR.sub.8-A.beta. (25-35) (panels H and K). The CD spectra were
recorded at the indicated incubation time. The TEM images were
taken after prolonged incubation. Panel I: The time course of
amyloidogenesis of A.beta.40 with and without the designed
bipartite peptides.
[0026] FIG. 11. Cell viability measurement by MTT assays. Panel A:
Neuro2a cells treated with DMSO (control), A.beta.40,
R.sub.8-A.beta.(25-35) or .sup.DR.sub.8-A.beta.(25-35). Panel B:
Neuro2a cells treated with DMSO (control), A.beta.40, and A.beta.40
with equal molar R.sub.8-A.beta.(25-35),
.sup.DR.sub.8-A.beta.(25-35), or A.beta.(25-35). Each bar was
generated by a triplicate experiment; the study was repeated 3
times. The statistics were performed with one way ANOVA corrected
with Fisher's LSD test. ***:p<0.001; ****p<0.0001; ns, not
significant.
[0027] FIG. 12. R.sub.8-A.beta.(25-35)-PEI improved memory as
measured by the Morris water maze assay. Panel A: Plot of the
escape latency period of wild type (WT) and APP.times.PS1
transgenic (Tg) mice of 8 months of age treated with either PEI or
R.sub.8-A.beta.(25-35) peptide from the age of 4 months to 8
months. Note a significant shortening of the latency in Tg mice by
the therapeutic peptide. Panel B: Percentage of time of WT or Tg
mice treated as indicated spent in swimming in the target quadrant
where the hidden platform used to be. The times of the indicated
mice crossing the target quadrant. R.sub.8-A.beta.(25-35)-PEI
peptide increased the time of Tg mice in the target quadrant. Panel
C: The percentage of time of the WT or Tg mice as indicated
crossing the target quadrant. Ten mice per group were used in this
study. The statistics were performed with two way ANOVA with
Fisher's LSD posthoc analysis. N=10/group *<0.05; ***<0.0005;
****<0.0001
[0028] FIG. 13. ELISA assay for the level of A.beta.40 and
A.beta.42 in the cortex (panel A) and hippocampus (panel B) in the
8 month-old Tg mice treated with PEI or R.sub.8-A.beta.(25-35)-PEI.
The therapeutic peptide significantly reduced the level of both
A.beta.40 and A.beta.42 in both regions. N=3/group; *:p<0.05;
**:p<0.01; ***:p<0.001; ****:p<0.0001; ns: not
significant. Statistics by Student's t-test.
[0029] FIG. 14. Effect of intranasally delivered
R.sub.8-A.beta.(25-35)-PEI on APP.times.PS1 mice on A.beta.
clearance after 4 months treatment. Wild type (WT) and
APP.times.PS1 transgenic (Tg) mice were treated with either PEI or
R.sub.8-A.beta.(25-35)-PEI from the age of 4 months to 8 months.
Panels A-E show ThS-staining of the brain slices.
[0030] FIG. 15. Assays for the level of level of IL-6 and
IL-1.beta. in the cortex (N=3/group) in the 8 month-old Tg mice
treated with PEI or R.sub.8-A.beta.25-35-PEI peptide from the age
of 4 months to 8 months. As shown, the therapeutic peptide
signficantly decreased the level of interleukin IL-6 and IL-1.beta.
in the cortex. N=3/group; **:P<0.01. Statistics by Student's
t-test.
[0031] FIG. 16. MicroPET amyloid images of 12 month-old wild type
(WT) control mice and transgenic (Tg) mice treated with PEI or
R.sub.8-A.beta.(25-35)-PEI peptide for 8 months. Panel A:
Representative PET images of the Tg mouse brains co-registered with
a mouse T2-weighted MRI brain template. As shown, the brain of the
PEI-treated Tg mouse had much higher amyloid signals at the cortex
(CT), hippocampus (HP), and amygdala (AMY) compared with that of
the WT mouse. R.sub.8-A.beta.(25-35)-PEI peptide treatment reduced
the amyloid signal of the Tg mouse. Panel B: Quantitation of the
signal of .sup.11C-labled Pittsburg compound B (PIB) in regions as
indicated. N=6 per group, **<0.001; ***<0.0005, statistics
were conducted with the Student's t test.
[0032] FIG. 17. ELISA assay of the level of total and insoluble
A.beta.40 and A.beta.42 in the cortex and hippocampus in the 12
month-old Tg mice treated with PEI or R.sub.8-A.beta.(25-35)-PEI
for 8 months. ELISA assay of total A.beta..sub.40 and
A.beta..sub.42 in the cortex (panel A) and hippocampus (panel B)
and insoluble A.beta..sub.40 and A.beta..sub.42 in the cortex
(panel C) and hippocampus (panel D). (N=5 per group). Statistics
were conducted with the Student's t test. **:p<0.01;
***:p<0.001; ns, not significant.
[0033] FIG. 18. Structural studies on V24P(1-28) and V24P(10-40).
The peptides at concentrations of 30 or 60 .mu.M were incubated at
25.degree. C. for the indicated times (0-12 days), then the CD
spectra (panels A and C) and fluorescence spectra after binding ThT
(panels B and D) were recorded. Panel E: Electron microscopy images
of 60 .mu.M V24P(1-28) and V24P(10-40) after incubation at
25.degree. C. for about 1 month.
[0034] FIG. 19. Structural studies on V24P(13-36), V24P(16-33), and
V24P(19-30). The peptides were dissolved at concentrations of 30,
60, or 90 .mu.M and their CD spectra (panel A) and fluorescence
spectra after binding ThT (panel B) were immediately recorded.
[0035] FIG. 20. Cell viability assay. The viability of mouse N2a
cells incubated with the indicated peptide(s) was measured using
the MTT assay. Panel A: Comparison of the viability of A.beta.40
and the designed peptides containing the V24.fwdarw..sup.DP
mutation. Panel B: Comparison of the viability of 30 .mu.M
A.beta.40 alone or together with 30 .mu.M designed peptide. The
standard deviations are shown as bars (compared with A.beta.40,
p<0.01 for all the other data by Student's t test). A: Cortex.
B: Hippocampus.
[0036] FIG. 21. Amyloid formation of hamster prion peptide
PrP(108-144) in the absence (panel A) or presence of V24P(10-40)
(panel B). Solutions of 50 .mu.M PrP(108-144) with (panel B) or
without (panel A) 50 .mu.M V24P(10-40) were incubated in 20 mM
NaOAc, pH 3.7/140 mM NaCl, at room temperature for different times.
Samples were then removed and amyloid formation was measured using
the ThT binding assay.
[0037] FIG. 22. Effect of V24P(10-40)-PEI on A.beta. peptide
levels. APP/PS1 mice were treated from the age of 4 months to 8
months with PEI (control) or V24P(10-40)-PEI as described in the
Examples. The levels of A.beta.40 and A.beta.42 levels in the
hippocampus and cortex were measured by ELISA. The data were
presented as the mean .+-.standard deviation for 3 mice per group
(*p<0.05; **p<0.01; ***p<0.001, Student's t test).
[0038] FIG. 23. V24P(10-40)-PEI decreases A.beta. plaque
accumulation in APP/PS1 mice. Panel A: Representative microPET
images of APP/PS1 mice taken after treatment with either PEI
(control) or V24P(10-40)-PEI for 8 months. A microPET image of a
wild type mouse (WT) is included for comparison. Panel B:
Quantitative analysis of [.sup.11C]PiB uptake in the cortex,
hippocampus, amydala, and olfactory bulb. The data are presented as
the mean .+-.standard deviation for 6 mice per group (*p<0.05;
**p<0.01; ***p<0.001, Student's t test).
DETAILED DESCRIPTION OF THE INVENTION
[0039] Neurodegenerative diseases are a group of neurological
disorders characterized by a gradual loss of neurons in association
with the formation of hallmark inclusion bodies (IBs), which
results in dysfunction of the nervous system and the eventual
demise of patients. Takalo et al., Am J Neuro Degener Dis (2013)
2:1-14. Neurodegenerative diseases such as Huntington's disease
(HD), Alzheimer's disease (AD), Parkinson's disease (PD), and
others are considered as diseases mediated by protein misfolding
because of the formation of hallmark inclusion bodies (IBs). Yates,
Nature Reviews (2012) 11: 352-353.
[0040] For instance, HD is an autosomal dominantly-inherited
neurodegenerative disease caused by an expansion of the CAG
trinucleotide repeats in the gene Huntingtin (HTT), which encodes a
mutant Htt (mHtt) protein containing a prolonged polyglutamine
(polyQ) stretch. Zheng et al., Progress in Mol Biol and
Translational Sci (2012) 107:189-214. Although having a seemingly
simple etiology, HD in fact, is very complex in its pathogenesis
(Li et al., Mol Neurodengener (2006) 1:19) which involves a large
number of important biological processes and signaling pathways
that have gone awry. Munoz-Sanjuan et al., J Clin Invest (2011)
121:476-83. Currently, no disease-modifying treatment for HD or
other neurodegenerative diseases is available. Thus, finding an
effective therapeutic regimen is a focus of the neurodegenerative
disease community. The aberrant pathways or processes may serve as
valuable therapeutic targets (Munoz-Sanjuan et al., J Clin Invest
(2011) 121:476-83); however, attempts to target these processes
would produce undesired side effects as illustrated in the
Semagacestat clinical trial for AD. Doody et al., N Eng J Med
(2013) 369:341-50.
[0041] Alzheimer's disease (AD), pathologically defined by the
amyloid plaques and neurofibrillary tangles (Nelson et al., J
Neuropathol Exp Neurol (2012) 71:362-81), is the most common
neurodegenerative disease that causes dementia across multiple
cognitive domains, and its incidence increases exponentially among
people >65 years of age. Reitz et al., Nat Rev Neurol (2011)
7:137-52. Despite the remarkable scientific advancements and the
huge amount of resources invested in drug development based on
these discoveries, no effective disease-modifying therapy is
currently available for AD. Castellani et al., Biochem Pharmacol
(2014) 88:671-6. Thus, it is one of the major unmet medical needs
worldwide.
[0042] Although the etiology of AD remains unclear, alterations in
multiple processes have been proposed as important causes and/or
contributors, including the amyloid cascade hypothesis. Yamashima
Prog Neurobiol (2013) 105:1-23; Swerdlow et al., Biochim Biophys
Acta (2014) 1842:1219-31; Erickson et al., J Cereb Blood Flow Metab
(2013) 33:1500-13; Clavaguera et al., Neuropharmacol (2014) 76 Pt
A:9-15; Castello et al., Ageing Res Rev (2013) 12:282-8; Sutherland
et al., Redox Rep (2013) 18:134-41; Tiiman et al., Neurochem Int
(2013) 62:367-78; Puglielli Neurobiol Aging (2008) 29:795-811;
Hardy, J Alzheimer's Dis (2006) 9:151-3; and Checler et al., J
Neurochem (2012) 120 Suppl 1:iii-iv. The amyloid cascade hypothesis
proposes that "amyloid .beta.-protein" (A.beta.), a peptide of
different lengths (39-43 amino acids) with variations and
modifications at both termini, plays a central and initiative role
in AD pathogenesis and/or progression. Hardy et al., Science (1992)
256:184-5; Hardy et al., Science (2002) 297:353-6; Tanzi et al.,
Cell (2005) 120:545-55. A.beta. is derived from amyloid precursor
protein (APP). During normal or non-amyloidogenic catabolism, APP
is cleaved by .alpha.- and .gamma.-secretases, while, in
amyloidogenic catabolism, it is cleaved by .beta.- and
.gamma.-secretases. The difference in length of the A.beta. peptide
is partially due to the variance in the cutting sites of
.gamma.-secretase. A.beta. peptides tend to self-aggregate into
amyloid fibrils and more cytotoxic oligomers. Walsh et al., J
Neurochem (2007) 101:1172-84. A.beta.40 and A.beta.42 are two main
species of A.beta. peptides recovered from amyloid plaques with the
latter being more prone to aggregate and cytotoxic.
[0043] Amyloid plaques belong to a large family of inclusion bodies
(IBs), which are characteristics of a variety of neurodegenerative
diseases, including Parkinson's disease (PD), Huntington's disease
(HD), and amyotrophic lateral sclerosis (ALS). In spite of the
differences in the constituent proteins and complexity of the
assembly mechanism, the co-existence of different neurodegenerative
diseases and associated IBs is well-recognized in a substantial
subset of patient cohorts. Keith-Rokosh Can J Neurol Sci (2008)
35:602-8; Tada et al., Acta Neuropathol (2012) 124:749-60; Schwab
et al., J Neuropathol Exp Neurol (2008) 67:1159-65; Amador-Ortiz et
al., Ann Neurol (2007) 61:435-45; Arai et al., Acta Neuropathol
(2009) 117:125-36; Szpak et al., Folia Neuropathol (2001) 39:63-71.
In addition, abundant evidence demonstrates that misfolded proteins
from different diseases can cross-seed each other to co-aggregate.
Jucker et al., Ann Neurol (2011) 70:532-40; Ma et al., J Mol Biol
(2012) 421:172-84; Guo et al., Cell (2013) 154:103-17; Waxman et
al., J Neurosci (2011) 31:7601-18; Vitrenko et al., J Biol Chem
(2007) 282:1779-87; Wasmer et al., J Mol Biol (2010) 402:311-25;
Yan et al., Am J Pathol (2007) 171:172-80. These findings suggest
that the formation of IBs may be governed, at least in part, by a
common set of thermodynamic principles (Jucker et al., Ann Neurol
(2011) 70:532-40), and interference with the association pathway
toward the most thermodynamically stable oligomers or fibrils sheds
light on amyloidosis therapy.
[0044] Conceptually, reducing the toxic species (the abnormal
aggregates) formed by the misfolded protein or its derivative may
be a practical and safe therapeutic strategy. Mielcarek et al.,
PLoS Biol (2013) 11:e1001717; Appl et al., Drug Discovery Today
(2012) 17:1217-23. In HD, the prolonged polyQ confers an aberrant
propensity for self-aggregation to form neurotoxic species to the
mHtt protein. Oligomer or large fibrillary aggregates in nuclei and
processes of neurons and glia are linked with HD pathogenesis.
Olshina et al., J Biol Chem (2010) 285:21807-16; Ren et al., Nat
Cell Biol (2009) 11:219-25; Hoffner et al., Prion (2007) 1:26-31;
Marcellin et al., PLoS One (2012) 7:e44457; and Legleiter et al., J
Biol Chem (2010) 285:14777-90. Indeed, this approach has been
tested in several neurodegenerative disease models including HD
(Kordasiewicz et al., Neuron (2012) 74:1031-44; Sontag et al., J
Neurosci (2012) 32:11109-19) and AD (Aisen et al., Current
Alzheimer Research (2007) 4:473-78; Frisardi et al., Current
Alzheimer Research (2010) 7:40-55) with encouraging results.
[0045] Since the formation of toxic misfolded proteins/derivatives
is governed by a common set of thermophysical laws across different
diseases, a common strategy as described herein be used to develop
therapeutic tools by reducing the formation of the misfolded
species in these neurodegenerative diseases.
[0046] Accordingly, described herein is a rational design of
therapeutic peptides that are expected to reverse the pathogenic
process of IB formation in a neurodegenerative disease as those
described herein. Here, novel bipartite molecules containing at
least one affinity module and at least one charged module were
designed and their efficacy was demonstrated using cellular,
APP/PS1 AD models, and R6/2 HD models. The bipartite molecules
would reduce toxic abnormal protein aggregates across various
neurodegenerative diseases. The rational design is built on a
principle of modular assembly of an affinity portion (e.g., a
peptide affinity moiety) and at least one charged moiety, e.g.,
positively charged or negatively charged. Without being bound by
theory, the affinity moiety would facilitate binding of such
therapeutic molecules (bipartite molecules) to a protein component
of an abnormal protein aggregate or a monomer of the abnormal
protein aggregate and the charged portion(s) would prevent or
reduce the formation of the abnormal protein aggregate by, e.g.,
the repulsion force of charges (FIG. 1).
[0047] The approach described herein possesses several unique
features and advantages as compared with current therapeutic
approaches for neurodegenerative diseases. Some examples are
provided below. First, the affinity moiety, which may be taken from
the pathogenic peptide/protein forming the abnormal aggregate not
only significantly reduces laborious work finding and optimizing
suitable peptide sequences, but also guarantees high affinity with
IBs through its self-aggregating property. Second, the multiple
charges in the charged moiety (e.g., a polyR fragment) render the
bipartite molecules described herein (a) soluble in an aqueous
environment, thereby simplifying the synthesis and delivery
processes and (b) cell-penetrable (Mitchell et al., J Pept Res
(2000) 56:318-25), making them suitable for inhibiting both
extracellular and intracellular IB formation. Further, the charged
moiety in the bipartite molecules can prevent or reduce IB
formation by charge repulsion after binding of the bipartite
molecules to the pathogenic peptide/protein or protein aggregate.
Third, the combination of the charged moiety with the affinity
moiety provides great feasibility and flexibility in applying this
design across different IB-containing diseases.
[0048] Accordingly, the present disclosure provides bipartite
molecules comprising an affinity moiety that binds an abnormal
protein aggregate associated with a disease or a component thereof
(e.g., a protein monomer that forms the abnormal protein aggregate)
and at least one charged moiety, and uses of such bipartite
molecules in preventing or reducing the formation of the abnormal
protein aggregates and/or in treating diseases (e.g.,
neurodegenerative diseases) involving such abnormal protein
aggregates.
I. Bipartite Molecules
[0049] The bipartite molecule described herein comprise (i) a
peptide affinity moiety, which is capable of binding to an abnormal
protein aggregate, or a component of that abnormal aggregate, and
(ii) at least one charged moiety (e.g., positively charged or
negatively charged), which facilitates the bipartite molecule to
cross cell membranes and prevent or decrease formation of the
abnormal protein aggregation associated with a disease, such as a
neurodegenerative disease as those described herein.
(i) Peptide affinity moiety
[0050] The peptide affinity moiety of the bipartite molecule can be
any peptide-based (e.g., comprising a peptide) molecule capable of
binding to a targeted abnormal protein aggregates or a protein
component thereof (e.g., a monomer capable of forming the abnormal
protein aggregates or a fragment thereof which is involved in the
aggregate formation). The peptide affinity moiety may contain a
peptide having up to 100 (e.g., 80, 60, 50, 40, 30, 20, or 10)
amino acid residues, which can contain either naturally-occurring
amino acids or modified ones such as D-amino acids, or a
combination thereof. In some examples, the peptide affinity moiety
may contain a peptide having 5-10, 5-20, 5-30, 10-15, 10-20, 10-30,
or 20-30 amino acid residues. The peptide affinity moiety may be
methylated or acetylated at the N-terminus, amidated at the
C-terminus, or both to enhance stability.
[0051] Disease-associated abnormal protein aggregates and the
corresponding protein constituents are known in the art. Some
examples are provided in Table 1 below:
TABLE-US-00002 TABLE 1 Exemplary Neurodegenerative Diseases and
Abnormal Protein Aggregates Associated with such Diseases Exemplary
Diseases involving abnormal protein Aggregate Associated protein
aggregates type components Alzheimer's disease [AD] amyloid amyloid
precursor plaques, protein, amyloid .beta., presenilin
neurofibrillary 1&2, tau, neurofilament tangles protein, alpha
B-crystallin, transthyretin Huntington's disease [HD] inclusion
uintingtin protein, bodies expanded polyglutamine tract in
huntingtin protein, alpha B- crystallin Parkinson's disease [PD]
Lewy alpha-synuclein, bodies ubiquitin, neurofilament protein,
alpha B-crystallin Dementia with Lewy body Lewy alpha-synuclein,
[DLB] bodies ubiquitin Amyotrophic lateral sclerosis inclusion
SOD-1, TAR DNA [ALS] bodies binding protein (TDP-43, or TARDBP),
FUS, ubiquitin, neurofilament protein Frontotemporal lobar
inclusion ubiquitin, TDP-43 degeneration [FTLD]-TDP-43 bodies
proteinopathy Frontotemporal lobar Pick tau, fused in sarcoma
degeneration [FTLD]-Tauopathy bodies (FUS), alpha B-crystallin
Cortical basal degeneration Astroglial tau [CBD] inclusions
Progressive supranuclear palsy neurofibrillary tau, Tau H1 halotype
[PSP] tangles, Lewy bodies Frontotemporal dementia and Lewy
microtubule-associated parkinsonism linked to chromosome 17 bodies
protein tau [MAPT], tau [FTDP-17] Creutzfeldt-Jacob disease
Aggregates prion protein (PRNP), [CJD] and prion in the Scrapie
form spongiform (PrP.sup.SC) change
[0052] In some embodiments, the peptide affinity moiety may contain
an amino acid sequence derived either from a disease protein, which
forms the abnormal protein aggregate or from the region of other
proteins that interact with the disease protein or the abnormal
protein aggregate formed thereby. In some embodiments, the peptide
affinity moiety comprises a fragment of the disease protein (e.g.,
any of those listed in Table 1 above) that is involved in
self-aggregation (formation of the abnormal protein
aggregates).
[0053] In some examples, the peptide affinity moiety comprises a
polyQ fragment. Such a peptide affinity moiety can bind to and
reduce/disrupt formation of protein aggregates involving polyQ, for
example, the aggregation of mHtt involved in HD. The polyQ moiety
may comprise at least 2, at least 5, at least 10, at least 15, at
least 20 at least 25, at least 30, or at least 50 glutamine (Q)
amino acid residues (e.g., 5-10, 5-15, 5-20, 5-30, or 10-20 Q
residues). In some embodiments the peptide affinity moiety is 5Q,
10Q, 15Q, or 20Q.
[0054] The protein affinity moiety may contain a fragment derived
from a wild-type disease protein (as those listed in Tables 1, 3,
and 4 and described herein) or from a mutant form or modified form
of the disease protein, particularly those involved in disease
pathogenesis. The modified form of the protein may include post
translational modifications such as phosphorylation. For example,
neurofibrillary tangles (NFTs) may be formed by
hyperphosphorylation of the microtubule-associated protein Tau.
Accordingly, the peptide affinity moiety may contain the
hyperphosphorylated form, or a portion of the hyperphosphorylated
form of Tau. Hyperphosphorylation may be achieved by employing the
use of various known kinases or by phosphomimetic amino acid
substitutions that mimic a phosphorylated protein. For example,
aspartic acid (D) is chemically similar to phospho-serine.
Therefore, when aspartic acid replaces a serine, it is a
phosphomimetic of phospho-serine.
[0055] In some embodiments, the peptide affinity moiety may contain
a fragment derived from amyloid .beta.-protein, SOD1, Tau, TDP-43,
.alpha.-synuclein, ubiquitin, neurofilament protein, alpha B
crystalline, PrP.sup.SC, or transthyretin. Such a fragment may
involve in formation of protein aggregate containing the disease
protein. For example, fragment A.beta.25-35 from amyloid
.beta.-protein, which may contain an N-methylated Gly33, can
interact with amyloid plaques. Hughes et al., J Biol Chem (2000)
275:25109-115. In another example, an A.beta.40 mutant peptide,
V24P, with the V24 replaced by D-form proline (.sup.DP), remains as
random coils in buffer and forms amorphous aggregates at a high
peptide concentration. Chang et al., J Mol Biol (2009) 385:1257-65.
Mixing V24P with A.beta.40 at a 1:1 molar ratio inhibits amyloid
formation and the cytotoxicity of A.beta.40. Iwata et al.,
Pharmacol Therapeut (2005) 108:129-48.
[0056] In some examples, the peptide affinity moiety may comprise
the amino acid sequence GSNKGAIIGLM (SEQ ID NO: 1), which is
derived from amyloid .beta.-protein. Alternatively, the affinity
moiety may comprise the amino acid sequence
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV (SEQ ID NO: 2), in which
.sup.DP refers to the D-form of proline. In another example, the
affinity moiety may comprise RPRTRLHTHRNR (SEQ ID NO: 8), which may
be an A.beta.42-binding 12-mer D-form peptide as described in
Wiesehan et al. (ChemBioChem (2003) 4:748-53), the relevant
teachings therein are incorporated by reference herein. Other
exemplary amyloid .beta.-protein fragments for use as the peptide
affinity moiety in making the bipartite molecules described herein
are provided in Table 3 below.
[0057] Any of the peptide affinity moieties as described herein can
be prepared by, e.g., chemical synthesis or recombinant
technology.
(ii) Charged Moiety
[0058] The charged moiety of the bipartite molecule may prevent or
reduce the formation of abnormal protein aggregates by misfolded
disease proteins or may lead to dissociation of existing abnormal
protein aggregates by the force of charge repulsion. See, e.g.,
FIG. 1. Further, the charged moiety may facilitate the bipartite
molecule containing such to cross cell membranes and/or blood brain
barrier (BBB).
[0059] In some embodiments, the charged moiety may contain a
peptide comprising up to 100 (e.g., 80, 60, 50, 40, 30, 20, or 10)
amino acid residues, which may contain at least 2, at least 4, at
least 5, at least 8, at least 10, at least 15, at least 20, at
least 25, or at least 30 charged amino acids. In some examples, the
charged moiety may be a peptide having all charged amino acid
residues. For example, the peptide may contain at least one (e.g.,
at least 2, 4, 5 or 10) negatively charged amino acid, e.g.,
aspartic acid (e.g., a polyD fragment) or glutamic acid (e.g., a
polyE fragment), or a combination of D and E amino acids.
Alternatively, the peptide may contain at least one (e.g., at least
2, 4, 5, or 10) positively charged amino acid, e.g., arginine,
histidine, lysine, or a combination thereof. In some examples, the
charged moiety contains a stretch of arginine (e.g., a polyR
fragment) or lysine residues (e.g., a polyK fragment). As used
herein, a polyR, polyK, polyD, or polyE fragment refers to a
stretch of R, K, D, or E residues. Such a fragment may contain up
to 30 (e.g., 25, 20, 15, 10 or 5) R, K, D, or E residues. In some
examples, the charged moiety may be a peptide containing a
combination of different negatively charges amino acids or a
combination of different positively charged amino acids.
[0060] In addition to preventing the misfolded target protein from
forming abnormal protein aggregates or to dissociate it from
existing aggregates, a charged moiety described herein, for
example, a polyR fragment, can facilitate a bipartite molecule
comprising such to penetrate through cell membranes, the blood
brain barrier (BBB), or both. In some embodiments, the charged
moiety has the amino acid sequence RRRRRRRR (SEQ ID NO: 9).
[0061] In some embodiments, the charged moiety can be a
cell-penetrating peptide (CPP) such as TAT, referring to a peptide
comprising the amino acid sequence of GRKKRRQRRRPQ (SEQ ID NO: 10),
which is derived from the transactivator of transcription (TAT) of
human immunodeficiency virus. Cell-penetrating peptides (CPPs) have
been used to overcome the lipophilic barrier of the cellular
membranes and deliver both large molecules and even small particles
(e.g., proteins, DNA, antibodies, contrast (imaging) agents,
toxins, and nanoparticular drug carriers including liposomes)
inside the cell for their biological actions. A peptide-based
charged moiety may be prepared by chemical synthesis or recombinant
technology.
[0062] In other embodiments, the charged moiety may be a
non-peptide polymer, such as a polyethylenimine (PEI) molecule.
Methods for synthesizing PEI-conjugated peptides are well known in
the art. For example, PEI conjugated peptides may be synthesized by
the batch fluorenylmethoxycarbonyl (fmoc)-polyamide method.
(iii) Configurations of the Affinity and Charged Moieties in the
Bipartite Molecules
[0063] In any of the bipartite molecules as described herein, the
peptide affinity moiety and the charged moiety or moieties are
linked (e.g., covalently) and may be configured in a suitable
manner. In some embodiments, the bipartite molecule contains a
peptide affinity moiety as described herein and a single charged
moiety. The charged moiety can be attached to either the N-terminus
or the C-terminus of the peptide affinity moiety, either directly
or via a suitable linker. If the charged moiety is also peptide
based, the peptide affinity moiety and the charged peptide may be
linked via a peptide bond. If the charged moiety is a non-peptide
polymer, such as PEI, the non-peptide polymer may be linked to
either the N-terminus or the C-terminus of the peptide affinity
moiety via a suitable bond (e.g., a suitable covalent bond).
[0064] In some embodiments, the bipartite molecule may contain two
charged moieties, which can be either identical or different. In
other examples, the two charged moieties are both polyR fragments.
For example, a poly-arginine (polyR) moiety having eight arginine
residues is linked to the amino-terminus of the peptide affinity
moiety and another poly-arginine (polyR) moiety having eight
arginine residues can be linked to the carboxy-terminus of the
peptide affinity moiety.
[0065] In some embodiments, the two charged moieties can be
different. For example, one of the charged moieties can be a
peptide-based moiety such as a polyR fragment and the other charged
moieties can be a non-peptide polymer such as PEI. The polyR
fragment and PEI may be linked to the N-terminus and C-terminus of
the peptide affinity moiety, respectively, or vice versa. In some
embodiments the two charged moieties may have opposing charges. For
example, a positively charged poly-arginine (polyR) moiety may be
linked to the amino-terminus and a negatively charged
poly-aspartate (polyD) moiety may be linked to the carboxy-terminus
of the peptide affinity moiety. In some examples, the charged
moieties may both carry a positive charge or both carry a negative
charge. For example, a positively charged poly-arginine (polyR)
moiety may be linked to the amino-terminus and a positively charged
poly-lysine (polyK) moiety may be linked to the carboxy-terminus of
the peptide affinity moiety. In some embodiments the bipartite
molecule is RRRRRRRRGSNKGAIIGLM-PEI (SEQ ID NO: 4). It should be
appreciated that the charged moiety, or moieties, of the bipartite
molecule, as described herein, may be configured in any number of
ways with respect to the peptide affinity moiety.
[0066] In some examples, the peptide moiety or moieties may be
modified at the C-terminus (e.g., acetylation), the N-terminus
(e.g., methylation), or both for at least enhancing stability of
the bipartite molecule containing such.
II. Pharmaceutical Compositions
[0067] The bipartite molecules described herein can be mixed with a
pharmaceutically acceptable carrier (excipient) to form a
pharmaceutical composition for use in treating a target disease.
"Acceptable" means that the carrier must be compatible with the
active ingredient of the composition (and preferably, capable of
stabilizing the active ingredient) and not deleterious to the
subject to be treated. Pharmaceutically acceptable excipients
(carriers) including buffers, which are well known in the art. See,
e.g., Remington: The Science and Practice of Pharmacy 20th Ed.
(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
[0068] The pharmaceutical compositions to be used in the present
methods can comprise pharmaceutically acceptable carriers,
excipients, or stabilizers in the form of lyophilized formulations
or aqueous solutions. (Remington: The Science and Practice of
Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E.
Hoover). Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations used, and
may comprise buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrans; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0069] In some examples, the pharmaceutical composition described
herein comprises liposomes containing the bipartite molecules (or
the encoding nucleic acids) which can be prepared by methods known
in the art, such as described in Epstein, et al., Proc. Natl. Acad.
Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA
77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556. Particularly useful liposomes can be generated by
the reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0070] The bipartite molecules may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are known in the art, see, e.g., Remington, The Science
and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
[0071] In other examples, the pharmaceutical composition described
herein can be formulated in sustained-release format. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the bipartite
molecule, which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0072] The pharmaceutical compositions to be used for in vivo
administration must be sterile. This is readily accomplished by,
for example, filtration through sterile filtration membranes.
Therapeutic bipartite molecule compositions may be placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0073] The pharmaceutical compositions described herein can be in
unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation.
[0074] For preparing solid compositions such as tablets, the
principal active ingredient can be mixed with a pharmaceutical
carrier, e.g. conventional tableting ingredients such as corn
starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium
stearate, dicalcium phosphate or gums, and other pharmaceutical
diluents, e.g. water, to form a solid preformulation composition
containing a homogeneous mixture of a compound of the present
invention, or a non-toxic pharmaceutically acceptable salt thereof.
When referring to these preformulation compositions as homogeneous,
it is meant that the active ingredient is dispersed evenly
throughout the composition so that the composition may be readily
subdivided into equally effective unit dosage forms such as
tablets, pills and capsules. This solid preformulation composition
is then subdivided into unit dosage forms of the type described
above containing from 0.1 to about 500 mg of the active ingredient
of the present invention. The tablets or pills of the novel
composition can be coated or otherwise compounded to provide a
dosage form affording the advantage of prolonged action. For
example, the tablet or pill can comprise an inner dosage and an
outer dosage component, the latter being in the form of an envelope
over the former. The two components can be separated by an enteric
layer that serves to resist disintegration in the stomach and
permits the inner component to pass intact into the duodenum or to
be delayed in release. A variety of materials can be used for such
enteric layers or coatings, such materials including a number of
polymeric acids and mixtures of polymeric acids with such materials
as shellac, cetyl alcohol and cellulose acetate.
[0075] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently comprise between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0076] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Intralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can comprise
fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im,
and have a pH in the range of 5.5 to 8.0.
[0077] The emulsion compositions can be those prepared by mixing
bipartite molecules with Intralipid.TM. or the components thereof
(soybean oil, egg phospholipids, glycerol and water).
[0078] Pharmaceutical compositions for inhalation or insufflation
include solutions and suspensions in pharmaceutically acceptable,
aqueous or organic solvents, or mixtures thereof, and powders. The
liquid or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect.
[0079] Compositions in preferably sterile pharmaceutically
acceptable solvents may be nebulised by use of gases. Nebulised
solutions may be breathed directly from the nebulising device or
the nebulising device may be attached to a face mask, tent or
intermittent positive pressure breathing machine. Solution,
suspension or powder compositions may be administered, preferably
orally or nasally, from devices which deliver the formulation in an
appropriate manner.
III. Methods of Treatment
[0080] Any of the bipartite molecules are useful in
preventing/reducing the formation of protein aggregates associated
with a disease such as a neurodegenerative disease as described
herein, or disrupting such a protein aggregate. The bipartite
molecule can also be used for treating a disease or disorder,
particularly a neurodegenerative disease or disorder, characterized
by abnormal protein aggregates.
[0081] To practice the method disclosed herein, an effective amount
of the pharmaceutical composition described herein can be
administered to a subject (e.g., a human) in need of the treatment
via a suitable route, such as intravenous administration, e.g., as
a bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerebrospinal, subcutaneous,
intra-articular, intrasynovial, intrathecal, oral, inhalation or
topical routes. Commercially available nebulizers for liquid
formulations, including jet nebulizers and ultrasonic nebulizers
are useful for administration. Liquid formulations can be directly
nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, the bipartite molecules as described
herein can be aerosolized using a fluorocarbon formulation and a
metered dose inhaler, or inhaled as a lyophilized and milled
powder. In one example, the bipartite molecule is administered via
an intranasal route, e.g., by nasal drops.
[0082] The subject to be treated by the methods described herein
can be a mammal, more preferably a human. Mammals include, but are
not limited to, farm animals, sport animals, pets, primates,
horses, dogs, cats, mice and rats. A human subject who needs the
treatment may be a human patient having, at risk for, or suspected
of having a target disease/disorder, such as Alzheimer's disease
(AD), Huntington's disease (HD), Parkinson's disease (PD), dementia
with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS),
frontotemporal lobar degeneration-TDP-43 proteinopathy,
frontotemporal lobar degeneration-tauopathy (Pick's disease,
cortical basal degeneration, progressive supranuclear palsy,
FTDP-17), Creutzfeldt-Jacob disease (CJD), or another disease. A
subject having a target disease or disorder can be identified by
routine medical examination, e.g., laboratory tests, organ
functional tests, CT scans, or ultrasounds. A subject suspected of
having any of such target disease/disorder might show one or more
symptoms of the disease/disorder. A subject at risk for the
disease/disorder can be a subject having one or more of the risk
factors for that disease/disorder.
[0083] As used herein, "an effective amount" refers to the amount
of each active agent required to confer therapeutic effect on the
subject, either alone or in combination with one or more other
active agents. In some embodiments, the therapeutic effect is
reduced abnormal protein aggregates, or the prevention of abnormal
protein aggregate formation. Determination of whether an amount of
the bipartite molecule achieved the therapeutic effect would be
evident to one of skill in the art. Effective amounts vary, as
recognized by those skilled in the art, depending on the particular
condition being treated, the severity of the condition, the
individual patient parameters including age, physical condition,
size, gender and weight, the duration of the treatment, the nature
of concurrent therapy (if any), the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose of the individual components or combinations thereof be used,
that is, the highest safe dose according to sound medical
judgment.
[0084] Empirical considerations, such as the half-life, generally
will contribute to the determination of the dosage. Frequency of
administration may be determined and adjusted over the course of
therapy, and is generally, but not necessarily, based on treatment
and/or suppression and/or amelioration and/or delay of a target
disease/disorder. Alternatively, sustained continuous release
formulations of a bipartite molecule may be appropriate. Various
formulations and devices for achieving sustained release are known
in the art.
[0085] In one example, dosages for a bipartite molecule as
described herein may be determined empirically in individuals who
have been given one or more administration(s) of the bipartite
molecule. Individuals are given incremental dosages of the
antagonist. To assess efficacy of the antagonist, an indicator of
the disease/disorder can be followed.
[0086] Generally, for administration of any of the bipartite
molecules described herein, an initial candidate dosage can be
about 2 mg/kg. For the purpose of the present disclosure, a typical
daily dosage might range from about any of 0.1 .mu.g/kg to 3
.mu.g/kg to 30 .mu.g/kg to 300 .mu.g/kg to 3 mg/kg, to 30 mg/kg to
100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of symptoms occurs or until sufficient therapeutic
levels are achieved to alleviate a target disease or disorder, or a
symptom thereof. An exemplary dosing regimen comprises
administering an initial dose of about 2 mg/kg, followed by a
weekly maintenance dose of about 1 mg/kg of the bipartite molecule,
or followed by a maintenance dose of about 1 mg/kg every other
week. However, other dosage regimens may be useful, depending on
the pattern of pharmacokinetic decay that the practitioner wishes
to achieve. For example, dosing from one-four times a week is
contemplated. In some embodiments, dosing ranging from about 3
.mu.g/mg to about 2 mg/kg (such as about 3 .mu.g/mg, about 10
.mu.g/mg, about 30 .mu.g/mg, about 100 .mu.g/mg, about 300
.mu.g/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some
embodiments, dosing frequency is once every week, every 2 weeks,
every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8
weeks, every 9 weeks, or every 10 weeks; or once every month, every
2 months, or every 3 months, or longer. The progress of this
therapy is easily monitored by conventional techniques and assays.
The dosing regimen (including the bipartite molecule used) can vary
over time.
[0087] In some embodiments, for an adult patient of normal weight,
doses ranging from about 0.3 to 5.00 mg/kg may be administered. The
particular dosage regimen, i.e., dose, timing and repetition, will
depend on the particular individual and that individual's medical
history, as well as the properties of the individual agents (such
as the half-life of the agent, and other considerations well known
in the art).
[0088] For the purpose of the present disclosure, the appropriate
dosage of a bipartite molecule as described herein will depend on
the specific bipartite molecule, bipartite molecules, the type and
severity of the disease/disorder, whether the bipartite molecule is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antagonist, and the discretion of the attending physician. A
clinician may administer a bipartite molecule, until a dosage is
reached that achieves the desired result. In some embodiments, the
desired result is a decrease in abnormal protein aggregates.
Methods of determining whether a dosage resulted in the desired
result would be evident to one of skill in the art.
[0089] Administration of one or more bipartite molecules can be
continuous or intermittent, depending, for example, upon the
recipient's physiological condition, whether the purpose of the
administration is therapeutic or prophylactic, and other factors
known to skilled practitioners. The administration of an bipartite
molecule may be essentially continuous over a preselected period of
time or may be in a series of spaced dose, e.g., either before,
during, or after developing a target disease or disorder. In some
examples, the amount of the bipartite molecule is effective in
reducing the formation of an abnormal protein aggregate by at least
20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or above.
[0090] As used herein, the term "treating" refers to the
application or administration of a composition including one or
more active agents to a subject, who has a target disease or
disorder, a symptom of the disease/disorder, or a predisposition
toward the disease/disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disorder, the symptom of the disease, or the predisposition
toward the disease or disorder.
[0091] Alleviating a target disease/disorder includes delaying the
development or progression of the disease, or reducing disease
severity. Alleviating the disease does not necessarily require
curative results. As used therein, "delaying" the development of a
target disease or disorder means to defer, hinder, slow, retard,
stabilize, and/or postpone progression of the disease. This delay
can be of varying lengths of time, depending on the history of the
disease and/or individuals being treated. A method that "delays" or
alleviates the development of a disease, or delays the onset of the
disease, is a method that reduces probability of developing one or
more symptoms of the disease in a given time frame and/or reduces
extent of the symptoms in a given time frame, when compared to not
using the method. Such comparisons are typically based on clinical
studies, using a number of subjects sufficient to give a
statistically significant result.
[0092] "Development" or "progression" of a disease means initial
manifestations and/or ensuing progression of the disease.
Development of the disease can be detectable and assessed using
standard clinical techniques as well known in the art. However,
development also refers to progression that may be undetectable.
For purpose of this disclosure, development or progression refers
to the biological course of the symptoms. "Development" includes
occurrence, recurrence, and onset. As used herein "onset" or
"occurrence" of a target disease or disorder includes initial onset
and/or recurrence.
[0093] In some embodiments, the bipartite molecules described
herein are administered to a subject in need of the treatment at an
amount sufficient to reduce abnormal protein aggregates by at least
5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater)
in vivo. In other embodiments, the bipartite molecules are
administered in an amount effective in reducing the activity level
of a target antigens by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or greater).
[0094] Conventional methods, known to those of ordinary skill in
the art of medicine, can be used to administer the pharmaceutical
composition to the subject, depending upon the type of disease to
be treated or the site of the disease. This composition can also be
administered via other conventional routes, e.g., administered
orally, parenterally, by inhalation spray, topically, rectally,
nasally, buccally, vaginally or via an implanted reservoir. The
term "parenteral" as used herein includes subcutaneous,
intracutaneous, intravenous, intramuscular, intraarticular,
intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional, and intracranial injection or infusion techniques.
In addition, it can be administered to the subject via injectable
depot routes of administration such as using 1-, 3-, or 6-month
depot injectable or biodegradable materials and methods. In some
examples, the pharmaceutical compositions is administered
intraocularlly or intravitreally.
[0095] Injectable compositions may contain various carriers such as
vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate,
ethyl carbonate, isopropyl myristate, ethanol, and polyols
(glycerol, propylene glycol, liquid polyethylene glycol, and the
like). For intravenous injection, water soluble bipartite molecules
can be administered by the drip method, whereby a pharmaceutical
formulation containing the bipartite molecule and a physiologically
acceptable excipients is infused. Physiologically acceptable
excipients may include, for example, 5% dextrose, 0.9% saline,
Ringer's solution or other suitable excipients. Intramuscular
preparations, e.g., a sterile formulation of a suitable soluble
salt form of the bipartite molecule, can be dissolved and
administered in a pharmaceutical excipient such as
Water-for-Injection, 0.9% saline, or 5% glucose solution.
[0096] In one embodiment, a bipartite molecule is administered via
site-specific or targeted local delivery techniques. Examples of
site-specific or targeted local delivery techniques include various
implantable depot sources of the bipartite molecule or local
delivery catheters, such as infusion catheters, an indwelling
catheter, or a needle catheter, synthetic grafts, adventitial
wraps, shunts and stents or other implantable devices, site
specific carriers, direct injection, or direct application. See,
e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No.
5,981,568.
[0097] Targeted delivery of therapeutic compositions containing an
antisense polynucleotide, expression vector, or subgenomic
polynucleotides can also be used. Receptor-mediated DNA delivery
techniques are described in, for example, Findeis et al., Trends
Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods
And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994);
Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem.
(1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990)
87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.
[0098] Therapeutic compositions containing a polynucleotide (e.g.,
those encoding the bipartite molecules described herein) are
administered in a range of about 100 ng to about 200 mg of DNA for
local administration in a gene therapy protocol. In some
embodiments, concentration ranges of about 500 ng to about 50 mg,
about 1 .mu.g to about 2 mg, about 5 .mu.g to about 500 .sub.lug,
and about 20 .mu.g to about 100 .mu.g of DNA or more can also be
used during a gene therapy protocol.
[0099] In some examples, patients with or at risk for Huntington's
disease may be treated with a bipartite molecule having an affinity
moiety that comprises polyQ attached to at least one charged
moiety. In some embodiments the bipartite molecule is
RRRRRRRRWDQQQQQQQQQQ (SEQ ID NO: 6), or RRRRRRRRWDQQQQQQQQQQQQQQQ
(SEQ ID NO: 7). Alternatively, a patient with or at risk for
Alzheimer's disease may be treated with a bipartite molecule having
an affinity moiety that comprises a portion of the amyloid
(3-protein attached to at least one charged moiety. In some
embodiments the bipartite molecule is RRRRRRRRGSNKGAIIGLM (SEQ ID
NO: 3), RRRRRRRRGSNKGAIIGLM-PEI (SEQ ID NO: 4), or
YEVHHQKLVFFAED.sup.DPGSNKGAIIGLMVGGVV-PEI (SEQ ID NO: 5).
[0100] The particular dosage regimen, i.e., dose, timing and
repetition, used in the method described herein will depend on the
particular subject and that subject's medical history.
[0101] In some embodiments, more than one bipartite molecule, or a
combination of a bipartite molecule and another suitable
therapeutic agent, may be administered to a subject in need of the
treatment. The bipartite molecule can also be used in conjunction
with other agents that serve to enhance and/or complement the
effectiveness of the agents.
[0102] Treatment efficacy for a target disease/disorder can be
assessed by, e.g., a method described in the Examples below.
IV. Kits for Use in Alleviating Neurodegenerative Diseases
Associated Abnormal Protein Aggregates
[0103] The present disclosure also provides kits for use in
alleviating diseases/disorders associated abnormal protein
aggregates, such as Alzheimer's disease (AD), Huntington's disease
(HD), Parkinson's disease (PD), dementia with Lewy bodies (DLB),
amyotrophic lateral sclerosis (ALS), frontotemporal lobar
degeneration-TDP-43 proteinopathy, frontotemporal lobar
degeneration-tauopathy (Pick's disease, cortical basal
degeneration, progressive supranuclear palsy, FTDP-17), or
Creutzfeldt-Jacob disease (CJD). Such kits can include one or more
containers comprising a bipartite molecule, e.g., any of those
described herein.
[0104] In some embodiments, the kit can comprise instructions for
use in accordance with any of the methods described herein. The
included instructions can comprise a description of administration
of the bipartite molecule to treat, delay the onset, or alleviate a
target disease as those described herein. The kit may further
comprise a description of selecting an individual suitable for
treatment based on identifying whether that individual has the
target disease. In still other embodiments, the instructions
comprise a description of administering a bipartite molecule to an
individual at risk of the target disease.
[0105] The instructions relating to the use of a bipartite molecule
generally include information as to dosage, dosing schedule, and
route of administration for the intended treatment. The containers
may be unit doses, bulk packages (e.g., multi-dose packages) or
sub-unit doses. Instructions supplied in the kits of the invention
are typically written instructions on a label or package insert
(e.g., a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0106] The label or package insert indicates that the composition
is used for treating, delaying the onset and/or alleviating a
disease or disorder associated with the presence of abnormal
protein aggregates, such as those described herein. Instructions
may be provided for practicing any of the methods described
herein.
[0107] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a bipartite molecule as those described
herein.
[0108] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container. In some embodiments, the invention provides articles
of manufacture comprising contents of the kits described above.
General Techniques
[0109] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook,
et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis
(M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana
Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed.,
1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed.,
1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.
E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,
Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular
Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase
Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: a practical approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
[0110] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications cited herein are incorporated by
reference for the purposes or subject matter referenced herein.
EXAMPLES
[0111] In order that the invention described herein may be more
fully understood, the following examples are set forth. The
examples described in this application are offered to illustrate
the molecules, pharmaceutical compositions, and methods provided
herein and are not to be construed in any way as limiting their
scope.
Example 1
Exemplary Bipartite Therapeutic Peptides and Uses thereof in
Treating Huntington's Disease
[0112] Huntington's disease (HD) is caused by an expansion of the
CAG trinucleotide repeats in the Huntingtin (HTT) gene. The
expanded CAG repeats encode an elongated polyglutamine stretch
(polyQ) within the mutant Htt (mHtt) protein, which leads to the
misfolding and aggregation of mHtt. Provided herein is a series of
therapeutic peptides , for example, 8R5Q, 8R10Q, 8R15Q and 8R20Q,
which contains polyarginines (e.g., 8R) and a short stretch of
polyQ (e.g., 5Q, 10Q, 15Q, or 20Q). The polyQ sequence was expected
to confer a specific affinity for the therapeutic peptides to bind
to mHtt and the polyarginine fragment possesses the capability to
penetrate neurons and prevent mHtt/peptide self-aggregation charge
repulsion. This was demonstrated by the observation that in Neuro2a
cells (mouse neuroblastoma), 8R10Q co-localized with 109QmHtt
aggregates. Both 8R10Q and 8R15Q significantly decreased the size
of 109QmHtt aggregates. In addition, 8R10Q reduced the level of
109QmHtt, which was blocked by the proteasome inhibitor MG132,
indicating that the peptide enhanced cellular ability to degrade
109QmHtt. Functionally, 8R10Q attenuated the 109QmHtt-induced
toxicity in the growth of undifferentiated Neuro2a cells by a MTT
assay and the retinoic acid-mediated neurite outgrowth of Neuro2a
cells. In vivo, 8R10Q significantly delayed the motor and memory
deterioration of R6/2 transgenic mice, a HD mouse model.
Histopathologically, 8R10Q also reduced the aggregation of mHtt and
neuronal loss, and ameliorated the activation of astrocytes and
microglia. Intriguingly, 8R10Q decreased the diabetic phenotype,
inflammatory index, and abnormal liver function of R6/2 mice.
Altogether, the therapeutic peptides designed by this modular
principle worked against HD, which may also shed light on the
development of a therapeutic strategy against other
neurodegenerative diseases.
Design and Synthesis of the Bipartite Peptides
[0113] The rational design of therapeutic peptides to reduce the
toxic misfolded proteins/derivatives across different
neurodegenerative diseases based on the principle of modular
assembly was attempted. The conjectured therapeutic peptides would
assume a bipartite or tripartite structure composed of a full or
partial sequence from the self-aggregating region of the misfolded
protein (e.g., mHtt or amyloid .beta. peptide, etc.) flanked either
upstream or downstream (bipartite) or both (tripartite) by a
stretch of charged amino acids. The rationale behind this design
was that the self-aggregating sequence would provide a specific
affinity between the peptide and the misfolded protein/derivative
through its self-aggregating property. The charged amino acid
chosen in the study may be arginine. Polyarginine stretch enabled
the therapeutic peptides to penetrate through cell membrane into
cytoplasmic and nuclear compartments (Mitchell, J Pept Res (2000)
56:318-25). It was also expected to prevent therapeutic peptides
from self-aggregation. Moreover, polyarginine can prevent the
mHtt/peptide hybrids from self-aggregation by charge repulsion
force (FIG. 1).
[0114] In this study, the rational design was tested for HD.
Several bipartite therapeutic peptides containing 8 consecutive
arginines (8R) attached to a stretch of consecutive glutamines
(polyQ) were synthesized and their biological activity of
decreasing the aggregation propensity of the mHtt protein was
investigated. The length of the polyQ of these bipartite peptides
was 5 (8R5Q), 10 (8R10Q), 15 (8R15Q) or 20 (8R20Q) (Table 2).
Peptides 8R5Q and 8R10Q were highly soluble in water or culture
medium (Table 2), and remained unchanged in their retention time
and amount for at least 28 days (FIG. 2, panels A and B),
indicating these two peptides were quite stable. In contrast, 8R15Q
was partially soluble, but 8R20Q was insoluble, and both underwent
a time-dependent decrease in the retention time and amount (FIG. 2,
panels A and B) during HPLC analysis, indicating their high
stability. Thus, 8R10Q was selected as the lead peptide for
subsequent studies given its superior biochemical properties.
TABLE-US-00003 TABLE 2 The sequence and biochemical properties of
therapeutic peptides. MALDI-TOF Solubility Peptide Sequence Formula
Calculated Found (DMEM/10%FBS) 8R NH.sub.2-(R).sub.8-W-D-CONH.sub.2
C.sub.63H.sub.113N.sub.35O.sub.13 1567.8 1568.0 Soluble 8R5Q
NH.sub.2-(R).sub.8-W-D-(Q).sub.5-CONH.sub.2
C.sub.88H.sub.153N.sub.45O.sub.23 2208.8 2208.5 Soluble 8R10Q
NH.sub.2-(R).sub.8-W-D-(Q).sub.10-CONH.sub.2
C.sub.113H.sub.193N.sub.55O.sub.33 2849.2 2849.2 Soluble 8R15Q
NH.sub.2-(R).sub.8-W-D-(Q).sub.15-CONH.sub.2
C.sub.138H.sub.233N.sub.65O.sub.43 3489.8 3489.8 aggregated 8R20Q
NH.sub.2-(R).sub.8-W-D-(Q).sub.20-CONH.sub.2
C.sub.163H.sub.273N.sub.75O.sub.53 4130.5 4130.5 aggregated s8R10Q
NH.sub.2- C.sub.113H.sub.193N.sub.55O.sub.33 2849.2 2849.5 Soluble
RQQRRQQDQRQWQRQRQQRR- CONH.sub.2 TAMRA-
TAMRA-NH-(R).sub.8-W-D-CONH.sub.2 C.sub.88H.sub.138N.sub.37O.sub.17
1982.3 1981.0 Soluble 8RWD TAMRA- TAMRA-NH-(R).sub.8-W-D-
C.sub.138H.sub.218N.sub.57O.sub.37 3264.7 3264.6 Soluble 8R10Q
(Q).sub.10-CONH.sub.2 TAMRA- TAMRA-NH-
C.sub.138H.sub.218N.sub.57O.sub.37 3264.7 3264.6 Soluble s8R10Q
RQQRRQQDQRQWQRQRQQRR- CONH.sub.2
s8R10Q: scrambled 8R10Q
[0115] The sequence in Table 2, from top to bottom, correspond to
SEQ ID NOs: 11-19.
Decreasing mHtt Aggregation by PolyR-PolyQ Peptide
[0116] The ability of 8R10Q to bind to the 109QmHtt was examined by
determining its co-localization with the 109QmHtt aggregates. The
8R10Q peptide was labeled with carboxytetramethylrhodamine (TAMRA)
dye (FIG. 3, panel A), and added into Neuro2a cells expressing
25QHtt or 109QmHtt. The TAMRA dye alone failed to associate with
the 109QmHtt aggregates, and the labeled 8R10Q also failed to
conform to the pattern of 25QHtt. In contrast, the TAMRA-labeled
8R10Q peptide was co-localized with the aggregates (FIG. 3, panel
B). These results clearly validated the ability of 8R10Q to
interact with the mHtt, as expected.
[0117] To probe into the details of the effect of peptide on the
mHtt aggregates, the parameters of the mHtt aggregates were
visualized and measured using the Total Internal Reflection
Fluorescence (TIRF) microscopy. As shown in FIG. 3, panel C, large
solid 109QmHtt aggregates were readily observed in the Neuro2a
cells treated with water, 8R, or a scrambled peptide (s8R10Q);
however, mainly small punctate aggregates were seen in cells
treated with 8R10Q, which remained so throughout the tested time
points. Quantitative data showed that the average size of the
aggregates from water, 8R or scrambled peptide-treated cells were
3.6 .mu.m.sup.2, 3.3 .mu.m.sup.2, 1.7 .mu.m.sup.2, respectively;
while those from the 8R10Q- or 8R15Q-treated cells were
dramatically reduced to 0.3 .mu.m.sup.2 and 0.2 .mu.m.sup.2,
respectively (FIG. 3, panel D). Notably, a subset of the aggregates
in the former control groups were larger than 10 .mu.m.sup.2, which
were never identified in the latter. The number of aggregates in 20
cells was also calculated. 8R10Q and 8R15Q increased the number of
the aggregates by 4-5 fold (FIG. 3, panel E). These results
suggested that 8R10Q or 8R15Q prevented the small punctate
structures from forming large conglomerates of 109QmHtt.
[0118] The small aggregates were tracked for 24 hours using live
cell imaging. As expected, multiple small punctuates within one
cell fused with each other with time, and eventually formed one
dominant or several large aggregates. 8R10Q or 8R15Q treatment
rendered these punctuates unchanged throughout the observation time
period.
[0119] Whether the peptides could modulate the aggregation
propensity of mHtt was next investigated. As shown in FIG. 4,
panels A and C, 20 .mu.M of peptide 8R10Q or 8R15Q, added at 8
hours (T1) or 8 plus 24 hours (T1+T2) after transfection
significantly reduced the aggregated 109QmHtt as compared with
water or the 8R peptide control by filter trap assay. However, when
the peptide was added at 24 hours alone, no obvious effects were
observed. These results showed that 8R10Q and 8R15Q peptides
effectively prevented the mHtt from aggregation.
[0120] For further characterization, Neuro2a lysates were separated
into the RIPA-soluble (sol) and insoluble (ins) fractions (FIG. 4,
panel B). 8R10Q peptide decreased the level of mHtt in both the
soluble and insoluble fractions as compared with water, 8R, or the
scrambled (s8R10Q) control (FIG. 4, panel D). Since the ubiquitin
proteasomal system (UPS) had been previously shown to be important
for mHtt degradation (Martin-Aparicio, J Neurosci (2001)
21:8772-81; Jana, Hum Mol Genet (2001) 10:1049-59), MG132, an
inhibitor of the UPS, was used to block the peptide-induced
decrease in the mHtt. MG132 reversed the level of the 109QmHtt in
the peptide group (FIG. 4, panel E). A quantitative assay showed
that MG132 treatment increased the level of mHtt across all groups,
indicating a basal turnover of 109QmHtt by UPS. However, the ratio
of the MG132:DMS0 109QmHtt was much higher for the 8R10Q treated
samples (FIG. 4, panel F). These results indicated that the peptide
reduced the mHtt through enhancement of its degradation by UPS.
Protection of Cells Against the Neurotoxicity of 109QmHtt and
H.sub.2O.sub.2 by Bipartite Peptides
[0121] It has long been reported that HD patients have higher
levels of oxidative stress linked with mitochondrial dysfunction
induced by mHtt. Browne, Brain Pathol (1999) 9:147-63; and Tasset,
Revista de Neurologia (2009) 49:424-9. mHtt and oxidative stress
may form a self-reinforcing vicious cycle in HD. In the present
study, approximately 83% of Neuro2a cells expressing 25QHtt treated
with 50 1.mu.M H.sub.2O.sub.2 with or without scrambled or
bipartite therapeutic peptides survived (FIG. 5, panel A). On the
other hand, the survival rate of Neuro2a cells expressing 109QmHtt
decreased to .about.72%, indicating that the 109QmHtt rendered
cells more vulnerable to the oxidative stress of H.sub.2O.sub.2.
8R10Q or 8R15Q treatment increased the survival rate to levels
above 80% (FIG. 5, panel A).
[0122] The expression of 109QmHtt reduced the growth of Neuro2a
cells, and so did the lower levels of H.sub.2O.sub.2 (12.5 .mu.M)
(FIG. 5, panel B). Peptide 8R10Q ameliorated the reduction in
growth induced not only by the toxicity of 109QmHtt, but also by
H.sub.2O.sub.2, as compared with 8R or the scrambled control.
[0123] Retinoic acid (RA) induced the differentiation of Neuro2a
cells with neurite outgrowth. 109QmHtt decreased the number of
cells bearing RA-induced neurites relative to 25QHtt (FIG. 5,
panels C and D). 8R10Q treatment significantly reversed the defect
in neurite outgrowth caused by 109QmHtt as compared with water or
the scrambled control. The 8R peptide also rescued the cells from
this toxicity to some extent, but the results were not
statistically significant. These results clearly showed that the
bipartite peptides could protect Neuro2a cells against the toxicity
caused either by 109QmHtt and/or by H.sub.2O.sub.2.
Therapeutic Effect of 8R10Q on R6/2 Transgenic Mice
[0124] To test the therapeutic effect of the peptide in vivo, the
8R10Q peptide was delivered 6 days/week into the wild type (WT) or
R6/2 transgenic mice through intranasal aspiration or continuously
with the Alzet osmotic minipumps into the neostriatum. The body
weights of the WT or R6/2 mice treated with 8R10Q through either
route remained comparable to those treated with PBS. As shown in
FIG. 6, panel A, the motor deterioration assessed by the rotarod of
R6/2 mice became significant from 11 weeks of age, but the 8R10Q
treatment effectively delayed the phenotype until 13 weeks. In
fact, the 8R10Q-treated R6/2 mice performed even better than the 11
week-old PBS-treated mice on average. The 8R10Q peptide delivered
intracerebrally exhibited a very similar therapeutic effect to that
given intranasally. These results showed that administration route
does not affect this effect. In addition, intranasal 8R10Q also
corrected the memory deficit of 13 week-old R6/2 mice as shown in
the T maze test (FIG. 6, panel B). The R6/2 mice were previously
reported to have diabetes. Bjorkqvist et al., Hum Mol Genet (2005)
14:565-74; and Hunt et al., Experimental Brain Res (2005)
166:220-9. The intranasal administration of 8R10Q peptide delayed
the rise and decreased the level of blood sugar (FIG. 6, panel C).
Similarly, 8R10Q given intracerebrally also significantly corrected
the diabetes phenotype. Lastly, all R6/2 mice treated with PBS died
before or at 16 weeks of age; however, 8R10Q given intranasally
prevented premature death by 41% at 16 weeks of age (FIG. 6, panel
D). To sum up, the 8R10Q peptide effectively delayed the onset of
disease and ameliorated the pathological phenotypes.
Decrease of Neuropathology by 8R10Q Peptide
[0125] R6/2 transgenic mice exhibited a decrease in cortical
thickness due to the loss of cortical neurons. Interestingly, 8R10Q
peptide prevented cortical thinning (FIG. 7, panels A and B),
suggesting that this peptide could rescue neurons from death.
Indeed, the neuronal count revealed that the loss of neurons in
both the cortex (FIG. 7, panels C and D) and striatum (FIG. 7,
panels E and F) of the R6/2 mice was significantly prevented by the
intranasal 8R10Q treatment.
[0126] To correlate the beneficial effect with the aggregates of
mHtt, immunohistochemistry was performed to calculate the areas
occupied by the aggregates per unit area (150 .mu.m.sup.2). The
method was selected because the frequent clustering of the
aggregates into conglomerates rendered an accurate count of the
aggregate number very difficult. As shown in FIG. 8, panels A and
B, the 8R10Q peptide decreased the areas of aggregates by
.about.60% in both the cortex and striatum as compared with the PBS
control. In addition, the intensity which represented the amount of
mHtt in the aggregates was also significantly decreased by the
8R10Q peptide in both areas. Corresponding Western blot analysis
revealed that the RIPA-insoluble or aggregate fraction was
decreased by the 8R10Q peptide as compared with PBS. The rescue
effect with respect to the neuronal loss and aggregation was
accompanied by a significant decrease in the numbers of
GFAP-positive astrocytes (FIG. 8, panels C and D) and that of
Iba-l-positive microglia (FIG. 8, panels E and F) in the cortex of
the 8R10Q-treated R6/2 mice. Furthermore, the R6/2 mice had weak
tau signals compared with the WT mice, but 8R10Q treatment
significantly enhanced this in both cortex and striatum, indicating
restoration of the axonal processes. Taken together, these results
showed that the 8R10Q peptide effectively decreased the aggregated
species of mHtt, and prevented the neurotoxicity induced by the
mHtt.
Discussion
[0127] In this study, bipartite therapeutic peptides like 8R10Q
were shown to bind to mHtt aggregates, decrease mHtt-mediated
toxicity, and delay the onset and progress of neurologic
dysfunction in HD models. In addition, the therapeutic effect of
8R10Q was observed in a mouse cell line transiently expressing
mHtt, R6/2 transgenic mice, and human HD iPS-derived neurons; its
therapeutic effect was independent of the animal species or length
of polyQ in mHtt.
[0128] In the bipartite peptide design, the charged sequence played
an essential role in preventing the therapeutic peptide itself or
misfolded target/peptide hybrid from aggregating. To identify
peptides that may decrease mHtt toxicity, studies using various
approaches, such as phage display (Kawasaki et al., Biosci,
Biotech, and Biochem (2012) 76:762-6; Lecerf et al., Proc Natl Acad
Sci USA (2001) 98:4764-9; Nagai et al., Hum Mol Genet (2003)
12:1253-9; Magai et al., J Biol Chem (2000) 275:10437-42) or other
screening methods (Arribat et al., PLoS One (2013) 8:e68775;
Lakhani et al., PLoS Computational Biol (2010) 6:e1000772; Skogen
et al., BMC Neurosci (2006) 7:65) and sequence modifications
(Lanning et al., Biochem (2010) 49:7108-18; Kazantsev et al., Nat
Genet (2002) 30:367-76) have previously been conducted. Compared
with those approaches, inclusion of the repulsion force by the
charged sequence into peptide design provides several advantages.
First, previous studies were mainly target- or disease-specific. In
contrast, the present study renders designing therapeutic peptides
against various neurodegenerative diseases possible by choosing a
partner sequence with a specific affinity for the misfolded
protein/derivative of the disease-of-interest. This possibility is
supported by a separate study, which showed that therapeutic
peptides designed by the same principle yielded a similar
beneficial outcome in APP/PS1 transgenic mice, a widely used mouse
model for AD. Second, it may significantly simplify the work spent
in searching for or in modifying candidate sequences for the
therapeutic peptides. Previous approaches required one to find
peptides of dual functions that would both bind to the misfolded
protein/derivative, and at the same time, stop the latter from
aggregation, which might require a considerable amount of effort.
Also, these efforts would likely need to be reinstated for a
different disease. In the present design, the affinity sequences
could be directly taken from the misfolded protein/derivative of
the particular disease-of-interest, as already demonstrated in both
of the studies. Thus, much work could be eliminated. Furthermore,
the strategy may be applied to the peptides identified in the
previous studies to further enhance their therapeutic effects.
Example 2
Exemplary Bipartite Therapeutic Peptides for Use in Delaying
Disease Onset in APP/PS1 Transgenic Mice
[0129] Adult neurodegenerative diseases (NDs) comprise a
heterogeneous group of neurological disorders characterized by
disease-specific inclusion bodies (IBs) formed by misfolded
peptides/proteins. Alzheimer's disease (AD) is the most common ND,
with signature IBs, amyloid plaques, and neurofibrillary tangles.
An imbalance in the production and clearance of misfolded amyloid
.beta. (A.beta.) peptide and its variants is considered the primary
cause for the pathogenesis of AD. A modular peptidic design
combining polyarginines (PolyR) and a peptide derived from the
pathogenic peptide/protein forming IBs is discussed below. The
designed bipartite peptides were expected to have target-specific
affinity and to be able to prevent misfolded peptide/protein
self-aggregation by the charge repulsion conferred by PolyR. A
designed bipartite peptide R.sub.8-A.beta.(25-35) and its D form
derivative .sup.DR.sub.8-A.beta.(25-35) were found to prevent
A.beta..sub.40 from forming amyloid fibrils by circular dichroism
and electron microscopy. When mixed at a 1:1 ratio, both peptides
significantly decreased the toxicity of A.beta..sub.40 to the
Neuro2a cells. Daily intranasal administration of PEI-conjugated
R.sub.8-A.beta.(25-35) peptide from 4 months of age significantly
ameliorated the memory deficits of the 8 month-old APPxPS1 double
transgenic mice compared with PEI-treated control group as
demonstrated by a Morris water maze test. The level of total
A.beta..sub.40 and A.beta..sub.42 in the cortex and hippocampus
were remarkably reduced; also, the number of amyloid plaques were
consistently reduced. Taken together, the modular design combining
polyR with a peptide from the pathogenic peptide/protein, like
R.sub.8-A.beta.(25-35), produced desirable therapeutic effects and
could be easily adopted to design therapeutic peptides for other
diseases characterized by IBs.
Methods and Materials
Peptide Synthesis
[0130] The peptides were prepared by the batch
fluorenylmethoxycarbonyl (fmoc)-polyamide method. Chen et al.,
Protein Sci (2001) 10:1794-1800. The sequence of A.beta. 40:
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 20). The
sequence of R8A .beta. 25-35: RRRRRRRRGSNKGAIIGLM (SEQ ID NO: 3);
.sup.DR8A .beta. 25-35 had the same sequence as R8A .beta. 25-35,
but the L-form arginines were replaced by D-form arginines. The
C-terminal carboxyl group was amidated using Rink Amide AM resin
(Novabiochem, Billerica, Mass., USA) as the solid support.
Fmoc-amino-acid derivatives (4 equivalents) (Anaspec, Freemont,
Calif., USA) were coupled on the resin (1 equivalent) using
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (4 equivalents) and 4.45% (v/v) N-methyl
morpholine in dimethylformamide (DMF). The Fmoc cleavage step was
performed using 20% piperidine in DMF. Side-chain deprotection and
peptide cleavage from the resin were performed simultaneously by
stirring the resin with a mixture of 9.4 ml of trifluoroacetic
acid, 0.25 ml of water, 0.25 ml of ethanedithiol, and 0.1 ml of
triisopropylsilane at room temperature for 1-2 hours, then the
resin was removed by passing the reaction mixture through a G2
glass funnel. The crude peptide was precipitated from the filtrate
by the addition of three volumes of ice-cold methyl t-butyl ether
(MTBE) and centrifugation at 2000 g for 15 minutes at 4.degree. C.,
then washed twice with MTBE, and dried under a vacuum. The
precipitated peptide was purified by reverse-phase HPLC using a
Vydac C18 column (10 mm.times.250mm) and acetonitrile-water
mixtures containing 0.1% trifluoroacetic acid. Peaks were analyzed
on a matrix-assisted laser desorption ionization (MALDI) mass
spectrometer and those containing the desired product were
lyophilized and stored at -20.degree. C. To synthesize the
PEI-conjugated peptide R.sub.8-A .beta. (25-35)-PEI, PEI was
conjugated to the C-terminal carboxyl group of the peptide.
Fmoc-Met-Wang resin (Anaspec) was used as the solid support during
synthesis. To avoid interference with PEI conjugation by the
N-terminal amino group, the N-terminal group of the peptide was
acetylated using 4 equivalents of acetic anhydride instead of an
amino acid derivative in the final synthetic step.
Circular Dichroism (CD) Spectroscopy
[0131] The peptide samples were dissolved in 75% trifluoroethanol
as 1.4 mM stock solutions, then diluted into 20 mM sodium phosphate
buffer with 150 mM KCl (pH 7) to a final peptide concentration of
30 .mu.M and incubated at 25.degree. C. After the indicated times,
the samples were placed in a 1-mm cell and the CD spectra between
200 and 250 nm were recorded on a J-715 CD spectrometer (JASCO,
Japan). The band width was set to 2 nm and the step resolution was
0.05 nm. Each sample was scanned twice and the recorded spectra
were averaged to get the final spectrum.
Transmission Electron Microscopy
[0132] The samples were deposited on carbon-coated 300-mesh copper
grids, incubated for 3 min for absorption, and then washed by
water. Negative staining was carried out by staining with 2% uranyl
acetate for 1.5 min. After air drying, the samples were viewed
using a Hitachi H-7000 electron microscope (Hitachi, Tokyo,
Japan).
Cell Viability Assay
[0133] Mouse N2a neuroblastoma cells (ATCC) were cultured in
Dulbecco's modified Eagle's medium (DMEM) (HyClone, USA)
supplemented with 10% fetal bovine serum (FBS; HyClone, USA) in 5%
CO.sub.2 at 37 .degree. C. For the cell viability assay, the cells
were harvested, suspended at a density of 350,000 cells/mL in DMEM,
and 100 .mu.L of each sample was plated in each well of a 96-well
CellBIND polystyrene microplate (Corning, USA). Because the
cytotoxicity experiments lasted for up to 4 days, cell
proliferation was blocked using a medium without FBS. The plates
were then incubated at 37.degree. C. under 5% CO.sub.2 for 24 h to
allow the cells to attach to the well. The peptides were dissolved
in DMSO as 6 mM stock solutions. Five microliters of the stock
solution (6 mM) was diluted with 95 .mu.L of PBS (20 mM sodium
phosphate buffer, 150 mM KCl, pH 7.0), and then the sample was
immediately added to 900 .mu.L of fresh DMEM medium to give a
peptide concentration of 30 .mu.M. To test the efficacy of the
peptide inhibitor, equal volumes of A.beta.40 and peptide inhibitor
stock solutions were pre-mixed and then diluted into PBS to make a
final concentration of 30 .mu.M for each peptide. The diluted
peptide solutions were pre-incubated for 24 h at room temperature
with shaking (50 rpm) before being added to the cultures. The
medium in the well of the 96-well plate was replaced with 100 .mu.L
of peptide-containing medium and the plate was incubated for 48 h.
Cell viability was determined using the MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
toxicity assay. Shearman et al., J Neurochem (1995) 65:218-27. Ten
microliters of 5 mg/mL of MTT in PBS was added to each well, and
then, after incubation for 4 h, the medium was removed and the MTT
crystals were dissolved in 100 .mu.L of 90% isopropanol, 0.5% SDS,
and 40 mM HCl, and their absorption at 570 nm was measured. Cell
viability was calculated by dividing the absorbance of the wells
containing peptide samples by that of the wells without any added
peptide. The experiment was repeated at least four times and eight
replicate wells were used for each sample and control in each
independent experiment.
Synthesis of PEI-Conjugated Peptide
[0134] Three milligrams of acetylated R.sub.8-A .beta. (25-35)
dissolved in 2.5 mL dimethyl sulfoxide (DMSO) was slowly mixed with
150 .mu.L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (600
mM in 0.1 M MES, 0.5 M NaCl, pH 6) and with 150 .mu.L
N-hydroxysuccinimide (NHS) (1200 mM in 0.1 M MES, 0.5 M NaCl, pH 6)
subsequently. The reaction mixture was reacted at room temperature
for 30 min with gentle shaking (70 rpm). To the mixture, 180 .mu.L
polyethylenimine (PEI) was added and reacted at room temperature
overnight with gentle shaking (70 rpm). The PEI-conjugated peptide,
named R.sub.8-A .beta. (25-35)-PEI, was separated from unreacted
PEI and R.sub.8-A .beta. (25-35) by reverse-phase HPLC using a
Vydac C18 column (10 mm.times.250 mm) and acetonitrile-water
mixtures containing 0.1% trifluoroacetic acid. Peaks were analyzed
on a matrix-assisted laser desorption ionization (MALDI) mass
spectrometer and those containing the desired product were
lyophilized and stored at -20 .degree. C.
Intranasal Administration
[0135] APP.times.PS1 transgenic mice
(B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax , purchased from Jackson
Laboratories (USA), were bred and genotyped following the vendor's
protocols. Borchelt et al., Neuron (1996) 17:1005-13; and Jankowsky
et al., Biomol Eng (2001) 17:157-65. The mice had access to food
and water ad libitum and were kept on a 12:12 h light-dark cycle.
PEI and R8A .beta. 25-35-PEI were dissolved in 100 mM
NaH.sub.2PO.sub.4/138 mM KCl (pH 5) to a final concentration of 400
.mu.M. For intranasal administration, 2.5 .mu.L PEI or R.sub.8-A
.beta. (25-35)-PEI was given to each nostril of one mouse when they
were 3 months of age for six days/week until they were 8 months of
age. Mice were then tested with a Morris water maze. Treatment were
resumed after a 6-week break for the water maze test, and continued
until the mice were 12 months of age.
ELISA Assays for Total A.beta.40 and A.beta.42
[0136] The levels of A.beta.40 and A.beta.42 in mouse brain
homogenate were detected using ELISA kits (Invitrogen, Md., USA)
according to the manufacturer's instructions. Briefly, the cortical
or hippocampal tissue was weighed and homogenized at 4.degree. C.
in the cell extraction buffer provided in the kit, supplemented
with protease inhibitor cocktail (Sigma, St. Louis, USA). The
homogenates were then centrifuged in Eppendorf tubes at 13000 rpm
at 4 .degree. C. for 10 min and the concentration of proteins in
the supernatant was measured using the microBCA protein assay
(Thermo, Ill., USA). The APP levels were adjusted in accordance
with the protein levels.
ELISA Assays for Insoluble A.beta.40 and A.beta.42
[0137] Half of the frozen cortical and hippocampal tissue samples
were homogenized in 1 mL tapered tissue grinders in 400 .mu.L of
ice-cold TBS containing a protease inhibitor cocktail (P8340,
Sigma). The homogenates were then transferred to 1.5 mL Eppendorf
tubes and centrifuged at 20000 g for 20 min at 4.degree. C. The
supernatant contained soluble A.beta., whereas the TBS-insoluble
pellet contained insoluble A.beta. proteins. The TBS-insoluble
pellet was suspended in 70% formic acid, sonicated for 1 min, and
then centrifuged at 20000 g for 20 min at 4.degree. C. The final
supernatant containing the solubilized A.beta. was removed and
neutralized with 20 volumes of 1 M Tris base. The protein
concentrations of the samples containing the solubilized
"insoluble" A.beta. was quantified using the Bradford protein assay
(Bio-Rad #500-0006).
Morris Water Maze Task
[0138] The maze was made of white opaque plastic with a diameter of
120 cm and contained 40 cm high walls. It was filled with
milk/water at 25 .degree. C. A small escape platform
(10.times.6.5.times.21.5 cm) was placed at a fixed position in the
center of one quadrant, 25 cm from the perimeter, and was hidden 1
cm beneath the water's surface. The room contained a number of
fixed visual cues on the walls. The acquisition trial phase
consisted of 5 training days (Days 1-5) and four trials per day
with a 15 min inter-trial interval. Four points equally spaced
along the circumference of the pool (North, South, East, and West)
served as the starting positions, which were randomized across the
four trials daily. If an animal did not reach the platform within
90 s, it was guided to the platform, where it stayed for 15 s. The
path length and escape latencies were recorded (n=10 per group).
Acquisition data, such as time taken to reach the escape platform
and path length were analyzed by two-way repeated measures ANOVA.
On Day 5, after finishing three trials, a probe trial was performed
in order to assess the mouse's spatial memory. The platform was
removed from the maze and animals were allowed to swim freely for
90 s and the swimming path was recorded and analyzed.
Cytometric Bead Array
[0139] A cytometric bead array (CBA, mouse inflammation kit; BD
Biosciences) was used to quantitatively measure cytokine expression
levels in the control and treated mouse brain tissue lysates. CBA
was also performed to measure the cytokines from different brain
regions (cortex and hippocampus). This method quantifies soluble
particles, in this case, cytokines, using a fluorescence-based
detection mechanism. The beads, coated with the desired cytokine,
IL-6 and IL-1.beta., reacted with the test lysates and standards,
to which fluorescence dyes were then added. The assay was performed
according to the manufacturer's instructions and analyzed on the
FACS Calibur (Becton Dickinson). Analysis was performed using CBA
software that allows the calculation of cytokine concentrations in
unknown samples. Soldan et al., J Neuroimmunol (2004)
146:209-15.
Thioflavin S Staining
[0140] The mice were perfused with ice-cold PBS (136.89 mM NaCl,
2.68 mM KCl, 1.62 mM KH.sub.2PO4, and 10.14 mM Na.sub.2HPO.sub.4,
pH 7.4) buffer and 4% paraformaldehyde (PFA)/PBS. Brains of the
perfused mice were then post-fixed in 4% PFA/PBS with gentle
shaking in a cold room for another 24 hours. Post-fixation, the
brain samples were washed with PBS buffer and preserved in a 70%
ethanol solution. PFA-fixed brain tissues were dehydrated by a
semi-enclosed benchtop tissue processor (Leica TP1020). Paraffin
blocks containing the dehydrated brain samples were prepared by a
Leica EG1150 H. Coronal sections with 5 .mu.m thickness were cut on
a microtome (Leica RM2235). Two brain sections were transferred
with a brush, put onto the surface of a water bath, floated onto
the surface of clean glass slides, and placed on a 34.degree. C.
warming block for several hours. Paraffin slides were
deparaffinized and rehydrated with xylene, absolute ethanol, 95%
ethanol, 70% ethanol, and water, sequentially. Rehydrated slides
were given 1% (w/v) thioflavin S (ThS) solution for 10 min at room
temperature while protected from light. The slides were washed with
80% ethanol and water to remove excess ThS and to facilitate
visualization. ThS-positive signals were then visualized with a
fluorescence microscope equipped for evaluation of green
fluorescence, and the plague number, plague area, and plague size
were analyzed by ImageJ.
Radiosynthesis of [.sup.11C] PIB
[0141] The radiosynthesis of [.sup.11C] PIB was performed by using
[.sup.11C] methyltriflate according to the method described
previously with minimal modification. Takalo et al., Am. J.
Neurodeger. Dis. (2013), 2:1-14. Briefly, [.sup.11C] methyl bromide
was produced by the multi-pass bromination of [.sup.11C] methane.
Subsequently, [.sup.11C] methyl bromide was eluted from a trap and
converted to [.sup.11C] methyltriflate by passing through a
preheated silver triflate column. [.sup.11C] methyltriflate was
carried by a helium stream (20 ml/min) into 350 .mu.l of anhydrous
methylethylketone containing 1.5 mg of
2-(4'-aminophenyl)-6-hydroxybenzothiazole. After the trapping was
finished, the reaction mixture was heated at 75.degree. C. for 2
min and then 0.4 ml of the HPLC mobile phase was added to the
reaction mixture for HPLC purification. HPLC purification was
performed on a Waters Bondapak column (10 m, 7.8 mm ID.times.300
mm) using a mobile phase of acetonitrile/0.01 M H.sub.3PO.sub.4
(40/60) at a flow rate of 5.0 ml/min. The radioactive fraction
corresponding to [.sup.11C] PIB was collected in a bottle
containing 30 ml of pure water and passed through a C18 Sep-Pak
Plus cartridge, then washed with 10 ml of pure water, eluted with 1
ml of ethanol and 10 ml of sterile normal saline, and passed
through a 0.22 .mu.m sterile filter for quality analysis and animal
experiments. Radiochemical purity was greater than 99%, as
determined by analytical HPLC. The specific activity was 152.+-.52
GBq/.mu.mol at the end of the synthesis.
In vivo Small-Animal Positron Emission Tomography Imaging
[0142] All PET scans were performed using Triumph pre-clinical
tri-modality (LabPET/X-SPECT/X-O CT) imaging system (TriFoil
Imaging, USA), which provides 31 transaxial slices 1.175 mm
(center-to-center) apart, a 100 mm transaxial FOV, and a 37 mm
axial FOV for the LabPET sub-system. The digital APD detector
technology delivers high spatial resolution better than 1 mm and a
high recovery coefficient. Before the scans, all of the mice were
kept warm with a heating lamp. After induction with 2.0%
isoflurane, the mice were placed with their heads in the center of
the field of view and were fixed in the prone position. A 20 min
static data acquisition was performed in the 3D list mode with an
energy window of 350-650 keV at 20 min following a [.sup.11C] PIB
(36.7.+-.2.6 MBq; volume <0.25 ml) injection via the tail vein.
The emission data were normalized and corrected for the tracer
decay time. All list mode data were sorted into 3D sinograms, which
were then single-slice Fourier rebinned into 2D sinograms.
Summation images from 20 to 40 min after the [.sup.11C] PIB
injection was reconstructed using a MLEM algorithm, and the
resulting image volume consisted of 240.times.240.times.31 voxels,
with voxel size of 0.25.times.0.25.times.1,175 mm.sup.3.
PET Data Analysis
[0143] All imaging data were processed and analyzed with PMOD 3.5
software package (Pmod Technologies, Ziirich, Switzerland). The PET
image dataset were converted to an absolute measure of
radioactivity concentration (kBq/cc) using a phantom-derived
calibration factor before being normalized to the injected dose
(ID) of [.sup.11C] PIB and the body mass of the animal. This
normalization enabled the comparison of the brain radioactivity
concentration of animals of different weights. Static PET images
were co-registered with a mouse T2-weighted MRI brain atlas based
on PMOD as an anatomic reference. Image origins were set to Bregma
(0, 0) according to the MRI atlas and the atlas was used for VOI
definition. [.sup.11C] PIB uptake was evaluated in the 4 regions of
interest, namely: the cortex, the hippocampus, the amygdala, and
the olfactory bulb. Standardized uptake values (SUV) were obtained
for each VOI by dividing the mean [.sup.11C] PIB activity by the
injection dose and the body weight (gram in grams). Thereafter,
regional [.sup.11C] PIB uptake in the target region was normalized
by [.sup.11C] PIB uptake in the cerebellum which was taken as the
reference region (ratio to cerebellum). Manook et al., PLoS One
(2012) 7:e31310; Poisnel et al., Neurobiol Aging (2012)
33:2561-71.
Results
Affinity Plus Charge Modular Design for Therapeutic Peptides
[0144] A rational design to synthesize therapeutic peptides that
might reduce toxic misfolded protein/derivative across various
neurodegenerative diseases was proposed. The design was built on a
principle of modular assembly of sequences with affinity and
charge. The conjectured peptide was composed of an affinity
sequence derived from the self-aggregating region of the misfolded
protein/derivative flanked on one side or both sides by a stretch
of charged amino acids. The rationale behind this design was that
the affinity sequence would facilitate the binding of therapeutic
peptides to the misfolded protein/derivative because of its
propensity to aggregate; the charged sequence would then prevent
the therapeutic peptide itself and the misfolded protein/peptide
hybrid from aggregation by the repulsion force of charges (FIG. 1).
Theoretically, an amino acid with either a positive or negative
charge could achieve the goal. In the present design, positively
charged arginine was chosen because polyarginine (poly-Arg) had
been shown to facilitate the penetration of therapeutic peptides
into the cytoplasmic and nuclear compartments of target cells or
organs. Mitchell et al., J Pept Res (2000) 56:318-25.
Conformational Study of A.beta..sub.40, R.sub.8-A.beta.(25-35), and
.sup.DR.sub.8-A.beta.(25-35)
[0145] Polyethylenimine (PEI) cationized proteins can transduce
across cell membranes. Futami et al., J Biosci Bioeng (2005)
99:95-103; Futami et al., Expert Opin Drug Discov (2007) 2:261-9;
Kitazoe et al., J Biochem (2005) 137:693-701; Kitazoe et al.,
Biotechnol J (2010) 5:385-92; Murata et al., J Biochem (2008)
144:447-55; Murata et al., J Biosci Bioeng (2008) 105:34-8. PEI is
protease resistant and has higher charger density than a poly-Arg
or poly-lysines fragment. PEI-conjugated green fluorescent protein
could pass through the blood-brain barrier, and enter into brain by
intranasal administration. Loftus et al., Neurosci (2006)
139:1061-7.
[0146] To investigate the biophysical property of these peptides
concerning amyloid formation, A.beta..sub.40,
R.sub.8-A.beta.(25-35), and .sup.DR.sub.8-A.beta.(25-35) dissolved
in 20 mM sodium phosphate buffer with 150 mM KCl (pH 7) were
individually incubated at 25.degree. C. The circular dichroism (CD)
spectra were recorded at various time points as indicated. As
expected, the intensity of the negative ellipticity at 218 nm of
A.beta..sub.40 spectrum increased with time (FIG. 10, panel A),
consistent with its known ability to form amyloid fibrils as shown
by the transmission electron microscopy (TEM) (FIG. 10, panel D).
In contrast, the CD spectra of R.sub.8-A.beta.(25-35) showed
negative ellipticity at 200 nm, indicative of random coil structure
(FIG. 10, panel B). Similarly, the .sup.DR.sub.8-A.beta.(25-35)
also had CD spectra consistent with random coil structure, which
lacked strong negative ellipticity at 200 nm due to the presence of
eight D-form arginines in the peptide (FIG. 10, panel C). The CD
spectra of both peptides remained largely unchanged with the
incubation time. The TEM showed that R.sub.8-A.beta.(25-35) and
.sup.DR.sub.8-A.beta.(25-35) formed amorphous aggregates in the
same condition (FIG. 10, panels E and F). No amyloid fibrils were
observed.
[0147] To examine whether R.sub.8-A.beta.(25-35) or
.sup.DR.sub.8-A.beta.(25-35) could interfere with the
amyloidogenesis of A.beta..sub.40, the CD spectra of A.beta..sub.40
mixed with R.sub.8-A.beta.(25-35) or .sup.DR.sub.8-A.beta.(25-35)
at a 1:1 ratio were measured. As shown in FIG. 10, panels G and H,
the signal at 218 nm is hardly changed when R.sub.8-A.beta.(25-35)
or .sup.DR.sub.8-A.beta.(25-35) co-incubated with A.beta..sub.40.
The kinetic traces of amyloidogenesis in FIG. 10, panel I suggested
that the existence of R.sub.8-A.beta.(25-35) or
.sup.DR.sub.8-A.beta.(25-35) largely increase the lag time. After
long time incubation, the TEM images of the peptide mixtures showed
hybrids of fibrils and amorphous aggregates (FIG. 10, panels J and
K). These results showed that R.sub.8-A.beta.(25-35) and
.sup.DR.sub.8-A.beta.(25-35) could interact with A.beta..sub.40 and
interfere with its self-aggregation, hence significantly delay or
decrease the formation of A.beta..sub.40 amyloid fibrils.
Attenuation of A.beta..sub.40 cytotoxicity by
R.sub.8-A.beta.(25-35) and .sup.DR.sub.8-A.beta.(25-35)
[0148] To test whether R.sub.8-A.beta.(25-35) or
.sup.DR.sub.8-A.beta.(25-35) could attenuate the cytotoxicity of A
.beta..sub.40, the MTT assays were performed to measure the
viability of Neuro2a cells (a mouse neuroblastoma cell line)
treated with peptides as indicated. A.beta..sub.40 (.mu.M) exerted
significant cytotoxicity and only 30% of cells survived. By
contrast, both R.sub.8-A.beta.(25-35) and
.sup.DR.sub.8-A.beta.(25-35) had no detectable toxicity to the N2a
cells (FIG. 11, panel A). Both R.sub.8-A.beta.(25-35) and
.sup.DR.sub.8-A.beta.(25-35) decreased A.beta..sub.40 toxicity and
increased the cell viability from 30% to 70-75%. For comparison,
when A.beta.40 was mixed with A.beta. (25-35), no significant
change in cell viability was observed. The data showed that both
R.sub.8-A.beta.(25-35) and .sup.DR.sub.8-A.beta.(25-35) peptides
had therapeutic potential in amyloid-induced toxicity.
Therapeutic Effect of R.sub.8-A.beta.(25-35) in APP/PS1 Transgenic
Mice
[0149] To test the effect of R.sub.8-A.beta.(25-35) to prevent the
deterioration of memory in vivo, PEI or PEI-conjugated
R.sub.8-A.beta.(25-35) was given intranasally to wild-type or
APP/PS1 transgenic mice starting from 3-4 months of age. The water
maze assay was performed when the mice reached 8 months of age. As
shown in FIG. 12, panel A, the wild-type mice treated with PEI and
R.sub.8-A.beta.(25-35)-PEI showed no clear difference in the
learning curve of finding the hidden platform. The
R.sub.8-A.beta.(25-35)-treated APP/PS1 mice, on the other hand,
exhibited significantly faster learning than transgenic mice
treated with PEI. In addition, R.sub.8-A.beta.(25-35)
peptide-treated APP/PS1 mice performed better at the probe test
evidenced by more crossings of (FIG. 12, panel C) and longer time
spent in the quadrant where the probe used to be than those treated
with PEI (FIG. 12, panel B).
[0150] The change in the level of A.beta. peptide was next examined
via ELISA. As shown in FIG. 13, at 8 months of age, the level of
A.beta..sub.40 and A.beta..sub.42 decreased by 86% and 30%,
respectively in the cortex of R.sub.8-A.beta.(25-35)
peptide-treated APP/PS1 mice as compared with that of PEI-treated
transgenic mice. Similarly, the levels of A.beta..sub.40 and
A.beta..sub.42 decreased by 73% and 60%, respectively in the
hippocampus of the former mice compared with the latter.
[0151] Consistently, histological sections stained with Thioflavin
S chemifluorescent dye revealed deposition of multiple
green-fluorescent amyloid plaques in both cortex and hippocampus of
the 8 month-old PEI-treated APP.times.PS1 mice, which was
significantly reduced in the age-matched peptide-treated
APP.times.PS1 mice (FIG. 14, panel A). As shown in FIG. 14, panel
B, the number of plaques was significantly reduced in the cortex
and hippocampus of the peptide-treated mice compared with that in
the corresponding regions of the PEI-treated mice. The total area
of amyloid plaques was correspondingly decreased in the both cortex
and hippocampus of the peptide-treated mice vs. that in the
PEI-treated controls (FIG. 14, panel C). To further eliminate the
effect that might contribute to the positive results by the
variation in the size of individual brain sections, the percentage
of the area of the cortex and hippocampus that harbored amyloid
plaques was calculated; the peptide treatment again significantly
decreased the percentage of the plaque areas of both regions in
comparison with the PEI control treatment (FIG. 14, panel D).
Whether the size of individual plaques were altered by treatment
was next examined; as shown in FIG. 14, panel E. Although the
peptide treatment decreased the average size of individual plaques,
the difference failed to reach statistical significance. The data
indicated that the peptide treatment effectively slowed down the
accumulation of amyloid plaques and the clinical impairment of the
memory. Amyloid deposition induced neuroinflammation which
contributed importantly to the diseases in these mice.
R.sub.8-A.beta.(25-35)-PEI effectively decreased the level of
pro-inflammatory cytokines interleukin (IL)-6 and IL-1.beta. in the
cortex (FIG. 15).
Therapeutic Effect of Peptide after a Suspension for 4 Weeks
[0152] During the water maze tests, the treatment was adjourned for
about 4 weeks. To examine whether the therapeutic effect could be
maintained or resumed after a suspension of treatment,
administration of PEI or peptide was resumed and continued until
these mice reached 12 months of age. The burden of amyloid plaques
was quantified with microPET using the tracer Pittsburg compound B
(PiB). As shown in FIG. 16, panels A and B, the
R.sub.8-A.beta.(25-35) peptide-treated APP.times.PS1 mice had a
much lower signal in the cortex, hippocampus, amygdala and
olfactory bulb as compared with the PEI-treated mice, which is
consistent with a beneficial therapeutic effect at this age. ELISA
analyses revealed a decrease in total A.beta..sub.40 by 16% and 21%
in the cortex and hippocampus, respectively (FIG. 17, panels A and
D). No statistical difference was detected in the level of total
A.beta..sub.42. Consistent with the results of microPET (17-35%
reduction), the insoluble pools of A.beta..sub.40 and
A.beta..sub.42 were both decreased by 25-30% in the cortex or
hippocampus of the peptide-treated APP XPS/mice compared with those
in PEI-treated mice (FIG. 17, panels C and D).
Discussion
[0153] In this study, it was demonstrated that the peptide
R.sub.8-A.beta.(25-35) or its D-form counterpart could reduce the
formation of amyloid fibrils by A.beta..sub.40 peptide in vitro,
and amyloid plaques and disease manifestation in vivo. Intranasal
administration of R.sub.8-A.beta.(25-35) peptide also exhibited
beneficial therapeutic effect in APP/PS1 transgenic mice. Thus, the
data indicates that therapeutic peptides designed based on the
affinity/charge modular principle described herein would be
expected to be therapeutically effective in treating
neurodegenerative diseases, particularly those that involve
abnormal protein aggregation.
[0154] In the present design, the charged sequence not only
enhanced cell permeability and bioavailability of the bipartite
peptides, but also had an essential role in the peptidic therapy by
providing a repulsion force to prevent the misfolded target from
further aggregation. The charge moieties were introduced at both N-
and C-ends of the peptide. The N-terminal positive charges were
introduced by the guanido groups in the side-chains of poly-Arg;
while the C-terminal positive charges, by the chemical modification
of C-terminal carboxyl group with polyethylenimine. In addition, in
comparison with another A.beta. aggregation-inhibiting PEI-coupled
V24P(10-40) peptide, R.sub.8-A.beta.(25-35) performed better.
[0155] Although many therapeutic peptides have been designed, only
few of them were tested in vivo (Shukla et al., FASEB J (2013)
27:174-86; Frydman-Marom et al., Angew Chem Int Ed (2009)
48:1981-6; Funke et al., ACS Chem Neurosci (2010) 1:639-48;
Permanne et al., FASEB J (2002) 16:860-2; van Groen et al.,
ChemMedChem (2008) 3:1848-52). In this study, the feasibility of
the administration of therapeutic peptidic prodrugs through an
intranasal route was demonstrated. When combined with technology in
delivery, the study showed a proof of a therapeutic principle for
neurodegenerative diseases through intranasal delivery. The dose
used in this study was estimated to be 2 nmoles per day, which was
quite low compared with previous studies. Frydman-Marom et al.,
Angew Chem Int Ed (2009) 48:1981-6; Funke et al., ACS Chem Neurosci
(2010) 1:639-48; Permanne et al., FASEB J (2002) 16:860-2; van
Groen et al., ChemMedChem (2008) 3:1848-52.
[0156] Given the fact that amyloid plaques form extraordinarily
rapidly in vivo, and might even do so as fast as in 1-2 days
(Meyer-Luehmann et al., Nature (2008) 451:720-4), it was likely
that after a 4-week interruption of treatment, deposition of
amyloid plaques in the R.sub.8-A.beta.(25-35)-PEI-treated mice had
approached or reached the level in PEI-treated mice. It was
encouraging that the resumption of peptide treatment remained
beneficial in 12 month-old mice. These findings indicate further
prevention of amyloid plaques from deposition can be attained, even
after a period of interruption. In fact, the preliminary tests for
liver and kidney function indicated no clear toxicity in the mice
receiving the peptides. In sum, intranasal administration of the
designed bipartite peptides provided an effective and user-friendly
preventative and therapeutic approach to treat aggregation-caused
neurodegenerative diseases.
Example 3
Intranasal Delivery of a Bipartite Peptide Reduced Amyloid Burden
in the Brains of APP/PS1 Transgenic Mice
[0157] A "scavenger peptide", V24P(10-40), designed to decrease
A.beta. accumulation in the brain, was conjugated to
polyethylenimine (PEI), and tested as a preventive and/or
therapeutic strategy for Alzheimer's disease (AD) in this study.
This PEI-conjugated V24P(10-40) peptide was delivered intranasally
to the APP/PS1 double transgenic mice of 4 months of age as nasal
drops for four months. Compared with control values, peptide
treatment reduced the amount of A.beta. peptides by 72% of
A.beta.40 and 40% of A.beta.42 in the hippocampus, and by 87% of
A.beta.40 and 32% of A.beta.42 in the cortex. After treatment for 8
months, amyloid load, as quantified by Pittsburg compound B
microPET imaging, was decreased significantly in the hippocampus,
cortex, amygdala, and olfactory bulb. The data demonstrate that the
intranasally delivered scavenger peptide is effective in decreasing
A.beta. plaque formation in the brain. Nasal application of peptide
drops is user-friendly, and could be further developed for
preventative and therapeutic purposes with respect to AD and other
neurodegenerative diseases, including those described herein.
Materials and Methods
Synthesis of the PEI-Conjugated Peptide
[0158] All peptides were synthesized by the batch
fluorenylmethoxycarbonyl (fmoc)-polyamide method. Chen et al.,
Protein Sci (2001) 10:1794-1800. To generate PEI-conjugated
V24P(10-40), PEI was conjugated to the C-terminal carboxyl group of
the peptide, and the N-terminal group of V24P(10-40) was acetylated
to avoid dimerization or cyclization of the peptide during
PEI-conjugation reaction and to provide protection against
exopeptidases. Acetylated V24P(10-40) (4.8 mg) dissolved in 4 mL of
dimethyl sulfoxide was slowly mixed with 240 .mu.L of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (600 mM in 0.1 M MES,
0.5 M NaCl, pH 6), then 240 .mu.L of N-hydroxysuccinimide (1200 mM
in 0.1 M MES, 0.5 M NaCl, pH 6) was added, and the mixture was
reacted at room temperature for 30 min with gentle shaking (70
rpm). PEI (288 .mu.L) was added and the mixture was incubated
overnight at room temperature with gentle shaking (70 rpm). The
PEI-conjugated peptide, V24P(10-40)-PEI, was separated from
unreacted PEI and V24P(10-40) by reverse-phase HPLC.
Animal Experiment
[0159] APP/PS1 transgenic mice
(B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax), purchased from Jackson
Laboratories (USA), were bred and genotyped as described on the
Jackson website. Jankowsky et al., Biomol Eng (2001) 17:157-65;
Borchelt et al., Neuron (1996) 17:1005-13. The mice had access to
food and water ad libitum and were kept on a 12:12 h light-dark
cycle. PEI and V24P(10-40)-PEI were dissolved at a concentration of
400 .mu.M in 100 mM NaH.sub.2PO.sub.4, 138 mM KCl, pH 5, and
4-month-old mice was given 2.5 .mu.L of PEI (as the control) or
V24P(10-40)-PEI in each nostril six days per week for the indicated
period.
ELISA Assays for A.beta.40 and A.beta.42
[0160] Concentrations of A.beta.40 and A.beta.42 in mouse brain
homogenate were measured using ELISA kits (Invitrogen, Md., USA)
according to the manufacturer's instructions. Briefly, the cortical
or hippocampal tissue from PEI-treated or V24P(10-40)-PEI-treated
APP/PS1 mice was weighed and homogenized at 4 .degree. C. in the
cell extraction buffer provided in the kit, supplemented with
protease inhibitor cocktail (Sigma, St. Louis, USA), then the
homogenates in Eppendorf tubes were centrifuged at 13000 rpm at 4
.degree. C. for 10 min and the concentration of protein in the
supernatant was measured using the microBCA protein assay (Thermo,
Ill., USA). To perform the ELISA, the supernatants were diluted
10-fold and the A.beta.40 and A.beta.42 concentrations were
normalized to the protein concentration and expressed as ng/mg of
protein.
In vivo MicroPET
[0161] [.sup.11C] PIB was generated using .sup.[11C] methyltriflate
using a previously described method with minimal modification.
Manook et al., PLoS One (2012) 7:e31310. PET scans were performed
using a Triumph pre-clinical tri-modality (LabPET/X-SPECT/X-O CT)
imaging system (TriFoil Imaging, USA). The mice were kept warm with
a heating lamp before scanning. After induction with 2.0%
isoflurane, the mice were placed with their heads in the center of
the field of view, fixed in the prone position, and then freshly
synthesized [.sup.11C]PiB (36.7.+-.2.6 MBq; volume <0.25 mL) was
injected via the tail vein. After 20 min, static data acquisition
was performed for 20 min in 3D list mode with an energy window of
350-650 keV. The emission data were normalized and corrected for
the tracer decay time. All list mode data were sorted into 3D
sinograms, which were then single-slice Fourier rebinned into 2D
sinograms. Summation images from 20 to 40 min after [.sup.11C]PiB
injection were reconstructed using a MLEM algorithm.
[0162] All imaging data were processed and analyzed using the PMOD
3.5 software package (Pmod Technologies, Zurich, Switzerland). The
PET image dataset was converted to an absolute measure of
radioactivity concentration (kBq/cc) using a phantom-derived
calibration factor before being normalized to the injected dose of
[.sup.11C]PiB and the body mass of the animal. Static PET images
were co-registered with the mouse T2-weighted MRI brain atlas based
on PMOD as anatomic reference. Image origins were set to bregma (0,
0) according to the MRI atlas, which was also used for VOI
definition. [.sup.11C]PiB uptake in the cortex, hippocampus,
amygdala, and olfactory bulb was evaluated. Standardized uptake
values were obtained for each VOI by dividing the mean
[.sup.11C]PiB activity by the injection dose and the body weight
(in grams). Thereafter, the regional [.sup.11C]PiB uptake in the
target region was normalized to [.sup.11C]PiB uptake in the
cerebellum, taken as the reference region. Manook et al., PLoS One
(2012) 7:e31310; Poisnel et al., Neurobiol Aging (2012)
33:2561-71.
Results
[0163] Several mutated A.beta.40 peptides with different N- and
C-terminal truncations were designed and synthesized (Table 3).
Their structural properties were first examined by Circular
Dichroism (CD) Spectroscopy, the Thioflavin T (ThT) binding assay,
and Transmission Electron Microscopy (TEM), and then their effects
on the formation of A.beta.40 fibrils and A.beta.40 cytotoxicity
using a cell viability assay in mouse neuroblastoma Neuro2a (N2a)
cells was performed.
TABLE-US-00004 TABLE 3 The synthesized peptides used in this study.
DP represents D-proline Peptide Serpence A.beta.40 DAEFRHDSGY
EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV V24P(1-28) DAEFRHDSGY EVHHQKLVFF
AED.sup.DPGSNK V24P(10-40) Y EVHHQKLVFF AED.sup.DPGSNKGA IIGLMVGGVV
V24P(13-36) HHQKLVFF AED.sup.DPGSNKGA IIGLMV V24P(16-33) KLVFF
AED.sup.DPGSNKGA IIG V24P(19-30) FF AED.sup.DPGSNKGA
[0164] The sequences in Table 3, from top to bottom, correspond to
SEQ ID NOs: 20-25.
C-Terminal-Truncated Peptide V24P(1-28) According to the structural
model of the Al3 40 fibrils (Petkova et al., Proc Natl Acad Sci
(2002) 99:16742-7), K28 is located at the beginning of the second
.beta. -strand. Without the C-terminal hydrophobic tail following
K28, V24P(1-28) had increased hydrophilicity compared with A .beta.
40. V24P(1-28) was dissolved in 20 mM sodium phosphate buffer, pH
7, containing 150 mM KCl (incubation buffer) and then incubated at
25.degree. C. At different time points (Day 0 was the time
immediately after dissolution), its CD spectra and fluorescence
spectra after ThT binding were recorded. The CD spectrum of the 30
.mu.M V24P(1-28) solution was typical of a random coil structure
and was identical at all tested time points (0-12 days) (FIG. 18,
panel A, left); consistently, the measurement of fluorescence
showed no ThT binding (FIG. 18, panel B, left). When the peptide
concentration was increased to 60 .mu.M, V24P(1-28) remained as a
random structure at days 0-3, but gradually formed a .beta.-sheet
structure (FIG. 18, panel A, right), which bound ThT, as shown by
the increased fluorescence (FIG. 18, panel B, right). TEM showed
that 60 .mu.M V24P(1-28) formed amyloid fibrils (FIG. 18, panel E,
left). The fibrils were straight and laterally associated, in
contrast with the twisted morphology commonly reported for A .beta.
40 fibrils. Chang et al., J Mol Biol (2009) 385:1257-65; Meinhardt
et al., J Mol Biol (2009) 386:869-77; Goldsbury et al., J Struct
Biol (2000) 130:217-31.
N-Terminal-Truncated Peptide V24P(10-40)
[0165] The N-terminal region of A.beta. 40 is hydrophilic and was
reported not to be involved in the amyloid cross-.beta. structure.
Petkova et al., Proc Natl Acad Sci USA (2002) 99:16742-7. To verify
whether this hydrophilic segment was important in the design of a
peptide inhibitor, a V24P mutant peptide, denoted V24P(10-40), with
the first 9 N-terminal amino acid residues truncated was chemically
synthesized and dissolved in the incubation buffer. Unlike 30 .mu.M
V24P forming a random coil structure (Chang et al., J Mol Biol
(2009) 385:1257-65), the CD spectra of 30 .mu.M V24P(10-40)
exhibited a characteristic .beta. -sheet signal evidenced by
negative ellipticity at 218 nm (FIG. 18, panel C, left) and,
consistently, fluorescence emission at 487 nm appeared in the ThT
binding assay (FIG. 18, panel D, left). These data showed that
V24P(10-40) was more prone to aggregate than V24P. At the
concentration of 60 .mu.M, more .beta. -aggregates were formed
(FIG. 18, panels C and D, right). TEM showed that V24P(10-40)
formed amorphous aggregates (FIG. 18, panel E, right).
Structural Studies on Shorter Peptide
[0166] In order to determine the shortest sequence for
self-aggregation, an additional three peptides with shorter
lengths, V24P(13-36), V24P(16-33), and V24P(19-30) were designed,
and their CD spectra and fluorescence spectra after binding ThT
immediately after dissolution at concentrations of 30, 60, and 90
.mu.M were examined.
[0167] The CD spectra showed that V24P(13-36) formed a random coil
structure at 30 .mu.M (FIG. 19, panel A, top) and the ThT
fluorescence spectra showed that .beta.-aggregates formed at 90
.mu.M (FIG. 19, panel B, top). V24P(13-36) lacked several
hydrophobic residues (Y10,V12,V39,V40) present in the V24P(10-40),
and thus required a higher peptide concentration (90 .mu.M) to form
.beta.-aggregates compared with V24P (60 .mu.M) or V24P(10-40) (30
.mu.M).
[0168] In contrast, the CD spectrum of V24P(16-33) showed a strong
peak at 204 nm and a trough at 228 nm (FIG. 19, panel A, center
panel), a pattern indicative of a type I .beta.-turn (Kelly et al.,
Curr Protein Pept Sci (2000) 1:349-84; Kelly et al., BBA-Protein
Proteom (2005) 1751:119-39). The weak fluorescence of 90 .mu.M
V24P(16-33) suggested that only a small amount of .beta.-aggregates
formed (FIG. 19, panel B, center).
[0169] V24P(19-30) showed a random coil structure. When the peptide
concentration was 60 .mu.Mor higher, the CD spectra showed a peak
at around 220 nm (FIG. 19, panel A, bottom), similar to that of an
extended 3.sub.10 helix or a poly(Pro) II helix (Kelly et al.,
BBA-Protein Proteom (2005) 1751:119-3930). No fluorescence emission
at 487 nm was observed at any concentration (FIG. 19, panel B,
bottom), suggesting that L17, V18, I31, and 132 were responsible
for ThT binding to V24P(16-33) aggregates.
Selection of Scavenger Peptide
[0170] A single substitution (V24.fwdarw..sup.DP) in A.beta.40
dramatically affects the structural behavior and decreases the
toxicity of A.beta. 40. (Chang et al., J Mol Biol (2009)
385:1257-65). FIG. 20, panel A shows that all of the
.sup.DP-containing peptides tested were much less cytotoxic than
A.beta. 40. Compared with V24P, V24P(10-40) formed .beta.
-aggregates at a lower peptide concentration, suggesting that it
had a higher propensity to aggregate and might have a greater
inhibitory effect on A.beta. 40 cytotoxicity. In the cytotoxicity
assay, A.beta. 40 peptide, when mixed with V24P(10-40) at an
equi-molar ratio, had the lowest cytotoxicity. FIG. 20, panel B.
Therefore, V24P(10-40) was chosen as the scavenger peptide in the
following animal studies.
The Inhibitory Effect of V24P(10-40) on Amyloid Formation was
Specific for A.beta.40
[0171] Many peptide inhibitors were designed based on a short
recognition sequence, such as "KLVFF" (SEQ ID NO: 26), but the
target specificity of this sequence for different amyloid-forming
peptides had rarely been examined. Therefore, hamster prion peptide
PrP(108-144) was used to explore the association specificity of
V24P(10-40). PrP(108-144) formed amyloid fibrils when incubated in
20 mM NaOAc buffer, pH 3.7/140 mM NaCl. Chen et al., Proc Natl Acad
Sci USA (2002) 99:12633-8. As shown in FIG. 21, whether equimolar
V24P(10-40) was added or not, the fluorescence intensity increase
in the ThT binding assay showed that PrP(108-144) formed amyloid
fibrils during incubation, and V24P(10-40) failed to inhibit
amyloid formation of PrP(108-144). These results indicated that the
inhibitory effect of V24P(10-40) was target-specific.
Scavenger Peptide V24P(10-40) Decreased A.beta. Accumulation in the
Brains of APP/PS1 Transgenic Mice
[0172] In the design of peptide inhibitors, D-form amino acids, end
capping, and methylation of amide hydrogens are often used to
combat digestions by exopeptidases and endopeptidases in the serum
in order to extend the lifetime of the peptide in vivo. Findeis et
al., Biochem (1999) 38:6791-800; Gordon et al., Biochem (2001)
40:8237-45; Gordon et al., J Pept Res (2002) 60:37-55.
Modifications to putrescine, a naturally occurring polyamine, have
been used to increase the ability of a designed peptide
D-YiA.beta.11 to cross the blood brain barrier. Poduslo et al., J
Neurobiol (1999) 39:371-82. Polyethylenimine (PEI) cationized
proteins were also found to be able to cross the cell membrane.
Futami et al. J. Biosci. Bioeng. (2005) 99(2):95-103; Futami et
al., Expert. Opin. Drug. Discov. (2007) 2:261-269; Kitazoe et al.,
J. Biochem. (2005) 137:693-701; Kitazoe et al., Biotechnol. J.
(2010) 5:385-392; Murata et al., J. Biochem. (2008) 144:447-455;
Murata et al., J. Biosci. Bioeng. (2008) 105:34-38.
[0173] In this study, PEI was added to the C-terminus of the
scavenger peptide V24P(10-40). 4-month-old APP/PS1 transgenic mice
were administered with V24P(10-40)-PEI or PEI alone by nasal drops
to both nostrils (1 nmole or 4 .mu.g per nostril) six times per
week. The mice were then sacrificed 4 months later and the A .beta.
contents of the brain were analyzed by ELISA. As shown in FIG. 22,
the mice treated with V24P(10-40)-PEI clearly had decreased A
.beta.40 and A .beta.42 levels in both the cortex (panel A) and
hippocampus (panel B), the effect being greater with A.beta.40,
probably because the scavenger peptide does not contain residues 41
and 42.
[0174] The effect of the scavenger peptide on reducing amyloid
plaque accumulation was examined in APP/PS1 mice treated with
V24P(10-40)-PEI or PEI from the age of 4 months to 12 months, using
micro positron emission tomography (microPET) with the radiotracer
.sup.11C-labeled Pittsburg compound B ([.sup.11C]PiB), which binds
to A .beta. plaques. The microPET images (FIG. 23, panel A) taken
at the end of treatment showed that A .beta. plaque deposition was
decreased significantly by the peptide treatment. Quantification of
[.sup.11C] PiB levels in the cortex, hippocampus, amygdala, and
olfactory bulb in APP/PS1 mice with or without peptide treatment
revealed that peptide treatment resulted in a significant reduction
in amyloid plaque formation in all four brain regions (FIG. 23,
panel B).
Discussion
[0175] All the peptides with the V24.fwdarw..sup.DP replacement
were less toxic than A.beta.40 for N2a cells, suggesting that V24
is an important residue for A.beta.40 to form toxic species. In
addition, the data showed that, without the C-terminal hydrophobic
tail, V24P(1-28) still retained the ability to form amyloid fibrils
as an A.beta. 40 peptide, whereas peptides without the N-terminal
hydrophilic tail increased the aggregation propensity of
V24P(10-40) to form amyloid-like .beta.-aggregates. Shorter
peptides V24P(13-36), V24P(16-33), and V24P(19-30) had lower
aggregation propensity and a higher peptide concentration was
required for them to form amyloid-like .beta.-aggregates.
[0176] The results of these peptide inhibitors tested in the
transgenic mouse models described herein were summarized and
compared in Table 4. In the present study, non-invasive intranasal
administration and less peptide (.about.200 .mu.g of
V24P(10-40)-PEI per mouse per month) were used. Treatment for four
months resulted in a reduction of 72% and 40% in the A.beta.40 and
A.beta.42 content of the hippocampus, respectively. In terms of
total peptide used in transgenic mouse models, 0.8 mg of
V24P(10-40)-PEI was used, while previous studies used 2.5 mg of
iA.beta.5p (icy infusion), 24 mg of iA.beta.5p (ip), 0.27 mg of D3
(hippocampal infusion), 28-56 mg of D3 (oral), or 3 mg of
D-Trp-Aib. The present peptide markedly reduced both the A.beta.
level and amyloid accumulation in the brain at a much lower dosage
when given via a non-invasive route. Thus, the present data shows
the therapeutic effects of the bipartite peptides described herein
for delaying the onset and/or reducing the progression of AD.
[0177] Questions have been raised about peptide therapy in terms of
immunogenicity, low stability, low solubility, poor
bioavailability, and low BBB permeability. The present study proved
that intranasal administration is an effective method of delivering
peptide drugs into the brain. Since this administration route is
much more user-friendly than the invasive routes such as
intracerebral infusion or ip injection, peptide drugs can be given
daily in this manner. Intranasal peptide administration could be
used to deliver other peptide inhibitors targeting other functions
in the brain, for example, the 24-aa peptide TFPS derived from p35
(the cdk5 activator) which has been reported to reduce cdk5
hyperactivation and inhibit tau hyperphosphorylation in mice via ip
injection. Shukla et al., FASEB J (2013) 27:74-86.
TABLE-US-00005 TABLE 4 Comparison of different peptides used in APP
transgenic mice. Administration route and mouse age at Total Total
A.beta. in start and end of peptide peptide hippocampus Peptide
treatment.sup.a (mg) (.mu.mol) (% of control) V24P (10-40)-
Intranasal delivery 0.8 0.2 A.beta.40 28%.sup.b PEI 6 times a week
for 4 A.beta.42 60% months; 4-m to 8-m V24P (10-40)- Intranasal
delivery 1.6 0.4 81%.sup.c PEI 6 times a week for 8 months; 4-m to
12-m iA.beta.5p (40) Icv infusion for 8 2.5 3.6 33%.sup.d weeks;
6/7-m to 8/9-m iA.beta.5p (40) Ip injection 24 34.6 54%.sup.d 3
times a week for 8 weeks; 8/9-m to 10/11-m D3 (42) Hippocampal 0.27
0.17 67%.sup.d infusion for 30 days; 8-m to 9-m D3 (43) Oral daily
for 8 28-56 17.5-35 68%.sup.d weeks; 4-m to 6-m D-Trp- Ip injection
3 10.4 53%.sup.e Aib (44) 3 times daily for 120 days; 4.5-m to
8.5-m .sup.aintracerebroventricular (icv); intraperitoneal (ip)
.sup.bquantified by ELISA .sup.cquantified by microPET
.sup.dquantified by immunohistochemistry .sup.equantified by
thioflavin S staining (40) Permanne et al. FASEB J. (2002) 16(8):
860-862. (42) van Groen et al., ChemMedChem (2008) 3: 1848-1852.
(43) Funke et al. ACS Chem. Neurosci. (2010) 1(9): 639-648. (44)
Frydman-Marom et al., Angew. Chem. Int. Ed. (2009) 48:
1981-1986.
[0178] (Relevant teachings of these references are incorporated
herein by reference.)
[0179] MicroPET images showed that THE amyloid load in the
olfactory bulb was significantly decreased. It has been reported
that senile plaques and neurofibrillary tangles accumulate in the
olfactory bulb and olfaction is damaged in the early stage of AD.
Christen-Zaech et al., Can J Neurol Sci (2003) 30:20-5; Arnold et
al., Ann Neurol (2010) 67:462-9. In APP transgenic mice,
non-fibrillar A.beta. deposition can be detected in the olfactory
bulb earlier than in other brain regions, and olfactory dysfunction
correlates with A .beta. burden. Wesson et al., J Neurosci (2010)
30:505-14. Thus, A .beta. deposition in the olfactory epithelium
may serve as a biomarker for identifying AD patients at the
preclinical stage. Attems et al., Acta Neuropathol (2014)
127:459-75. Since the present peptide was delivered to the brain
intranasally, it has the advantage of inhibiting A.beta. deposition
in the olfactory bulb at the early stage of AD progression.
OTHER EMBODIMENTS
[0180] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0181] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
Sequence CWU 1
1
26111PRTArtificial SequenceSynthetic Polypeptide 1Gly Ser Asn Lys
Gly Ala Ile Ile Gly Leu Met 1 5 10 232PRTArtificial
SequenceSynthetic Polypeptide 2Tyr Glu Val His His Gln Lys Leu Val
Phe Phe Ala Glu Asp Asp Pro 1 5 10 15 Gly Ser Asn Lys Gly Ala Ile
Ile Gly Leu Met Val Gly Gly Val Val 20 25 30 319PRTArtificial
SequenceSynthetic Polypeptide 3Arg Arg Arg Arg Arg Arg Arg Arg Gly
Ser Asn Lys Gly Ala Ile Ile 1 5 10 15 Gly Leu Met 419PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(19)..(19)Methionine
polyethylenimine-conjugated 4Arg Arg Arg Arg Arg Arg Arg Arg Gly
Ser Asn Lys Gly Ala Ile Ile 1 5 10 15 Gly Leu Met 531PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(15)..(15)Xaa is the
D-form of prolineMISC_FEATURE(31)..(31)Valine is
polyethylenimine-conjugated 5Tyr Glu Val His His Gln Lys Leu Val
Phe Phe Ala Glu Asp Xaa Gly 1 5 10 15 Ser Asn Lys Gly Ala Ile Ile
Gly Leu Met Val Gly Gly Val Val 20 25 30 620PRTArtificial
SequenceSynthetic Polypeptide 6Arg Arg Arg Arg Arg Arg Arg Arg Trp
Asp Gln Gln Gln Gln Gln Gln 1 5 10 15 Gln Gln Gln Gln 20
725PRTArtificial SequenceSynthetic Polypeptide 7Arg Arg Arg Arg Arg
Arg Arg Arg Trp Asp Gln Gln Gln Gln Gln Gln 1 5 10 15 Gln Gln Gln
Gln Gln Gln Gln Gln Gln 20 25 812PRTArtificial SequenceSynthetic
Polypeptide 8Arg Pro Arg Thr Arg Leu His Thr His Arg Asn Arg 1 5 10
98PRTArtificial SequenceSynthetic Polypeptide 9Arg Arg Arg Arg Arg
Arg Arg Arg 1 5 1012PRTArtificial SequenceSynthetic Polypeptide
10Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln 1 5 10
1110PRTArtificial SequenceSynthetic Polypeptide 11Arg Arg Arg Arg
Arg Arg Arg Arg Trp Asp 1 5 10 1215PRTArtificial SequenceSynthetic
Polypeptide 12Arg Arg Arg Arg Arg Arg Arg Arg Trp Asp Gln Gln Gln
Gln Gln 1 5 10 15 1320PRTArtificial SequenceSynthetic Polypeptide
13Arg Arg Arg Arg Arg Arg Arg Arg Trp Asp Gln Gln Gln Gln Gln Gln 1
5 10 15 Gln Gln Gln Gln 20 1425PRTArtificial SequenceSynthetic
Polypeptide 14Arg Arg Arg Arg Arg Arg Arg Arg Trp Asp Gln Gln Gln
Gln Gln Gln 1 5 10 15 Gln Gln Gln Gln Gln Gln Gln Gln Gln 20 25
1530PRTArtificial SequenceSynthetic Polypeptide 15Arg Arg Arg Arg
Arg Arg Arg Arg Trp Asp Gln Gln Gln Gln Gln Gln 1 5 10 15 Gln Gln
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 20 25 30
1619PRTArtificial SequenceSynthetic Polypeptide 16Arg Gln Gln Arg
Arg Gln Gln Asp Gln Arg Gln Trp Gln Arg Gln Arg 1 5 10 15 Gln Gln
Arg 1710PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Arginine is TAMRA-tagged 17Arg Arg
Arg Arg Arg Arg Arg Arg Trp Asp 1 5 10 1820PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(1)..(1)Arginine is
TAMRA-tagged 18Arg Arg Arg Arg Arg Arg Arg Arg Trp Asp Gln Gln Gln
Gln Gln Gln 1 5 10 15 Gln Gln Gln Gln 20 1920PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(1)..(1)Arginine is
TAMRA-tagged 19Arg Gln Gln Arg Arg Gln Gln Asp Gln Arg Gln Trp Gln
Arg Gln Arg 1 5 10 15 Gln Gln Arg Arg 20 2040PRTArtificial
SequenceSynthetic Polypeptide 20Asp Ala Glu Phe Arg His Asp Ser Gly
Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp
Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly
Gly Val Val 35 40 2128PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(24)..(24)Xaa is the D-form of proline 21Asp
Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10
15 Leu Val Phe Phe Ala Glu Asp Xaa Gly Ser Asn Lys 20 25
2231PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(15)..(15)Xaa is the D-form of proline 22Tyr
Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Xaa Gly 1 5 10
15 Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 20
25 30 2324PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(12)..(12)Xaa is the D-form of proline 23His
His Gln Lys Leu Val Phe Phe Ala Glu Asp Xaa Gly Ser Asn Lys 1 5 10
15 Gly Ala Ile Ile Gly Leu Met Val 20 2418PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(9)..(9)Xaa is the D-form
of proline 24Lys Leu Val Phe Phe Ala Glu Asp Xaa Gly Ser Asn Lys
Gly Ala Ile 1 5 10 15 Ile Gly 2512PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(6)..(6)Xaa is the D-form of proline 25Phe
Phe Ala Glu Asp Xaa Gly Ser Asn Lys Gly Ala 1 5 10 265PRTArtificial
SequenceSynthetic Polypeptide 26Lys Leu Val Phe Phe 1 5
* * * * *