U.S. patent application number 14/785204 was filed with the patent office on 2016-03-10 for treatment agent for cognitive impairment induced by amyloid beta-protein, therapeutic agent for alzheimer's disease, and treatment method and pathological analysis method related to these.
This patent application is currently assigned to NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY. The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY, NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY. Invention is credited to Saori Ban, Tsuyoshi Inoue, Toshiharu Suzuki.
Application Number | 20160067306 14/785204 |
Document ID | / |
Family ID | 51731374 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160067306 |
Kind Code |
A1 |
Inoue; Tsuyoshi ; et
al. |
March 10, 2016 |
TREATMENT AGENT FOR COGNITIVE IMPAIRMENT INDUCED BY AMYLOID
BETA-PROTEIN, THERAPEUTIC AGENT FOR ALZHEIMER'S DISEASE, AND
TREATMENT METHOD AND PATHOLOGICAL ANALYSIS METHOD RELATED TO
THESE
Abstract
The present invention aims to provide an anti-Alzheimer's
disease agent based on an action mechanism associated with amyloid
.beta. protein, which action mechanism is different from
conventional action mechanisms. The therapeutic drug for
Alzheimer's disease according to the present invention contains a
therapeutic agent for cognitive impairment induced by amyloid
.beta. protein, which therapeutic agent comprises a peptide having
the amino acid sequence represented by SEQ ID NO:1 or a peptide
similar to this peptide, especially a peptide containing the amino
acid sequence represented by SEQ ID NO:2, which is a partial
sequence of SEQ ID NO:1. TABLE-US-00001 (SEQ ID NO: 1)
VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT (SEQ ID NO: 2)
HRGHQPPPEMA
Inventors: |
Inoue; Tsuyoshi;
(Okayama-shi, Okayama, JP) ; Suzuki; Toshiharu;
(Sapporo-shi, Hokkaido, JP) ; Ban; Saori;
(Sapporo-shi, Hokkaido, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY |
Okayama-shi, Okayama
Sapporo-shi, Hokkaido |
|
JP
JP |
|
|
Assignee: |
NATIONAL UNIVERSITY CORPORATION
HOKKAIDO UNIVERSITY
Sapporo-shi, Hokkaido
JP
NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
Okayama-shi, Okayama
JP
|
Family ID: |
51731374 |
Appl. No.: |
14/785204 |
Filed: |
April 15, 2014 |
PCT Filed: |
April 15, 2014 |
PCT NO: |
PCT/JP2014/060665 |
371 Date: |
October 16, 2015 |
Current U.S.
Class: |
800/3 ; 435/29;
514/17.8; 530/324; 530/327 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 38/00 20130101; A61P 25/28 20180101; A61P 25/00 20180101; C07K
7/08 20130101; C07K 7/06 20130101; C07K 14/4711 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 19, 2013 |
JP |
2013-088319 |
Claims
1. A therapeutic agent for cognitive impairment induced by amyloid
.beta. protein, comprising as an effective component a peptide
corresponding to any one of the following (I) to (VI): (I) a
peptide consisting of the amino acid sequence represented by SEQ ID
NO:1: TABLE-US-00006 (SEQ ID NO: 1)
VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT;
(II) a peptide consisting of the same amino acid sequence as the
amino acid sequence represented by SEQ ID NO:1 except that one to
three amino acids are altered by any one or more of deletion,
addition, substitution, and side-chain modification; (III) a
peptide consisting of a partial sequence of the amino acid sequence
represented by SEQ ID NO:1, comprising the amino acid sequence
represented by SEQ ID NO:2: TABLE-US-00007 HRGHQPPPEMA; (SEQ ID NO:
2)
(IV) a peptide consisting of the same amino acid sequence as a
partial sequence of the amino acid sequence represented by SEQ ID
NO:1 comprising the amino acid sequence represented by SEQ ID NO:2,
except that one or two amino acids are altered by any one or more
of deletion, addition, substitution, and side-chain modification;
(V) a peptide consisting of an amino acid sequence comprising the
amino acid sequence represented by SEQ ID NO:2, wherein a total of
1 to 50 amino acids are added to the N-terminus and/or the
C-terminus of the amino acid sequence represented by SEQ ID NO:2
(excluding amino acid sequences which are identical to all or part
of the amino acid sequence represented by SEQ ID NO:1); and (VI) a
peptide consisting of an amino acid sequence comprising the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO:2 except that one or two amino acids are altered by any one
or more of deletion, addition (excluding addition to the N-terminus
and/or the C-terminus), substitution, and side-chain modification,
wherein a total of 1 to 50 amino acids are added to the N-terminus
and/or the C-terminus of said altered amino acid sequence.
2. A therapeutic drug for Alzheimer's disease, comprising the
therapeutic agent according to claim 1.
3. A method for treatment of cognitive impairment induced by
amyloid .beta. protein, comprising a step of administering the
therapeutic agent according to claim 1 to a mammal (excluding
human) which developed cognitive impairment induced by amyloid
.beta. protein or to a model animal thereof.
4. A method for treatment of Alzheimer's disease, comprising a step
of administering the therapeutic drug according to claim 2 to a
mammal (excluding human) which developed Alzheimer's disease or to
a model animal thereof.
5. A method for pathological analysis of cognitive impairment
induced by amyloid .beta. protein, comprising a step of
administering a peptide corresponding to any one of the above (I)
to (VI) to a mammal (excluding human) which developed cognitive
impairment induced by amyloid .beta. protein or to a model animal
thereof, or a step of adding said peptide to a cultured nerve
cell(s) as a model cell(s) thereof or to a biomolecule(s) expressed
in a nerve cell(s).
6. A method for pathological analysis of Alzheimer's disease,
comprising a step of administering a peptide corresponding to any
one of the above (I) to (VI) to a mammal (excluding human) which
developed Alzheimer's disease or to a model animal thereof, or a
step of adding said peptide to a cultured nerve cell(s) as a model
cell(s) thereof or to a biomolecule(s) expressed in a nerve
cell(s).
Description
TECHNICAL FIELD
[0001] The present invention relates to a peptide having a
therapeutic effect on cognitive impairment induced by amyloid
.beta. protein, which is a symptom of Alzheimer's disease, and use
of the peptide.
BACKGROUND ART
[0002] Alzheimer's disease is a nervous disease which exhibits a
decline in cognitive function (for example, memory disorder) as a
main symptom. Although Alzheimer's disease frequently develops in
the elderly, there is not effective therapeutic drug, and this has
become a big problem in developed countries in which the society is
aging. In postmortem brains of patients with Alzheimer's disease,
senile plaques are found. These are known to be aggregates of
"amyloid .beta. protein". The fact that amyloid .beta. protein is
the main cause of Alzheimer's disease is widely accepted based on a
number of studies.
[0003] Amyloid .beta. protein is produced from its precursor,
"amyloid precursor protein". Amyloid precursor protein is a
membrane protein present on the nerve cell membrane. In normal
brain, cleavage of the extracellular domain of amyloid precursor
protein with .alpha.-secretase followed by cleavage of the domain
inside the cell membrane with .gamma.-secretase causes production
and extracellular release of a peptide called p3. This is called
"non-amyloidgenic pathway", and amyloid .beta. protein is not
produced in the pathway (Non-patent Document 1). In normal brain,
only several percent of the extracellular domain undergoes cleavage
by .beta.-secretase, to cause extracellular secretion of amyloid
.beta. protein. On the other hand, it has been thought that the
brain of a patient developing Alzheimer's disease exhibits
increased production of amyloid .beta. protein, or production of a
molecular species of amyloid .beta. protein having higher
aggregability. Due to the property of amyloid protein to cause
aggregation, senile plaques (amyloid aggregates) are finally formed
inpatients with Alzheimer's disease, and these senile plaques are
found as deposition in the brain.
[0004] After being cleaved out, amyloid .beta. protein gradually
aggregates to finally form senile plaques (amyloid aggregates). The
"oligomer (which is composed of several amyloid .beta. protein
units associated with each other)" produced during the aggregation
process is known to have strong neurotoxicity. From both in vitro
and in vivo studies, the oligomer of amyloid .beta. protein is
reported to inhibit synaptic plasticity, which is a neurological
phenomenon indispensable for memory and learning (Non-patent
Documents 2 and 3). It is also reported that administration of the
oligomer into the brain of a mouse causes loss of memory and
learning ability (Non-patent Documents 4 and 5). A recent study
also reported that long-term exposure to the oligomer causes
neuronal death and the like (Non-patent Document 6). The oligomer
is therefore attracting attention as a causative substance for
development of Alzheimer's disease.
[0005] Thus, in spite of the fact that amyloid .beta. protein is
widely recognized as a cause of Alzheimer's disease, the
therapeutic drugs for Alzheimer's disease that are clinically used
at present are not designed such that they act on amyloid .beta.
protein. More specifically, donepezil (Patent Document 1) and
memantine (Patent Document 2), which are used as therapeutic drugs
for Alzheimer's disease, act as an acetylcholine esterase inhibitor
and an NMDA receptor inhibitor, respectively, and do not interact
with amyloid .beta. protein. Since these are the so called
symptomatic drugs, they have no drastic action to ameliorate
Alzheimer's disease at present.
[0006] Under such circumstances, development of novel therapeutic
drugs for Alzheimer's disease based on the action mechanism
(associated with amyloid protein) is in progress. In an attempt to
suppress production of amyloid .beta. protein from amyloid
precursor protein, secretase regulating agents are being developed
(Non-patent Document 1). In particular, development of
.gamma.-secretase inhibitors is most rapidly progressing, and
Semagacestat (Patent Document 3), Begacestat (Patent Document 4),
and the like have been clinically developed one after another.
Another therapeutic method which is drawing attention is an
antibody therapy based on recognition and elimination of amyloid
.beta. protein in the brain by an antibody. Bapineuzumab (Patent
Document 5), solanezumab, and the like have been actually
clinically developed so far.
[0007] However, the therapeutic drugs for Alzheimer's disease
having action mechanisms associated with amyloid protein which are
mainly developed at present are known to have problems. In terms of
.gamma.-secretase inhibitors, it is known that the substrate for
.gamma.-secretase is originally not limited to amyloid precursor
protein, and that there are about 100 kinds of its substrates
(Non-patent Document 7). Since the Notch receptor, which is
important for cell differentiation, is included in such substrates
(Non-patent Document 8), there is a concern that side effects may
occur. Actually, development of Semagacestat, which had been a
major candidate for a .gamma.-secretase inhibitor, was stopped
after the phase III clinical trial in 2010. The antibody therapy
using an anti-amyloid .beta. antibody may cause angiitis, vasogenic
cerebral edema, and/or the like. Actually, development of
Bapineuzumab, which had been a promising agent for antibody
therapy, was stopped in 2012.
[0008] Some .gamma.-secretase substrates are known to release their
extracellular domains after cleavage, similarly to the amyloid
peptide (Non-patent Documents 9 and 10). Since these peptides can
be used as indices of .gamma.-secretase, there are several
inventions that attempt to use the cleaved fragments as biomarkers
for Alzheimer's disease (Patent Documents 6 and 7). It is also
known that the membrane protein called Alcadein-.beta. in the
living body is cleaved both by .gamma.-secretase and by
.alpha.-secretase, resulting in production of a peptide composed of
37 amino acids (VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT) (Non-patent
Document 11).
[0009] However, an idea to use such a peptide as a regulating agent
for Alzheimer's disease is difficult to expect under the current
levels of knowledge and technology, and, accordingly, there has
been no report on such an idea.
PRIOR ART DOCUMENTS
Patent Documents
[0010] [Patent Document 1] EP 0296560 A2 [0011] [Patent Document 2]
U.S. Pat. No. 3,391,142 A [0012] [Patent Document 3] WO 2002/40451
A2 [0013] [Patent Document 4] US 2004/198778 A1 [0014] [Patent
Document 5] WO 2009/017467 A1 [0015] [Patent Document 6] U.S. Pat.
No. 7,666,982 B2 [0016] [Patent Document 7] U.S. Pat. No. 7,807,777
B2
Non-Patent Documents
[0016] [0017] [Non-patent Document 1] De Strooper et al., Nat Rev
Neurol 6, 99-107, 2010 [0018] [Non-patent Document 2] Lambert et
al., Proc Natl Acad Sci USA 95, 6448-6453, 1998 [0019] [Non-patent
Document 3] Walsh et al., Nature 416, 535-539, 2002 [0020]
[Non-patent Document 4] Cleary et al., Nat Neurosci 8, 79-84, 2005
[0021] [Non-patent Document 5] Balducci et al., Proc Natl Acad Sci
USA 107, 2295-2300, 2010 [0022] [Non-patent Document 6] Brouillette
et al., J Neurosci 32, 7852-7861, 2012 [0023] [Non-patent Document
7] Haapasalo and Kovacs, J Alzheimers Dis 25, 3-28, 2011 [0024]
[Non-patent Document 8] De Strooper et al., Nature 398, 518-522,
1999 [0025] [Non-patent Document 9] Okochi et al., EMBO J 21,
5408-5416, 2002 [0026] [Non-patent Document 10] Araki et al., J
Biol Chem 279, 24343-24354, 2004 [0027] [Non-patent Document 11]
Hata et al., J Biol Chem 284, 36024-36033, 2009
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0028] Under the circumstances described above, an anti-Alzheimer's
disease agent based on an action mechanism associated with amyloid
.beta. protein, which action mechanism is different from
conventional action mechanisms, has been demanded, and the present
invention aims to provide such an anti-Alzheimer's disease
agent.
Means for Solving the Problems
[0029] As described above, .gamma.-secretase is known to cleave
membrane proteins other than amyloid precursor protein. The present
inventors made a daring hypothesis that peptide fragments
themselves "other than" the amyloid .beta. protein, produced by
cleavage by .gamma.-secretase might exhibit actions to ameliorate
Alzheimer's disease. In view of this, the present inventors
discovered that, among such peptide fragments, the above-described
peptide composed of 37 amino acids
(VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT) produced by cleavage of
Alcadein-.beta. both by .gamma.-secretase and by .alpha.-secretase
(p3-Alc.beta.37 peptide) drastically ameliorates cognitive
impairment due to amyloid .beta. protein, and that even a peptide
composed of only 11 amino acids (HRGHQPPPEMA) contained in the
p3-Alc.beta.37 peptide (p3-Alc[9-19] peptide) has the action to
ameliorate cognitive impairment, thereby completing the present
invention.
[0030] That is, in one aspect of the present invention, a
therapeutic agent for cognitive impairment induced by amyloid
.beta. protein and a therapeutic drug for Alzheimer's disease are
provided. In another aspect of the present invention, a method for
treatment of cognitive impairment induced by amyloid .beta. protein
and a method for treatment of Alzheimer's disease are provided. In
still another aspect of the present invention, a method for
pathological analysis of cognitive impairment induced by amyloid
.beta. protein and a method for pathological analysis of
Alzheimer's disease are provided. These present inventions include
the following inventions.
[1] A therapeutic agent for cognitive impairment induced by amyloid
.beta. protein, comprising as an effective component a peptide
corresponding to any one of the following (I) to (VI):
[0031] (I) a peptide consisting of the amino acid sequence
represented by SEQ ID NO:1:
TABLE-US-00002 (SEQ ID NO: 1)
VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT;
[0032] (II) a peptide consisting of the same amino acid sequence as
the amino acid sequence represented by SEQ ID NO:1 except that one
to three amino acids are altered by any one or more of deletion,
addition, substitution, and side-chain modification;
[0033] (III) a peptide consisting of a partial sequence of the
amino acid sequence represented by SEQ ID NO:1, comprising the
amino acid sequence represented by SEQ ID NO:2:
TABLE-US-00003 HRGHQPPPEMA; (SEQ ID NO: 2)
[0034] (IV) a peptide consisting of the same amino acid sequence as
a partial sequence of the amino acid sequence represented by SEQ ID
NO: 1 comprising the amino acid sequence represented by SEQ ID
NO:2, except that one or two amino acids are altered by any one or
more of deletion, addition, substitution, and side-chain
modification;
[0035] (V) a peptide consisting of an amino acid sequence
comprising the amino acid sequence represented by SEQ ID NO:2,
wherein a total of 1 to 50 amino acids are added to the N-terminus
and/or the C-terminus of the amino acid sequence represented by SEQ
ID NO:2 (excluding amino acid sequences which are identical to all
or part of the amino acid sequence represented by SEQ ID NO:1);
and
[0036] (VI) a peptide consisting of an amino acid sequence
comprising the same amino acid sequence as the amino acid sequence
represented by SEQ ID NO:2 except that one or two amino acids are
altered by any one or more of deletion, addition (excluding
addition to the N-terminus and/or the C-terminus), substitution,
and side-chain modification, wherein a total of 1 to 50 amino acids
are added to the N-terminus and/or the C-terminus of the altered
amino acid sequence.
[2] A therapeutic drug for Alzheimer's disease, comprising the
therapeutic agent according to [1]. [3] A method for treatment of
cognitive impairment induced by amyloid .beta. protein, comprising
a step of administering the therapeutic agent according to [1] to a
mammal (excluding human) which developed cognitive impairment
induced by amyloid .beta. protein or to a model animal thereof. [4]
A method for treatment of Alzheimer's disease, comprising a step of
administering the therapeutic drug according to [2] to a mammal
(excluding human) which developed Alzheimer's disease or to a model
animal thereof. [5] A method for pathological analysis of cognitive
impairment induced by amyloid .beta. protein, comprising a step of
administering a peptide corresponding to any one of the above (I)
to (VI) to a mammal (excluding human) which developed cognitive
impairment induced by amyloid .beta. protein or to a model animal
thereof, or a step of adding the peptide to a cultured nerve
cell(s) as a model cell(s) thereof or to a biomolecule(s) expressed
in a nerve cell(s). [6] A method for pathological analysis of
Alzheimer's disease, comprising a step of administering a peptide
corresponding to any one of the above (I) to (VI) to a mammal
(excluding human) which developed Alzheimer's disease or to a model
animal thereof, or a step of adding the peptide to a cultured nerve
cell(s) as a model cell(s) thereof or to a biomolecule(s) expressed
in a nerve cell(s).
[0037] It is evident to those skilled in the art that the
above-described inventions can be converted to inventions
represented as, for example, use of the specific peptide as an
effective component of a therapeutic agent for cognitive impairment
induced by amyloid .beta. protein or a therapeutic drug for
Alzheimer's disease (in production of these agents).
Effect of the Invention
[0038] Since there is no effective therapeutic drug for Alzheimer's
disease at present, a novel therapeutic drug is demanded. As
described in Background Art, novel therapeutic drugs based on
amyloid .beta. protein are being clinically developed, but they are
still commercially unavailable because of their side effects and
the like. The anti-Alzheimer's disease agent provided by the
present invention has a novel action mechanism that is different
from those of conventional anti-Alzheimer's disease agents, and
uses a peptide which is produced by cleavage by .gamma.-secretase
and actually present in the living body (p3-Alc.beta.37) per se or
a partial peptide thereof (p3-Alc.beta.[9-19]), or a peptide having
an amino acid sequence similar to these peptides, so that the agent
can be an anti-Alzheimer's disease agent which has no side effect
and whose high safety can be expected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 shows the effect of the p3-Alc.beta.37 peptide on
memory disorder induced by an amyloid .beta. protein oligomer. Each
graph shows the exploration times for a novel object and a familiar
object measured in Examples. A (ACSF): Artificial cerebrospinal
fluid alone (control). B (A.beta. oligomer): Artificial
cerebrospinal fluid in which 1 .mu.M amyloid .beta. protein
oligomer is dissolved. C (A.beta. oligomer+p3-Alc.beta.37):
Artificial cerebrospinal fluid in which 1 .mu.M amyloid .beta.
protein oligomer and 10 .mu.M p3-Alc.beta.37 peptide are dissolved.
**: P<0.01.
[0040] FIG. 2 shows the change in the discrimination index due to
administration of the p3-Alc.beta.37 peptide. The graph shows the
discrimination index calculated from the results of Examples (FIG.
1) according to the following equation:
Discrimination index=([exploration time for novel
object]-[exploration time for familiar object])/([exploration time
for novel object]+[exploration time for familiar object]).
**: P<0.01.
[0041] FIG. 3 shows the effect of the p3-Alc.beta.37 peptide on
memory disorder induced by MK-801. Each graph shows the exploration
times for a novel object and a familiar object measured in
Examples. A (MK-801+ACSF): Administration of an artificial
cerebrospinal fluid (control) to the cerebral ventricle, and
intraperitoneal administration of MK-801. B
(MK-801+p3-Alc.beta.37): Administration, to the cerebral ventricle,
of an artificial cerebrospinal fluid in which 10 .mu.M
p3-Alc.beta.37 peptide is dissolved, and intraperitoneal
administration of MK-801.
[0042] FIG. 4 shows the effects of partial p3-Alc.beta.37 peptides
on memory disorder induced by an amyloid .beta. protein oligomer.
A: Comparison of the amino acid sequence among the following
partial peptides used in the experiment: p3-Alc.beta.[1-19],
p3-Alc.beta.[20-37], p3-Alc.beta.[1-11], and p3-Alc.beta.[9-19]. B
(A.beta. oligomer+p3-Alc.beta.[1-19]): Artificial cerebrospinal
fluid in which 1 .mu.M amyloid .beta. protein oligomer and 10 .mu.M
p3-Alc.beta.[1-19] peptide are dissolved. **: P<0.01. C (A.beta.
oligomer+p3-Alc.beta.[20-37]): Artificial cerebrospinal fluid in
which 1 .mu.M amyloid .beta. protein oligomer and 10 .mu.M
p3-Alc.beta.[20-37] peptide are dissolved. D (A.beta.
oligomer+p3-Alc.beta.[1-11]): Artificial cerebrospinal fluid in
which 1 .mu.M amyloid .beta. protein oligomer and 10 .mu.M
p3-Alc.beta.[1-11] peptide are dissolved. E (A.beta.
oligomer+p3-Alc.beta.[9-19]): Artificial cerebrospinal fluid in
which 1 .mu.M amyloid .beta. protein oligomer and 10 .mu.M
p3-Alc.beta.[9-19] peptide are dissolved. **: P<0.01.
MODE FOR CARRYING OUT THE INVENTION
--Therapeutic Agent--
[0043] The therapeutic agent for cognitive impairment induced by
amyloid .beta. protein according to the present invention includes
the first to sixth embodiments described below.
[0044] The "amyloid .beta. protein" herein is a polypeptide
generally known to accumulate in the brain as Alzheimer's disease
progresses, and may be in any of the form of a monomer composed of
a single polypeptide chain, the form of an oligomer composed of
several (usually, 2 to 6) polypeptide chains, and the form of an
aggregate composed of a plurality of such oligomers. The "cognitive
impairment induced by amyloid .beta. protein" includes memory
disorder, disorientation, learning disability, disturbance of
attention, and disorders of spatial cognitive function and
problem-solving abilities, which are generally known as symptoms of
Alzheimer's disease.
[0045] In the first embodiment, a peptide consisting of the amino
acid sequence represented by SEQ ID NO:1 is used as an effective
component of the therapeutic agent of the present invention.
VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT (SEQ ID NO:1)
[0046] In the second embodiment, a peptide consisting of the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO:1 except that one to three amino acids are altered by any one
or more of deletion, addition, substitution, and side-chain
modification (corresponding to a non-natural amino acid,
post-translationally modified amino acid, or the like) is used as
an effective component of the therapeutic agent of the present
invention. The deletion includes not only deletions at the
N-terminus and/or the C-terminus of the amino acid sequence
represented by SEQ ID NO:1, but also deletions inside the amino
acid sequence represented by SEQ ID NO:1. The addition includes not
only additions at the N-terminus and/or the C-terminus of the amino
acid sequence represented by SEQ ID NO: 1, but also additions
inside, that is, insertions into, the amino acid sequence
represented by SEQ ID NO:1. In the second embodiment, the number of
the amino acids that are altered is usually 1 to 3, preferably 1 to
2, more preferably 1. In cases where the amino acid sequence is
altered by a combination of two or more of deletion, addition,
substitution, and side-chain modification, the number described
above indicates their total number. As long as the action and
effect of the present invention are not deteriorated, such
deletion, addition (insertion), substitution, and/or side-chain
modification in the second embodiment may be carried out either in
the part corresponding to the amino acid sequence represented by
SEQ ID NO:2 (HRGHQPPPEMA) contained in the amino acid sequence
represented by SEQ ID NO:1, or in a part other than the amino acid
sequence represented by SEQ ID NO:2.
[0047] In the third embodiment, a peptide consisting of a partial
sequence of the amino acid sequence represented by SEQ ID NO:1,
especially a peptide consisting of a partial sequence comprising
the amino acid sequence represented by SEQ ID NO:2, is used as an
effective component of the therapeutic agent of the present
invention.
TABLE-US-00004 HRGHQPPPEMA (SEQ ID NO: 2)
[0048] Here, the partial sequence is a partial continuous amino
acid sequence in the amino acid sequence represented by SEQ ID NO:1
(that is, the entire amino acid sequence represented by SEQ ID NO:1
is not included in the definition of "partial sequence"), and can
be prepared by removing at least one amino acid from the N-terminus
and/or the C-terminus of a peptide consisting of the amino acid
sequence represented by SEQ ID NO:1. Such a peptide having the
partial sequence may be a peptide consisting of only the amino acid
sequence represented by SEQ ID NO:2, or may be a peptide consisting
of an amino acid sequence in which an amino acid sequence(s) is/are
added to the N-terminus and/or the C-terminus of the amino acid
sequence represented by SEQ ID NO:2 correspondingly to SEQ ID
NO:1.
[0049] It should be noted that the peptides used in the third
embodiment may overlap with (have the same meaning as) the peptides
used in the second embodiment. For example, amino acid sequences
which are the same as the amino acid sequence represented by SEQ ID
NO:1 (37 amino acids in length) except that a total of 1 to 3 amino
acids at the N-terminus and/or the C-terminus are deleted (34 to 36
amino acids in length), which are used in the second embodiment,
may overlap with amino acid sequences which are the same as the
amino acid sequence represented by SEQ ID NO:2 (11 amino acids in
length) except that a total of 23 to 25 amino acids are added to
the N-terminus and/or the C-terminus (34 to 36 amino acids in
length). If necessary, the peptides used in the third embodiment
may be defined such that the peptides used in the second embodiment
are excluded. This also applies to the peptides used in the fourth
embodiment.
[0050] In the fourth embodiment, a peptide consisting of the same
amino acid sequence as a partial sequence of the amino acid
sequence represented by SEQ ID NO: 1, especially a partial sequence
comprising the amino acid sequence represented by SEQ ID NO:2 (that
is, the partial sequence defined in the third embodiment), except
that one or two amino acids are altered by any one or more of
deletion, addition, substitution, and side-chain modification, is
used as an effective component of the therapeutic agent of the
present invention. The deletion includes not only deletions at the
N-terminus and/or the C-terminus of the partial sequence of the
amino acid sequence represented by SEQ ID NO:1, but also deletions
inside the amino acid sequence represented by SEQ ID NO:1. The
addition includes not only additions at the N-terminus and/or the
C-terminus of the partial sequence of the amino acid sequence
represented by SEQ ID NO:1, but also additions inside, that is,
insertions into, the partial sequence. In the fourth embodiment,
the number of the amino acids that are altered is usually 1 to 2,
preferably 1. In cases where the amino acid sequence is altered by
a combination of two or more of deletion, addition, substitution,
and side-chain modification, the number described above indicates
their total number. As long as the action and effect of the present
invention are not deteriorated, such deletion, addition
(insertion), substitution, and/or side-chain modification in the
fourth embodiment may be carried out either in the part
corresponding to the amino acid sequence represented by SEQ ID NO:2
(HRGHQPPPEMA), or in apart other than the amino acid sequence
represented by SEQ ID NO:2.
[0051] In the fifth embodiment, a peptide consisting of an amino
acid sequence comprising the amino acid sequence represented by SEQ
ID NO:2, wherein a total of 1 to 50 amino acids are added to the
N-terminus and/or the C-terminus of the amino acid sequence
represented by SEQ ID NO:2, is used as an effective component of
the therapeutic agent of the present invention. The number of the
amino acids added in the fifth embodiment is usually 1 to 50,
preferably 1 to 26 (this corresponds to a total peptide length of
up to 37 after the addition, which is the same as the length of
p3-Alc.beta.37), more preferably 1 to 8 (this corresponds to a
total peptide length of up to 19 after the addition, which is the
same as the length of p3-Alc.beta.[1-19]).
[0052] It should be noted that the peptides used in the fifth
embodiment may overlap with (have the same meaning as) the peptides
used in the first to fourth embodiments. For example, the amino
acid sequence represented by SEQ ID NO:1 (37 amino acids in
length), which is used in the first embodiment, can also be said to
be an amino acid sequence comprising the amino acid sequence
represented by SEQ ID NO:2 (11 amino acids in length; the 9th to
19th amino acids in SEQ ID NO: 1), wherein 9 amino acids (the 1st
to 8th amino acids in SEQ ID NO:1) are added to the N-terminus and
18 amino acids (the 20th to 37th amino acids in SEQ ID NO:1) are
added to the C-terminus of the amino acid sequence represented by
SEQ ID NO:2. If necessary, the peptides used in the fifth
embodiment may be defined such that the peptides used in the first
to fourth embodiment are excluded. This also applies to the
peptides used in the sixth embodiment described below.
[0053] In the sixth embodiment, a peptide consisting of an amino
acid sequence comprising the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO:2 except that one or two
amino acids are altered by any one or more of deletion, addition,
substitution, and side-chain modification, wherein a total of 1 to
50 amino acids are added to the N-terminus and/or the C-terminus of
the altered amino acid sequence, is used as an effective component
of the therapeutic agent of the present invention. The deletion
includes not only deletions at the N-terminus and/or the C-terminus
of the amino acid sequence represented by SEQ ID NO:2, but also
deletions inside the amino acid sequence represented by SEQ ID
NO:2. The addition includes not only additions at the N-terminus
and/or the C-terminus of the amino acid sequence represented by SEQ
ID NO:2 (in this case, the addition of "1 to 50" amino acids
described in the latter half of the recitation of the sixth
embodiment may be carried out in addition to the addition of "one
or two" amino acids described in the first half of the recitation),
but also additions inside, that is, insertions into, the partial
sequence. In the sixth embodiment, the number of the amino acids
that are further added to the N-terminus and/or the C-terminus of
the altered amino acid sequence is usually 1 to 50, preferably 1 to
26 (this corresponds to a total peptide length of up to 37 after
the addition, which is the same as the length of p3-Alc.beta.37;
however, in cases where one or two amino acids are deleted from or
added to the amino acid sequence represented by SEQ ID NO:2, the
decrease or increase in the number of amino acids due to the
deletion or addition may be taken into account), more preferably 1
to 8 (this corresponds to a total peptide length of up to 19 after
the addition, which is the same as the length of
p3-Alc.beta.[1-19]; however, in cases where one or two amino acids
are deleted from or added to the amino acid sequence represented by
SEQ ID NO:2, the decrease or increase in the number of amino acids
due to the deletion or addition may be taken into account).
[0054] Those skilled in the art can select, without requiring
excessive trial and error, a peptide that exerts the action and
effect to treat cognitive impairment induced by amyloid .beta.
protein among the peptides having the amino acid sequences defined
in the second to sixth embodiments, and use the selected peptide in
the present invention.
[0055] The peptides used in the first to sixth embodiments can be
prepared by a known method, and the method of preparation is not
limited. For example, a peptide consisting of a desired amino acid
sequence can be prepared by sequentially linking amino acids using
the Fmoc-peptide solid-phase synthesis, which is a conventionally
used method for peptide synthesis.
[0056] The therapeutic agent according to the present invention
described above, or the predetermined peptide contained therein,
can be suitably used as an effective component of therapeutic drugs
for Alzheimer's disease. However, the embodiment of the therapeutic
agent according to the present invention or the predetermined
peptide contained therein is not limited to the uses for such drugs
(pharmaceuticals). For example, the therapeutic agent according to
the present invention or the predetermined peptide contained
therein may be used in an embodiment in which the therapeutic agent
or the predetermined peptide is administered to the body of human
or a mammal other than human who developed cognitive impairment
induced by amyloid .beta. protein, or a model animal thereof (e.g.,
mouse, rat, guinea pig, rabbit, goat, cat, dog, pig, or monkey) to
allow the therapeutic agent or the predetermined peptide to act in
vivo in order to study details of its therapeutic effect on the
cognitive impairment or its action mechanism, or an embodiment in
which the therapeutic agent or the predetermined peptide is added
to model cells prepared from cultured nerve cells derived from any
of these animals to allow the therapeutic agent or the
predetermined peptide to act in vitro.
[0057] The therapeutic agent according to the present invention may
be prepared as an agent containing only a predetermined peptide as
an effective component, and the peptide alone or a solution of the
peptide in an appropriate solvent (for example, artificial
cerebrospinal fluid) may be used for the administration or the
addition. Alternatively, the therapeutic agent according to the
present invention may be prepared as a formulation according to the
therapeutic drug described below, to be used for the administration
or the addition.
--Therapeutic Drug--
[0058] The therapeutic drug for Alzheimer's disease according to
the present invention contains the therapeutic agent according to
the present invention, and may optionally contain, for example, an
effective component other than the therapeutic agent (predetermined
peptide) of the present invention, a pharmaceutically acceptable
carrier suitable for the dosage form, and/or other pharmaceutical
additives used for common pharmaceuticals. That is, the therapeutic
drug for Alzheimer's disease according to the present invention may
be prepared as a pharmaceutical composition containing such a
component(s).
[0059] Examples of the dosage form of the therapeutic drug which
may be selected include dosage forms for oral administration, such
as tablets, capsules, soft capsules, granules, powders, fine
granules, (dry) syrups, solutions, and suspensions; and dosage
forms for parenteral administration, such as injections for
subcutaneous, intramuscular, or intravenous administration, drops,
suppositories, and transnasal agents.
[0060] The pharmaceutical compositions having such dosage forms can
be produced by common production methods. For example, a dosage
form for oral administration can be produced by mixing the
effective component with one or more of additives such as vehicles,
disintegrators, binders, lubricants, suspending agents, isotonic
agents, emulsifiers, sweeteners, flavoring agents, and coloring
agents by a conventional method, and molding the resulting mixture.
Examples of the vehicles include cellulose derivatives (for
example, crystalline cellulose, methylcellulose, hydroxypropyl
cellulose, and hydroxypropyl methyl cellulose);
polyvinylpyrrolidone; dextrin; starch; lactose; mannitol; sorbitol;
vegetable oils (for example, corn oil, cottonseed oil, coconut oil,
almond oil, olive oil, and peanut oil); oily esters such as
medium-chain fatty acid glyceride oils; mineral oil; glycerin
esters such as tricaprilin and triacetin; alcohols such as ethanol;
physiological saline; propylene glycol; polyethylene glycol; animal
fats and oils; and vaseline.
[0061] The injection can be produced by dissolving the effective
component in an appropriate diluent (physiological saline, glucose
injection, lactose injection, mannitol injection, or the like) and
subjecting the resulting solution to sterilization treatment such
as sterilization by filtration, followed by filling the sterilized
solution into a sealable container such as an ampule.
Alternatively, the injection can be produced in the form of a
product freeze-dried according to the Japanese Pharmacopoeia, or as
a powder for injection prepared by mixing with sodium chloride. As
the additive, an adjuvant such as polyethylene glycol or a
surfactant; or a carrier such as ethanol, liposome, or
cyclodextrine; may be used.
[0062] The content of the effective component in the pharmaceutical
composition may be controlled within an appropriate range. The
content of the effective component is usually 0.05 to 99 wt %,
preferably 0.1 to 70 wt %, more preferably 0.1 to 50 wt % with
respect to the total weight of the pharmaceutical composition.
--Therapeutic Method--
[0063] The therapeutic agent described above may be used for
treating cognitive impairment induced by amyloid .beta. protein.
The therapeutic drug described above may be used for treating
Alzheimer's disease.
[0064] That is, the method for treatment of cognitive impairment
induced by amyloid .beta. protein according to the present
invention comprises a step of administration to a mammal (human, or
a mammal other than human) which developed cognitive impairment
induced by amyloid .beta. protein or to a model animal (a mammal
other than human) thereof. The method for treatment of Alzheimer's
disease according to the present invention comprises a step of
administration to a mammal (excluding human) which developed
Alzheimer's disease or to a model animal thereof.
[0065] The cognitive impairment induced by amyloid .beta. protein
to be treated with the therapeutic agent according to the present
invention typically means symptoms accompanying Alzheimer's
disease, but may be symptoms accompanying a disease which has not
been definitively diagnosed with Alzheimer's disease, or symptoms
in a subject in the preclinical stage including mild cognitive
impairment (MCI) or in a model animal. The model animal of
cognitive impairment induced by amyloid .beta. protein can be
prepared using a known method. For example, a transgenic mouse
which exhibits excessive expression of amyloid .beta. protein or a
mouse to which a solution of amyloid .beta. protein in an
artificial cerebrospinal fluid is administered can be used as such
a model animal.
[0066] The "treatment" with the therapeutic agent or the
therapeutic drug includes both therapeutic treatment and
prophylactic treatment. The effect of the treatment includes
prevention of occurrence or recurrence of the disease, amelioration
of symptoms, suppression of progression of the disease,
amelioration or reduction of symptoms, and recovery. The extent of
the effect is not limited, and can be evaluated by known methods
related to cognitive impairment induced by amyloid .beta. protein
or to Alzheimer's disease.
[0067] The method of administration of the therapeutic agent or the
therapeutic drug according to the present invention is not limited
as long as a desired effect can be obtained. Usually, as an in vivo
method, an administration method which enables delivery of a
predetermined peptide as an effective component to the cerebral
ventricle, where amyloid .beta. protein accumulates, is used. As an
in vitro method, an addition method in which a predetermined
peptide is added to a cell culture liquid to bring the peptide into
contact with cultured nerve cells or the like is used.
[0068] Another possible therapeutic method is use of a virus vector
for expressing p3-Alc.beta.37 or a part of its peptide sequence in
cerebral nerve cells. As such a virus vector, for example,
recombinant adeno-associated virus serotype 9 (AAV9), which can
pass through the blood-brain barrier, may be used. Expression of a
gene in central nerves and the glial cell system using AAV9 vector
administered into blood is known. In cases where nerve
cell-specific expression of p3-Alc.beta.37 is necessary, for
example, the virus vector AAV9 in which the promoter region of the
synapsin I gene is incorporated may be used. In order to allow
expression of p3-Alc.beta.37 in cells and its secretion from the
cells, for example, a precursor peptide sequence which is prepared
by cleaving Alcadein .beta. at the .epsilon. cleavage site and
linked to a signal peptide sequence of 19 amino acids for Alcadein
.beta. is expressed. After the expression in the cells, the signal
sequence is removed from the precursor peptide by cleavage by
signal peptidase, and cleavage by .gamma.-secretase proceeds from
the .epsilon.-site to the .gamma.-site, resulting in secretion of
p3-Alc.beta.37 composed of 37 amino acids (Piao et al., PLoS One 8,
e62431, 2013). Of course, as a method for allowing the expression
and the secretion of the peptide, a known method other than this
method, for example, a method in which a gene encoding the 37 amino
acids produced by the cleavage at the .gamma.-site is used from the
beginning, may be used. In consideration of the efficiency of the
expression and the secretion, a method effective for the treatment
may be selected. By using a method in which a virus vector is
introduced into blood, p3-Alc.beta.37 can be noninvasively
introduced into the central nerve system, and the therapeutic
effect can be assayed by a known method.
[0069] The dose of the therapeutic drug, that is, the amount of the
effective component per administration and the number (frequency)
of doses, may be controlled within an appropriate range taking into
account the object; the age and the body weight of the subject to
which the therapeutic drug is administered; the severity of the
disease; the administration route; the pharmacokinetics; and/or the
like.
--Method for Pathological Analysis--
[0070] The predetermined peptides in the present invention, that
is, the peptides used in the first to sixth embodiments described
above, may also be used in methods for pathological analysis of
cognitive impairment induced by amyloid .beta. protein, or
Alzheimer's disease. That is, the method for pathological analysis
of cognitive impairment induced by amyloid .beta. protein according
to the present invention comprises a step of administration of the
predetermined peptide used in the present invention to a mammal
(human, or a mammal other than human) which developed cognitive
impairment induced by amyloid .beta. protein or to a model animal
(a mammal other than human) thereof, or a step of adding the
peptide to a cultured nerve cell(s) as a model cell(s) thereof or
to a biomolecule(s) expressed in a nerve cell(s). The method for
pathological analysis of Alzheimer's disease according to the
present invention comprises a step of administration of the
predetermined peptide used in the present invention to a mammal
(human, or a mammal other than human) which developed Alzheimer's
disease or to a model animal (a mammal other than human) thereof,
or a step of adding the peptide to a cultured nerve cell(s) as a
model cell(s) thereof or to a biomolecule(s) expressed in a nerve
cell(s).
[0071] Examples of the biomolecules expressed in nerve cells
include proteins such as receptors expressed on the cell surface,
and proteins associated with signal transduction expressed in the
cytoplasm.
[0072] The model cells are cells which reproduce the disease state
of, for example, degeneration of nerve cells (e.g., neurons) or the
like caused by cognitive impairment induced by amyloid .beta.
protein or Alzheimer's disease. The model cells can be prepared
from cultured nerve cells derived from human or a mammal other than
human using a known method.
[0073] In such methods for pathological analysis, by observing and
evaluating the reaction caused by administering or adding the
predetermined peptide used in the present invention, the action
mechanism of the therapeutic agent or the therapeutic drug using
the peptide can be studied in detail. This enables development of
means for enhancing the effects of therapeutic methods using them,
and development of novel therapeutic agents and therapeutic
drugs.
Examples
Methods
[0074] Preparation of a mouse model of memory disorder by
administration of the amyloid .beta. protein oligomer, and
evaluation of the ability of memory acquisition by a novel object
recognition test were carried out according to an earlier paper
(Balducci et al., Proc Natl Acad Sci USA 107, 2295-2300, 2010).
[0075] ICR mice (6 to 7 weeks old) were anesthetized with ketamine
(100 mg/kg) and xylazine (40 mg/kg), and each mouse was fixed on a
brain stereotaxic apparatus. After removal of the scalp, small
holes were made on the skull on the lateral ventricles, and guide
cannulae (23 gage) were bilaterally embedded at the positions of
.+-.1.0 mm lateral and 0.65 mm posterior to the bregma. The guide
cannulae were fixed with a dental resin. After a period of four to
five days for recovery from the surgery, each mouse was allowed to
move freely for 10 minutes in order to allow acclimatization to the
environment in a test cage (39 cm.times.22 cm) (habituation). This
was continued for 3 days. On the next day, an artificial
cerebrospinal fluid (control), artificial cerebrospinal fluid in
which 1 .mu.M amyloid .beta. protein oligomer is dissolved, or
artificial cerebrospinal fluid in which 1 .mu.M amyloid .beta.
protein oligomer and 10 .mu.M p3-Alc.beta.37 peptide or a partial
peptide thereof are dissolved, was bilaterally administered (3.5
.mu.l/side) to the lateral ventricles through the guide cannulae
embedded in the head, using an injection cannulae (30 gage, 1.7 mm
from the brain surface).
[0076] Two hours after the administration, two identical objects
(glass vials each having a plastic cap [objects A]: 3-cm
diameter.times.6.5-cm height) were placed in the test cage for
which the habituation had been carried out, such that the objects
were positioned about 20 cm distant from each other. In the test
cage, each mouse was allowed to move freely for 10 minutes to allow
learning of the object A (training session). On the next day, 24
hours after the learning, a test was carried out to investigate if
the mouse remembered the object A, which was presented on the
previous day (test session). More specifically, in the same test
cage, the object A presented on the previous day (that is, a
familiar object) and a novel object (a metal cube [object B]: 4
cm.times.4 cm.times.4 cm) were placed at the same positions as
those used on the previous day, and the mouse was allowed to move
freely for 10 minutes in the test cage. In normal mice, since they
naturally show interest in novel objects, the time for exploring
the novel object (object B) is longer than the time for exploring
the familiar object (object A). In contrast, mice whose ability to
remember is impaired show almost the same length of exploration
time for the object A and the object B in the test session, since
the mice do not remember the object presented on the previous day
(object A). The exploratory behavior for each object was recorded
using a video camera, and the total exploration time for each
object was calculated. The time spent for contacting or approach of
the head to the object (including sniffing), and contacting of a
forelimb or hindlimb with the object was included in the
exploration time. The ability of memory acquisition of the mice in
each group was represented using the discrimination index. The
discrimination index was defined as follows: ([exploration time for
novel object]-[exploration time for familiar object])/([exploration
time for novel object]+[exploration time for familiar object]). In
cases where discrimination between the novel object and the
familiar object is impossible, the discrimination index is 0, while
in cases where the exploration time for the novel object is longer
than the exploration time for the familiar object, the
discrimination index is positive. When a novel object is presented
to a mouse, the mouse becomes less interested in the object (shows
less exploratory behavior for the novel object) as time passes.
Therefore, the calculation of the exploration time and the
discrimination index was carried out based on the behavior observed
during the first 5 minutes after the placement of the mouse in the
test cage.
[0077] Also for a mouse model of memory disorder prepared by
administration of an NMDA receptor inhibitor MK-801, evaluation of
the ability of memory acquisition was carried out by a novel object
recognition test. The experiment was carried out by same method as
described above except for the following two conditions: (1) an
artificial cerebrospinal fluid (control), or an artificial
cerebrospinal fluid in which 10 .mu.M p3-Alc.beta.37 peptide is
dissolved, was administered to the cerebral ventricle; and (2) one
and a half hours after the administration to the cerebral ventricle
(that is, 30 minutes before placing the mouse into the test cage),
(+)-MK-801 maleate (2 mg/kg) was intraperitoneally administered to
induce NMDA receptor-dependent memory disorder.
[0078] In terms of the reagents used, the composition of the
artificial cerebrospinal fluid was as follows: 137 mM NaCl, 3 mM
KCl, 1 mM MgCl.sub.2, 1.2 mM CaCl.sub.2, 2.5 mM glucose, and 2 mM
sodium phosphate buffer (pH 7.4). The amyloid .beta. protein
oligomer was prepared according to an earlier literature (Balducci
et al., Proc Natl Acad Sci USA 107, 2295-2300, 2010). A stock
solution of amyloid .beta. protein (300 .mu.M) was prepared as an
aqueous solution in 0.02% trifluoroacetic acid, and stored at
-25.degree. C. The stock solution was used within one month after
the preparation. The preparation of the amyloid .beta. protein
oligomer was carried out at the time of use as follows. After
melting the amyloid .beta. protein stock solution, the solution was
alkalized with a solution of NaOH:NH.sub.3=1:3 (pH>10.5), and
the amyloid .beta. protein was diluted with 50 mM
phosphate-buffered saline (pH 7.4) to 100 .mu.M. The resulting
dilution was incubated at 22.degree. C. for 18 hours. The solution
was then diluted with the artificial cerebrospinal fluid to 1
.mu.M, and administered to the cerebral ventricle as an amyloid
.beta. protein oligomer. The mixture of the oligomer and the
p3-Alc.beta.37 peptide or the partial peptide thereof was prepared
in the same manner as the procedure described above except that the
p3-Alc.beta.37 peptide or the partial peptide thereof was also
added to a final concentration of 10 .mu.M when the incubated 100
.mu.M amyloid .beta. protein oligomer was diluted to 1 .mu.M.
[0079] The sequences of the peptides used were as follows.
TABLE-US-00005 amyloid-.beta.42:
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA p3-Alc.beta.37:
VLSSQQFLHRGHQPPPEMAGHSLASSHRNSMIPSAAT p3-Alc.beta.[1-19]:
VLSSQQFLHRGHQPPPEMA p3-Alc.beta.[20-37]: GHSLASSHRNSMIPSAAT
p3-Alc.beta.[1-11]: VLSSQQFLHRG p3-Alc.beta.[9-19]: HRGHQPPPEMA
[0080] The peptides used were obtained from the following:
amyloid-.beta.42 (Keck Biotechnology Resource Laboratory, Yale
University), p3-Alc.beta.37 (Keck Biotechnology Resource
Laboratory, Yale University), p3-Alc.beta.[1-19] (Genemed
Synthesis, and Peptide Institute), p3-Alc.beta.[20-37] (Genemed
Synthesis, and Peptide Institute), p3-Alc.beta.[1-11] (Peptide
Institute), and p3-Alc.beta.[9-19] (Peptide Institute).
Results
[0081] In the mice to which the artificial cerebrospinal fluid was
administered, the exploration time for the novel object B was
significantly longer than the exploration time for the familiar
object A (FIG. 1A; n=11 mice, P<0.01, paired t-test). That is,
the memory was normally acquired. On the other hand, in the mice to
which the amyloid .beta. protein oligomer was administered, the
exploration time was almost the same between the familiar object A
and the novel object B (FIG. 1B; n=20 mice, P>0.05, paired
t-test). That is, it was found that acquisition of the memory was
completely impaired. The present inventors also found that the mice
to which the mixture of the amyloid .beta. protein oligomer and the
p3-Alc.beta.37 peptide was administered showed remarkable recovery
of the ability to remember (FIG. 1C; n=14 mice, P<0.01, paired
t-test). In FIG. 2, the ability of memory acquisition in each group
is represented using the discrimination index. Relative to the
group in which the artificial cerebrospinal fluid was administered,
the group in which the oligomer was administered showed a
significant decrease in the discrimination index (FIG. 2;
P<0.01, Tukey test). Relative to the group in which the oligomer
was administered, the group in which the oligomer/p3-Alc.beta.37
mixture was administered showed a significant increase in the
discrimination index (FIG. 2; P<0.01, Tukey test). No
significant difference in the discrimination index was found
between the group in which the artificial cerebrospinal fluid was
administered and the group in which the oligomer/p3-Alc.beta.37
mixture was administered (FIG. 2; P>0.05, Tukey test). These
results indicate that the memory disorder induced by the amyloid
.beta. protein oligomer is drastically ameliorated by intracerebral
administration of the p3-Alc.beta.37 peptide.
[0082] Subsequently, whether the action of p3-Alc.beta.37 to allow
recovery from memory disorder is specific to the memory disorder
caused by amyloid .beta. protein, or the action also allows
recovery from other memory disorders, was studied. It is well known
that the NMDA receptor is indispensable for memory and learning. It
is reported that gene knockout of the NMDA receptor (Rampon et al.,
Nat Neurosci 3, 238-244, 2000) or administration of an NMDA
receptor inhibitor MK-801 (de Lima et al., Behav Brain Res 156,
139-143, 2005) causes impairment in novel object recognition tests.
In view of this, also in the present study, MK-801 was
intraperitoneally administered in the state where the artificial
cerebrospinal fluid was administered to the cerebral ventricle. It
was found, as a result, that the exploration time became almost the
same between the familiar object A and the novel object B (FIG. 3A;
n=8 mice, P>0.05, paired t-test). That is, acquisition of the
memory was found to be impaired. Subsequently, MK-801 was
intraperitoneally administered in the state where the
p3-Alc.beta.37 peptide was administered to the cerebral ventricle.
However, no remarkable difference in the exploration time was found
between the familiar object A and the novel object B (FIG. 3B; n=9
mice, P>0.05, paired t-test), so that the effect to ameliorate
memory disorder was not found. Thus, it was shown that, although
the p3-Alc.beta.37 peptide ameliorates memory disorder caused by
amyloid .beta. protein, the peptide is not capable of ameliorating
memory disorder caused by inhibition of the NMDA receptor. This
indicates that the p3-Alc.beta.37 peptide relatively selectively
acts on amyloid .beta. protein to ameliorate memory disorder.
[0083] Finally, whether a partial peptide of p3-Alc.beta.37 is also
capable of allowing recovery from memory disorder caused by the
amyloid .beta. protein oligomer was studied. In mice to which a
mixture of the amyloid .beta. protein oligomer and the partial
peptide corresponding to positions 1 to 19 of p3-Alc.beta.37
(p3-Alc.beta.37[1-19], FIG. 4A) was administered, remarkable
recovery of the ability to remember was found (FIG. 4B; n=15 mice,
P<0.01, paired t-test). In contrast, in mice to which a mixture
of the oligomer and the partial peptide corresponding to positions
20 to 37 of p3-Alc.beta.37 (p3-Alc.beta.37[20-37], FIG. 4A) was
administered, no recovery of the ability to remember was found
(FIG. 4C; n=16 mice, P>0.05, paired t-test). Subsequently, in
view of this, the p3-Alc.beta.37[1-19] peptide was further
fragmented to prepare the partial peptide corresponding to
positions 1 to 11 (p3-Alc.beta.37[1-11], FIG. 4A) and the partial
peptide corresponding to positions 9 to 19 (p3-Alc.beta.37[9-19],
FIG. 4A), and the prepared partial peptides were subjected to the
study. As a result, while the ability to remember remained impaired
in the case of the oligomer/p3-Alc.beta.37[1-11] (FIG. 4D; n=14
mice, P>0.05, paired t-test), the ability to remember showed
remarkable recovery in the case of the
oligomer/p3-Alc.beta.37[9-19] (FIG. 4E; n=16 mice, P<0.01,
paired t-test). These results indicate that p3-Alc.beta.37[9-19]
(11 amino acids), which is a partial peptide of p3-Alc.beta.37, is
also capable of ameliorating memory disorder caused by amyloid
.beta. protein.
SEQUENCE LISTING FREE TEXT
[0084] SEQ ID NO:1 p3-Alc.beta.37 SEQ ID NO:2 p3-Alc.beta.[9-19]/a
part of p3-Alc beta 37 (positions 9-19) SEQ ID NO:3
p3-Alc.beta.[1-11]/a part of p3-Alc beta 37 (positions 1-11) SEQ ID
NO:4 p3-Alc.beta.[1-19]/a part of p3-Alc beta 37 (positions 1-19)
SEQ ID NO:5 p3-Alc.beta.[20-37]/a part of p3-Alc beta 37 (positions
20-37) SEQ ID NO:6 amyloid-.beta.42
Sequence CWU 1
1
6137PRTHomo sapiens 1Val Leu Ser Ser Gln Gln Phe Leu His Arg Gly
His Gln Pro Pro Pro 1 5 10 15 Glu Met Ala Gly His Ser Leu Ala Ser
Ser His Arg Asn Ser Met Ile 20 25 30 Pro Ser Ala Ala Thr 35
211PRTArtificial Sequencea part of p3-Alc beta 37 (position 9-19)
2His Arg Gly His Gln Pro Pro Pro Glu Met Ala 1 5 10
311PRTArtificial Sequencea part of p3-Alc beta 37 (position 1-11)
3Val Leu Ser Ser Gln Gln Phe Leu His Arg Gly 1 5 10
419PRTArtificial Sequencea part of p3-Alc beta 37 (position 1-19)
4Val Leu Ser Ser Gln Gln Phe Leu His Arg Gly His Gln Pro Pro Pro 1
5 10 15 Glu Met Ala 518PRTArtificial Sequencea part of p3-Alc beta
37 (position 20-37) 5Gly His Ser Leu Ala Ser Ser His Arg Asn Ser
Met Ile Pro Ser Ala 1 5 10 15 Ala Thr 642PRTHomo sapiens 6Asp 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 Ile Ala 35 40
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