U.S. patent application number 12/491120 was filed with the patent office on 2009-12-03 for method of measuring neprilysin activity.
This patent application is currently assigned to Riken. Invention is credited to Nobuhisa Iwata, Tadashi Nakaya, Takaomi Saido, Takashi Saito, Yoshie Takaki, Satoshi Tsubuki.
Application Number | 20090298775 12/491120 |
Document ID | / |
Family ID | 29272368 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090298775 |
Kind Code |
A1 |
Saido; Takaomi ; et
al. |
December 3, 2009 |
METHOD OF MEASURING NEPRILYSIN ACTIVITY
Abstract
The present invention provides a method of measuring the
activity of neprilysin, etc. More specifically, the present
invention provides a method of measuring the activity of neprilysin
in nerve cells; a method of screening a protein, a peptide or a
compound enhancing the activity or expression of neprilysin in
nerve cells by measuring the activity of neprilysin; a method of
enhancing the activity or expression of neprilysin; and so on.
Thus, the compound enhancing the activity and/or expression of
neprilysin, which is obtained by the screening method characterized
by using the method of measuring the activity of neprilysin in
accordance with the present invention, is useful as a preventive
and/or therapeutic agent for Alzheimer's disease. The method of
measuring the activity of the present invention can be used for
presymptomatic diagnosis of Alzheimer's disease.
Inventors: |
Saido; Takaomi; (Saitama,
JP) ; Iwata; Nobuhisa; (Saitama, JP) ;
Tsubuki; Satoshi; (Saitama, JP) ; Takaki; Yoshie;
(Saitama, JP) ; Saito; Takashi; (Saitama, JP)
; Nakaya; Tadashi; (Hokkaido, JP) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Riken
Saitama
JP
Takeda Pharmaceutical Company, Ltd.
Osaka
JP
|
Family ID: |
29272368 |
Appl. No.: |
12/491120 |
Filed: |
June 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10512588 |
Jul 15, 2005 |
7572574 |
|
|
PCT/JP03/05239 |
Apr 24, 2003 |
|
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12491120 |
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Current U.S.
Class: |
514/2.4 |
Current CPC
Class: |
G01N 2333/96486
20130101; A61P 43/00 20180101; G01N 33/6896 20130101; A61P 25/28
20180101; C12Q 1/37 20130101; G01N 2500/00 20130101 |
Class at
Publication: |
514/12 ; 514/14;
514/2 |
International
Class: |
A61K 38/31 20060101
A61K038/31; A61K 38/10 20060101 A61K038/10; A61K 38/12 20060101
A61K038/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 26, 2002 |
JP |
2002-126257 |
Sep 6, 2002 |
JP |
2002-261250 |
Claims
1-27. (canceled)
28. A method of enhancing the activity of neprilysin, which
comprises administering to a mammal an effective dose of
somatostatin, a protein containing substantially the same amino
acid sequence as somatostatin or its partial peptide, an agonist of
somatostatin receptor or a salt thereof.
29. A method of enhancing the expression of neprilysin, which
comprises administering to a mammal an effective dose of
somatostatin, a protein containing substantially the same amino
acid sequence as somatostatin or its partial peptide, an agonist of
somatostatin receptor or a salt thereof.
30. A method of enhancing the activity or expression of neprilysin,
which comprises promoting the activity of somatostatin receptor in
a mammal.
31-49. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of promoting the
degradation of amyloid .beta. protein (A.beta.) deeply associated
with the onset/progression of Alzheimer's disease to reduce A.beta.
levels in the brain and improve the symptoms of Alzheimer's
disease. That is, the present invention relates to a method of
enhancing the activity or expression of neprilysin, which is an
enzyme responsible for the degradation of A.beta. in the brain; a
method of measuring the activity of neprilysin in nerve cells; and
a method of screening a protein, a peptide or a compound enhancing
the activity or expression of neprilysin in nerve cells by
measuring the activity of neprilysin. The present invention further
relates to a pharmaceutical composition comprising the protein,
peptide or compound controlling the degradation of A.beta., which
is found by the screening method; a method of treating disease and
a method of preventing disease.
BACKGROUND ART
[0002] Alzheimer's disease is a neurodegenerative disorder of which
the main symptom is dementia. In Alzheimer's disease patients,
atrophy of the cerebral cortex is found and pathologically, the
characteristic lesions including senile plaques, changes in
neurofibrillary tangles, etc. are observed, in addition to severe
loss of nerve cells. Among them, the pathological change observed
from a relatively early stage is the formation of senile plaques
and since a major component in the senile plaques is amyloid .beta.
protein (A.beta.), it is considered that abnormalities in the
formation or degradation of A.beta. would be closely associated
with the onset/progression of Alzheimer's disease. A.beta. is
cleaved with and produced from the A.beta. precursor protein
(.beta.APP) by .beta.-secretase (Science, 286, 735-741, 1999) and
.gamma.-secretase, which belong to aspartic proteases. With regard
to .gamma.-secretase, it has been revealed that a familial
Alzheimer's disease (FAD)-pathogenic gene, presenilin or a
presenilin-containing complex takes part in expressing the activity
(Nature, 398, 513-517, 1999; Nature, 405,689-694, 2000).
[0003] This A.beta. is steadily synthesized/secreted in vivo and it
is considered that under normal conditions A.beta. will be rapidly
degraded but not accumulated. When this degradability decreases for
some reason, it results in accumulation of A.beta. and conversely
when the degradability is enhanced, the accumulation of A.beta. can
be prevented. Recently, it was clarified that the major enzyme
involved in the degradation of A.beta. is a neutral endopeptidase,
neprilysin (Nature Med., 6, 143-151, 2000). In neprilysin knockout
mice, the A.beta. level elevated in the brain and the elevation was
most remarkable in the hippocampus (Science, 292, 1550-1552, 2001).
It was further clarified that the expression of neprilysin declined
in the brain of Alzheimer's disease patients (Neuroscience Lett.,
297, 97-100, 2001).
[0004] It is considered that when the activity or expression of
neprilysin enhances to increase degradability of A.beta. clearance
of A.beta. in the brain will increase to downregulate the
accumulation of A.beta.. However, the mechanism concerning the
downregulation of neprilysin expression is not very clear.
Neprilysin is a neutral endopeptidase and has an action of
degrading various peptides in the brain. Since a number of
physiologically active peptides are present in the brain, there is
a possibility that a peptide regulating the expression of
neprilysin mediated by GPCR (G-protein coupled receptor) as a
receptor or by a nuclear receptor would be present among them. Once
the peptide in the brain which regulates the expression of
neprilysin can be found, a method of searching a novel drug
promoting A.beta. degradation can be provided and furthermore,
application to novel pharmaceuticals such as therapeutic or
preventive agents for Alzheimer's disease, etc. can be
provided.
DISCLOSURE OF THE INVENTION
[0005] In order to explore a brain peptide regulating the activity
of neprilysin in nerve cells, the present inventor found a method
of visualizing the activity of neprilysin in mouse primary nerve
cells using a fluorescent substrate. Using this measuring system,
various brain peptides were acted on to explore a peptide affecting
the activity of neprilysin. As a result, the inventor has found
that somatostatin enhances the activity of neprilysin in primary
nerve cells and has come to accomplish the present invention.
[0006] That is, the present invention provides the following
features. [0007] (1) In a method of measuring the activity of
neprilysin using a fluorescent substance, the method of measuring
the activity of neprilysin which comprises using an isolated nerve
cell or an immobilized nerve cell; [0008] (2) The method of
measuring the activity of neprilysin according to (1), wherein an
isolated and immobilized nerve cell is used; [0009] (3) The method
of measuring the activity of neprilysin according to (1), wherein a
nerve cell immobilized with paraformaldehyde is used; [0010] (4)
The measuring method according to (1), wherein the nerve cell is a
cultured cell; [0011] (5) The measuring method according to (4),
wherein the cultured cell is a primary cultured cell; [0012] (6)
The measuring method according to (1), wherein dendrites of the
nerve cell are used; [0013] (7) The measuring method according to
(1), wherein an axon of the nerve cell is used; [0014] (8) The
measuring method according to (1), wherein the synaptic terminal of
the nerve cell is used; [0015] (9) The measuring method according
to (1), wherein synaptic vesicles of the nerve cell are used;
[0016] (10) The measuring method according to (1), wherein the
nerve cell prepared from the cerebral cortex or the hippocampus is
used; [0017] (11) The measuring method according to (1), wherein
the fluorescent substance is the reaction product of
4-methoxy-2-naphthylamide and nitrosalicylaldehyde; [0018] (12) The
measuring method according to (11), wherein
4-methoxy-2-naphthylamide is derived from a substrate of
neprilysin; [0019] (13) The measuring method according to (12),
wherein the substrate of neprilysin is
glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide;
[0020] (14) The measuring method according to (11), wherein
4-methoxy-2-naphthylamide is obtained by treating
glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide with
neprilysin and then with an aminopeptidase; [0021] (15) The
measuring method according to (1), wherein the fluorescent
substance is 7-amino-4-methylcoumarine; [0022] (16) The measuring
method according to (15), wherein 7-amino-4-methylcoumarine is
obtained by treating succinyl-alanyl-alanyl-phenylalanine
4-methylcoumarin-7-amide with neprilysin; [0023] (17) A method of
screening a compound or its salt enhancing the activity of
neprilysin, which comprises using the measuring method according to
(1); [0024] (18) A compound or its salt enhancing the activity of
neprilysin, which is obtainable using the screening method
according to (17); [0025] (19) A pharmaceutical comprising the
compound or its salt according to (18); [0026] (20) The
pharmaceutical according to (19), which is a preventive and/or
therapeutic agent, a symptom-improving agent or a
progression-retarding agent for Alzheimer's disease; [0027] (21) A
method of screening a compound or its salt enhancing the expression
of neprilysin, which comprises using the measuring method according
to (1); [0028] (22) A compound or its salt enhancing the activity
of neprilysin, -which is obtainable using the screening method
according to (21); [0029] (23) A pharmaceutical comprising the
compound or its salt according to (22); [0030] (24) The
pharmaceutical according to (23), which is a preventive and/or
therapeutic agent, a symptom-improving agent or a
progression-retarding agent for Alzheimer's disease; [0031] (25) A
method of diagnosis for Alzheimer's disease, which comprises using
the method of measuring the activity according to (1); [0032] (26)
An agent for enhancing the activity of neprilysin comprising
somatostatin, a protein containing substantially the same amino
acid sequence as somatostatin or its partial peptide, an agonist of
somatostatin receptor or a salt thereof; [0033] (27) An agent for
enhancing the expression of neprilysin comprising somatostatin, a
protein containing substantially the same amino acid sequence as
somatostatin or its partial peptide, an agonist of somatostatin
receptor or a salt thereof; [0034] (28) A method of enhancing the
activity of neprilysin, which comprises administering to a mammal
an effective dose of somatostatin, a protein containing
substantially the same amino acid sequence as somatostatin or its
partial peptide, an agonist of somatostatin receptor or a salt
thereof; [0035] (29) A method of enhancing the expression of
neprilysin, which comprises administering to a mammal an effective
dose of somatostatin, a protein containing substantially the same
amino acid sequence as somatostatin or its partial peptide, an
agonist of somatostatin receptor or a salt thereof; [0036] (30) A
method of enhancing the activity or expression of neprilysin, which
comprises promoting the activity of somatostatin receptor in a
mammal; [0037] (31) Use of somatostatin, a protein containing
substantially the same amino acid sequence as somatostatin or its
partial peptide, an agonist of somatostatin receptor or a salt
thereof to manufacture an agent for enhancing the activity or
expression of neprilysin; [0038] (32) An agent for enhancing the
activity of neprilysin comprising substance P, a protein containing
substantially the same amino acid sequence as substance P or its
partial peptide, an agonist of substance P receptor or a salt
thereof; [0039] (33) An agent for enhancing the expression of
neprilysin comprising substance P, a protein containing
substantially the same amino acid sequence as substance P or its
partial peptide, an agonist of substance P receptor or a salt
thereof; [0040] (34) A method of enhancing the activity of
neprilysin, which comprises administering to a mammal an effective
dose of substance P, a protein containing substantially the same
amino acid sequence as substance P or its partial peptide, an
agonist of substance P receptor or a salt thereof; [0041] (35) A
method of enhancing the expression of neprilysin, which comprises
administering to a mammal an effective dose of substance P, a
protein containing substantially the same amino acid sequence as
substance P or its partial peptide, an agonist of substance P
receptor or a salt thereof; [0042] (36) A method of enhancing the
activity or expression of neprilysin, which comprises promoting the
activity of substance P receptor in a mammal; [0043] (37) Use of
substance P, a protein containing substantially the same amino acid
sequence as substance P or its partial peptide, an agonist of
substance P receptor or a salt thereof to manufacture an agent for
enhancing the activity or expression of neprilysin; [0044] (38) An
agent for enhancing the activity of neprilysin comprising an
antagonist of pituitary adenylate cyclase-activating polypeptide
receptor, or a salt thereof; [0045] (39) An agent for enhancing the
expression of neprilysin comprising an antagonist of pituitary
adenylate cyclase-activating polypeptide receptor, or a salt
thereof; [0046] (40) A method of enhancing the activity of
neprilysin, which comprises administering an effective dose of an
antagonist of pituitary adenylate cyclase-activating polypeptide
receptor, or a salt thereof, to a mammal; [0047] (41) A method of
enhancing the expression of neprilysin, which comprises
administering an effective dose of an antagonist of pituitary
adenylate cyclase-activating polypeptide receptor, or a salt
thereof, to a mammal; [0048] (42) A method of enhancing the
activity or expression of neprilysin, which comprises inhibiting
the activity of pituitary adenylate cyclase-activating polypeptide
receptor in a mammal; [0049] (43) Use of an antagonist of pituitary
adenylate cyclase-activating polypeptide receptor or a salt thereof
to manufacture an agent for enhancing the activity or expression of
neprilysin; [0050] (44) An agent for enhancing the activity of
neprilysin comprising an antagonist of vasoactive intestinal
peptide receptor or a salt thereof; [0051] (45) An agent for
enhancing the expression of neprilysin comprising an antagonist of
vasoactive intestinal peptide receptor or a salt thereof; [0052]
(46) A method of enhancing the activity of neprilysin, which
comprises administering an effective dose of vasoactive intestinal
peptide receptor or a salt thereof to a mammal; [0053] (47) A
method of enhancing the expression of neprilysin, which comprises
administering an effective dose of vasoactive intestinal peptide
receptor or a salt thereof to a mammal; [0054] (48) A method of
enhancing the activity or expression of neprilysin, which comprises
inhibiting the activity of vasoactive intestinal peptide receptor
in a mammal; [0055] (49) Use of an antagonist of vasoactive
intestinal peptide receptor or a salt thereof to manufacture an
agent for enhancing the activity or expression of neprilysin; and
the like.
[0056] The present invention further provides: [0057] (50) a method
of preventing and/or treating Alzheimer's disease, which comprises
enhancing the activity or expression of neprilysin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 is a microscopic photograph showing the results from
the activity staining of the neprilysin activity in mouse primary
cultured nerve cells.
[0059] FIG. 2 is a microscopic photograph showing the effects of
somatostatin and thiorphan against the neprilysin activity in mouse
primary cultured nerve cells.
[0060] FIG. 3 shows the results of quantification of the stained
images obtained in EXAMPLE 2 using image analysis software.
[0061] FIG. 4 is a microscopic photograph showing the effects of
PACAP and thiorphan against the neprilysin activity in mouse
primary cultured nerve cells.
[0062] Hereinafter the present invention is described in
detail.
BEST MODE FOR CARRYING OUT THE INVENTION
[0063] (1) Method of measuring the activity of neprilysin
[0064] In a first embodiment of the present invention, it is
provided a method of measuring the activity of neprilysin using a
fluorescent substance, characterized by using an isolated nerve
cell (viable cell) or an immobilized nerve cell (preferably, an
isolated and immobilized nerve cell).
[0065] The nerve cell used in the measuring method of the present
invention is normally collected from any of brain regions (e.g.,
olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus,
thalamus, hypothalamus, subthalamic nucleus, cerebral cortex,
medulla oblongata, cerebellum, occipital lobes, frontal lobe,
lateral lobe, putamen, caudate nucleus, corpus callosum, substantia
nigra), using publicly known methods (methods described in, e.g.,
Culturing Nerve Cells (The MIT press, 1991), etc.). Preferably, the
nerve cell collected from the cerebral cortex or hippocampus is
used in the present invention.
[0066] In the present invention, the nerve cell collected/isolated
as described above can be used as it is, but usually, the collected
cell is cultured and the resulting cultured cell is used. Since the
property of cell becomes stable and sticky by culturing the cell, a
primary cultured cell is preferably used. Further in view of the
function of nerve cells, it is preferred to use the dendrites,
axons, synaptic terminals, synaptic vesicles, etc, of nerve cells,
not the whole nerve cell.
[0067] Culture is performed under conditions appropriately selected
by one skilled in the art. For example, the cells collected from
the regions as described above are cultured oh a microtiter plate
or a chamber slide in a medium such as serum-supplemented
Neurobasal Medium (LifeTech), serum-supplemented DEME,
serum-supplemented MEM, etc. at 37.degree. C. in the presence of 5%
CO.sub.2 for 4 to 6 days. Alternatively, culture may be performed
for about 21 to about 28 days. As will be later described, the
cells are ordinarily observed microscopically in the measuring
method of the present invention and, it is advantageous to culture
the nerve cells obtained on a chamber slide.
[0068] In the measuring method of the present invention, a test
compound, etc. are added to the cells and after incubation for a
given period of time, the cells are preferably immobilized using a
fixing agent. As the cell fixing agent used in the present
invention, there are paraformaldehyde, formalin, acetone, etc. The
cell fixing agent advantageously used in the present invention is
paraformaldehyde. Conditions for the immobilization can be
appropriately selected by one skilled in the art (see the
literature such as Current Protocols in Cell Biology (John Wiley
& Sons, NIH), etc.). The immobilization is effected by treating
the cells, e.g., in a paraformaldehyde solution (1.5%
paraformaldehyde/50 mM phosphate buffer, pH 6.8) for 10 to 40
minutes.
[0069] In one embodiment of the measuring method of the present
invention, a test compound and a substrate solution are reacted
with the isolated/immobilized cells as described above, followed by
further adding a solution mixture of aminopeptidase and
phosphoramidone and a nitrosalicylaldehyde solution to the mixture
and reacting them. Examples of the substrate used here are
synthetic substrates such as
glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide,
glutaryl-alanyl-alanyl-phenylalanyl-2-napthylamide,
succinyl-alanyl-alanyl-phenylalanine 4-methylcoumarin-7-amide, etc.
A preferred substrate is
glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide.
Reaction conditions for the reactions described above may be
suitably chosen by one skilled in the art, depending upon cells
used, substrate, kind/amount of test compound, etc. For example,
where the nerve cell prepared from the cerebral cortex,
hippocampus, etc. is used as the cell and
glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide is
used as the substrate, the reaction of the cell with a test
compound and a substrate solution is carried out at 0 to 20.degree.
C. for 1 to 72 hours. The following reaction with the solution
mixture of aminopeptidase and phosphoramidone and a
nitrosalicylaldehyde solution is carried out, e.g., at 20 to
40.degree. C. for 10 to 60 minutes.
[0070] In the reactions described above, a compound used as the
substrate is degraded by the neprilysin and aminopeptidase
treatment and the product obtained by the degradation is reacted
with, e.g., nitrosalicyl aldehyde to form a fluorescent substance.
When glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide
is used as the substrate, this is degraded to form free
4-methoxy-2-naphthylamine (MNA). This free MNA is reacted with
nitrosalicylaldehyde to form an insoluble yellow fluorescent
substance. On the other hand, when
succinyl-alanyl-alanyl-phenylalanine 4-methylcoumarin-7-amide is
used as the substrate, the amide is degraded to form
7-amino-4-methylcoumarine.
[0071] In the measuring method of the present Invention, the
reactions as described above are carried out and positive stained
images by the produced fluorescent substance are observed, e.g.,
under a confocal laser scanning microscope with an Argon laser and
a filter for rhodamine.
[0072] The enhanced neprilysin activity or gene expression can be
quantified from the stained images obtained in the activity
measurement described above by digitalizing its fluorescence
intensity, using software for image analysis. As the software,
there are used MetaVue from Nippon Roper Co., Ltd., and the like.
[0073] (2) Method of screening a compound enhancing the activity
and/or expression of neprilysin using neprilysin activity
staining
[0074] In another embodiment of the present invention, there is
provided a method of screening a compound or its salt enhancing the
activity of neprilysin, which comprises using the method of
measuring the activity of neprilysin described above. That is, a
compound (e.g., a peptide, a protein, a non-peptide compound, a
synthetic compound, a fermentation product, etc.) enhancing the
activity and/or expression of neprilysin, or a salt thereof can be
efficiently screened using the neprilysin activity staining of the
present invention.
[0075] Such a compound includes (a) a compound having the effect of
promoting (or enhancing) the neprilysin activity, (b) a compound
having the effect of Increasing (or enhancing) the expression of
neprilysin gene, and the like.
[0076] Specifically, in the screening method of the present
invention, a compound enhancing the activity or expression of
neprilysin, or a salt thereof, is screened by the method, which
comprises comparing (i) the case that a nerve cell alone is used
without using a test compound and (ii) the case that the nerve cell
is treated with the test compound, in terms of the neprilysin
activity (e.g., the activity determined from neprilysin inhibition
induced by thiorphan, which is a specific enzyme inhibitor of
neprilysin; etc.).
[0077] More specifically, the nerve cell prepared as described
above is first incubated on a chamber slide. After incubation of
the cell, a test compound is added to the cell in an amount
sufficiently reacting with the cell, though the amount depends on
kind or concentration of the test compound. After incubation for a
given period of time (e.g., for 24 hours in a final concentration
of 1 .mu.M in the case of somatostatin later described), the cell
is immobilized with paraformaldehyde. The immobilized cell is
reacted with a substrate solution
(glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide) and
an aminopeptidase-phosphoramidone solution mixture and a
nitrosalicylaldehyde solution are further added to and reacted with
the reaction mixture. Reaction conditions for these reactions are
the same as described above. After the reaction, positive stained
images are observed under a confocal laser scanning microscope
using an Argon laser and a filter for rhodamine.
[0078] In the screening method of the present invention, for
example, peptides (for example, angiotensin, bombesin, canavinoid,
cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y,
opioids, purines, vasopressin, oxytocin, substance P, PACAP,
secretin, glucagon, calcitonin, adrenomedulin, somatostatin, GHRH,
CRF, ACTH, GRP, PTH, VIP, dopamine, motilin, amylin, bradykinin,
CGRP (calcitonin gene-related peptide), pancreastatin, thromboxane,
adrenaline, the chemokine superfamily, endothelin, enterogastrin,
histamine, neurotensin, TRH, pancreatic polypeptide, galanin),
proteins, non-peptide compounds, synthetic compounds, fermentation
products, cell extracts, vegetable extracts, animal tissue
extracts, etc. are employed as the test compound. These compounds
may be novel compounds or publicly known compounds. Also, at least
two of these compounds may be mixed and the mixture may be provided
as a sample. Preferred test compounds in the screening method of
the present invention are a protein containing substantially the
same amino acid sequence as somatostatin or its partial peptide, an
agonist of somatostatin receptor or its salt, a protein containing
substantially the same amino acid sequence as substance P or its
partial peptide, an agonist of substance P receptor or its salt
and, PACAP and VIP receptor antagonists or salts thereof, which
will be later described.
[0079] In the screening method, when the nerve cell is treated with
a test compound and the neprilysin activity increases by about 10%
or more, preferably by about 30% or more and more preferably by
about 50% or more, the test compound can be selected as a compound
having the effect of enhancing the activity or expression of
neprilysin. [0080] (3) Pharmaceutical comprising the compound
obtained as a result of screening
[0081] The compound obtained using the screening method is a
compound selected from the test compounds described above and has
the effect of enhancing the activity or expression of neprilysin.
Thus, the compound can be used as a pharmaceutical, such as a safe
and low toxic preventive and/or therapeutic agent for Alzheimer's
disease, etc. In addition, compounds derived from the compound
obtained by the screening described above can be used as well.
[0082] (4) Method for diagnosis of Alzheimer's disease
[0083] By using the activity measuring method of the present
invention, presymptomatic diagnosis of Alzheimer's disease can be
made. That is, the results obtained by the activity measurement of
the present invention are quantified and the quantified values are
compared with those obtained with control. It is thus possible to
predict a risk of developing Alzheimer's disease in its subject.
[0084] (5) Agent for enhancing the neprilysin activity
[0085] In a still other embodiment of the present invention, there
is provided the agent for enhancing the activity of neprilysin or
the agent for enhancing the expression of neprilysin comprising
somatostatin, a protein containing substantially the same amino
acid sequence as somatostatin or its partial peptide, or an agonist
of somatostatin receptor or its salt. Somatostatin is a publicly
known protein (peptide) composed of 14 amino acids
(Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys) and is
disclosed in, e.g., Proc. Natl. Acad. Sci. USA, 80, 6932-6936,
1983; Science, 224, 168-171, 1984, etc.
[0086] Herein, "substantially the same amino acid sequence" is used
to mean an amino acid sequence having at least about 50% homology,
preferably at least about 60% homology, more preferably at least
about 70% homology, much more preferably at least about 80%
homology, particularly preferably at least about 90% homology and
most preferably at least about 95% homology, to the amino acid
sequence to be compared. Also, the "partial peptide" may be any
peptide so far as it is a partial peptide of somatostatin but
preferably include a peptide having the sequence of at least 3,
preferably at least 5, and more preferably at least 7 amino acids,
in the constituent amino acid sequence, and the like.
[0087] By administering to a mammal an effective dose of
somatostatin, a protein containing substantially the same amino
acid sequence as somatostatin or its partial peptide, an agonist of
somatostatin receptor or its salt, the activity of neprilysin or
the expression of neprilysin in the target mammal can be
enhanced.
[0088] In a still other embodiment of the present invention, there
is provided the agent for enhancing the activity of neprilysin or
the agent for enhancing the expression of neprilysin comprising
substance P, a protein containing substantially the same amino acid
sequence as substance P or its partial peptide, or an agonist of
substance P receptor or its salt. Substance P is a publicly known
peptide composed of 11 amino acids
(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) and is disclosed in,
e.g., Neuropeptides: Regulators of Physiological Processes, (Fleur
L. Strand, ed.), The MIT Press, Massachusetts, 1999, etc.
[0089] Herein, "substantially the same amino acid sequence" is used
to mean an amino acid sequence having at least about 50% homology,
preferably at least about 60% homology, more preferably at least
about 70% homology, much more preferably at least about 80%
homology, particularly preferably at least about 90% homology and
most preferably at least about 95% homology, to the amino acid
sequence to be compared. Also, the "partial peptide" may be any
peptide so far as it is a partial peptide of substance P but
preferably include a peptide having the sequence of at least 3,
preferably at least 5, and more preferably at least 7 amino acids,
in the constituent amino acid sequence, and the like.
[0090] By administering to a mammal an effective dose of substance
P, a protein containing substantially the same amino acid sequence
as substance P or its partial peptide, an agonist of substance P
receptor or its salt, the activity of neprilysin or the expression
of neprilysin in the target mammal can be enhanced.
[0091] In a still other embodiment of the present invention, there
is provided the agent for enhancing the activity of neprilysin or
the agent for enhancing the expression of neprilysin comprising the
antagonist of PACAP receptor, VIP receptor, etc., or a salt
thereof. These are publicly known peptides disclosed in
Neuropeptides: Regulators of Physiological Processes, (Fleur L.
Strand, ed.), The MIT Press, Massachusetts, 1999, etc.
[0092] PACAP or VIP inhibitorily acts against the neprilysin
activity or its gene expression. Therefore, by administering to a
mammal an effective dose of antagonist of PACAP receptor, VIP
receptor, etc., or a salt thereof, the activity of neprilysin or
the expression of neprilysin can be enhanced in the target
mammal.
[0093] In the present invention, a novel agent for enhancing the
activity or expression of neprilysin can be screened using the
known agents for enhancing the activity or expression of neprilysin
described above as probes. That is, a novel agent for enhancing the
activity or expression of neprilysin can be screened by comparing
the level of the activity or expression of neprilysin when a test
compound is added to neprilysin, with the level of the neprilysin
activity or expression when no test compound is added to
neprilysin, the activity or expression of neprilysin being enhanced
by somatostatin, the agonist of somatostatin receptor, substance P,
the agonist of substance P receptor, the agonists to PACAP and VIP
receptors, or the like. Alternatively, a novel agent for enhancing
the activity or expression of neprilysin can be screened by
comparing the level of the activity or expression of neprilysin
enhanced by somatostatin, the agonist of somatostatin receptor,
substance P, the agonist of substance P receptor, the agonists to
PACAP and VIP receptors, or the like, with the level of the
activity or expression of neprilysin enhanced by a test compound.
As the test compound, there are used, for example, peptides (e.g.,
angiotensin, bombesin, canavinoid, cholecystokinin, glutamine,
serotonin, melatonin, neuropeptide Y, opioids, purines,
vasopressin, oxytocin, substance P, PACAP, secretin, glucagon,
calcitonin, adrenomedulin, somatostatin, GHRH, CRF, ACTH, GRP, PTH,
VIP, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin
gene-related peptide), pancreastatin, thromboxane, adrenaline, the
chemokine superfamily, endothelin, enterogastrin, histamine,
neurotensin, TRH, pancreatic polypeptide, galanin), proteins,
non-peptide compounds, synthetic compounds, fermentation products,
cell extracts, vegetable extracts, animal tissue extracts, etc. are
employed as the test compound. These compounds may be novel
compounds or publicly known compounds. Also, at least two of these
compounds may be mixed and the mixture may be provided as a sample.
[0094] (6) Pharmaceutical preparations, dose, administration
method, etc.
[0095] When it is intended to use the compounds obtained by the
screening method using the activity measuring method of the present
invention as agents for promoting A.beta. degradation or removing
A.beta. and furthermore as preventive and/or therapeutic agents for
Alzheimer's disease, utilizing the neprilysin activity, the
compounds can be prepared into pharmaceutical preparations in
conventional manners.
[0096] Where the compounds described above can form
pharmaceutically acceptable salts thereof, such salts may be
formed. As these salts, there may be used salts with
physiologically acceptable acids (e.g., inorganic acids, organic
acids, etc.) or bases (e.g., alkali metal salts, etc.), preferably
in the form of physiologically acceptable acid addition salts.
Examples of such salts are salts with inorganic acids (e.g.,
hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric
acid, etc.), salts with organic acids (e.g., acetic acid, formic
acid, propionic acid, fumaric acid, maleic acid, succinic acid,
tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
methanesulfonic acid, benzenesulfonic acid, etc.) and the like.
[0097] The compounds described above (including salts thereof) can
be used orally in the form of tablets which, if necessary, may be
coated with sugar, capsules, elixirs, microcapsules, etc., or
parenterally in the form of injectable preparations such as a
sterile solution or a suspension in water or with other
pharmaceutically acceptable liquid. These preparations can be
manufactured, e.g., by mixing the active compounds described above
with publicly known carriers recognized to be physiologically
acceptable, flavoring agents, excipients, vehicles, antiseptics,
stabilizers, binders, etc., in a unit dosage form required in a
generally accepted manner applied to making pharmaceutical
preparations. The active ingredient in the preparation is
controlled in such an amount that an appropriate dose is obtained
within the specified range given.
[0098] Additives miscible with tablets, capsules, etc. include
binders such as gelatin, corn starch, tragacanth or gum arabic,
excipients such as crystalline cellulose, swelling agents such as
com starch, gelatin, alginic acid, etc., lubricants such as
magnesium stearate, sweetening agents such as sucrose, lactose or
saccharin, flavoring agents such as peppermint, akamono oil or
cherry, and the like. When the unit dosage is in the form of
capsules, liquid carriers such as oils and fats may further be used
together with the additives described above. A sterile composition
for injection may be formulated following a conventional manner
used to make pharmaceutical compositions, e.g., by dissolving or
suspending the active ingredients in a vehicle such as water for
injection with a naturally occurring vegetable oil such as sesame
oil, coconut oil, etc. to prepare the pharmaceutical composition.
Examples of an aqueous medium for injection include physiological
saline, an isotonic solution containing glucose and other auxiliary
agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) or the
like, which may be used in combination with an appropriate
dissolution aid such as an alcohol (e.g., ethanol), a polyalcohol
(e.g., propylene glycol, polyethylene glycol), a nonionic
surfactant (e.g., polysorbate 80.TM. and HCO-50), etc. As an oily
medium, for example, sesame oil, soybean oil or the like may be
used, which can be used in combination with a dissolution aid such
as benzyl benzoate, benzyl alcohol, etc.
[0099] Furthermore, the preventive and/or therapeutic agents
described above may also be formulated with buffers (e.g.,
phosphate buffer, sodium acetate buffer), soothing agents (e.g.,
benzalkonium chloride, procaine hydrochloride, etc.), stabilizers
(e.g., human serum albumin, polyethylene glycol, etc.),
preservatives (e.g., benzyl alcohol, phenol, etc.), antioxidants,
etc. The thus prepared liquid for injection is normally filled in
an appropriate ampoule.
[0100] The dose of the compound obtained in the present invention
varies depending on subject to be administered, conditions, route
for administration, etc.; in oral administration, the dose for a
patient (as 60 kg) is normally about 0.1 mg to about 100 mg,
preferably about 1.0 to about 50 mg, and more preferably about 1.0
to about 20 mg per day. In parenteral administration, the single
dose may vary depending on subject to be administered, conditions,
route for administration, etc. but it is advantageous to administer
the compound intravenously to a patient (as 60 kg) in a daily dose
of about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg,
and more preferably about 0.1 to about 10 mg. For other animal
species, the corresponding dose as converted per 60kg can be
administered.
[0101] The thus obtained pharmaceutical preparation can be
administered to human or other mammals (e.g., mice, rats, rabbits,
sheep, swine, bovine, cats, dogs, monkeys, etc.).
[0102] In the specification and drawings, where the codes of bases,
amino acids, etc. are denoted in abbreviations, they are based on
the abbreviations in accordance with the IUPAC-IUB Commission on
Biochemical Nomenclature or by the common codes in the art. For
amino acids that may have the optical isomer, L form is presented
unless otherwise indicated.
EXAMPLES
Example 1
Neprilysin Activity Staining in Primary Cultured Nerve Cells
[0103] Nerve cells were prepared from the mouse fetal cerebral
cortex and hippocampus and seeded on a poly-L-coated chamber slide
(8 chambers, IWAKI 4730-040) at a density of 1.times.10.sup.5
cells/chamber, followed by incubation for 7 days. After completion
of the incubation, the cells were washed with TBS (pH 7.4) and
fixed for 12.5 minutes at 20.degree. C. with a paraformaldehyde
solution (1.5% paraformaldehyde/50 mM phosphate buffer, pH 6.8).
After the cells were washed twice with TBS, 0.2 ml of substrate
solution
(glutaryl-alanyl-alanyl-phenylalanyl-4-methoxy-2-naphthylamide,
Sigma G3769, final concentration: 0.5 mM; 0.05 M Tris-HCl buffer,
pH 7.4 (pH was adjusted to 7.4 at 4.degree. C.)) was added to the
cells, followed by reacting at 4.degree. C. for 48 hours. After
Completion of the reaction, 25 .mu.l of the solution mixture of
aminopeptidase (Sigma L-5006, final concentration: 20.4 .mu.g/ml)
and phosphoramidone (Peptide Inst. 4082, final concentration: 10
.mu.M) and 25 .mu.l of nitrosalicylaldehyde solution (6 mM) were
further added thereto sequentially. The mixture was reacted at
37.degree. C. for 30 minutes. The reaction was completed by washing
the cells twice with TBS. Using argon laser and a filter for
rhodamine, positive stained images were observed under a confocal
laser scanning microscope (FIG. 1). When the substrate is degraded
by neprilysin, an insoluble fluorescent reaction product is formed
so that the sites where neprilysin is present are visualized in
yellow. By this method it was confirmed that the enzyme activity of
neprilysin was present in the primary cultured nerve cells of the
mouse fetal cerebral cortex and hippocampus. The sites where
neprilysin was present were prominent in the protruding parts of
the nerve cells.
Example 2
Induction of the Neprilysin Enzyme Activity by Somatostatin
[0104] Nerve cells were prepared from the mouse fetal cerebral
cortex and hippocampus, followed by incubation as in EXAMPLE 1. On
day 7 of the incubation, somatostatin (somatostatin 14 and 28,
final concentration: 1 .mu.M) was added to the cells, followed by
incubation for further 24 hours. After completion of the
incubation, the neprilysin activity in the mouse primary cultured
nerve cells was visualized and observed by the procedure shown in
EXAMPLE 1 (FIG. 2). By the addition of somatostatin, the neprilysin
activity in the nerve cells markedly increased as compared to
control added with no somatostatin. It was confirmed that this
neprilysin enzyme activity was specific to neprilysin, since the
neprilysin enzyme activity almost completely disappeared by adding
a neprilysin-specific enzyme inhibitor or thiorphan (Sigma T6031,
final concentration: 10 .mu.M) to the substrate solution. To
investigate in further detail, these stained images were quantified
by image analysis software (Nippon Roper Co., Ltd., MetaVue) and
examined. As a result, it was confirmed that the enzyme activity of
neprilysin increased by about 2.5 times with somatostatin (FIG.
3).
Example 3
Regulation of the Neprilysin Enzyme Activity by Neuropeptides
[0105] Nerve cells were prepared from the mouse fetal cerebral
cortex and hippocampus, followed by incubation as in EXAMPLE 1. On
day 7 of the incubation, various neuropeptides such as substance P,
PACAP, VIP, etc. (final concentration: 1 .mu.M) was added to the
cells, followed by incubation for further 24 hours. After
completion of the incubation, the neprilysin activity in the mouse
primary cultured nerve cells was visualized and observed by the
procedure shown in EXAMPLE 1. The cells with markedly changed
(enhanced or inhibited) neprilysin activity by the addition of the
peptides as compared to control added with no peptide were
selected.
[0106] PACAP (PACAP 38, final concentration: 1 .mu.M) was added and
treated as in EXAMPLE 2. The results are shown in FIG. 4. By the
addition of PACAP, the neprilysin activity of the nerve cells
markedly decreased when compared to control added with no PACAP.
Also, it was confirmed that this neprilysin enzyme activity was
specific to neprilysin, since the neprilysin enzyme activity almost
completely disappeared by adding a neprilysin-specific enzyme
inhibitor or thiorphan (Sigma T6031, final concentration: 10 .mu.M)
to the substrate solution.
INDUSTRIAL APPLICABILITY
[0107] The compounds enhancing the activity and/or expression of
neprilysin, which are obtained by the screening method
characterized by using the method of measuring the activity of
neprilysin according to the present invention, are useful as agents
for promoting A.beta. degradation or removing A.beta. and
furthermore as preventive and/or therapeutic agents for Alzheimer's
disease, utilizing the neprilysin activity. Also, the activity
measuring method of the present invention can be used for
presymptomatic diagnosis of Alzheimer's disease.
Sequence CWU 1
1
2114PRTUnknown OrganismDescription of Unknown Organism Somatostatin
peptide 1Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 1
5 10211PRTUnknown OrganismDescription of Unknown Organism Substance
P peptide 2Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10
* * * * *