U.S. patent application number 10/552014 was filed with the patent office on 2006-09-28 for novel screening method.
Invention is credited to Ryo Fujii, Tadatoshi Hashimoto, Shuji Hinuma, Yasuaki Ito, Makoto Kobayashi, Yasuhiro Tanaka.
Application Number | 20060216286 10/552014 |
Document ID | / |
Family ID | 33410077 |
Filed Date | 2006-09-28 |
United States Patent
Application |
20060216286 |
Kind Code |
A1 |
Ito; Yasuaki ; et
al. |
September 28, 2006 |
Novel screening method
Abstract
By using a G protein-coupled receptor protein comprising an
amino acid sequence, which is the same or substantially the same as
the amino acid sequence represented by SEQ ID NO: 1, or a salt
thereof, and an ionizable metal element or a salt thereof, an
agonist for or an antagonist to the above receptor protein or a
salt thereof can be efficiently screened.
Inventors: |
Ito; Yasuaki; (Ibaraki,
JP) ; Fujii; Ryo; (Ibaraki, JP) ; Kobayashi;
Makoto; (Osaka, JP) ; Hinuma; Shuji; (Ibaraki,
JP) ; Hashimoto; Tadatoshi; (Osaka, JP) ;
Tanaka; Yasuhiro; (Ibaraki, JP) |
Correspondence
Address: |
TAKEDA PHARMACEUTICALS NORTH AMERICA, INC;INTELLECTUAL PROPERTY DEPARTMENT
475 HALF DAY ROAD
SUITE 500
LINCOLNSHIRE
IL
60069
US
|
Family ID: |
33410077 |
Appl. No.: |
10/552014 |
Filed: |
April 23, 2004 |
PCT Filed: |
April 23, 2004 |
PCT NO: |
PCT/JP04/05947 |
371 Date: |
October 12, 2005 |
Current U.S.
Class: |
424/143.1 ;
435/7.1 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 33/68 20130101; G01N 33/5008 20130101; G01N 33/5041 20130101;
G01N 2333/726 20130101; G01N 2500/00 20130101 |
Class at
Publication: |
424/143.1 ;
435/007.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 2003 |
JP |
2003-122464 |
Claims
1. A method of screening a compound or element or a salt thereof
that changes the binding property or signal transduction of a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, to an
ionizable metal element or a salt thereof, which comprises using
(1) said receptor protein, its partial peptide, or a salt thereof
and (2) said metal element or a salt thereof.
2. The screening method according to claim 1, wherein said G
protein-coupled receptor protein is a G protein-coupled receptor
protein consisting of the amino acid sequence represented by SEQ ID
NO: 1, SEQ ID NO: 6 or SEQ ID NO: 8.
3. A kit for screening a compound or element or a salt thereof that
changes the binding property or signal transduction of a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, to an
ionizable metal element or a salt thereof, which comprises (1) said
receptor protein, its partial peptide, or a salt thereof and (2)
said metal element or a salt thereof.
4. A pharmaceutical, which comprises a compound or element or a
salt thereof that changes the binding property or signal
transduction of an ionizable metal element or a salt thereof to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof.
5. A prophylactic/therapeutic agent for, wounds, burn, declining of
learning ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, diabetes mellitus, respiratory
disturbances, indigestion, cardiac disturbances or hypothyroidism,
which comprises an agonist for a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or a salt
thereof.
6. A prophylactic/therapeutic agent for hypotonic bladder induced
by sensory decrease of the bladder or hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs, which
comprises an agonist for a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or a salt
thereof.
7. A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction or sensory neuropathy, which comprises an
antagonist to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof.
8. A prophylactic/therapeutic agent for overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises an antagonist to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or a salt
thereof.
9. A method of screening an agonist for a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, which comprises assaying an
intracellular Ca.sup.2+ level increasing activity when a test
compound or element or a salt thereof is brought in contact with a
cell containing said receptor protein.
10. A method of screening an agonist for or an antagonist to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises using (1) said receptor protein or its partial peptide,
or a salt thereof and (2) a compound or element or a salt thereof
that changes the binding property of said receptor protein or a
salt thereof to an ionizable metal element or a salt thereof.
11. A kit for screening an agonist for or an antagonist to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises (1) said receptor protein or its partial peptide, or a
salt thereof and (2) a compound or element or a salt thereof that
changes the binding property of said receptor protein or a salt
thereof to an ionizable metal element or a salt thereof.
12. A prophylactic/therapeutic agent for wounds, burn, declining of
learning ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, diabetes mellitus, respiratory
disturbances, indigestion, cardiac disturbances or hypothyroidism,
which comprises a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
or a salt thereof.
13. A prophylactic/therapeutic agent for hypotonic bladder induced
by sensory decrease of the bladder or hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs, which
comprises a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, or a
salt thereof.
14. A prophylactic/therapeutic agent for, wounds, burn, declining
of learning ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, diabetes mellitus, respiratory
disturbances, indigestion, cardiac disturbances or hypothyroidism,
which comprises a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
15. A prophylactic/therapeutic agent for hypotonic bladder induced
by sensory decrease of the bladder or hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs, which
comprises a polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
16. A diagnostic agent for wounds, burn, declining of learning
ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, diabetes mellitus, respiratory
disturbances, indigestion, cardiac disturbances, hypothyroidism,
renal dysfunction, pulmonary dysfunction or sensory neuropathy,
which comprises a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
17. A diagnostic agent for hypotonic bladder induced by sensory
decrease of the bladder, hypotonic bladder induced by postsurgical
bladder anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises a polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
18. A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction or sensory neuropathy, which comprises an
antibody to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
or a salt thereof.
19. A prophylactic/therapeutic agent for overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises an antibody to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, or a salt thereof.
20. A diagnostic agent for wounds, burn, declining of learning
ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, diabetes mellitus, respiratory
disturbances, indigestion, cardiac disturbances, hypothyroidism,
renal dysfunction, pulmonary dysfunction or sensory neuropathy,
which comprises an antibody to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, or a salt thereof.
21. A diagnostic agent for hypotonic bladder induced by sensory
decrease of the bladder, hypotonic bladder induced by postsurgical
bladder anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises an antibody to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, or a salt thereof.
22. A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction or sensory neuropathy, which comprises a
polynucleotide containing the entire or part of a base sequence
complementary to a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
23. A prophylactic/therapeutic agent for overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises a polynucleotide containing the entire or part of a base
sequence complementary to a polynucleotide comprising a
polynucleotide encoding a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide.
24. A method of screening a compound or its salt that changes the
expression level of a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1 to prevent/treat wounds,
burn, declining of learning ability, hypogonadism, dysgeusia,
anosmia, hyperplasia, arteriosclerosis, myocardial infarction,
apoplexy, cirrhosis, cholesterol accumulation, cancer, diabetes
mellitus, respiratory disturbances, indigestion, cardiac
disturbances, hypothyroidism, renal dysfunction, pulmonary
dysfunction or sensory neuropathy, which comprises using a
polynucleotide comprising a polynucleotide encoding said receptor
protein or its partial peptide.
25. A method of screening a compound or a salt thereof that changes
the expression level of a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, to
prevent/treat hypotonic bladder induced by sensory decrease of the
bladder, hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises using a polynucleotide comprising a polynucleotide
encoding said receptor protein or its partial peptide.
26. A kit for screening a compound or its salt that changes the
expression level of a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1 to prevent/treat wounds,
burn, declining of learning ability, hypogonadism, dysgeusia,
anosmia, hyperplasia, arteriosclerosis, myocardial infarction,
apoplexy, cirrhosis, cholesterol accumulation, cancer, diabetes
mellitus, respiratory disturbances, indigestion, cardiac
disturbances, hypothyroidism, renal dysfunction, pulmonary
dysfunction or sensory neuropathy, which comprises a polynucleotide
comprising a polynucleotide encoding said receptor protein or its
partial peptide.
27. A kit for screening a compound or a salt thereof that changes
the expression level of a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, to
prevent/treat hypotonic bladder induced by sensory decrease of the
bladder, hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises a polynucleotide comprising a polynucleotide encoding
said receptor protein or its partial peptide.
28. A prophylactic/therapeutic agent for wounds, burn, declining of
learning ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, hard labor, diabetes mellitus,
respiratory disturbances, indigestion, cardiac disturbances or
hypothyroidism, which comprises a compound or its salt that
increases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide.
29. A prophylactic/therapeutic agent for hypotonic bladder induced
by sensory decrease of the bladder or hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs, which
comprises a compound or its salt that increases the expression
level of a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
30. A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction or sensory neuropathy, which comprises a
compound or its salt that decreases the expression level of a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
31. A prophylactic/therapeutic agent for overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises a compound or its salt that decreases the expression
level of a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
32. A method of preventing/treating wounds, burn, declining of
learning ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, cancer, diabetes mellitus, respiratory
disturbances, indigestion, cardiac disturbances or hypothyroidism,
which comprises administering to a mammal an effective dose of (i)
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, its partial peptide, or a
salt thereof, (ii) a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an agonist for a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that increases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
33. A method of preventing/treating hypotonic bladder induced by
sensory decrease of the bladder or hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs, which
comprises administering to a mammal an effective dose of (i) a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, its partial peptide, or a
salt thereof, (ii) a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an agonist for a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that increases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
34. A method of preventing/treating renal dysfunction, pulmonary
dysfunction or sensory neuropathy, which comprises administering to
a mammal an effective dose of (i) an antibody to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, its partial peptide, or a
salt thereof, (ii) a polynucleotide comprising the entire or part
of a base sequence complementary to a polynucleotide comprising a
polynucleotide encoding a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, (iii) an antagonist to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, or (iv) a compound or its salt that
decreases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide.
35. A method of preventing/treating overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, which
comprises administering to a mammal an effective dose of (i) an
antibody to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, its partial peptide, or
a salt thereof, (ii) a polynucleotide comprising the entire or part
of a base sequence complementary to a polynucleotide comprising a
polynucleotide encoding a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, (iii) an antagonist to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, or (iv) a compound or its salt that
decreases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide.
36. Use of (i) a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, its partial peptide, or
a salt thereof, (ii) a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an agonist for a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that increases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, to
manufacture A prophylactic/therapeutic agent for wounds, burn,
declining of learning ability, hypogonadism, dysgeusia, anosmia,
hyperplasia, arteriosclerosis, myocardial infarction, apoplexy,
cirrhosis, cholesterol accumulation, cancer, diabetes mellitus,
respiratory disturbances, indigestion, cardiac disturbances or
hypothyroidism.
37. Use of (i) a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, its partial peptide, or
a salt thereof, (ii) a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an agonist for a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that increases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, to
manufacture a prophylactic/therapeutic agent for hypotonic bladder
induced by sensory decrease of the bladder or hypotonic bladder
induced by postsurgical bladder anesthesia of the pelvic
organs.
38. Use of (i) an antibody to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, its partial
peptide, or a salt thereof, (ii) a polynucleotide comprising the
entire or part of a base sequence complementary to a polynucleotide
comprising a polynucleotide encoding a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide, (iii) an antagonist to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, or (iv) a compound or its salt that
decreases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide, to manufacture a prophylactic/therapeutic
agent for renal dysfunction, pulmonary dysfunction or sensory
neuropathy.
39. Use of (i) an antibody to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, its partial
peptide, or a salt thereof, (ii) a polynucleotide comprising the
entire or part of a base sequence complementary to a polynucleotide
comprising a polynucleotide encoding a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide, (iii) an antagonist to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, or (iv) a compound or its salt that
decreases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide, to manufacture a prophylactic/therapeutic
agent for overactive bladder-induced pollakiuria, nocturia,
cystitis-induced pollakiuria, prostatic hyperplasia-induced
pollakiuria, urinary incontinence, urinary urgency, pelvic visceral
pain, coital pain, bladder irritation symptoms or various disorders
caused by urinary calculus.
Description
TECHNICAL FIELD
[0001] The present invention relates to use of a G protein-coupled
receptor protein (GPR39).
BACKGROUND ART
[0002] Physiological active substances such as various hormones,
neurotransmitters, etc. regulate the biological function via
specific receptor proteins present on cell membranes. Many of these
receptor proteins are coupled with guanine nucleotide-binding
protein (hereinafter sometimes simply referred to as G protein) and
mediate the intracellular signal transduction via activation of G
protein. These receptor proteins possess the common structure
containing seven transmembrane domains and are thus collectively
referred to as G protein-coupled receptor proteins (GPCR) or
seven-transmembrane receptor proteins (7TMR).
[0003] G protein-coupled receptor proteins are present on the cell
surface of each functional cell and organ in the body, and play a
vitally important physiological role as the target of the molecules
that regulate the functions of the cells and organs, e.g.,
physiologically active substances such as peptide hormones, nucleic
acids, amines, lipids, etc. Receptor proteins transmit signals into
cells via binding with physiologically active substances, and the
signals induce various reactions such as activation and inhibition
of the cells.
[0004] To clarify the relationship between substances that regulate
complex biological functions in various cells and organs in the
body, and their specific receptor proteins, in particular, G
protein-coupled receptor proteins will elucidate the functional
mechanisms in various cells and organs in the body to provide a
very important means for development of drugs closely associated
with these functions.
[0005] For example, in various organs, their physiological
functions are controlled in vivo through regulation by many
hormones, hormone-like substances, neurotransmitters or
physiologically active substances. In particular, physiologically
active substances are found in numerous sites of the body and
regulate the physiological functions through their corresponding
receptor proteins. Many unknown hormones, neurotransmitters or
other physiologically active substances still exist in the body
and, as to their receptor proteins, many of these proteins have not
yet been reported. In addition, a lot remains yet to be established
if there are subtypes of known receptor proteins or if there are
differences depending on species.
[0006] It is very important for development of drugs to clarify the
relationship between substances that regulate elaborated functions
in vivo and their specific receptor proteins. Furthermore, for
efficient screening of agonists for and antagonists to receptor
proteins in development of drugs, it is required to clarify
functional mechanisms of receptor protein genes expressed in vivo
and express the genes in an appropriate expression system.
[0007] In recent years, random analysis of cDNA sequences has been
actively studied as a means for analyzing genes expressed in vivo.
The sequences of cDNA fragments thus obtained have been registered
on and published to databases as Expressed Sequence Tag (EST).
Also, it has been extensively attempted to predict unknown genes
from the analysis of genome sequences. However, since many ESTs or
genome sequences contain sequence information only, it is difficult
to predict their functions.
[0008] The amino acid sequence of human-derived GPR39 and the DNA
encoding the same are reported (Genomics 1997, December
15;46(3):426-34, WO2002/61087, US2002/004491, WO2002/39885,
WO2002/71928, WO2001/81634, WO2002/79492, WO2002/86443). However,
functions of these G protein-coupled receptor proteins and their
physiological ligands remain unresolved.
[0009] Heretofore, substances that inhibit the binding of G
protein-coupled receptor proteins to physiologically active
substances (i.e., ligands) or substances that bind and induce
signals similar to those physiologically active substances (i.e.,
ligands) have been utilized for pharmaceuticals as antagonists or
agonists specific to these receptor proteins that regulate the
biological functions. Therefore, it is a very important means in
search for agonists and antagonists that can be targeted for
pharmaceutical development to determine specific ligands for G
protein-coupled receptor proteins.
[0010] However, many G protein-coupled receptor proteins with
unknown functions and many so-called orphan receptor proteins in
which the corresponding ligands are yet unidentified are present
even at this point of time. Thus, search of ligands for G
protein-coupled receptor proteins and elucidation of their
functions are eagerly awaited.
[0011] G protein-coupled receptor proteins are useful in search for
a novel physiological active substance (i.e., ligand) using the
signal transduction activity as an indicator and in search for
agonists and antagonists to the receptor protein. On the other
hand, even though no physiological ligand is found, agonists and
antagonists to the receptor protein may be prepared by analyzing
the physiological action of the receptor protein through
inactivation experiment of the receptor protein (knockout animal).
Ligands, agonists or antagonists, etc. of these receptor proteins
are expected to be utilized as preventive/therapeutic agents or
diagnostic agents for diseases associated with dysfunction or
hyperfunction of the G protein-coupled receptor proteins.
[0012] Furthermore, the decreased or increased functions of G
protein-coupled receptor proteins due to genetic aberration of the
G protein-coupled receptor proteins in the body often cause some
disorders. In this case, the G protein coupled receptor proteins
may be used not only for administration of antagonists or agonists
for the receptor proteins, but also for gene therapy by introducing
the receptor protein gene into the body (or some particular organs)
or by introducing the antisense nucleic acid of the receptor
protein gene. In this case, information on the base sequence of the
receptor protein is essentially required for investigating deletion
or mutation on the gene. The receptor protein gene is also
applicable as preventive/therapeutic agents or diagnostic agents
for diseases associated with dysfunction of the receptor
protein.
[0013] The present invention relates to determination of ligands
for orphan G protein-coupled receptor proteins with unknown
functions and use of the G protein-coupled receptor proteins and
their ligands. That is, the present invention aims at providing a
method of screening a compound (an antagonist or agonist) or its
salt that changes the binding property of the ligand to the G
protein-coupled receptor protein; a kit for the screening; a
compound (an antagonist or agonist) or its salt that changes the
binding property of the ligand to the G protein-coupled receptor
protein, which is obtainable by using the screening method or the
screening kit; a pharmaceutical comprising a compound (an
antagonist or agonist) or its salt that changes the binding
property of the ligand to the G protein-coupled receptor protein or
a compound that changes the expression level of the G
protein-coupled receptor protein; and so on.
DISCLOSURE OF THE INVENTION
[0014] The present inventors made extensive investigations and as a
result, found that ligands for human-derived GPR39 are particular
metal elements or salts thereof. Based on these findings, the
inventors have continued further investigations and come to
accomplish the present invention.
[0015] That is, the present invention provides the following
features.
[0016] [1] A method of screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, to an
ionizable metal element or a salt thereof, which comprises using
(1) said receptor protein, its partial peptide, or a salt thereof
and (2) said metal element or a salt thereof.
[0017] [2] The screening method according to [I], wherein said G
protein-coupled receptor protein is a G protein-coupled receptor
protein consisting of the amino acid sequence represented by SEQ ID
NO: 1, SEQ ID NO: 6 or SEQ ID NO: 8.
[0018] [3] A kit for screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, to an
ionizable metal element or a salt thereof, which comprises (1) said
receptor protein, its partial peptide, or a salt thereof and (2)
said metal element or a salt thereof.
[0019] [4] A compound or element or a salt thereof that changes the
binding property or signal transduction of a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, to an ionizable metal element or a
salt thereof, which is obtainable by using the screening method
according to [1] or the screening kit according to [3].
[0020] [5] A pharmaceutical, which comprises a compound or element
or a salt thereof that changes the binding property or signal
transduction of an ionizable metal element or a salt thereof to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof.
[0021] [6] A prophylactic/therapeutic agent for growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression or
menoxenia, which comprises an agonist for a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof.
[0022] [7] A prophylactic/therapeutic agent for hypotonic bladder
induced by sensory decrease of the bladder or hypotonic bladder
induced by postsurgical bladder anesthesia of the pelvic organs, or
an agent for promoting the secretion of cytokines (e.g., IL-8),
which comprises an agonist for a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or a salt
thereof.
[0023] [8] A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises an antagonist to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof.
[0024] [9] A prophylactic/therapeutic agent for overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus; an agent for suppressing the secretion of cytokines
(e.g., IL-8); or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy,
macroangiopathy, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), which comprises an
antagonist to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof.
[0025] [10] A method of screening an agonist for a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises assaying an intracellular Ca.sup.2+ level increasing
activity when a test compound or element or a salt thereof is
brought in contact with a cell containing said receptor
protein.
[0026] [11] A method of screening an agonist for or an antagonist
to a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises using (1) said receptor protein or its partial peptide,
or a salt thereof and (2) a compound or element or a salt thereof
that changes the binding property of said receptor protein or a
salt thereof to an ionizable metal element or a salt thereof.
[0027] [12] A kit for screening an agonist for or an antagonist to
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises (1) said receptor protein or its partial peptide, or a
salt thereof and (2) a compound or element or a salt thereof that
changes the binding property of said receptor protein or a salt
thereof to an ionizable metal element or a salt thereof.
[0028] [13] An agonist for or an antagonist to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, which is obtainable by using the
screening method according to [11] or the screening kit according
to [12].
[0029] [14] A pharmaceutical comprising an agonist for or an
antagonist to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof.
[0030] [15] A prophylactic/therapeutic agent for growth
retardation, wounds, burn, cold constitution, declining of learning
ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia, which comprises a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, or a
salt thereof.
[0031] [16] A prophylactic/therapeutic agent for hypotonic bladder
induced by sensory decrease of the bladder or hypotonic bladder
induced by postsurgical bladder anesthesia of the pelvic organs, or
an agent for promoting the secretion of cytokines (e.g., IL-8),
which comprises a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
or a salt thereof.
[0032] [17] A prophylactic/therapeutic agent for growth
retardation, wounds, burn, cold constitution, declining of learning
ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia, which comprises a
polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0033] [18] A prophylactic/therapeutic agent for hypotonic bladder
induced by sensory decrease of the bladder or hypotonic bladder
induced by postsurgical bladder anesthesia of the pelvic organs, or
an agent for promoting the secretion of cytokines (e.g., IL-8),
which comprises a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0034] [19] A diagnostic agent for growth retardation, wounds,
burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression or
menoxenia, renal dysfunction, pulmonary dysfunction, allergic
dermatitis, sensory neuropathy or Wilson's disease, which comprises
a polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0035] [20] A diagnostic agent for hypotonic bladder induced by
sensory decrease of the bladder, hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs, overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus, inflammatory diseases (e.g., diabetic complications such
as neuropathy, macroangiopathy, etc.; inflammatory bowel diseases
such as ulcerative colitis, etc.; cystitis; irritable bowel
syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis or chronic obstructive pulmonary disease (COPD)), which
comprises a polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0036] [21] A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises an antibody to a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, or a
salt thereof.
[0037] [22] A prophylactic/therapeutic agent for overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus, an agent for suppressing the secretion of cytokines
(e.g., IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy,
macroangiopathy, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), which comprises an
antibody to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
or a salt thereof.
[0038] [23] A diagnostic agent for growth retardation, wounds,
burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression or
menoxenia, renal dysfunction, pulmonary dysfunction, allergic
dermatitis, sensory neuropathy or Wilson's disease, which comprises
an antibody to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
or a salt thereof.
[0039] [24] A diagnostic agent for overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus or
inflammatory diseases (e.g., diabetic complications such as
neuropathy, macroangiopathy, etc.; inflammatory bowel diseases such
as ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), which comprises an
antibody to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
or a salt thereof.
[0040] [25] A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises a polynucleotide containing the
entire or part of a base sequence complementary to a polynucleotide
comprising a polynucleotide encoding a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide.
[0041] [26] A prophylactic/therapeutic agent for overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus, an agent for suppressing the secretion of cytokines
(e.g., IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy,
macroangiopathy, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), which comprises a
polynucleotide containing the entire or part of a base sequence
complementary to a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0042] [27] A method of screening a compound or its salt that
changes the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1 to
prevent/treat growth retardation, wounds, burn, cold constitution,
declining of learning ability, hypogonadism, dysgeusia, anosmia,
hyperplasia, arteriosclerosis, myocardial infarction, apoplexy,
cirrhosis, cholesterol accumulation, lowered resistance to
infection, gout, cancer, hard labor, diabetes mellitus, brown
spots, anemia, alopecia, respiratory disturbances, indigestion,
cardiac disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia, renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises using a polynucleotide comprising
a polynucleotide encoding said receptor protein or its partial
peptide.
[0043] [28] A method of screening a compound or a salt thereof that
changes the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, to
prevent/treat hypotonic bladder induced by sensory decrease of the
bladder, hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, a drug to
regulate the secretion of cytokines (e.g., IL-8), or a compound or
a salt thereof to prevent/treat inflammatory diseases (e.g.,
diabetic complications such as neuropathy, macroangiopathy, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia), allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)), which comprises using a polynucleotide comprising
a polynucleotide encoding said receptor protein or its partial
peptide.
[0044] [29] A kit for screening a compound or its salt that changes
the expression level of a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1 to
prevent/treat growth retardation, wounds, burn, cold constitution,
declining of learning ability, hypogonadism, dysgeusia, anosmia,
hyperplasia, arteriosclerosis, myocardial infarction, apoplexy,
cirrhosis, cholesterol accumulation, lowered resistance to
infection, gout, cancer, hard labor, diabetes mellitus, brown
spots, anemia, alopecia, respiratory disturbances, indigestion,
cardiac disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia, renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises a polynucleotide comprising a
polynucleotide encoding said receptor protein or its partial
peptide.
[0045] [30] A kit for screening a compound or a salt thereof that
changes the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, to
prevent/treat hypotonic bladder induced by sensory decrease of the
bladder, hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, a drug to
regulate the secretion of cytokines (e.g., IL-8), or a drug to
prevent/treat inflammatory diseases (e.g., diabetic complications
such as neuropathy, macroangiopathy, etc.; inflammatory bowel
diseases such as ulcerative colitis, etc.; cystitis; irritable
bowel syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis or chronic obstructive pulmonary disease (COPD)), which
comprises a polynucleotide comprising a polynucleotide encoding
said receptor protein or its partial peptide.
[0046] [31] A compound or its salt that changes the expression
level of a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide to
prevent/treat growth retardation, wounds, burn, cold constitution,
declining of learning ability, hypogonadism, dysgeusia, anosmia,
hyperplasia, arteriosclerosis, myocardial infarction, apoplexy,
cirrhosis, cholesterol accumulation, lowered resistance to
infection, gout, cancer, hard labor, diabetes mellitus, brown
spots, anemia, alopecia, respiratory disturbances, indigestion,
cardiac disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia, renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which is obtainable by using the screening method
according to [27] or the screening kit according to [29].
[0047] [32] A compound or a salt thereof that changes the
expression level of a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
to prevent/treat hypotonic bladder induced by sensory decrease of
the bladder, hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, a drug to
regulate the secretion of cytokines (e.g., IL-8), or a compound or
a salt thereof to prevent/treat inflammatory diseases (e.g.,
diabetic complications such as neuropathy, macroangiopathy, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia), allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)), which is obtainable by using the screening method
according to [28] or the screening kit according to [30].
[0048] [33] A prophylactic/therapeutic agent for growth
retardation, wounds, burn, cold constitution, declining of learning
ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia, which comprises a compound
or its salt that increases the expression level of a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0049] [34] A prophylactic/therapeutic agent for hypotonic bladder
induced by sensory decrease of the bladder or hypotonic bladder
induced by postsurgical bladder anesthesia of the pelvic organs, or
an agent for promoting the secretion of cytokines (e.g., IL-8),
which comprises a compound or its salt that increases the
expression level of a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial
peptide.
[0050] [35] A prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises a compound or its salt that
decreases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide.
[0051] [36] A prophylactic/therapeutic agent for overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus, an agent for suppressing the secretion of cytokines
(e.g., IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy,
macroangiopathy, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), which comprises a
compound or its salt that decreases the expression level of a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0052] [37] A method of preventing/treating growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression or
menoxenia, which comprises administering to a mammal an effective
dose of (i) a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, its partial peptide, or
a salt thereof, (ii) a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an agonist for a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that increases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0053] [38] A method of preventing/treating hypotonic bladder
induced by sensory decrease of the bladder or hypotonic bladder
induced by postsurgical bladder anesthesia of the pelvic organs, or
a method of promoting the secretion of cytokines (e.g., IL-8),
which comprises administering to a mammal an effective dose of (i)
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, its partial peptide, or a
salt thereof, (ii) a polynucleotide comprising a polynucleotide
encoding a G protein-coupled receptor protein comprising the same
or substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an agonist for a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that increases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0054] [39] A method of preventing/treating renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease, which comprises administering to a mammal an
effective dose of (i) an antibody to a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1,
its partial peptide, or a salt thereof, (ii) a polynucleotide
comprising the entire or part of a base sequence complementary to a
polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an antagonist to a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that decreases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide.
[0055] [40] A method of preventing/treating overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus, a method of suppressing the expression of cytokines
(e.g., IL-8), or a method of preventing/treating inflammatory
diseases (e.g., diabetic complications such as neuropathy,
macroangiopathy, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), which comprises
administering to a mammal an effective dose of (i) an antibody to a
G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, its partial peptide, or a
salt thereof, (ii) a polynucleotide comprising the entire or part
of a base sequence complementary to a polynucleotide comprising a
polynucleotide encoding a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, (iii) an antagonist to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, or (iv) a compound or its salt that
decreases the expression level of a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
its partial peptide.
[0056] [41] Use of (i) a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, its partial
peptide, or a salt thereof, (ii) a polynucleotide comprising a
polynucleotide encoding a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, (iii) an agonist for a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
a salt thereof, or (iv) a compound or its salt that increases the
expression level of a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
to manufacture a prophylactic/therapeutic agent for growth
retardation, wounds, burn, cold constitution, declining of learning
ability, hypogonadism, dysgeusia, anosmia, hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression or menoxenia.
[0057] [42] Use of (i) a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, its partial
peptide, or a salt thereof, (ii) a polynucleotide comprising a
polynucleotide encoding a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or its
partial peptide, (iii) an agonist for a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
a salt thereof, or (iv) a compound or its salt that increases the
expression level of a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or its partial peptide,
to manufacture a prophylactic/therapeutic agent for hypotonic
bladder induced by sensory decrease of the bladder or hypotonic
bladder induced by postsurgical bladder anesthesia of the pelvic
organs, or an agent for promoting the secretion of cytokines (e.g.,
IL-8).
[0058] [43] Use of (i) an antibody to a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1,
its partial peptide, or a salt thereof, (ii) a polynucleotide
comprising the entire or part of a base sequence complementary to a
polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an antagonist to a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that decreases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, to
manufacture a prophylactic/therapeutic agent for renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy or
Wilson's disease.
[0059] [44] Use of (i) an antibody to a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1,
its partial peptide, or a salt thereof, (ii) a polynucleotide
comprising the entire or part of a base sequence complementary to a
polynucleotide comprising a polynucleotide encoding a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, (iii)
an antagonist to a G protein-coupled receptor protein comprising
the same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, or
(iv) a compound or its salt that decreases the expression level of
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or its partial peptide, to
manufacture a prophylactic/therapeutic agent for overactive
bladder-induced pollakiuria, nocturia, cystitis-induced
pollakiuria, prostatic hyperplasia-induced pollakiuria, urinary
incontinence, urinary urgency, pelvic visceral pain, coital pain,
bladder irritation symptoms or various disorders caused by urinary
calculus, an agent for suppressing the secretion of cytokines
(e.g., IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy,
macroangiopathy, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)).
[0060] The present invention further provides the following
features and so on.
[0061] [45] The screening method according to [1], wherein
comparison is made between (i) the case where a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1 (hereinafter briefly referred to as GPR39), its partial
peptide, or a salt thereof is brought in contact with an ionizable
metal element (hereinafter briefly referred to as a metal element)
or a salt thereof and (ii) the case where GPR39, its partial
peptide, or a salt thereof is brought in contact with a metal
element or a salt thereof and a test compound or element or a salt
thereof.
[0062] [46] The screening method according to [1], wherein the
binding amount of a metal element to GPR39, its partial peptide, or
a salt thereof is assayed (i) when a radioisotope of the metal
element is brought in contact with GPR39, its partial peptide, or a
salt thereof and (ii) when a radioisotope of the metal element and
a test compound or element or a salt thereof are brought in contact
with GPR39, its partial peptide, or a salt thereof, followed by
comparison of (i) and (ii).
[0063] [47] The screening method according to [1], wherein the
binding amount of a radioisotope of metal element to a cell
containing GPR39 is assayed (i) when the radioisotope of metal
element is brought in contact with a cell containing GPR39 and (ii)
when the radioisotope of metal element and a test compound or
element or a salt thereof are brought in contact with the cell,
followed by comparison of (i) and (ii).
[0064] [48] The screening method according to [1], wherein the
binding amount of a radioisotope of metal element to a membrane
fraction of a cell containing GPR39 is assayed (i) when the
radioisotope of metal element is brought in contact with a membrane
fraction of the cell containing GPR39 and (ii) when the
radioisotope of metal element and a test compound or element or a
salt thereof are brought in contact with a membrane fraction of the
cell, followed by comparison of (i) and (ii).
[0065] [49] The screening method according to [I], wherein the
binding amount of a radioisotope of metal element to GPR39 is
assayed (i) when the radioisotope of metal element is brought in
contact with GPR39 expressed on the cell membrane of a transformant
by culturing the transformant transformed with a recombinant vector
bearing a DNA comprising a DNA encoding GPR39 and (ii) when the
radioisotope of metal element and a test compound or element or a
salt thereof are brought in contact with GPR39 expressed on the
cell membrane of said transformant, followed by comparison of (i)
and (ii).
[0066] [50] The screening method according to [1], wherein the cell
stimulating activity mediated by GPR39 is assayed (i) when a
compound or element or a salt thereof that activates GPR39 is
brought in contact with a cell containing GPR39 and (ii) when a
compound or element or a salt thereof that activates GPR39 and a
test compound or element or a salt thereof are brought in contact
with the cell containing GPR39, followed by comparison of (i) and
(ii).
[0067] [51] The screening method according to [1], the cell
stimulating activity mediated by GPR39 is assayed (i) when a
compound or element or a salt thereof that activates GPR39 is
brought in contact with GPR39 expressed on the cell membrane of a
transformant by culturing the transformant transformed with a
recombinant vector bearing a DNA comprising a DNA encoding GPR39
and (ii) when a compound or element or a salt thereof that
activates GPR39 and a test compound or element or a salt thereof
are brought in contact with GPR39 expressed on the cell membrane of
a transformant by culturing the transformant transformed with a
recombinant vector bearing a DNA comprising a DNA encoding GPR39,
followed by comparison of (i) and (ii).
[0068] [52] The screening method according to [50] or [51], wherein
the element that activates GPR39 is an ionizable metal element.
[0069] [53] The screening kit according to [3] or [10], which
comprises a cell containing GPR39 or its membrane fraction.
[0070] [54] The screening kit according to [3] or [10], which
comprises GPR39 expressed on the cell membrane of a transformant by
culturing the transformant transformed with a recombinant vector
bearing a DNA comprising a DNA encoding GPR39.
[0071] [55] A method for confirming that a drug for
preventing/treating growth retardation, wounds, burn, cold
constitution, declining of learning ability, hypogonadism,
dysgeusia, anosmia, hyperplasia, arteriosclerosis, myocardial
infarction, apoplexy, cirrhosis, cholesterol accumulation, lowered
resistance to infection, gout, cancer, hard labor, diabetes
mellitus, brown spots, anemia, alopecia, respiratory disturbances,
indigestion, cardiac disturbances, gray hair, puffiness, wrinkles,
saggings, hypothyroidism, depression or menoxenia, renal
dysfunction, pulmonary dysfunction, allergic dermatitis, sensory
neuropathy or Wilson's disease binds to a G protein-coupled
receptor protein comprising the same or substantially the same
amino acid sequence as the amino acid sequence represented by SEQ
ID NO: 1, or a salt thereof, which comprises using said receptor
protein or a salt thereof.
[0072] [56] A method for confirming that a drug for
preventing/treating hypotonic bladder induced by sensory decrease
of the bladder, hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, an agent
for regulating the secretion of cytokines (e.g., IL-8) or a drug
for preventing/treating inflammatory diseases (e.g., diabetic
complications such as neuropathy, macroangiopathy, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia) or allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)) binds to a G protein-coupled receptor protein
comprising the same or substantially the same amino acid sequence
as the amino acid sequence represented by SEQ ID NO: 1, or a salt
thereof, which comprises using said receptor protein, or a salt
thereof.
[0073] [57] A method for confirming that a drug for
preventing/treating growth retardation, wounds, burn, cold
constitution, declining of learning ability, hypogonadism,
dysgeusia, anosmia, hyperplasia, arteriosclerosis, myocardial
infarction, apoplexy, cirrhosis, cholesterol accumulation, lowered
resistance to infection, gout, cancer, hard labor, diabetes
mellitus, brown spots, anemia, alopecia, respiratory disturbances,
indigestion, cardiac disturbances, gray hair, puffiness, wrinkles,
saggings, hypothyroidism, depression or menoxenia is an agonist for
a G protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises using said receptor protein or a salt thereof.
[0074] [58] A method for confirming that a drug for
preventing/treating hypotonic bladder induced by sensory decrease
of the bladder or hypotonic bladder induced by postsurgical bladder
anesthesia of the pelvic organs or a drug for promoting the
secretion of cytokines (e.g., IL-8) is an agonist for a G
protein-coupled receptor protein comprising the same or
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises using said receptor protein or a salt thereof.
[0075] [59] A method for confirming that a drug for
preventing/treating renal dysfunction, pulmonary dysfunction,
allergic dermatitis, sensory neuropathy or Wilson's disease is an
antagonist to a G protein-coupled receptor protein comprising the
same or substantially the same amino acid sequence as the amino
acid sequence represented by SEQ ID NO: 1, or a salt thereof, which
comprises using said receptor protein or a salt thereof.
[0076] [60] A method for confirming that a drug for
preventing/treating overactive bladder-induced pollakiuria,
nocturia, cystitis-induced pollakiuria, prostatic
hyperplasia-induced pollakiuria, urinary incontinence, urinary
urgency, pelvic visceral pain, coital pain, bladder irritation
symptoms or various disorders caused by urinary calculus, a drug
for suppressing the secretion of cytokines (e.g., IL-8) or a drug
for preventing/treating inflammatory diseases (e.g., diabetic
complications such as neuropathy, macroangiopathy, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia) or allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)) is an antagonist to a G protein-coupled receptor
protein comprising the same or substantially the same amino acid
sequence as the amino acid sequence represented by SEQ ID NO: 1, or
a salt thereof, which comprises using said receptor protein or a
salt thereof.
[0077] [61] The method for confirming according to [55] through
[60], wherein the binding amount of each drug to said receptor
protein, its partial peptide, or a salt thereof is assayed when
each drug is brought in contact with said receptor protein, its
partial peptide, or a salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1 shows the results obtained by monitoring changes in
intracellular calcium levels when cadmium chloride was added to
human GPR39-GFP expression vector-transfected CHO cell line
(CHO/hGPR39-GFP). "Time (sec)" designates a time period (second)
after the addition. "Counts" designates changes in intracellular
calcium levels. Symbol .quadrature. (open square) designates the
case where 300 .mu.M of cadmium chloride was added, symbol (open
triangle) designates the case where 100 .mu.M of cadmium chloride
was added and symbol .largecircle. designates the case where 10
.mu.M of cadmium chloride was added.
[0079] FIG. 2 shows the results obtained by monitoring changes in
intracellular calcium levels when zinc chloride was added to human
GPR39-GFP expression vector-transfected CHO cell line
(CHO/hGPR39-GFP). "Time (sec)" designates a time period (second)
after the addition. "Counts" designates changes in intracellular
calcium levels. Symbol .quadrature. (open square) designates the
case where 300 .mu.M of zinc chloride was added, symbol (open
triangle) designates the case where 100 .mu.M of zinc chloride was
added and symbol .largecircle. designates the case where 10 .mu.M
of zinc chloride was added.
[0080] FIG. 3 shows the results obtained by monitoring changes in
intracellular calcium levels when copper chloride was added to
human GPR39-GFP expression vector-transfected CHO cell line
(CHO/hGPR39-GFP). "Time (sec)" designates a time period (second)
after the addition. "Counts" designates changes in intracellular
calcium levels. Symbol .quadrature. (open square) designates the
case where 300 .mu.M of copper chloride was added, symbol (open
triangle) designates the case where 100 .mu.M of copper chloride
was added and symbol .largecircle. (open circle) designates the
case where 10 .mu.M of copper chloride was added.
[0081] FIG. 4 shows the results obtained by monitoring changes in
intracellular calcium levels when nickel chloride was added to
human GPR39-GFP expression vector-transfected CHO cell line
(CHO/hGPR39-GFP). "Time (sec)" designates a time period (second)
after the addition. "Counts" designates changes in intracellular
calcium levels. Symbol .quadrature. (open square) designates the
case where 300 .mu.M of nickel chloride was added, symbol (open
triangle) designates the case where 100 .mu.M of nickel chloride
was added and symbol .largecircle. (open circle) designates the
case where 10 .mu.M of nickel chloride was added.
[0082] FIG. 5 shows the expression distribution of GPR39 mRNA in
human tissues. "Copies/25 ng total RNA" designates the number of
copies per 25 ng of total RNA.
[0083] FIG. 6 shows the expression level of GPR39 mRNA in human
primary culture cells. "Copies/25 ng total RNA" designates the
number of copies per 25 ng of total RNA.
[0084] FIG. 7 shows the expression of GPR39 mRNA in human colon
cancer cell line. "Copies/25 ng total RNA" designates the number of
copies per 25 ng of total RNA.
[0085] FIG. 8 shows the results obtained by monitoring pH changes
in extracellular fluids using a cytosensor, when nickel chloride
was added to human GPR39 expression vector-transfected CHO cell
line (CHO/hGPR39). "Cycles" designates the number of detecting the
rate of pH change after the addition. "Change of Acidification Rate
(%)" designates pH changes (acidification rate) in extracellular
fluids and reflects the cell respiration rate. Symbols
.diamond-solid. (closed diamond), .tangle-solidup. (closed
triangle) and .quadrature. (open square) designate GPR39-expressed
cells, TGR7-expressed cells and mock cells, respectively.
[0086] FIG. 9 shows the results obtained by monitoring changes in
intracellular calcium levels when cobalt chloride was added to rat
GPR39 expression vector-transfected CHO cell line (CHO/rGPR39).
"Time (sec)" designates a time period (second) after the addition.
"Counts" designates changes in intracellular calcium levels. Symbol
(open diamond) designates the case where 3000 .mu.M of cobalt
chloride was added, symbol .quadrature. (open square) designates
the case where 1000 .mu.M of cobalt chloride was added, and symbol
(open triangle) designates the case where 300 .mu.M of cobalt
chloride was added.
[0087] FIG. 10 shows the results obtained by monitoring changes in
intracellular calcium levels when nickel chloride was added to rat
GPR39 expression vector-transfected CHO cell line (CHO/rGPR39).
"Time (sec)" designates a time period (second) after the addition.
"Counts" designates changes in intracellular calcium levels. Symbol
(open diamond) designates the case where 100 .mu.M of nickel
chloride was added, symbol .quadrature. (open square) designates
the case where 30 .mu.M of nickel chloride was added, and symbol
(open triangle) designates the case where 10 .mu.M of nickel
chloride was added.
[0088] FIG. 11 shows the results obtained by monitoring changes in
intracellular calcium levels when copper chloride was added to rat
GPR39 expression vector-transfected CHO cell line (CHO/rGPR39).
"Time (sec)" designates a time period (second) after the addition.
"Counts" designates changes in intracellular calcium levels. Symbol
(open diamond) designates the case where 100 .mu.M of copper
chloride was added, symbol .quadrature. (open square) designates
the case where 30 .mu.M of copper chloride was added, and symbol
(open triangle) designates the case where 10 .mu.M of copper
chloride was added.
[0089] FIG. 12 shows the results obtained by monitoring changes in
intracellular calcium levels when zinc chloride was added to rat
GPR39 expression vector-transfected CHO cell line (CHO/rGPR39).
"Time (sec)" designates a time period (second) after the addition.
"Counts" designates changes in intracellular calcium levels. Symbol
(open diamond) designates the case where 100 .mu.M of zinc chloride
was added, symbol .quadrature. (open square) designates the case
where 30 .mu.M of zinc chloride was added, and symbol (open
triangle) designates the case where 10 .mu.M of zinc chloride was
added.
[0090] FIG. 13 shows the results obtained by monitoring changes in
intracellular calcium levels when cadmium chloride was added to rat
GPR39 expression vector-transfected CHO cell line (CHO/rGPR39).
"Time (sec)" designates a time period (second) after the addition.
"Counts" designates changes in intracellular calcium levels. Symbol
(open diamond) designates the case where 3000 .mu.M of cadmium
chloride was added, symbol .quadrature. (open square) designates
the case where 1000 .mu.M of cadmium chloride was added, and symbol
(open triangle) designates the case where 300 .mu.M of cadmium
chloride was added.
[0091] FIG. 14 shows the results obtained by monitoring changes in
intracellular calcium levels when nickel chloride was added to
mouse GPR39 expression vector-transfected CHO cell line
(CHO/mGPR39). "Time (sec)" designates a time period (second) after
the addition. "Counts" designates changes in intracellular calcium
levels. Symbol (open diamond) designates the case where 300 .mu.M
of nickel chloride was added, symbol .quadrature. (open square)
designates the case where 100 .mu.M of nickel chloride was added,
and symbol (open triangle) designates the case where 30 .mu.M of
nickel chloride was added.
[0092] FIG. 15 shows the results obtained by monitoring changes in
intracellular calcium levels when cobalt chloride was added to
mouse GPR39 expression vector-transfected CHO cell line
(CHO/mGPR39). "Time (sec)" designates a time period (second) after
the addition. "Counts" designates changes in intracellular calcium
levels. Symbol (open diamond) designates the case where 3000 .mu.M
of cobalt chloride was added, symbol .quadrature. (open square)
designates the case where 1000 .mu.M of cobalt chloride was added,
and symbol (open triangle) designates the case where 300 .mu.M of
cobalt chloride was added.
[0093] FIG. 16 shows the results obtained by monitoring changes in
intracellular calcium levels when copper chloride was added to
mouse GPR39 expression vector-transfected CHO cell line
(CHO/mGPR39). "Time (sec)" designates a time period (second) after
the addition. "Counts" designates changes in intracellular calcium
levels. Symbol (open diamond) designates the case where 300 .mu.M
of copper chloride was added, symbol .quadrature. (open square)
designates the case where 100 .mu.M of copper chloride was added,
and symbol (open triangle) designates the case where 30 .mu.M of
copper chloride was added.
[0094] FIG. 17 shows the results obtained by monitoring changes in
intracellular calcium levels when zinc chloride was added to mouse
GPR39 expression vector-transfected CHO cell line (CHO/mGPR39).
"Time (sec)" designates a time period (second) after the addition.
"Counts" designates changes in intracellular calcium levels. Symbol
(open diamond) designates the case where 300 .mu.M of zinc chloride
was added, symbol .quadrature. (open square) designates the case
where 100 .mu.M of zinc chloride was added, and symbol (open
triangle) designates the case where 30 .mu.M of zinc chloride was
added.
[0095] FIG. 18 shows the results obtained by monitoring changes in
intracellular calcium levels when cadmium chloride was added to
mouse GPR39 expression vector-transfected CHO cell line
(CHO/mGPR39). "Time (sec)" designates a time period (second) after
the addition. "Counts" designates changes in intracellular calcium
levels. Symbol (open diamond) designates the case where 3000 .mu.M
of cadmium chloride was added, symbol .quadrature. (open square)
designates the case where 1000 .mu.M of cadmium chloride was added,
and symbol (open triangle) designates the case where 300 .mu.M of
cadmium chloride was added.
[0096] FIG. 19 shows the tissue distribution of GPR39 mRNA in rats
by RT-PCR. "Copies/25 ng total RNA" designates the number of copies
per 25 ng of total RNA.
[0097] FIG. 20 shows the expression level of GPR39 mRNA in cell
lines by RT-PCR. "Cell lines" on the abscissa designates cell
lines, and "human," "mouse" and "rat" designate human, mouse and
rat, respectively. "Copies/25 ng total RNA" designates the number
of copies per 25 ng of total RNA.
BEST MODE FOR CARRYING OUT THE INVENTION
[0098] The G protein-coupled receptor protein GPR39 used in the
present invention (hereinafter sometimes briefly referred to as
GPR39) is a receptor protein comprising the same or substantially
the same amino acid sequence as the amino acid sequence represented
by SEQ ID NO: 1.
[0099] GPR39 may be any protein derived from, for example, any cell
of human and mammal (e.g., guinea pig, rat, mouse, rabbit, swine,
sheep, bovine, monkey, etc.); any cell (e.g., splenocyte, nerve
cells, glial cells, .beta. cells of pancreas, bone marrow cells,
mesangial cells, Langerhans' cells, epidermic cells, epithelial
cells, endothelial cells, fibroblasts, fibrocytes, myocytes, fat
cells, immune cells (e.g., macrophage, T cells, B cells, natural
killer cells, mast cells, neutrophils, basophils, eosinophils,
monocytes), megakaryocytes, synovial cells, chondrocytes, bone
cells, osteoblasts, osteoclasts, mammary gland cells, hepatocytes
or interstitial cells; or the corresponding precursor cells, stem
cells, cancer cells, etc.) or cells originated from blood cells; or
any tissues where such cells are present, such as brain or any of
brain regions (e.g., olfactory bulb, amygdaloid nucleus, basal
ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex,
medulla oblongata, cerebellum, occipital pole, frontal lobe,
temporal lobe, putamen, caudate nucleus, corpus callosum,
substantia nigra), spinal cord, hypophysis, stomach, pancreas,
kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal
gland, skin, muscle, lung, gastrointestinal tract (e.g., large
intestine and small intestine), intestinal tract, blood vessel,
heart, thymus, spleen, submandibular gland, peripheral blood,
peripheral blood cells, prostate, testicle, testis, ovary,
placenta, uterus, bone, joint, skeletal muscle, bladder, etc.; in
particular, GPR39 may be proteins derived from bladder, central
nervous tissues, hypophysis, kidney, gastrointestinal tract
tissues, fetal brain, primary cultured cells from blood vessels,
primary cultured cells from the kidney or colon cancer cell lines,
or may be synthetic proteins.
[0100] The amino acid sequence comprising substantially the same
amino acid sequence as the amino acid sequence includes, for
example, an amino acid sequence having at least about 80% homology,
preferably at least about 90% homology and more preferably at least
about 95% homology, to the amino acid sequence represented by SEQ
ID NO: 1, and the like.
[0101] Examples of the protein of the present invention comprising
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1 include a protein comprising
substantially the same amino acid sequence as the amino acid
sequence represented by SEQ ID NO: 1 and preferably having the
activity substantially equivalent to the protein consisting of the
amino acid sequence represented by SEQ ID NO: 1, etc.
[0102] Homology of the amino acid sequences can be measured under
the following conditions (Expectation value=10; gaps are allowed;
matrix=BLOSUM62; filtering=OFF) using a homology scoring algorithm
NCBI BLAST (National Center for Biotechnology Information Basic
Local Alignment Search Tool).
[0103] Examples of the substantially equivalent activity include a
ligand binding activity, a signal transduction activity, etc. The
term substantially equivalent is used to mean that the activities
are the same in nature. Although it is preferred that activities
such as the ligand binding and signal transduction activities, etc.
be equivalent (e.g., about 0.01- to about 100-fold, preferably
about 0.5- to about 20-fold, more preferably about 0.5- to about
2-fold), quantitative factors such as a level of these activities,
a molecular weight of the protein, etc. may differ.
[0104] The activities such as ligand binding and signal
transduction activities or the like can be determined according to
publicly known methods, for example, by the ligand determination
methods or the screening methods that will be later described.
[0105] Also, proteins having the following amino acid sequences are
used as GPR39: a) amino acid sequences wherein at least 1 or 2
amino acids (preferably approximately 1 to 30 amino acids, more
preferably approximately 1 to 10 amino acids and most preferably
several (1 to 5) amino acids) are deleted of the amino acid
sequence represented by SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 8;
b) amino acid sequences wherein at least 1 or 2 amino acids
(preferably approximately 1 to 30 amino acids, more preferably
approximately 1 to 10 amino acids and most preferably several (1 to
5) amino acids) are added to the amino acid sequence represented by
SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 8; c) amino acid sequences
wherein at least 1 or 2 amino acids (preferably approximately 1 to
30 amino acids, more preferably approximately 1 to 10 amino acids
and most preferably several (1 to 5) amino acids) in the amino acid
sequence represented by SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 8
are substituted by other amino acids; or d) combination of these
amino acid sequences described above; and the like.
[0106] Throughout the present specification, GPR39 is represented
in accordance with the conventional way of describing peptides,
that is, the N-terminus (amino terminus) at the left hand and the
C-terminus (carboxyl terminus) at the right hand. In GPR39
including GPR39 comprising the amino acid sequence shown by SEQ ID
NO: 1, the C-terminus may be in any form of a carboxyl group
(--COOH), carboxylate (--COO.sup.-), an amide (--CONH.sub.2) or an
ester (--COOR).
[0107] Examples of the ester group shown by R include a C.sub.1-6
alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl,
etc.; a C.sub.3-8 cycloalkyl group such as cyclopentyl, cyclohexyl,
etc.; a C.sub.6-12 aryl group such as phenyl, .alpha.-naphthyl,
etc.; a C.sub.7-14 aralkyl group such as a phenyl-C.sub.1-2-alkyl
group, e.g., benzyl, phenethyl, etc., or an
.alpha.-naphthyl-C.sub.1-2-alkyl group such as
.alpha.-naphthylmethyl, etc.; and the like. In addition,
pivaloyloxymethyl or the like, which is used widely as an ester for
oral administration, may also be used.
[0108] Where GPR39 contains a carboxyl group (or a carboxylate) at
a position other than the C-terminus, it may be amidated or
esterified and such an amide or ester is also included within
GPR39. The ester group may be the same group as that described with
respect to the C-terminus described above.
[0109] Furthermore, examples of GPR39 include variants of the above
proteins, wherein the amino group at the N-terminal methionine
residue of the protein supra is protected with a protecting group
(for example, a C.sub.1-6 acyl group such as a C.sub.2-6 alkanoyl
group, e.g., formyl group, acetyl group, etc.); those wherein the
N-terminal region is cleaved in vivo and the glutamyl group thus
formed is pyroglutaminated; those wherein a substituent (e.g.,
--OH, --SH, amino group, imidazole group, indole group, guanidino
group, etc.) on the side chain of an amino acid in the molecule is
protected with a suitable protecting group (e.g., a C.sub.1-6 acyl
group such as a C.sub.2-6 alkanoyl group, e.g., formyl group,
acetyl group, etc.), or conjugated proteins such as glycoproteins
bound to sugar chains.
[0110] Specific examples of GPR39 include human-derived GPR39
consisting of the amino acid sequence represented by SEQ ID NO: 1,
mouse-derived GPR39 consisting of the amino acid sequence
represented by SEQ ID NO: 6, rat-derived GPR39 consisting of the
amino acid sequence represented by SEQ ID NO: 8, etc. Human-derived
GPR39 is a publicly known protein described in Genomics, 1997 Dec.
15; 46(3): 426-34, WO2002/61087, US2002/004491, WO2002/39885,
WO2002/71928, WO2001/81634, WO2002/79492 or WO2002/86443.
[0111] As the partial peptide of GPR39 (hereinafter sometimes
referred to as the partial peptide), any partial peptide can be
used so long as it can be a partial peptide of the aforesaid GPR39.
Among GPR39 protein molecules, for example, those having a site
exposed to the outside of a cell membrane and having substantially
the same receptor binding activity can be used.
[0112] Specifically, the partial peptide of GPR39 having the amino
acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID
NO: 8 is a peptide containing the parts analyzed to be
extracellular domains (hydrophilic domains) in the hydrophobic
plotting analysis. A peptide containing a hydrophobic domain in
part can be used as well. In addition, the peptide may contain each
domain separately or plural domains together.
[0113] In the partial peptides of the present invention, preferred
are peptides having at least 20, preferably at least 50, and more
preferably at least 100 amino acids, in the amino acid sequence
which constitutes the receptor protein of the present invention
described above.
[0114] The amino acid sequence having substantially the same amino
acid sequence includes an amino acid sequence having at least about
85% homology, preferably at least about 90% homology and more
preferably at least about 95% homology, to these amino acid
sequences.
[0115] Homology in the amino acid sequence can be measured under
the same conditions using the homology scoring algorithm NCBI BLAST
as described above.
[0116] Herein the term "substantially the same receptor activity"
has the same significance as described above. The "substantially
the same receptor activity" can be assayed by the same manner as
described above.
[0117] In the amino acid sequence described above, the partial
peptide of the present invention may contain amino acid sequences,
of which at least 1 or 2 amino acids (preferably approximately 1 to
10 amino acids, more preferably several (1 to 5) amino acids) are
deleted; to which at least 1 or 2 amino acids (preferably
approximately 1 to 20 amino acids, more preferably approximately 1
to 10 amino acids, and most preferably several (1 to 5) amino
acids) are added; or, in which at least 1 or 2 amino acids
(preferably approximately 1 to 10 amino acids, more preferably
several and most preferably approximately 1 to 5 amino acids) are
substituted by other amino acids.
[0118] In the partial peptide of the present invention, the
C-terminus may be in any form of a carboxyl group (--COOH),
carboxylate (--COO.sup.-), an amide (--CONH.sub.2) or an ester
(--COOR). Where the partial peptide contains a carboxyl group (or a
carboxylate) at a position other than the C-terminus, it may be
amidated or esterified and such an amide or ester is also included
within the partial peptide of the present invention. In this case,
the ester group may be the same group as that described with
respect to the C-terminus described above.
[0119] As in GPR39 described above, the partial peptide of the
present invention further includes those in which the amino group
of the amino acid residue of the N-terminal methionine residue is
protected by a protecting group, those in which the N-terminal
residue is cleaved in vivo and the produced glutamine residue is
pyroglutaminated, those in which substituents on the side chains of
amino acids in the molecule are protected by appropriate protecting
groups, conjugated peptides such as so-called glycopeptides, to
which sugar chains are bound, and the like.
[0120] For salts of GPR39 or its partial peptide, preferred are
physiologically acceptable salts with acids or bases, especially
physiologically acceptable acid addition salts. Examples of the
salts include salts with, for example, inorganic acids (e.g.,
hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric
acid); 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) and the like.
[0121] GPR39 or salts thereof may be manufactured by a publicly
known method used to purify receptor proteins from human or
mammalian cells or tissues described above, or by culturing a
transformant that contains the DNA encoding GPR39, as will be later
described. Furthermore, GPR39 or its salts may also be manufactured
by the methods for synthesizing proteins or by modifications
thereof, which will also be described hereinafter.
[0122] Where GPR39 or its salts are produced from human or
mammalian tissues or cells, human or mammalian tissues or cells are
homogenized, then extracted with an acid or the like, and the
extract is isolated and purified by a combination of chromatography
techniques such as reverse phase chromatography, ion exchange
chromatography, and the like.
[0123] To synthesize GPR39 or its partial peptides, or salts or
amides thereof, commercially available resins that are used for
protein synthesis may be used. Examples of such resins include
chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin,
aminomethyl resin, 4-benzyloxybenzyl alcohol resin,
4-methylbenzhydrylamine resin, PAM resin,
4-hydroxymethylmehtylphenyl acetamidomethyl resin, polyacrylamide
resin, 4-(2',4'-dimethoxyphenylhydroxy-methyl)phenoxy resin,
4-(2',4'-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin, etc. Using
these resins, amino acids in which .alpha.-amino groups and
functional groups on the side chains are appropriately protected
are condensed on the resin in the order of the sequence of the
objective protein according to various condensation methods
publicly known in the art. At the end of the reaction, the protein
is cut out from the resin and at the same time, the protecting
groups are removed. Then, intramolecular disulfide bond-forming
reaction is performed in a highly diluted solution to obtain the
objective protein or its amides.
[0124] For condensation of the protected amino acids described
above, a variety of activation reagents for protein synthesis may
be used, and carbodiimides are particularly preferable. Examples of
such carbodiimides include DCC, N,N'-diisopropylcarbodiimide,
N-ethyl-N'-(3-dimethylaminoprolyl)carbodiimide, etc. For activation
by these reagents, the protected amino acids in combination with a
racemization inhibitor (e.g., HOBt, HOOBt) are added directly to
the resin, or the protected amino acids are previously activated in
the form of symmetric acid anhydrides, HOBt esters or HOOBt esters,
followed by adding the thus activated protected amino acids to the
resin.
[0125] Solvents suitable for use to activate the protected amino
acids or condense with the resin may be chosen from solvents known
to be usable for protein condensation reactions. Examples of such
solvents are acid amides such as N,N-dimethylformamide,
N,N-dimethylacetamide, N-methylpyrrolidone, etc.; halogenated
hydrocarbons such as methylene chloride, chloroform, etc.; alcohols
such as trifluoroethanol, etc.; sulfoxides such as
dimethylsulfoxide, etc.; ethers such as pyridine, dioxane,
tetrahydrofuran, etc.; nitriles such as acetonitrile,
propionitrile, etc.; esters such as methyl acetate, ethyl acetate,
etc.; and appropriate mixtures of these solvents. The reaction
temperature is appropriately chosen from the range known to be
applicable to protein binding reactions and is usually selected in
the range of approximately -20.degree. C. to 50.degree. C. The
activated amino acid derivatives are used generally in an excess of
1.5 to 4 times. The condensation is examined by a test using the
ninhydrin reaction; when the condensation is insufficient, the
condensation can be completed by repeating the condensation
reaction without removal of the protecting groups. When the
condensation is yet insufficient even after repeating the reaction,
unreacted amino acids are acetylated with acetic anhydride or
acetylimidazole.
[0126] Examples of the protecting groups used to protect the amino
groups of the starting compounds include Z, Boc,
t-pentyloxycarbonyl, isobornyloxycarbonyl,
4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,
trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl,
diphenylphosphinothioyl, Fmoc, etc.
[0127] A carboxyl group can be protected by, e.g., alkyl
esterification (in the form of linear, branched or cyclic alkyl
esters of the alkyl moiety such as methyl, ethyl, propyl, butyl,
t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
2-adamantyl, etc.), aralkyl esterification (e.g., esterification in
the form of benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl
ester, 4-chlorobenzyl ester, benzhydryl ester, etc.), phenacyl
esterification, benzyloxycarbonyl hydrazidation, t-butoxycarbonyl
hydrazidation, trityl hydrazidation, or the like.
[0128] The hydroxyl group of serine can be protected through, for
example, its esterification or etherification. Examples of groups
appropriately used for the esterification include a lower alkanoyl
group, such as acetyl group, an aroyl group such as benzoyl group,
and a group derived from carbonic acid such as benzyloxycarbonyl
group, ethoxycarbonyl group, etc. Examples of a group appropriately
used for the etherification include benzyl group, tetrahydropyranyl
group, t-butyl group, etc.
[0129] Examples of groups for protecting the phenolic hydroxyl
group of tyrosine include Bzl, Cl.sub.2-Bzl, 2-nitrobenzyl, Br-Z,
t-butyl, etc.
[0130] Examples of groups used to protect the imidazole moiety of
histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl,
DNP, benzyloxymethyl, Burn, Boc, Trt, Fmoc, etc.
[0131] Examples of the activated carboxyl groups used in the
starting compounds include the corresponding acid anhydrides,
azides, activated esters [esters with alcohols (e.g.,
pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol,
cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccimide,
N-hydroxyphthalimide, HOBt)], etc.
[0132] As the activated amino acids, in which the amino groups are
activated in the starting material, for example, the corresponding
phosphoric amides are employed.
[0133] To eliminate (split off) the protecting groups, there are
used catalytic reduction under hydrogen gas flow in the presence of
a catalyst such as Pd-black, Pd-carbon, etc.; an acid treatment
with anhydrous hydrogen fluoride, methanesulfonic acid,
trifluoromethane-sulfonic acid or trifluoroacetic acid, or a
mixture solution of these acids; a treatment with a base such as
diisopropylethylamine, triethylamine, piperidine, piperazine, etc.;
and reduction with sodium in liquid ammonia. The elimination of the
protecting group by the acid treatment described above is carried
out generally at a temperature of approximately -20.degree. C. to
40.degree. C. In the acid treatment, it is efficient to add a
cation scavenger such as anisole, phenol, thioanisole, m-cresol,
p-cresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol,
etc. Furthermore, 2,4-dinitrophenyl group known as the protecting
group for the imidazole of histidine is removed by a treatment with
thiophenol. Formyl group used as the protecting group of the indole
of tryptophan is eliminated by the aforesaid acid treatment in the
presence of 1,2-ethanedithiol, 1,4-butanedithiol, etc. as well as
by a treatment with an alkali such as a dilute sodium hydroxide
solution, dilute ammonia, etc.
[0134] Protection of the functional groups that should not be
involved in the reaction of the starting materials, protecting
groups, elimination of the protecting groups, activation of
functional groups involved in the reaction, or the like may be
appropriately selected from publicly known groups and publicly
known means.
[0135] In another method for obtaining the amides of the protein,
for example, the .alpha.-carboxyl group of the carboxy terminal
amino acid is first protected by amidation; the peptide (protein)
chain is then extended from the amino group side to a desired
length. Thereafter, a protein in which only the protecting group of
the N-terminal .alpha.-amino group in the peptide chain has been
eliminated from the protein and a protein in which only the
protecting group of the C-terminal carboxyl group has been
eliminated are prepared. The two proteins are condensed in a
mixture of the solvents described above. The details of the
condensation reaction are the same as described above. After the
protected protein obtained by the condensation is purified, all the
protecting groups are eliminated by the method described above to
give the desired crude protein. This crude protein is purified by
various known purification means. Lyophilization of the major
fraction gives the amide of the desired protein.
[0136] To prepare the esterified protein, for example, the
.alpha.-carboxyl group of the carboxy terminal amino acid is
condensed with a desired alcohol to prepare the amino acid ester,
which is followed by procedure similar to the preparation of the
amidated protein above to give the ester form of the desired
protein.
[0137] The partial peptide or its salts in GPR39 can be
manufactured by publicly known methods for peptide synthesis, or by
cleaving GPR39 with an appropriate peptidase. For the methods for
peptide synthesis, for example, either solid phase synthesis or
liquid phase synthesis may be used. That is, the partial peptide or
amino acids that can construct GPR39 are condensed with the
remaining part. Where the product contains protecting groups, these
protecting groups are removed to give the desired peptide. Publicly
known methods for condensation and elimination of the protecting
groups are described in a)-e) below. [0138] a) M. Bodanszky &
M. A. Ondetti: Peptide Synthesis, Interscience Publishers, New York
(1966) [0139] b) Schroeder & Luebke: The Peptide, Academic
Press, New York (1965) [0140] c) Nobuo Izumiya, et al.: Peptide
Gosei-no-Kiso to Jikken (Basics and experiments of peptide
synthesis), published by Maruzen Co. (1975) [0141] d) Haruaki
Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza (Biochemical
Experiment) 1, Tanpakushitsu no Kagaku (Chemistry of Proteins) IV,
205 (1977) [0142] e) Haruaki Yajima, ed.: Zoku Iyakuhin no Kaihatsu
(A sequel to Development of Pharmaceuticals), Vol. 14, Peptide
Synthesis, published by Hirokawa Shoten
[0143] After completion of the reaction, the product may be
purified and isolated by a combination of conventional purification
methods such as solvent extraction, distillation, column
chromatography, liquid chromatography and recrystallization to give
the partial peptide of the present invention. When the partial
peptide obtained by the methods above is in a free form, the
peptide can be converted into an appropriate salt by a publicly
known method; conversely when the peptide is obtained in a salt
form, it can be converted into a free form by a publicly known
method.
[0144] The polynucleotide encoding GPR39 may be any polynucleotide
so long as it contains the base sequence (DNA or RNA, preferably
DNA) encoding GPR39 described above. Such a polynucleotide may also
be any one of DNA encoding GPR39, RNA such as mRNA, etc., and may
be double-stranded or single-stranded. Where the polynucleotide is
double-stranded, it may be double-stranded DNA, double-stranded RNA
or DNA:RNA hybrid. Where the polynucleotide is single-stranded, it
may be a sense strand (i.e., a coding strand) or an antisense
strand (i.e., a non-coding strand).
[0145] Using the polynucleotide encoding GPR39, mRNA of GPR39 can
be quantified by, for example, the publicly known method published
in separate volume of Jikken Igaku, 15 (7) "New PCR and its
application" (1997), or by its modifications.
[0146] The DNA encoding GPR39 may be any of genomic DNA, genomic
DNA library, cDNA derived from the cells and tissues described
above, cDNA library derived from the cells and tissues described
above and synthetic DNA. The vector to be used for the library may
be any of bacteriophage, plasmid, cosmid and phagemid. The DNA may
also be directly amplified by reverse transcriptase polymerase
chain reaction (hereinafter abbreviated as RT-PCR) using the total
RNA or mRNA fraction prepared from the cells and tissues described
above.
[0147] Specifically, the DNA encoding GPR39 may be any DNA, so long
as it is, for example, a DNA containing the base sequence
represented by SEQ ID NO: 2, or any DNA having a base sequence
hybridizable to the base sequence represented by SEQ ID NO: 2 under
high stringent conditions and encoding a receptor protein, which
has the activity substantially equivalent to that of GPR39
consisting of the amino acid sequence as the amino acid sequence
represented by SEQ ID NO: 1 (e.g., the ligand-binding activities,
the signal transduction activities, cytokine (e.g., IL-8)
secretion-promoting activities, etc.).
[0148] Examples of the DNA hybridizable to the DNA containing the
base sequence represented by SEQ ID NO: 2 include a DNA containing
a base sequence having at least about 80% homology, preferably at
least about 90% homology and more preferably at least about 95%
homology, to the base sequence represented by SEQ ID NO: 2.
[0149] Homology of the base sequences can be measured under the
following conditions (Expectation value=10; gaps are allowed;
filtering=ON; match score=1; mismatch score=-3) using a homology
scoring algorithm NCBI BLAST (National Center for Biotechnology
Information Basic Local Alignment Search Tool).
[0150] The hybridization can be carried out by publicly known
methods or by modifications of these methods, for example,
according to the method described in Molecular Cloning, 2nd (J.
Sambrook et al., Cold Spring Harbor Lab. Press, 1989). A
commercially available library may also be used according to the
instructions of the attached manufacturer's protocol. Preferably,
the hybridization can be carried out under highly stringent
conditions.
[0151] The highly stringent conditions used herein are, for
example, those in a sodium concentration at about 19 to 40 mM,
preferably about 19 to 20 mM at a temperature of about 50 to
70.degree. C., preferably about 60 to 65.degree. C. In particular,
hybridization conditions in a sodium concentration of about 19 mM
at a temperature of about 65.degree. C. are most preferred.
[0152] More specifically, for the DNA encoding human GPR39
containing the amino acid sequence represented by SEQ ID NO: 1,
there may be employed a DNA containing the base sequence
represented by SEQ ID NO: 2; etc. For the DNA encoding mouse GPR39
containing the amino acid sequence represented by SEQ ID NO: 6,
there may be employed a DNA containing the base sequence
represented by SEQ ID NO: 7; etc. For the DNA encoding rat GPR39
containing the amino acid sequence represented by SEQ ID NO: 8,
there may be employed a DNA containing the base sequence
represented by SEQ ID NO: 9; etc.
[0153] The term polynucleotide comprising a part of the base
sequence of the DNA encoding GPR39 or a part of the base sequence
complementary to the DNA is used to mean that the polynucleotide
embraces not only the DNA encoding the partial peptide of the
present invention described below but also RNA.
[0154] According to the present invention, antisense
polynucleotides (nucleic acids) that can inhibit the replication or
expression of GPR39 gene can be designed and synthesized based on
the base sequence information of the cloned or determined DNA
encoding GPR39. Such a polynucleotide (nucleic acid) is capable of
hybridizing to RNA of GPR39 gene to inhibit the synthesis or
function of said RNA or capable of modulating or controlling the
expression of GPR39 gene via interaction with GPR39-associated RNA.
Polynucleotides complementary to the selected sequences of
GPR39-associated RNA and polynucleotides specifically hybridizable
to the GPR39-associated RNA are useful in modulating or controlling
the expression of GPR39 gene in vivo and in vitro, and useful for
the treatment or diagnosis of diseases. The term "corresponding" is
used to mean homologous to or complementary to a particular
sequence of the nucleotide, base sequence or nucleic acid including
the gene. The term "corresponding" between nucleotides, base
sequences or nucleic acids and peptides (proteins) usually refer to
amino acids of a peptide (protein) under the order derived from the
sequence of nucleotides (nucleic acids) or their complements. In
GPR39 genes, the 5' end hairpin loop, 5' end 6-base-pair repeats,
5' end untranslated region, polypeptide translation initiation
codon, protein coding region, ORF translation initiation codon, 3'
end untranslated region, 3' end palindrome region, and 3' end
hairpin loop, may be selected as preferred target regions, though
any other region may be selected as a target in GPR39 genes.
[0155] The relationship between the targeted nucleic acids and the
polynucleotides complementary to at least a part of the target,
specifically the relationship between the target and the
polynucleotides hybridizable to the target, can be denoted to be
"antisense". Examples of the antisense polynucleotides include
polydeoxyribonucleotide containing 2-deoxy-D-ribose,
polyribonucleotide containing D-ribose, any other type of
polynucleotides which are N-glycosides of a purine or pyrimidine
base, or other polymers containing non-nucleotide backbones (e.g.,
protein nucleic acids and synthetic sequence-specific nucleic acid
polymers commercially available) or other polymers containing
nonstandard linkages (provided that the polymers contain
nucleotides having such a configuration that allows base pairing or
base stacking, as is found in DNA or RNA), etc. They may be
double-stranded DNA, single-stranded DNA, single-stranded RNA or a
DNA:RNA hybrid, and may further include unmodified polynucleotides
(or unmodified oligonucleotides), those with publicly known types
of modifications, for example, those with labels known in the art,
those with caps, methylated polynucleotides, those with
substitution of one or more naturally occurring nucleotides by
their analogue, those with intramolecular modifications of
nucleotides such as those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.)
and those with charged linkages or sulfur-containing linkages
(e.g., phosphorothioates, phosphorodithioates, etc.), those having
side chain groups such as proteins (nucleases, nuclease inhibitors,
toxins, antibodies, signal peptides, poly-L-lysine, etc.),
saccharides (e.g., monosaccharides, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylating agents, those with modified linkages (e.g.,
.alpha. anomeric nucleic acids, etc.), and the like. Herein the
terms "nucleoside", "nucleotide" and "nucleic acid" are used to
refer to moieties that contain not only the purine and pyrimidine
bases, but also other heterocyclic bases, which have been modified.
Such modifications may include methylated purines and pyrimidines,
acylated purines and pyrimidines and other heterocyclic rings.
Modified nucleotides and modified nucleotides also include
modifications on the sugar moiety, wherein, for example, one or
more hydroxyl groups may optionally be substituted with a halogen
atom(s), an aliphatic group(s), etc., or may be converted into the
corresponding functional groups such as ethers, amines, or the
like.
[0156] The antisense polynucleotide (nucleic acid) of the present
invention is RNA, DNA or a modified nucleic acid (RNA, DNA).
Specific examples of the modified nucleic acid are, but not limited
to, sulfur and thiophosphate derivatives of nucleic acids and those
resistant to degradation of polynucleoside amides or
oligonucleoside amides. The antisense nucleic acids of the present
invention can be modified preferably based on the following design,
that is, by increasing the intracellular stability of the antisense
nucleic acid, increasing the cellular permeability of the antisense
nucleic acid, increasing the affinity of the nucleic acid to the
targeted sense strand to a higher level, or minimizing the
toxicity, if any, of the antisense nucleic acid.
[0157] Many of such modifications are known in the art, as
disclosed in J. Kawakami, et al., Pharm. Tech. Japan, Vol. 8, pp.
247, 1992; Vol. 8, pp. 395, 1992; S. T. Crooke, et al. ed.,
Antisense Research and Applications, CRC Press, 1993; etc.
[0158] The antisense nucleic acid of the present invention may
contain changed or modified sugars, bases or linkages. The
antisense nucleic acid may also be provided in a specialized form
such as liposomes, microspheres, or may be applied to gene therapy,
or may be provided in combination with attached moieties. Such
attached moieties include polycations such as polylysine that act
as charge neutralizers of the phosphate backbone, or hydrophobic
moieties such as lipids (e.g., phospholipids, cholesterols, etc.)
that enhance the interaction with cell membranes or increase uptake
of the nucleic acid. Preferred examples of the lipids to be
attached are cholesterols or derivatives thereof (e.g., cholesteryl
chloroformate, cholic acid, etc.). These moieties may be attached
to the nucleic acid at the 3' or 5' ends thereof and may also be
attached thereto through a base, sugar, or intramolecular
nucleoside linkage. Other moieties may be capping groups
specifically placed at the 3' or 5' ends of the nucleic acid to
prevent degradation by nucleases such as exonuclease, RNase, etc.
Such capping groups include, but are not limited to, hydroxyl
protecting groups known in the art, including glycols such as
polyethylene glycol, tetraethylene glycol and the like.
[0159] The inhibitory action of the antisense nucleic acid can be
examined using the transformant of the present invention, the gene
expression system of the present invention in vivo and in vitro, or
the translation system of the G protein-coupled receptor protein in
vivo and in vitro. The nucleic acid can be applied to cells by a
variety of publicly known methods.
[0160] The DNA encoding the partial peptide of the present
invention may be any DNA so long as it contains the base sequence
encoding the partial peptide of the present invention described
above. The DNA may also be any of genomic DNA, genomic DNA library,
cDNA derived from the cells and tissues described above, cDNA
library derived from the cells and tissues described above and
synthetic DNA. The vector to be used for the library may be any of
bacteriophage, plasmid, cosmid and phagemid. The DNA may also be
directly amplified by reverse transcriptase polymerase chain
reaction (hereinafter abbreviated as RT-PCR) using mRNA fraction
prepared from the cells and tissues described above.
[0161] Specifically, the DNA encoding the partial peptide of the
present invention may be any one of, for example, (1) a DNA
containing a partial base sequence of the DNA having the base
sequence represented by SEQ ID NO: 2 or (2) any DNA containing a
partial base sequence of the DNA having a base sequence
hybridizable to the base sequence represented by SEQ ID NO: 2 under
highly stringent conditions and encoding the receptor protein which
has the activities substantially equivalent to those of GPR39
(e.g., a ligand-biding activity, signal transduction activity,
cytokine (e.g., IL-8) secretion-promoting activity, etc.); etc.
[0162] Examples of the DNA that is hybridizable to the base
sequence represented by SEQ ID NO: 2 include DNA comprising a base
sequence having at least about 80% homology, preferably at least
about 90% homology and more preferably at least about 95% homology,
to the base sequence represented by SEQ ID NO: 2.
[0163] Homology in the amino acid sequence can be measured under
the same conditions using the homology scoring algorithm NCBI BLAST
described above.
[0164] The method and conditions of the hybridization is same to
the described above.
[0165] For cloning of the DNA that completely encodes GPR39 or its
partial peptide (hereinafter sometimes collectively referred to as
GPR39), the DNA may be amplified by PCR using synthetic DNA primers
containing a part of the base sequence of DNA encoding GPR39, or
the DNA inserted into an appropriate vector can be selected by
hybridization with a labeled DNA fragment or synthetic DNA that
encodes a part or entire region of GPR39. The hybridization can be
carried out, for example, according to the method described in
Molecular Cloning, 2nd, J. Sambrook et al., Cold Spring Harbor Lab.
Press, 1989. The hybridization may also be performed using
commercially available library in accordance with the protocol
described in the attached instructions.
[0166] Conversion of the base sequence of the DNA can be effected
by publicly known methods such as the ODA-LA PCR method, the Gapped
duplex method or the Kunkel method or its modification by using a
publicly known kit available as Mutan.TM.-superExpressKm
(manufactured by Takara Shuzo Co., Ltd.), Mutan.TM.-K (manufactured
by TaKaRa Shuzo Co., Ltd.), etc.
[0167] The cloned DNA encoding GPR39 can be used as it is,
depending upon purpose or, if desired, after digestion with a
restriction enzyme or after the addition of a linker thereto. The
DNA may contain ATG as a translation initiation codon at the 5' end
thereof and may further contain TAA, TGA or TAG as a translation
termination codon at the 3' end thereof. These translation
initiation and termination codons may also be added by using an
appropriate synthetic DNA adapter.
[0168] The expression vector for GPR39 can be manufactured, for
example, by (a) excising the desired DNA fragment from the DNA
encoding GPR39, and then (b) ligating the DNA fragment with an
appropriate expression vector downstream a promoter in the
vector.
[0169] Examples of the vector include plasmids derived form E. coli
(e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from
Bacillus subtilis (e.g., pUB110, pTP5, pC194), plasmids derived
from yeast (e.g., pSH19, pSH15), bacteriophages such as .lamda.
phage, etc., animal viruses such as retrovirus, vaccinia virus,
baculovirus, etc. as well as pA1-11, pXT1, pRc/CMV, pRc/RSV,
pcDNAI/Neo, etc.
[0170] The promoter used in the present invention may be any
promoter if it matches well with a host to be used for gene
expression. In the case of using animal cells as the host, examples
of the promoter include SR.alpha. promoter, SV40 promoter, LTR
promoter, CMV promoter, HSV-TK promoter, etc.
[0171] Among them, CMV promoter or SR.alpha. promoter is preferably
used. Where the host is bacteria of the genus Escherichia,
preferred examples of the promoter include trp promoter, lac
promoter, recA promoter, .lamda.P.sub.L promoter, lpp promoter,
etc. In the case of using bacteria of the genus Bacillus as the
host, preferred example of the promoter are SPO1 promoter, SPO2
promoter, penP promoter, etc. When yeast is used as the host,
preferred examples of the promoter are PHO5 promoter, PGK promoter,
GAP promoter, ADH promoter, etc. When insect cells are used as the
host, preferred examples of the promoter include polyhedrin
prompter, P10 promoter, etc.
[0172] In addition to the foregoing examples, the expression vector
may further optionally contain an enhancer, a splicing signal, a
polyA addition signal, a selection marker, SV40 replication origin
(hereinafter sometimes abbreviated as SV40ori) etc. Examples of the
selection marker include dihydrofolate reductase (hereinafter
sometimes abbreviated as dhfr) gene [methotrexate (MTX)
resistance], ampicillin resistant gene (hereinafter sometimes
abbreviated as Amp.sup.r), neomycin resistant gene (hereinafter
sometimes abbreviated as Neo.sup.r, G418 resistance), etc. In
particular, when dhfr gene is used as the selection marker in CHO
(dhfr.sup.-) cells, selection can also be made on thymidine free
media.
[0173] If necessary, a signal sequence that matches with a host is
added to the N-terminus of the receptor protein of the present
invention. Examples of the signal sequence that can be used are Pho
A signal sequence, OmpA signal sequence, etc. in the case of using
bacteria of the genus Escherichia as the host; .alpha.-amylase
signal sequence, subtilisin signal sequence, etc. in the case of
using bacteria of the genus Bacillus as the host; MF.alpha. signal
sequence, SUC2 signal sequence, etc. in the case of using yeast as
the host; and insulin signal sequence, .alpha.-interferon signal
sequence, antibody molecule signal sequence, etc. in the case of
using animal cells as the host, respectively.
[0174] Using the vector containing the DNA encoding GPR39 thus
constructed, transformants can be manufactured.
[0175] Examples of the host, which may be employed, are bacteria
belonging to the genus Escherichia, bacteria belonging to the genus
Bacillus, yeast, insect cells, insects and animal cells, etc.
[0176] Specific examples of the bacteria belonging to the genus
Escherichia include Escherichia coli K12 DH1 [Proc. Natl. Acad.
Sci. U.S.A., 60, 160 (1968)), JM103 (Nucleic Acids Research, 9, 309
(1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)],
HB101 [Journal of Molecular Biology, 41, 459 (1969)), C600
(Genetics, 39, 440 (1954)], etc.
[0177] Examples of the bacteria belonging to the genus Bacillus
include Bacillus subtilis M114 [Gene, 24, 255 (1983)], 207-21
[Journal of Biochemistry, 95, 87 (1984)], etc.
[0178] Examples of yeast include Saccharomyces cereviseae AH22,
AH22R.sup.-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe
NCYC1913, NCYC2036, Pichia pastoris, etc.
[0179] Examples of insect cells include, for the virus AcNPV,
Spodoptera frugiperda cells (Sf cells), MG1 cells derived from
mid-intestine of Trichoplusia ni, High Five.TM. cells derived from
egg of Trichoplusia ni, cells derived from Mamestra brassicae,
cells derived from Estigmena acrea, etc.; and for the virus BmNPV,
Bombyx mori N cells (BmN cells), etc. are used. Examples of the Sf
cell which can be used are Sf9 cells (ATCC CRL1711) and Sf21 cells
(both cells are described in Vaughn, J. L. et al., In Vivo, 13,
213-217 (1977)).
[0180] As the insect, for example, a larva of Bombyx mori can be
used [Maeda, et al., Nature, 315, 592 (1985)].
[0181] Examples of animal cells include monkey cells COS-7, Vero,
Chinese hamster cells CHO (hereinafter referred to as CHO cells),
dhfr gene deficient Chinese hamster cells CHO (hereinafter simply
referred to as CHO(dhfr.sup.-) cell), mouse L cells, mouse AtT-20,
mouse myeloma cells, rat GH3, human FL cells, etc.
[0182] Bacteria belonging to the genus Escherichia can be
transformed, for example, by the method described in Proc. Natl.
Acad. Sci. U.S.A., 69, 2110 (1972), Gene, 17, 107 (1982), etc.
[0183] Bacteria belonging to the genus Bacillus can be transformed,
for example, by the method described in Molecular & General
Genetics, 168, 111 (1979).
[0184] Yeast can be transformed, for example, by the method
described in Methods in Enzymology, 194, 182-187 (1991), Proc.
Natl. Acad. Sci. U.S.A., 75, 1929 (1978), etc.
[0185] Insect cells or insects can be transformed, for example,
according to the method described in Bio/Technology, 6,
47-55(1988), etc.
[0186] Animal cells can be transformed, for example, according to
the method described in Saibo Kogaku (Cell Engineering), extra
issue 8, Shin Saibo Kogaku Jikken Protocol (New Cell Engineering
Experimental Protocol), 263-267 (1995), published by Shujunsha, or
Virology, 52, 456 (1973).
[0187] Thus, the transformant transformed with the expression
vector containing the DNA encoding GPR39 can be obtained.
[0188] Where the host is bacteria belonging to the genus
Escherichia or the genus Bacillus, the transformant can be
appropriately incubated in a liquid medium which contains materials
required for growth of the transformant such as carbon sources,
nitrogen sources, inorganic materials, and so on. Examples of the
carbon sources include glucose, dextrin, soluble starch, sucrose,
etc. Examples of the nitrogen sources include inorganic or organic
materials such as ammonium salts, nitrate salts, corn steep liquor,
peptone, casein, meat extract, soybean cake, potato extract, etc.
Examples of the inorganic materials are calcium chloride, sodium
dihydrogenphosphate, magnesium chloride, etc. In addition, yeast
extract, vitamins, growth promoting factors etc. may also be added
to the medium. Preferably, pH of the medium is adjusted to about 5
to about 8.
[0189] A preferred example of the medium for incubation of the
bacteria belonging to the genus Escherichia is M9 medium
supplemented with glucose and Casamino acids (Miller, Journal of
Experiments in Molecular Genetics, 431-433, Cold Spring Harbor
Laboratory, New York, 1972). If necessary, a chemical such as
3.beta.-indolylacrylic acid can be added to the medium thereby to
activate the promoter efficiently.
[0190] Where the bacteria belonging to the genus Escherichia are
used as the host, the transformant is usually cultivated at about
15 to 43.degree. C. for about 3 hours to about 24 hours. If
necessary, the culture may be aerated or agitated.
[0191] Where the bacteria belonging to the genus Bacillus are used
as the host, the transformant is cultivated generally at about 30
to 40.degree. C. for about 6 to 24 hours. If necessary, the culture
can be aerated or agitated.
[0192] Where yeast is used as the host, the transformant is
cultivated, for example, in Burkholder's minimal medium [Bostian,
K. L. et al., Proc. Natl. Acad. Sci. U.S.A., 77, 4505 (1980)] or in
SD medium supplemented with 0.5% Casamino acids [Bitter, G. A. et
al., Proc. Natl. Acad. Sci. U.S.A., 81, 5330 (1984)]. Preferably,
pH of the medium is adjusted to about 5 to about 8. In general, the
transformant is cultivated at about 20.degree. C. to about
35.degree. C. for about 24 to 72 hours. If necessary, the culture
can be aerated or agitated.
[0193] Where insect cells or insects are used as the host, the
transformant is cultivated in, for example, Grace's Insect Medium
(Grace, T. C. C., Nature, 195, 788 (1962)) to which an appropriate
additive such as immobilized 10% bovine serum is added. Preferably,
pH of the medium is adjusted to about 6.2 to about 6.4. Normally,
the transformant is cultivated at about 27.degree. C. for about 3
days to about 5 days and, if necessary and desired, the culture can
be aerated or agitated.
[0194] Where animal cells are employed as the host, the
transformant is cultivated in, for example, MEM medium containing
about 5% to about 20% fetal bovine serum [Science, 122, 501
(1952)], DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium
[The Journal of the American Medical Association, 199, 519 (1967)],
199 medium [Proceeding of the Society for the Biological Medicine,
73, 1 (1950)], etc. Preferably, pH of the medium is adjusted to
about 6 to about 8. The transformant is usually cultivated at about
30.degree. C. to about 40.degree. C. for about 15 hours to about 60
hours and, if necessary and desired, the culture can be aerated or
agitated.
[0195] As described above, GPR39 can be produced into the cell, in
the cell membrane or out of the cell of the transformant.
[0196] GPR39 can be separated and purified from the culture
described above by the following procedures.
[0197] When GPR39 is extracted from the culture or cells, after
cultivation the transformants or cells are collected by a publicly
known method and suspended in an appropriate buffer. The
transformants or cells are then disrupted by publicly known methods
such as ultrasonication, a treatment with lysozyme and/or
freeze-thaw cycling, followed by centrifugation, filtration, etc.
Thus, the crude extract of GPR39 can be obtained. The buffer used
for the procedures may contain a protein modifier such as urea or
guanidine hydrochloride, or a surfactant such as Triton X-100.TM.,
etc. When GPR39 is secreted in the culture, after completion of the
cultivation the supernatant can be separated from the transformants
or cells to collect the supernatant by a publicly known method.
[0198] GPR39 contained in the supernatant or the extract thus
obtained can be purified by appropriately combining the publicly
known methods for separation and purification. Such publicly known
methods for separation and purification include a method utilizing
difference in solubility such as salting out, solvent
precipitation, etc.; a method utilizing mainly difference in
molecular weight such as dialysis, ultrafiltration, gel filtration,
SDS-polyacrylamide gel electrophoresis, etc.; a method utilizing
difference in electric charge such as ion exchange chromatography,
etc.; a method utilizing difference in specific affinity such as
affinity chromatography, etc.; a method utilizing difference in
hydrophobicity such as reverse phase high performance liquid
chromatography, etc.; a method utilizing difference in isoelectric
point such as isoelectrofocusing electrophoresis; and the like.
[0199] In the case that GPR39 thus obtained is in a free form, it
can be converted into the salt by publicly known methods or
modifications thereof. On the other hand, when GPR39 is obtained in
the form of a salt, it can be converted into the free form or in
the form of a different salt by publicly known methods or
modifications thereof.
[0200] GPR39 produced by the recombinant can be treated, prior to
or after the purification, with an appropriate protein modifying
enzyme so that GPR39 can be appropriately modified to partially
remove a polypeptide. Examples of the protein-modifying enzyme
include trypsin, chymotrypsin, arginyl endopeptidase, protein
kinase, glycosidase or the like.
[0201] The activity of the thus produced GPR39 can be determined by
a binding experiment to a labeled ligand, by an enzyme immunoassay
using a specific antibody, or the like.
[0202] Antibodies to GPR39 may be any of polyclonal antibodies and
monoclonal antibodies, as long as they are capable of recognizing
GPR39.
[0203] The antibodies to GPR39 may be manufactured by publicly
known methods for manufacturing antibodies or antisera, using as
antigens GPR39.
[Preparation of Monoclonal Antibody]
(a) Preparation of Monoclonal Antibody-Producing Cells
[0204] GPR39 is administered to mammals either solely or together
with carriers or diluents to the site where the production of
antibody is possible by the administration. In order to potentiate
the antibody productivity upon the administration, complete
Freund's adjuvants or incomplete Freund's adjuvants may be
administered. The administration is usually carried out once in
every two to six weeks and 2 to 10 times in total. Examples of the
applicable mammals are monkeys, rabbits, dogs, guinea pigs, mice,
rats, sheep and goats, with mice and rats being preferred.
[0205] In the preparation of monoclonal antibody-producing cells,
warm-blooded animals, e.g., mice, immunized with an antigen wherein
the antibody titer is noted is selected, then the spleen or lymph
node is collected after 2 to 5 days from the final immunization and
antibody-producing cells contained therein are fused with myeloma
cells to give monoclonal antibody-producing hybridomas. Measurement
of the antibody titer in antisera may be made, for example, by
reacting a labeled form of the receptor protein, which will be
described later, with the antiserum followed by assaying the
binding activity of the labeling agent bound to the antibody. The
fusion may be operated, for example, by the known Koehler and
Milstein method [Nature, 256, 495 (1975)]. Examples of the fusion
accelerator are polyethylene glycol (PEG), Sendai virus, etc., of
which PEG is preferably employed.
[0206] Examples of the myeloma cells are NS-1, P3U1, SP2/0, etc. In
particular, P3U1 is preferably employed. A preferred ratio of the
count of the antibody-producing cells used (spleen cells) to the
count of myeloma cells is within a range of approximately 1:1 to
20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added in a
concentration of approximately 10 to 80% followed by incubating at
about 20 to about 40.degree. C., preferably at about 30 to about
37.degree. C. for about 1 to about 10 minutes, an efficient cell
fusion can be carried out.
[0207] Various methods can be used for screening of a monoclonal
antibody-producing hybridoma. Examples of such methods include a
method which comprises adding the supernatant of hybridoma to a
solid phase (e.g., microplate) adsorbed with an antigen of the
receptor protein directly or together with a carrier, adding an
anti-immunoglobulin antibody (when mouse cells are used for the
cell fusion, anti-mouse immunoglobulin antibody is used) labeled
with a radioactive substance or an enzyme, or Protein A and
detecting the monoclonal antibody bound to the solid phase, and a
method which comprises adding the supernatant of hybridoma to a
solid phase adsorbed with an anti-immunoglobulin antibody or
Protein A, adding the receptor protein labeled with a radioactive
substance or an enzyme and detecting the monoclonal antibody bound
to the solid phase.
[0208] The monoclonal antibody can be selected by publicly known
methods or by modifications of these methods. In general, the
selection can be effected in a medium for animal cells supplemented
with HAT (hypoxanthine, aminopterin and thymidine). Any selection
and growth medium can be employed as far as the hybridoma can grow
therein. For example, RPMI 1640 medium containing 1% to 20%,
preferably 10% to 20% fetal bovine serum, GIT medium (Wako Pure
Chemical Industries, Ltd.) containing 1% to 10% fetal bovine serum,
a serum free medium for cultivation of a hybridoma (SFM-101, Nissui
Seiyaku Co., Ltd.) and the like can be used for the selection and
growth medium. The cultivation is carried out generally at
20.degree. C. to 40.degree. C., preferably at about 37.degree. C.,
for 5 days to 3 weeks, preferably 1 to 2 weeks. The cultivation can
be conducted normally in 5% CO.sub.2. The antibody titer of the
culture supernatant of hybridomas can be determined as in the assay
for the antibody titer in antisera described above.
(b) Purification of Monoclonal Antibody
[0209] Separation and purification of a monoclonal antibody can be
carried out by methods applied to conventional separation and
purification of immunoglobulins, as in the conventional methods for
separation and purification of polyclonal antibodies [e.g.,
salting-out, alcohol precipitation, isoelectric point
precipitation, electrophoresis, adsorption and desorption with ion
exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a
specific purification method which comprises collecting only an
antibody with an activated adsorbent such as an antigen-binding
solid phase, Protein A, Protein G. etc. and dissociating the
binding to obtain the antibody].
[Preparation of Polyclonal Antibody]
[0210] The polyclonal antibody of the present invention can be
manufactured by publicly known methods or modifications thereof.
For example, a complex of immunogen (GPR39 antigen) and a carrier
protein is prepared, and a mammal is immunized with the complex in
a manner similar to the method described above for the manufacture
of monoclonal antibodies. The product containing the antibody to
GPR39 is collected from the immunized animal followed by separation
and purification of the antibody.
[0211] In the complex of an immunogen and a carrier protein used to
immunize a mammal, the type of carrier protein and the mixing ratio
of a carrier to hapten may be any type and in any ratio, as long as
the antibody is efficiently produced to the hapten immunized by
crosslinking to the carrier. For example, bovine serum albumin,
bovine thyroglobulins, keyhole limpet hemocyanin, etc. is coupled
to hapten in a carrier-to-hapten weight ratio of approximately 0.1
to 20, preferably about 1 to about 5.
[0212] A variety of condensing agents can be used for the coupling
of a carrier to hapten. Glutaraldehyde, carbodiimide, maleimide
activated ester, activated ester reagents containing thiol group or
dithiopyridyl group, etc. are used for the coupling.
[0213] The condensation product is administered to warm-blooded
animals either solely or together with carriers or diluents to the
site in which the antibody can be produced by the administration.
In order to potentiate the antibody productivity upon the
administration, complete Freund's adjuvant or incomplete Freund's
adjuvant may be administered. The administration is usually made
once approximately in every 2 to 6 weeks and about 3 to about 10
times in total.
[0214] The polyclonal antibody can be collected from the blood,
ascites, etc., preferably from the blood of mammals immunized by
the method described above.
[0215] The polyclonal antibody titer in antiserum can be assayed by
the same procedure as that for the determination of serum antibody
titer described above. The separation and purification of the
polyclonal antibody can be carried out, following the method for
the separation and purification of immunoglobulins performed as
applied to the separation and purification of monoclonal antibodies
described hereinabove.
[0216] The ligand for GPR39 is an ionizable metal element or salts
thereof.
[0217] Examples of the ionizable metal element include zinc,
copper, nickel, cadmium, and so on.
[0218] As salts of these metal elements, for example, salts with
acids such as hydrochloric acid, sulfuric acid, nitric acid, acetic
acid, etc., specifically preferred are cadmium chloride, zinc
chloride, zinc acetate, copper chloride, copper sulfate, nickel
chloride and nickel sulfate, etc. are used and more preferred are
cadmium chloride, zinc chloride, copper chloride and nickel
chloride.
[0219] In the present specification, the ionizable metal elements
include (1) metal elements in an ionic state (e.g., zinc ions,
copper ions, nickel ions and cadmium ions); (2) compounds
containing the ionizable metal elements (e.g., oxides such as zinc
oxide, copper oxide, nickel oxide and cadmium oxide), and the
like.
[0220] Hereinafter, the ionizable metal elements such as cadmium,
zinc, copper, nickel, etc., or salts thereof are merely referred to
as the "metal elements" throughout the specification.
[0221] GPR39, the DNA encoding GPR39 (hereinafter sometimes
referred to briefly as the DNA of the present invention), the
antibody to GPR39 (hereinafter sometimes referred to briefly as the
antibody of the present invention) and the antisense DNA to the DNA
of the present invention (hereinafter sometimes referred to briefly
as the antisense DNA of the present invention) have the following
applications.
(1) Agent for Preventing/Treating Diseases Associated with
Dysfunction of GPR39
[0222] a) GPR39 or b) the DNA encoding GPR39 can be used as
pharmaceutical drugs such as agents for preventing/treating
diseases associated with dysfunction of GPR39, etc.
[0223] For example, when the physiological activities of the metal
element as the ligand cannot be expected in a patient (deficiency
of GPR39) due to a decrease of GPR39 in the body, the amount of
GPR39 can be increased in the body of the patient a) by
administering GPR39 to the patient thereby to supplement the amount
of GPR39; or b) (a) by administering the DNA encoding GPR39 to the
patient to express the same, or (ii) by inserting and expressing
the DNA encoding GPR39 in the objective cells and then
transplanting the cells to the patient, whereby the activities of
the ligand can be sufficiently exhibited. That is, the DNA encoding
GPR39 is useful as a safe and low toxic agent for
preventing/treating diseases associated with dysfunction of
GPR39.
[0224] Specifically, GPR39 or the DNA of the present invention can
be used as a prophylactic/therapeutic agent for metal deficiency
symptoms such as growth retardation, wounds, burn, cold
constitution, declining of learning ability, hypogonadism,
dysgeusia, anosmia, prostatic hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression,
menoxenia, hypotonic bladder induced by sensory decrease of the
bladder by diabetes mellitus, etc., hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs; etc., or an
agent for promoting the secretion of cytokines (e.g., IL-8).
[0225] Where GPR39 is used as the preventive/therapeutic agent
described above, GPR39 can be prepared into a pharmaceutical
composition in a conventional manner.
[0226] On the other hand, where the DNA of the present invention is
used as the preventive/therapeutic agent described above, the DNA
itself is administered alone; alternatively, the DNA is inserted
into an appropriate vector such as retrovirus vector, adenovirus
vector, adenovirus-associated virus vector, etc. and then
administered in a conventional manner. The DNA of the present
invention may also be administered as naked DNA, or with adjuvants
to assist its uptake by gene gun or through a catheter such as a
catheter with a hydrogel.
[0227] For example, a) GPR39 or b) the DNA of the present invention
can be used orally, for example, in the form of tablets which may
be sugar coated if necessary, capsules, elixirs, microcapsules
etc., or parenterally in the form of injectable preparations such
as a sterile solution and a suspension in water or with other
pharmaceutically acceptable liquid. These preparations can be
manufactured by mixing a) GPR39 or b) the DNA of the present
invention with a physiologically acceptable known carrier, a
flavoring agent, an excipient, a vehicle, an antiseptic agent, a
stabilizer, a binder, etc. in a unit dosage form required in a
generally accepted manner that is applied to making pharmaceutical
preparations. The effective component in the preparation is
controlled in such a dose that an appropriate dose is obtained
within the specified range given.
[0228] Additives miscible with tablets, capsules, etc. include a
binder such as gelatin, corn starch, tragacanth and gum arabic, an
excipient such as crystalline cellulose, a puffiness agent such as
corn starch, gelatin and alginic acid, a lubricant such as
magnesium stearate, a sweetening agent such as sucrose, lactose and
saccharin, and a flavoring agent such as peppermint, akamono oil
and cherry. 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 by conventional procedures 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 and coconut
oil, etc. to prepare the pharmaceutical composition. Examples of an
aqueous medium for injection include physiological saline and an
isotonic solution containing glucose and other auxiliary agents
(e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) and may be
used in combination with an appropriate dissolution aid such as an
alcohol (e.g., ethanol or the like), a polyalcohol (e.g., propylene
glycol and polyethylene glycol), a nonionic surfactant (e.g.,
polysorbate 80.TM. and HCO-50), etc. Examples of the oily medium
include sesame oil and soybean oil, which may also be used in
combination with a dissolution aid such as benzyl benzoate and
benzyl alcohol.
[0229] The preventive/therapeutic agent described above may further
be formulated with a buffer (e.g., phosphate buffer, sodium acetate
buffer, etc.), a soothing agent (e.g., benzalkonium chloride,
procaine hydrochloride, etc.), a stabilizer (e.g., human serum
albumin, polyethylene glycol, etc.), a preservative (e.g., benzyl
alcohol, phenol, etc.), an antioxidant, etc. The thus-prepared
liquid for injection is normally filled in an appropriate
ampoule.
[0230] Since the thus obtained pharmaceutical preparation is safe
and low toxic, the preparation can be administered to human or
mammals (e.g., rats, mice, rabbits, sheep, swine, bovine, cats,
dogs, monkeys, etc.).
[0231] The dose of GPR39 varies depending on the subject to be
administered, target organ, conditions, method for administration,
etc.; in oral administration for the patient (as 60 kg body weight)
with, e.g., cancer, the dose 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 varies depending on the subject to be administered,
target organ, conditions, method for administration, etc. but in
the form of injectable preparation, it is advantageous to
administer GPR39 intravenously to the patient (as 60 kg body
weight) with, e.g., cancer, 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 60 kg can be administered.
[0232] The dose of the DNA of the present invention varies
depending on the subject to be administered, target organ,
conditions, method for administration, etc.; in oral administration
for the patient (as 60 kg body weight) with, e.g., cancer, the dose
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 varies depending on
the subject to be administered, target organ, conditions, method
for administration, etc. but in the form of injectable preparation,
it is advantageous to administer the DNA intravenously to the
patient (as 60 kg body weight) with, e.g., cancer 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 60 kg can be
administered.
(2) Gene Diagnostic Agent
[0233] By using the DNA and antisense DNA as probes, an abnormality
(gene abnormality) of the DNA or mRNA encoding GPR39 or its partial
peptides in human or mammals (e.g., rats, mice, rabbits, sheep,
swine, bovine, cats, dogs, monkeys, etc.) can be detected, and are
thus useful as gene diagnostic agents for the damage against the
DNA or mRNA, its mutation, or its reduced expression, or increased
expression or overexpression of the DNA or mRNA.
[0234] The gene diagnosis described above using the DNA, antisense
DNA of the present invention can be performed by, for example, the
publicly known Northern hybridization assay or the PCR-SSCP assay
(Genomics, 5, 874-879 (1989); Proceedings of the National Academy
of Sciences of the United States of America, 86, 2766-2770 (1989)),
etc.
[0235] Where a reduced expression or overexpression of GPR39 is
detected by, e.g., northern hybridization, it can be diagnosed that
one suffers from diseases associated with, for example, the
dysfunction or excessive action of GPR39, or it is highly likely to
suffer from these disease in the future.
[0236] The diseases associated with the dysfunction of GPR39
include metal deficiency symptoms such as growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc.
[0237] The diseases caused by the excessive action of GPR39
include, for example, excess metal-induced symptoms such as renal
dysfunction, pulmonary dysfunction, allergic dermatitis, sensory
neuropathy, Wilson's disease, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria including
interstitial cystitis, prostatic hyperplasia induced-pollakiuria,
urinary incontinence, urinary urgency, pelvic visceral pain, coital
pain, bladder irritation symptoms, various disorders caused by
urinary calculus, etc., or inflammatory diseases (e.g., diabetic
complications such as neuropathy, large vascular disorders, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia), allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)); or the like.
(3) Pharmaceutical Drug Comprising a Compound or its Salt that
Changes the Expression Level of GPR39
[0238] By using the DNA of the present invention as a probe, the
DNA can be used for screening a compound or its salt that changes
the expression level of GPR39.
[0239] That is, the present invention provides methods of screening
the compound or its salt that changes the expression level of
GPR39, which comprises measuring the amount of mRNA in GPR39
contained in, for example, (i) a) blood, b) particular organs, c)
tissues or cells isolated from the organs of non-human mammal or in
(ii) transformants, etc.
[0240] Specifically, the amount of mRNA in GPR39 can be measured as
follows.
[0241] (i) Normal or disease models of non-human mammals (e.g.,
mice, rats, rabbits, sheep, swine, bovine, cats, dogs, monkeys,
more specifically, immunodeficiency model rats, mice, rabbits,
etc.) receive a drug (e.g., an immunomodulator, etc.) or physical
stress (e.g., soaking stress, electric shock, light and darkness,
low temperature, etc.), and the blood, particular organs (e.g.,
brain, liver, kidney, etc.), or tissues or cells isolated from the
organs are obtained after a specified period of time.
[0242] The mRNA of GPR39 contained in the thus obtained cells is
extracted from the cells, for example, in a conventional manner and
quantified by means of, e.g., TaqManPCR, or may also be analyzed by
northern blot technique by publicly known methods.
[0243] (ii) Transformants that express GPR39 are prepared according
to the methods described above, and the mRNA of GPR39 can be
quantified and analyzed, as described above.
[0244] The compound that changes the expression level of GPR39 can
be screened by the following procedures.
[0245] (i) To a normal or disease model of non-human mammal, a test
compound is administered at a specified period of time before (30
minutes to 24 hours before, preferably 30 minutes to 12 hours
before, more preferably 1 hour to 6 hours before), at a specified
time after (30 minutes to 3 days after, preferably 1 hour to 2 days
after, more preferably 1 hour to 24 hours after), or simultaneously
with a drug or physical stress. At a specified time (30 minute to 3
days, preferably 1 hour to 2 days, more preferably 1 hour to 24
hours) after administration of the test compound, the amount of
mRNA in GPR39 contained in cells are quantified and analyzed.
[0246] (ii) Transformants are cultured in a conventional manner and
a test compound is mixed in the culture medium. After a specified
time (after 1 day to 7 days, preferably after 1 day to 3 days, more
preferably after 2 to 3 days), the amount of mRNA in GPR39
contained in the transformants can be quantified and analyzed.
[0247] The test compounds include peptides, proteins, non-peptide
compounds, synthetic compounds, fermentation products, cell
extracts, plant extracts, animal tissue extracts, blood plasma,
etc. They may be novel or known compounds.
[0248] The test compound may form salts and may be used in the form
of salts with physiologically acceptable acids (e.g., inorganic
acids, etc.) or bases (e.g., organic acids, etc.), preferably
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.
[0249] The compound, which is obtained by the screening methods of
the present invention, is the compound that changes the expression
level of GPR39. Specifically, it is (a) the compound that
potentiates the cell stimulating activities mediated by GPR39
(e.g., the activities that promote or suppress arachidonic acid
release, acetylcholine release, intracellular Ca.sup.2+ release,
intracellular cAMP production, intracellular cGMP production,
inositol phosphate production, alters in cell membrane potential,
phosphorylation of intracellular proteins, activation of c-fos, pH
reduction, etc.; an action of promoting the secretion of cytokines
(e.g., IL-8), etc.; in particular, the intracellular Ca.sup.2+
level increasing activity (intracellular Ca.sup.2+ release
activity), action of promoting the secretion of cytokines (e.g.,
IL-8)) by increasing the expression level of GPR39; and (b) the
compound that attenuates the cell-stimulating activities by
reducing the expression level of GPR39.
[0250] The compounds obtained by using the screening method of the
present invention include peptides, proteins, non-peptide
compounds, synthetic compounds, fermentation products, etc. They
may be novel or known compounds.
[0251] As salts of the compound obtained by the screening method of
the present invention, salts with physiologically acceptable acids
(e.g., inorganic acids, etc.) or bases (e.g., organic acids, etc.)
may be used and preferably used are 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.
[0252] The ligand for GPR39 is the ionizable metal element such as
cadmium, zinc, copper and nickel, or salts thereof.
[0253] Thus, the compound or its salts, which are obtained by the
screening method described above and change the expression level of
GPR39, can be used as a prophylactic/therapeutic agent for diseases
associated with the dysfunction of GPR39 (for example, metal
deficiency symptoms such as growth retardation, wounds, burn, cold
constitution, declining of learning ability, hypogonadism,
dysgeusia, anosmia, prostatic hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression,
menoxenia, hypotonic bladder induced by sensory decrease of the
bladder by diabetes mellitus, etc., hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs; etc.), or
diseases associated with the excessive action of GPR39 (for
example, excess metal-induced symptoms such as renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy,
Wilson's disease, overactive bladder-induced pollakiuria, nocturia,
cystitis-induced pollakiuria including interstitial cystitis,
prostatic hyperplasia induced-pollakiuria, urinary incontinence,
urinary urgency, pelvic visceral pain, coital pain, bladder
irritation symptoms, various disorders caused by urinary calculus,
etc.), an agent for regulating the secretion of cytokines (e.g.,
IL-8), or inflammatory diseases (e.g., diabetic complications such
as neuropathy, large vascular disorders, etc.; inflammatory bowel
diseases such as ulcerative colitis, etc.; cystitis; irritable
bowel syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis or chronic obstructive pulmonary disease (COPD)), and so
on.
[0254] Specifically, the compound or its salts that increase the
expression level of GPR39 can be used as a prophylactic/therapeutic
agent for, for example, metal deficiency symptoms such as growth
retardation, wounds, burn, cold constitution, declining of learning
ability, hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc., or as an agent for promoting the secretion of
cytokines (e.g., IL-8).
[0255] On the other hand, the compound or its salts that decrease
the expression level of GPR39 can be used as a
prophylactic/therapeutic agent for, for example, excess
metal-induced symptoms such as renal dysfunction, pulmonary
dysfunction, allergic dermatitis, sensory neuropathy, Wilson's
disease, overactive bladder-induced pollakiuria, nocturia,
cystitis-induced pollakiuria including interstitial cystitis,
prostatic hyperplasia induced-pollakiuria, urinary incontinence,
urinary urgency, pelvic visceral pain, coital pain, bladder
irritation symptoms, various disorders caused by urinary calculus,
etc., an agent for suppressing the secretion of cytokines (e.g.,
IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy, large
vascular disorders, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), and the like.
[0256] Where the compound or its salt, which is obtained by the
screening methods of the present invention, is used as a
pharmaceutical composition, the compound or its salt can be
prepared into a pharmaceutical preparation in a conventional
manner.
[0257] For example, the compound can be used orally in the form of
tablets which may be tablets, if necessary, 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
compound, with a physiologically acceptable known carrier,
flavoring agent, excipient, vehicle, antiseptic, stabilizer,
binder, 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.
[0258] Additives miscible with tablets, capsules, etc. include a
binder such as gelatin, corn starch, tragacanth or gum arabic, an
excipient such as crystalline cellulose, a puffiness agent such as
corn starch, gelatin, alginic acid, etc., a lubricant such as
magnesium stearate, a sweetening agent such as sucrose, lactose or
saccharin, a flavoring agent 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.
[0259] Furthermore, the preventive/therapeutic agent described
above may also be formulated with a buffer (e.g., phosphate buffer,
sodium acetate buffer), a soothing agent (e.g., benzalkonium
chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human
serum albumin, polyethylene glycol, etc.), a preservative (e.g.,
benzyl alcohol, phenol, etc.), an antioxidant, etc. The thus
prepared liquid for injection is normally filled in an appropriate
ampoule.
[0260] Since the thus obtained pharmaceutical preparation is safe
and low toxic, the preparation can be administered to human or
mammal (e.g., rats, mice, rabbits, sheep, swine, bovine, cats,
dogs, monkeys, etc.).
[0261] The dose of the compound or its salt varies depending on the
subject to be administered, target organ, conditions, method for
administration, etc.; in oral administration for the patient (as 60
kg body weight) with, e.g., cancer, the dose of the compound or its
salt that increases the expression level of GPR39 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 varies depending on the subject to
be administered, target organ, conditions, method for
administration, etc. but in the form of injectable preparation, it
is advantageous to administer the compound or its salt
intravenously to the patient (as 60 kg body weight) with, e.g.,
cancer 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 60 kg can be administered.
(4) Method for Diagnosis Using the Antibody of the Present
Invention
[0262] The antibody of the present invention is capable of
specifically recognizing GPR39. Therefore, the antibody can be used
to detect or neutralize GPR39 in a test fluid.
[0263] That is, the present invention provides, for example, the
following quantification methods:
[0264] (i) a method for quantification of GPR39 in a test fluid,
which comprises competitively reacting the antibody of the present
invention with the test fluid and a labeled form of GPR39, and
measuring a ratio of the labeled GPR39 bound to the antibody;
and,
[0265] (ii) a method for quantification of GPR39 in a test fluid,
which comprises reacting the test fluid with the antibody of the
present invention immobilized on a carrier and a labeled form of
the antibody of the present invention simultaneously or
sequentially, and measuring the activity of the label on the
immobilized carrier.
[0266] In the quantification method (ii) described above, it is
preferred that one antibody recognizes the N-terminal region of
GPR39, and another antibody reacts with the C-terminal region of
GPR39.
[0267] Using the monoclonal antibodies to GPR39, GPR39 can be
assayed and in addition, can be detected by tissue staining or the
like. For this purpose, the antibody molecule itself may be used,
or F(ab').sub.2, Fab' or Fab fractions of the antibody molecule may
also be used.
[0268] The quantification methods of GPR39 using the antibodies are
not particularly limited. Any quantification method can be used, so
long as the amount of antibody, antigen, or antibody-antigen
complex corresponding to the amount of antigen (e.g., the amount of
GPR39) in the test fluid can be detected by chemical or physical
means and the amount of the antigen can be calculated from a
standard curve prepared from standard solutions containing known
amounts of the antigen. For example, nephrometry, competitive
methods, immunometric method, and sandwich method are appropriately
used, with the sandwich method described below being most
preferable in terms of sensitivity and specificity.
[0269] As the labeling agent for the methods using labeled
substances, there are employed, for example, radioisotopes,
enzymes, fluorescent substances, luminescent substances, etc. For
the radioisotope, for example, [.sup.125I], [.sup.131I], [.sup.3H],
[.sup.14C] and the like are used. As the enzyme described above,
stable enzymes with high specific activity are preferred; for
example, .beta.-galactosidase, .beta.-glucosidase, alkaline
phosphatase, peroxidase, malate dehydrogenase and the like are
used. Examples of the fluorescent substance used are fluorescamine
and fluorescein isothiocyanate are used. For the luminescent
substance, there are used, for example, luminol, luminol
derivatives, luciferin, and lucigenin. Furthermore, the
biotin-avidin system may be used for binding antibody or antigen to
the label.
[0270] For immobilization of antigen or antibody, physical
adsorption may be used. Chemical binding methods conventionally
used for insolubilization or immobilization of GPR39, enzymes or
the like may also be used. For the carrier, for example, insoluble
polysaccharides such as agarose, dextran, cellulose, etc.;
synthetic resin such as polystyrene, polyacrylamide, silicon, etc.,
and glass or the like are used.
[0271] In the sandwich method, the immobilized monoclonal antibody
of the present invention is reacted with a test fluid (primary
reaction), then with the labeled monoclonal antibody of the present
invention (secondary reaction), and the activity of the label on
the immobilizing carrier is measured, whereby the amount of GPR39
in the test fluid can be quantified. The order of the primary and
secondary reactions may be reversed, and the reactions may be
performed simultaneously or with an interval. The methods of
labeling and immobilization can be performed by the methods
described above. In the immunoassay by the sandwich method, the
antibody used for immobilized or labeled antibodies is not
necessarily one species, but a mixture of two or more species of
antibody may be used to increase the measurement sensitivity.
[0272] In the methods of assaying GPR39 by the sandwich method,
antibodies that bind to different sites of GPR39 are preferably
used as the monoclonal antibodies of the present invention for the
primary and secondary reactions. That is, in the antibodies used
for the primary and secondary reactions are, for example, when the
antibody used in the secondary reaction recognizes the C-terminal
region of GPR39, it is preferable to use the antibody recognizing
the region other than the C-terminal region for the primary
reaction, e.g., the antibody recognizing the N-terminal region.
[0273] The monoclonal antibodies of the present invention can be
used for the assay systems other than the sandwich method, for
example, competitive method, immunometric method, nephrometry,
etc.
[0274] In the competitive method, antigen in a test fluid and the
labeled antigen are competitively reacted with antibody, and the
unreacted labeled antigen (F) and the labeled antigen bound to the
antibody (B) are separated (B/F separation). The amount of the
label in B or F is measured, and the amount of the antigen in the
test fluid is quantified. This reaction method includes a liquid
phase method using a soluble antibody as an antibody, polyethylene
glycol for B/F separation and a secondary antibody to the soluble
antibody, and an immobilized method either using an immobilized
antibody as the primary antibody, or using a soluble antibody as
the primary antibody and immobilized antibody as the secondary
antibody.
[0275] In the immunometric method, antigen in a test fluid and
immobilized antigen are competitively reacted with a definite
amount of labeled antibody, the immobilized phase is separated from
the liquid phase, or antigen in a test fluid and an excess amount
of labeled antibody are reacted, immobilized antigen is then added
to bind the unreacted labeled antibody to the immobilized phase,
and the immobilized phase is separated from the liquid phase. Then,
the amount of the label in either phase is measured to quantify the
antigen in the test fluid.
[0276] In the nephrometry, insoluble precipitate produced after the
antigen-antibody reaction in gel or solution is quantified. When
the amount of antigen in the test fluid is small and only a small
amount of precipitate is obtained, laser nephrometry using
scattering of laser is advantageously employed.
[0277] For applying these immunological methods to the measurement
methods of the present invention, any particular conditions or
procedures are not required. Systems for measuring GPR39 are
constructed by adding the usual technical consideration in the art
to the conventional conditions and procedures. For the details of
these general technical means, reference can be made to the
following reviews and texts.
[0278] For example, Hiroshi Irie, ed. "Radioimmunoassay" (Kodansha,
published in 1974), Hiroshi Irie, ed. "Sequel to the
Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa, et
al. ed. "Enzyme immunoassay" (Igakushoin, published in 1978), Eiji
Ishikawa, et al. ed. "Immunoenzyme assay" (2nd ed.) (Igakushoin,
published in 1982), Eiji Ishikawa, et al. ed. "Immunoenzyme assay"
(3rd ed.) (Igakushoin, published in 1987), Methods in ENZYMOLOGY,
Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73
(Immunochemical Techniques (Part B)), ibid., Vol. 74
(Immunochemical Techniques (Part C)), ibid., Vol. 84
(Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.,
Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies
and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical
Techniques (Part 1: Hybridoma Technology and Monoclonal
Antibodies)) (all published by Academic Press Publishing).
[0279] As described above, GPR39 can be quantified with high
sensitivity, using the antibody of the present invention.
[0280] Where a reduction in GPR39 level is detected by quantifying
GPR39 level using the antibody of the present invention, it can be
diagnosed that one suffers from, for example, diseases associated
with dysfunction of GPR39, or it is highly likely to suffer from
these disease in the future.
[0281] Where an increase in the GPR39 level is detected, it can be
diagnosed that one suffers from, for example, diseases caused by
the excessive action of GPR39, or it is highly likely to suffer
from these disease in the future.
[0282] The diseases associated with the dysfunction of GPR39
include metal deficiency symptoms such as growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc.
[0283] The diseases associated with the excessive action of GPR39
include, for example, excess metal-induced symptoms such as renal
dysfunction, pulmonary dysfunction, allergic dermatitis, sensory
neuropathy, Wilson's disease, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria including
interstitial cystitis, prostatic hyperplasia induced-pollakiuria,
urinary incontinence, urinary urgency, pelvic visceral pain, coital
pain, bladder irritation symptoms, various disorders caused by
urinary calculus, etc., or inflammatory diseases (e.g., diabetic
complications such as neuropathy, large vascular disorders, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia), allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)), etc.
(5) Method of Screening the Agonist for GPR39
[0284] Since the specific metal element as the ligand binds to
GPR39, increased intracellular Ca.sup.2+ level and promoted
secretion of IL-8 are observed. Thus, GPR39 is useful as a reagent
for searching or determining the agonist (including a naturally
occurring ligand, synthetic ligand and element) for GPR39 other
than the metal elements described above, using the intracellular
signal as an indicator.
[0285] That is, the present invention provides a method of
determining the agonist for GPR39, which comprises assaying the
intracellular Ca.sup.2+ level increasing activity or the IL-8
secretion promoting action mediated by GPR39, when a test compound
or element or a salt thereof are brought in contact with a cell
containing GPR39.
[0286] Examples of test compounds used include publicly known
ligands (for example, angiotensin, bombesin, canavinoid,
cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y,
opioids, purines, vasopressin, oxytocin, PACAP (e.g., PACAP27,
PACAP38), secretin, glucagon, calcitonin, adrenomedulin,
somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal
and related polypeptide), somatostatin, dopamine, motilin, amylin,
bradykinin, CGRP (calcitonin gene-related peptide), leukotrienes,
pancreastatin, prostaglandins, thromboxane, adenosine, adrenaline,
the chemokine superfamily (e.g., the CXC chemokine subfamily such
as IL-8, GRO.alpha., GRO.beta., GRO.gamma., NAP-2, ENA-78, GCP-2,
PF4, IP-10, Mig, PBSF/SDF-1, etc.; the CC chemokine subfamily such
as MCAF/MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIP-1.alpha.,
MIP-1.beta., HCC-1, MIP-3.alpha./LARC, MIP-3.beta./ELC, I-309,
TARC, MIPF-1, MIPF-2/eotaxin-2, MDC, DC-CK1/PARC, SLC, etc.; the C
chemokine subfamily such as lymphotactin, etc.; the CX3C chemokine
subfamily such as fractalkine, etc.), endothelin, enterogastrin,
histamine, neurotensin, TRH, pancreatic polypeptide, galanin,
lysophosphatidic acid (LPA), sphingosine 1-phosphate, etc.) as well
as other substances, for example, tissue extracts from human or
mammal (e.g., mice, rats, swine, bovine, sheep, monkeys, etc.),
cell culture supernatants, low molecular synthetic compounds, or
the like. For example, the tissue extract, cell culture supernatant
or the like is added to GPR39, which is fractionated while assaying
the cell-stimulating activity and a single ligand can be finally
obtained.
[0287] As salts of the test compound, salts with physiologically
acceptable acids (e.g., inorganic acids, etc.) or bases (e.g.,
organic acids, etc.) may be used and preferably used are
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.
[0288] As the test elements, the elements described in, e.g., the
Periodic Table, preferably, metal elements are used.
[0289] Examples of the salts of the test elements include salts
with acids such as hydrochloric acid, sulfuric acid, nitric acid,
acetic acid, etc.
[0290] (6) Method of Screening a Compound or Element or a Salt
Thereof (Agonist, Antagonist, etc.) that Changes the Binding
Property or Signal Transduction of GPR39 to the Metal Element and a
Pharmaceutical Comprising the Compound or Element or a Salt Thereof
that Changes the Binding Property or Signal Transduction of GPR39
to the Metal Element
[0291] Either by using GPR39, or by constructing the recombinant
GPR39 expression system and using the receptor-binding assay system
via the resulting expression system, the compound (e.g., a peptide,
protein, non-peptide compound, synthetic compound or fermentation
product, etc.), the compound or element or salts thereof that
change the binding property or signal transduction of the metal
element, which is a ligand, to GPR39 can be screened
efficiently.
[0292] Examples of such a compound or element or a salt thereof
include (a) a compound or element or a salt thereof showing the
cell stimulating activities mediated by GPR39 (a so-called agonist
for GPR39), (b) a compound or element or a salt thereof that
inhibits the cell stimulating activities (a so-called antagonist to
GPR39), (c) a compound or element or a salt thereof that
potentiates the binding property of the metal element to GPR39, or
(d) a compound or element or a salt thereof that decreases the
binding property of the metal element to GPR39, and the like.
[0293] That is, the present invention provides a method of
screening a compound or element or a salt thereof or a salt thereof
that changes the binding property or signal transduction of GPR39
to the metal element, which comprises comparing the following two
cases: (i) the case wherein GPR39 is brought in contact with the
metal element; and (ii) the case wherein GPR39 is brought in
contact with the metal element and a test compound.
[0294] According to the screening method of the present invention,
the method is characterized by assaying, e.g., the binding amount
of the metal element to GPR39, the cell-stimulating activities,
etc. in (i) and (ii) and comparing (i) and (ii).
[0295] The cell-stimulating activities include the activities that
promote or suppress, for example, arachidonic acid release,
acetylcholine release, intracellular Ca.sup.2+ release (increase in
intracellular Ca.sup.2+ level), intracellular cAMP production,
intracellular cGMP production, inositol phosphate production, cell
membrane potential, phosphorylation of intracellular proteins,
activation of c-fos, pH reduction, etc., an action of promoting the
secretion of cytokines (e.g., IL-8) from the GPR39 expression cell
UM-UC-3, etc. Among them, the intracellular Ca.sup.2+ level
increasing activity, action of promoting the secretion of cytokines
(e.g., IL-8) from the GPR39 expression cell UM-UC-3, etc. are
preferred.
[0296] More specifically, the present invention provides the
following methods.
[0297] a) A method of screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a metal element to GPR39, which comprises measuring the binding
amount of a radioisotope of the metal element to GPR39 in the case
wherein the radioisotope of the metal element is brought in contact
with GPR39 and in the case wherein the radioisotope of the metal
element and a test compound or element or a salt thereof are
brought in contact with GPR39, and comparing the cases.
[0298] b) A method of screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a metal element to GPR39, which comprises measuring the binding
amount of a radioisotope of the metal element to a cell containing
GPR39 or a membrane fraction of said cell, in the case wherein the
radioisotope of the metal element is brought in contact with the
cell containing GPR39 or a membrane fraction of said cell and in
the case wherein the radioisotope of the metal element and a test
compound or element or a salt thereof are brought in contact with
the cell containing GPR39 or said membrane fraction, and comparing
the cases.
[0299] c) A method of screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a metal element to GPR39, which comprises measuring the amount of a
radioisotope of the metal element bound to GPR39, in the case
wherein the radioisotope of the metal element is brought in contact
with GPR39 expressed on a cell membrane by culturing a transformant
containing the DNA of the present invention and in the case wherein
the radioisotope of the metal element and a test compound or
element or a salt thereof are brought in contact with GPR39
expressed on the cell membrane by culturing a transformant
containing the DNA of the present invention, and comparing the
cases.
[0300] d) A method of screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a metal element to GPR39, which comprises assaying GPR39-mediated
cell stimulating activities in the case wherein a compound (e.g., a
metal element or its radioisotope, etc.) that activates GPR39 is
brought in contact with a cell containing GPR39 and in the case
wherein a compound that activates GPR39 and a test compound or
element or a salt thereof are brought in contact with the cell
containing GPR39, and comparing the cell stimulating activities
between the two cases.
[0301] e) A method of screening a compound or element or a salt
thereof that changes the binding property or signal transduction of
a metal element to GPR39, which comprises assaying the
receptor-mediated cell stimulating activities in the case wherein a
compound (e.g., a metal element or its radioisotope, etc.) that
activates GPR39 is brought in contact with GPR39 expressed on a
cell membrane by culturing a transformant containing the DNA of the
present invention and in the case wherein a compound that activates
GPR39 and a test compound or element or a salt thereof are brought
in contact with GPR39 expressed on a cell membrane by culturing a
transformant containing the DNA of the present invention, and
comparing the cell stimulating activities between the two
cases.
[0302] In addition, a compound or element or a salt thereof (a
synthetic low molecular compound, especially a synthetic low
molecular agonist) that changes the binding property of the metal
element to GPR39 can be used as the ligand for GPR39 in place of
the metal element described above. The compound or element or a
salt thereof that changes the binding property of the metal element
to GPR39 can be obtained by performing the screening methods later
described using, e.g., the metal element as a ligand. In the
following screening method, the compound or element or a salt
thereof that changes the binding property of the metal element to
GPR39 is all merely referred to as the ligand.
[0303] Hereinafter the screening method of the present invention
will be described more specifically.
[0304] First, GPR39 used for the screening method of the present
invention may be any one, so long as it contains GPR39 described
above, though GPR39-containing membrane fractions from mammalian
organs are preferably employed. Since it is very difficult to
obtain human-derived organs especially, human-derived GPR39, etc.
expressed abundantly by use of recombinants are suitable for use in
the screening.
[0305] In producing GPR39, the methods described above can be used
and preferably, it is produced by expressing the DNA of the present
invention expressed on mammalian cells or insect cells. As the DNA
fragment encoding the target protein region, a complementary DNA
may be used but is not limited thereto. For example, gene fragments
or a synthetic DNA may also be used as the DNA fragment. In order
to introduce the DNA fragment encoding GPR39 into host animal cells
and express the same efficiently, the DNA fragment is preferably
incorporated into a polyhedron promoter of nuclear polyhedrosis
virus (NPV) belonging to the Baculovirus, an SV40-derived promoter,
a promoter of retrovirus, a metallothionein promoter, a human heat
shock promoter, a cytomegalovirus promoter, SR.alpha. promoter,
etc. at the downstream thereof. The quantity and quality of the
thus expressed receptors can be examined by a publicly known
method, for example, by the method described in the literature
[Nambi, P. et al., J. Biol. Chem., 267, 19555-19559, 1992].
[0306] Accordingly, in the screening method of the present
invention, the substance containing GPR39 may be GPR39 purified by
publicly known methods, or a cell containing said GPR39 or a
membrane fraction of the cell containing said GPR39 may be used as
well.
[0307] Where the GPR39-containing cell is used in the screening
method of the present invention, the cell may be fixed with
glutaraldehyde, formalin, etc. The fixation may be carried out by a
publicly known method.
[0308] The cell containing GPR39 refers to a host cell expressing
the GPR39. Examples of such a host cell include Escherichia coli,
Bacillus subtilis, yeast, insect cells, animal cells, etc.
[0309] The cell membrane fraction refers to a fraction abundant in
cell membrane obtained by cell disruption and subsequent
fractionation by publicly known methods. Cell disruption methods
include cell squashing using a Potter-Elvehjem homogenizer,
disruption using a Waring blender or Polytron (manufactured by
Kinematica Inc.), disruption by ultrasonication, disruption by cell
spraying through thin nozzles under an increased pressure using a
French press, or the like. Cell membrane fractionation is effected
mainly by fractionation using a centrifugal force, such as
centrifugation for fractionation and density gradient
centrifugation. For example, cell disruption fluid is centrifuged
at a low speed (500 rpm to 3,000 rpm) for a short period of time
(normally about 1 to about 10 minutes), the resulting supernatant
is then centrifuged at a higher speed (15,000 rpm to 30,000 rpm)
normally for 30 minutes to 2 hours. The precipitate thus obtained
is used as the membrane fraction. The membrane fraction is abundant
in GPR39 expressed and membrane components such as cell-derived
phospholipids, membrane proteins, etc.
[0310] The amount of GPR39 in the GPR39-containing cell or membrane
fraction is preferably 10.sup.3 to 10.sup.8 molecules per cell,
more preferably 10.sup.5 to 10.sup.7 molecules per cell. As the
level of expression increases, the ligand binding activity per unit
of membrane fraction (specific activity) increases so that not only
the highly sensitive screening system can be constructed but also
large quantities of samples can be assayed with the same lot.
[0311] To perform a) through c) described above for screening the
compound or its salt that changes the binding property or signal
transduction of the ligand to GPR39, an appropriate GPR39 fraction
and a labeled ligand are required.
[0312] The GPR39 fraction is preferably a fraction of naturally
occurring type GPR39 or a fraction of recombinant GPR39 having an
activity equivalent thereto. Herein, the equivalent activity is
intended to mean the ligand binding activity or the signal
transduction activity, which is equivalent to the activity
possessed by naturally occurring type GPR39.
[0313] As the labeled ligand, radioisotopes of the ionizable metal
element such as cadmium, zinc, copper, nickel, etc. are employed.
For example, zinc 65 (.sup.65 Zn), nickel 63 (.sup.63Ni), etc. are
used. Among them, nickel 63 (.sup.63Ni) is preferred.
[0314] Specifically, the compound or element or a salt thereof that
changes the binding property or signal transduction of the ligand
to GPR39 is screened by the following procedures. First, a GPR39
preparation is prepared by suspending a cell containing GPR39 or a
membrane fraction of the cell in a buffer appropriate for use in
the screening method. Any buffer can be used so long as it does not
interfere the ligand-GPR39 binding, including a phosphate buffer or
a Tris-HCl buffer, having pH of 4 to 10 (preferably pH of 6 to 8),
etc. For the purpose of minimizing non-specific binding, a
surfactant such as CHAPS, Tween-80.TM. (Kao-Atlas Inc.), digitonin,
deoxycholate, etc., may optionally be added to the buffer. Further
for the purpose of suppressing the degradation of the receptor or
ligand by a protease, a protease inhibitor such as PMSF, leupeptin,
E-64 (manufactured by Peptide Institute, Inc.), pepstatin, etc. may
also be added. A given amount (5,000 cpm to 500,000 cpm) of the
labeled ligand is added to 0.01 ml to 10 ml of the receptor
solution, in which 10.sup.4 M to 10.sup.-10 M of a test compound is
co-present. To determine the amount of non-specific binding (NSB),
a reaction tube containing an unlabeled ligand in a large excess is
also provided. The reaction is carried out at approximately
0.degree. C. to 50.degree. C., preferably approximately 4.degree.
C. to 37.degree. C. for about 20 minutes to about 24 hours,
preferably about 30 minutes to 3 hours. After completion of the
reaction, the reaction mixture is filtrated through glass fiber
filter paper, etc. and washed with an appropriate volume of the
same buffer. The residual radioactivity on the glass fiber filter
paper is then measured by means of a liquid scintillation counter
or .gamma.-counter. When nonspecific binding (NSB) is subtracted
from the count (B.sub.0) where any antagonizing substance is absent
and the resulting count (B.sub.0 minus NSB) is made 100%, the test
compound showing the specific binding amount (B minus NSB) of,
e.g., 50% or less may be selected as a candidate compound.
[0315] The method d) or e) described above for screening the
compound or element or a salt thereof that changes the binding
property or signal transduction of the ligand to GPR39 or signal
transduction can be performed as follows. For example,
GPR39-mediated cell stimulating activities can be determined by a
publicly known method, or using an assay kit commercially
available.
[0316] Specifically, the cells containing GPR39 are first cultured
in a multiwell plate, etc. Prior to screening, the medium is
replaced with fresh medium or with an appropriate non-cytotoxic
buffer, followed by incubation for a given period of time in the
presence of a test compound, etc. Subsequently, the cells are
extracted or the supernatant is recovered and the resulting product
is quantified by appropriate procedures. Where it is difficult to
detect the production of the cell-stimulating activity indicator
(e.g., cAMP, arachidonic acid, etc.) due to a degrading enzyme
contained in the cells, an inhibitor against such as a degrading
enzyme may be added prior to the assay. For detecting the
activities such as the cAMP production suppression activity, the
baseline production in the cells is increased by forskolin or the
like and the suppressing effect on the increased baseline
production can be detected.
[0317] For screening through the assay for the cell stimulating
activities, cells, in which an appropriate GPR39 has been
expressed, are necessary. Preferred cells, in which GPR39 has been
expressed, are a naturally occurring type cell line containing
GPR39 and the aforesaid cell line, in which recombinant type GPR39
has been expressed.
[0318] Examples of the test compounds include peptides, proteins,
non-peptide compounds, synthetic compounds, fermentation products,
cell extracts, plant extracts, animal tissue extracts, blood
plasma, etc. These test compounds may be either novel or publicly
known compounds.
[0319] As salts of the test compound, salts with physiologically
acceptable acids (e.g., inorganic acids, etc.) or bases (e.g.,
organic acids, etc.) may be used and preferably used are
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.
[0320] As the test elements, the elements described in, e.g., the
Periodic Table, are used. Among them, metal elements are
preferred.
[0321] Salts of the test elements include, for example, salts with
acids such as hydrochloric acid, sulfuric acid, nitric acid, acetic
acid, etc.
[0322] The test compound which is preferably used is a compound
designed to bind to the ligand-binding pocket, based on the atomic
coordinate and the position of the ligand-binding pocket in the
active site of GPR39. The atomic coordinate and the position of the
ligand-binding pocket in the active site of GPR39 can be determined
by publicly known methods or modifications thereof.
[0323] By using the method of screening the agonist for GPR39
described above, it can be confirmed whether the compound that
changes the binding property of the ligand to GPR39 is either an
agonist or an antagonist.
[0324] Specifically, the confirmation can be made by (i) or (ii)
below.
[0325] (i) By binding assay as shown by the screening methods a) to
c) described above, the compound that changes the binding property
of the metal element to GPR39 (especially inhibits the binding) is
obtained. It is then determined whether or not the compound
possesses the cell stimulating activity described above. When the
compound or its salt has the cell stimulating activity, it is an
agonist for GPR39, whereas the compound or its salt having no cell
stimulating activity is an antagonist to GPR39.
[0326] (ii) (a) A test compound is brought in contact with a cell
containing GPR39 to assay the cell stimulating activity described
above. The test compound or its salt having the cell stimulating
activity is an agonist for GPR39.
[0327] (b) The cell stimulating activity is assayed in the case
wherein a compound that activates GPR39 (e.g., a metal element) is
brought in contact with a cell containing GPR39 and in the case
wherein a compound that activates GPR39 and a test compound are
brought in contact with a cell containing GPR39, and comparison is
made therebetween. The test compound, which can decrease the cell
stimulating activity mediated by the compound or its salt that
activates GPR39, is an antagonist to GPR39.
[0328] The kit for screening the compound or its salt that changes
the binding property or signal transduction of the ligand to GPR39
is a kit comprising GPR39, a cell containing GPR39, or a membrane
fraction of the cell containing GPR39, and the like.
[0329] Examples of the screening kit of the present invention
include the following.
1. Reagent for Screening
a) Assay Buffer and Wash Buffer
[0330] Hanks' balanced salt solution (manufactured by Gibco, Inc.)
supplemented with 0.05% bovine serum albumin (manufactured by
Sigma, Inc.)
[0331] The solution is sterilized by filtration through a 0.45
.mu.m filter, and stored at 4.degree. C. or may be prepared at
use.
b) GPR39 Preparation
[0332] CHO cells wherein GPR39 has been expressed are passaged in a
12-well plate at a density of 5.times.10.sup.5 cells/well followed
by culturing at 37.degree. C. under 5% CO.sub.2 and 95% air for 2
days.
c) Labeled Ligand
[0333] A radioisotope such as zinc 65 (.sup.65Zn), nickel 63
(.sup.63Ni), etc.
[0334] An aqueous solution is stored at 4.degree. C. or -20.degree.
C. and diluted to 1 .mu.M with the assay buffer upon use.
d) Standard Ligand Solution
[0335] The ligand is dissolved in and adjusted to 1 mM with PBS
containing 0.1% bovine serum albumin (manufactured by Sigma, Inc.)
and stored at -20.degree. C.
2. Assay Method
[0336] a) CHO cells wherein GPR39 has been expressed are cultured
in a 12-well culture plate and washed twice with 1 ml of the assay
buffer, and 490 .mu.l of the assay buffer is added to each
well.
[0337] b) After adding 5 .mu.l of 10.sup.-3-10.sup.-10 M test
compound solution, 5 .mu.l of a labeled ligand is added to the
mixture, and the cells are incubated at room temperature for an
hour. To determine the amount of the non-specific binding, 5 .mu.l
of the non-labeled ligand is added in place of the test
compound.
[0338] c) The reaction solution is removed, and the wells are
washed 3 times with the washing buffer. The labeled ligand bound to
the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of
liquid scintillator A (manufactured by Wako Pure Chemical
Industries, Ltd.)
[0339] d) The radioactivity is measured using a liquid
scintillation counter (manufactured by Beckman Co.), and the
percent maximum binding (PMB) is calculated by the equation below.
PMB=[(B-NSB)/(B.sub.0-NSB)].times.100 PMB: Percent maximum binding
B: Value obtained in the presence of a test compound NSB:
Non-specific binding B.sub.0: Maximum binding
[0340] The compound or element or a salt thereof, which is obtained
by using the screening method or the screening kit of the present
invention, is the compound or element or a salt thereof, which has
the action of changing the binding property or signal transduction
of the metal element to GPR39 or signal transduction. Specifically,
the compound is: (a) a compound or element or a salt thereof having
the cell-stimulating activity mediated by GPR39 (a so-called
agonist for GPR39); (b) a compound or element or a salt thereof
having no cell stimulating activity (a so-called antagonist to
GPR39); (c) a compound or element or a salt thereof that
potentiates the binding affinity of the metal element to GPR39; or
(d) a compound or element or a salt thereof that reduces the
binding affinity of the metal element to GPR39.
[0341] The compounds obtained by using screening method of the
present invention include peptides, proteins, non-peptide
compounds, synthetic compounds, fermentation products, etc. These
compounds may be novel or known compounds.
[0342] As salts of the compound, salts with physiologically
acceptable acids (e.g., inorganic acids, etc.) or bases (e.g.,
organic acids, etc.) may be used and preferably used are
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.
[0343] As the elements obtained by the screening method or
screening kit of the present invention, for example, the elements
described in, e.g., the Periodic Table, are used. Among them, metal
elements are preferred.
[0344] The salts of the elements include, for example, salts with
acid such as hydrochloric acid, sulfuric acid, nitric acid, acetic
acid, etc.
[0345] Since the agonists for GPR39 have the same physiological
activities as those of the metal element, which is a ligand for
GPR39, the agonists are useful as safe and low toxic
pharmaceuticals, correspondingly to the physiological activity
possessed by the metal element.
[0346] Since the antagonists to GPR39 can suppress the
physiological activities possessed by the metal element, which is a
ligand for GPR39, the antagonists are useful as safe and low toxic
pharmaceuticals to suppress the physiological activities of the
metal element.
[0347] The compound or element or a salt thereof that potentiates
the binding affinity of the metal element to GPR39 can potentiate
the physiological activity the metal element has, and is thus
useful as a safe and low toxic pharmaceutical correspondingly to
the physiological activity possessed by the metal element.
[0348] The compound or element or a salt thereof that reduces the
binding affinity of the metal element to GPR39, element or its salt
can reduce the physiological activities the metal element has, and
is useful as a safe and low toxic pharmaceutical to suppress the
physiological activity possessed by the metal element.
[0349] Specifically, the agonist for GPR39 or the compound or
element or a salt thereof that potentiates the binding affinity of
the metal element to GPR39 is useful as a prophylactic/therapeutic
agent for diseases, for example, metal deficiency symptoms such as
growth retardation, wounds, burn, cold constitution, declining of
learning ability, hypogonadism, dysgeusia, anosmia, prostatic
hyperplasia, arteriosclerosis, myocardial infarction, apoplexy,
cirrhosis, cholesterol accumulation, lowered resistance to
infection, gout, cancer, hard labor, diabetes mellitus, brown
spots, anemia, alopecia, respiratory disturbances, indigestion,
cardiac disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc., or as an agent for promoting the secretion of
cytokines (e.g., IL-8).
[0350] On the other hand, the antagonist to GPR39 or the compound
or element or a salt thereof that reduces the binding affinity of
the metal element to GPR39 is useful as a prophylactic/therapeutic
agent for diseases, for example, excess metal-induced symptoms such
as renal dysfunction, pulmonary dysfunction, allergic dermatitis,
sensory neuropathy, Wilson's disease, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria including
interstitial cystitis, prostatic hyperplasia induced-pollakiuria,
urinary incontinence, urinary urgency, pelvic visceral pain, coital
pain, bladder irritation symptoms, various disorders caused by
urinary calculus, etc., an agent for suppressing the secretion of
cytokines (e.g., IL-8), or a prophylactic/therapeutic agent for
inflammatory diseases (e.g., diabetic complications such as
neuropathy, large vascular disorders, etc.; inflammatory bowel
diseases such as ulcerative colitis, etc.; cystitis; irritable
bowel syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis or chronic obstructive pulmonary disease (COPD)),
etc.
[0351] Where the compound or element or a salt thereof, which is
obtained by the screening method of the present invention, is used
as a pharmaceutical composition above, the compound or element or a
salt thereof can be prepared into a pharmaceutical preparation in a
conventional manner.
[0352] For example, the compound or its salt can be used orally in
the form of tablets which may be tablets, if necessary, 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
compound, with a physiologically acceptable known carrier,
flavoring agent, excipient, vehicle, antiseptic, stabilizer,
binder, 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.
[0353] Additives miscible with tablets, capsules, etc. include a
binder such as gelatin, corn starch, tragacanth or gum arabic, an
excipient such as crystalline cellulose, a puffiness agent such as
corn starch, gelatin, alginic acid, etc., a lubricant such as
magnesium stearate, a sweetening agent such as sucrose, lactose or
saccharin, a flavoring agent 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, and may be used in combination with a dissolution aid such as
benzyl benzoate, benzyl alcohol, etc.
[0354] Furthermore, the preventive/therapeutic agent described
above may also be formulated with a buffer (e.g., phosphate buffer,
sodium acetate buffer), a soothing agent (e.g., benzalkonium
chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human
serum albumin, polyethylene glycol, etc.), a preservative (e.g.,
benzyl alcohol, phenol, etc.), an antioxidant, etc. The thus
prepared liquid for injection is normally filled in an appropriate
ampoule.
[0355] Since the thus obtained pharmaceutical preparation is safe
and low toxic, the preparation can be administered to human or
mammal (e.g., rats, mice, rabbits, sheep, swine, bovine, cats,
dogs, monkeys, etc.).
[0356] The dose of the compound or element or a salt thereof varies
depending on subject to be administered, target organ, conditions,
method for administration, etc.; in oral administration, the
agonist for GPR39 is administered to the patient (as 60 kg body
weight) with, e.g., cancer normally in a dose of 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, target organ, conditions, method for administration,
etc. but in the form of injectable preparation, the agonist for
GPR39 is advantageously administered intravenously to the patient
(as 60 kg body weight) with, e.g., cancer 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 60 kg can be administered.
(7) Methods for Elucidation of the Action Mechanism of Various
Drugs
[0357] By using GPR39, it can be confirmed whether or not various
drugs exhibit GPR39-mediated pharmacological effects.
[0358] That is, the present invention provides the following
methods.
[0359] (1) A method for confirming that a preventive/therapeutic
drug for metal deficiency symptoms such as growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc.; excess metal-induced symptoms such as renal
dysfunction, pulmonary dysfunction, allergic dermatitis, sensory
neuropathy, Wilson's disease, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria including
interstitial cystitis, prostatic hyperplasia induced-pollakiuria,
urinary incontinence, urinary urgency, pelvic visceral pain, coital
pain, bladder irritation symptoms, various disorders caused by
urinary calculus, etc., an agent for regulating the secretion of
cytokines (e.g., IL-8), a drug for regulating the secretion of
cytokines (e.g., IL-8), or a preventive/therapeutic drug for
inflammatory diseases (e.g., diabetic complications such as
neuropathy, large vascular disorders, etc.; inflammatory bowel
diseases such as ulcerative colitis, etc.; cystitis; irritable
bowel syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis or chronic obstructive pulmonary disease (COPD)) binds
to GPR39, which comprises using GPR39.
[0360] (2) A method for confirming that a preventive/therapeutic
drug for metal deficiency symptoms such as growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc., or a drug for promoting the secretion of
cytokines (e.g., IL-8) is an agonist for GPR39, which comprises
using GPR39.
[0361] (3) A method for confirming that a preventive/therapeutic
drug for excess metal-induced symptoms such as renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy,
Wilson's disease, overactive bladder-induced pollakiuria, nocturia,
cystitis-induced pollakiuria including interstitial cystitis,
prostatic hyperplasia induced-pollakiuria, urinary incontinence,
urinary urgency, pelvic visceral pain, coital pain, bladder
irritation symptoms, various disorders caused by urinary calculus,
etc., a drug for suppressing the secretion of cytokines (e.g.,
IL-8), or a preventive/therapeutic drug for inflammatory diseases
(e.g., diabetic complications such as neuropathy, large vascular
disorders, etc.; inflammatory bowel diseases such as ulcerative
colitis, etc.; cystitis; irritable bowel syndrome; neuralgia),
allergic diseases (e.g., asthma, atopic dermatitis or chronic
obstructive pulmonary disease (COPD)), etc. is an antagonist to
GPR39, which comprises using GPR39.
[0362] (4) The screening method according to (1) to (3), wherein
the binding amount of each drug to GPR39 is measured when the drug
is brought in contact with GPR39.
[0363] This confirmation method can be performed by using the drug
described above in place of the test compound in the above methods
of screening the compound that changes the binding property of the
metal element to GPR39.
[0364] The kit used for the confirmation method of the present
invention comprises the drug described above in place of the test
compound, in the above kits for screening the compound that changes
the binding property of the metal element to GPR39.
[0365] By using the confirmation method of the present invention as
described above, it can be confirmed that various drugs
commercially available or under development exhibit the
GPR39-mediated pharmacological effects.
(8) Pharmaceutical Comprising the Compound or its Salt that Changes
the Amount of GPR39 or its Partial Peptide in Cell Membrane
[0366] The antibody of the present invention is capable of
specifically recognizing GPR39 and can be used for screening the
compound or its salt that changes the amount of GPR39 in the cell
membrane.
[0367] That is, the present invention provides, for example, the
following methods:
[0368] (i) a method of screening the compound or its salt that
changes the amount of GPR39 in the cell membrane, which comprises
measuring the amount of GPR39 contained in a) blood, b) particular
organs or c) a cell membrane fraction isolated after disrupting
tissues or cells isolated from the organs of non-human mammals;
[0369] (ii) a method of screening the compound or its salt that
changes the amount of GPR39 in the cell membrane, which comprises
disrupting transformants, etc. expressing GPR39, isolating the cell
membrane fraction and quantifying GPR39 contained in the cell
membrane fraction; and,
[0370] (iii) a method of screening the compound or its salt that
changes the amount of GPR39 in the cell membrane, which comprises
preparing a slice of a) blood, b) particular organs or c) tissues,
cells, etc. isolated from organs of non-human mammals and
quantifying the stained receptor protein on the cell surface using
immunostaining assay thereby to confirm the protein on the cell
membrane.
[0371] The present invention provides:
[0372] (iv) a method of screening the compound or its salt that
changes the amount of GPR39 in the cell membrane, which comprises
preparing a slice of a transformant expressing GPR39 and
quantifying the stained receptor protein on the cell surface using
immunostaining assay thereby to confirm the protein on the cell
membrane.
[0373] Specifically, the GPR39 contained in the cell membrane
fraction can be measured as follows.
[0374] (i) Normal or disease models of non-human mammals (e.g.,
mice, rats, rabbits, sheep, swine, bovine, cats, dogs, monkeys,
more specifically, rats, mice or rabbits with immunodeficiency,
etc.) receive a drug (e.g., an immunomodulator, etc.) or physical
stress (e.g., soaking stress, electric shock, light and darkness,
low temperature, etc.), and the blood, particular organs (e.g.,
brain, liver, kidney, etc.), or tissues or cells isolated from the
organs are obtained after a specified period of time. The obtained
organs, tissues, cells or the like are suspended in, for example,
an appropriate buffer (e.g., Tris hydrochloride buffer, phosphate
buffer, HEPES buffer, etc.), and the organs, tissues or cells are
disrupted, and the cell membrane fraction is obtained using
surfactants (e.g., Triton-X 100.TM., Tween 20.TM.) and further
using techniques such as centrifugal separation, filtration, column
fractionation, etc.
[0375] The cell membrane fraction refers to a fraction abundant in
cell membrane obtained by cell disruption and subsequent
fractionation by publicly known methods. Cell disruption methods
include cell squashing using a Potter-Elvehjem homogenizer,
disruption using a Waring blender or Polytron (manufactured by
Kinematica Inc.), disruption by ultrasonication, disruption by cell
spraying through thin nozzles under an increased pressure using a
French press, or the like. Cell membrane fractionation is effected
mainly by fractionation using a centrifugal force, such as
centrifugation for fractionation and density gradient
centrifugation. For example, cell disruption fluid is centrifuged
at a low speed (500 rpm to 3,000 rpm) for a short period of time
(normally about 1 to about 10 minutes), the resulting supernatant
is then centrifuged at a higher speed (15,000 rpm to 30,000 rpm)
normally for 30 minutes to 2 hours. The precipitate thus obtained
is used as the membrane fraction. The membrane fraction is abundant
in GPR39 expressed and membrane components such as cell-derived
phospholipids, membrane proteins, etc.
[0376] The GPR39 contained in the cell membrane fraction can be
quantified by, for example, the sandwich immunoassay, western blot
analysis, etc. using the antibody of the present invention.
[0377] The sandwich immunoassay can be performed as described
above, and the western blotting can be performed by publicly known
methods.
[0378] (ii) Transformants expressing GPR39 are prepared by the
method described above, and the GPR39 contained in the cell
membrane fraction can be quantified.
[0379] The compound or its salt that changes the amount of GPR39 in
cell membranes can be screened as follows.
[0380] (i) Normal non-human mammal or disease model of non-human
mammal is administered with a test compound at a specified period
of time before (30 minutes to 24 hours before, preferably 30
minutes to 12 hours before, more preferably 1 hour to 6 hours
before), at a specified time after (30 minutes to 3 days after,
preferably 1 hour to 2 days after, more preferably 1 hour to 24
hours after), or simultaneously with a drug or physical stress. At
a specified time (30 minute to 3 days, preferably 1 hour to 2 days,
more preferably 1 hour to 24 hours) after administration of the
test compound, the amount of GPR39 in the cell membranes can be
quantified.
[0381] (ii) Transformants are cultured in a conventional manner and
a test compound is mixed in the culture medium. After a specified
time (after 1 day to 7 days, preferably after 1 day to 3 days, more
preferably after 2 to 3 days), the amount of GPR39 in the cell
membranes can be quantified.
[0382] Specifically, the GPR39 contained in cell membrane fractions
is confirmed as follows.
[0383] (iii) Normal non-human mammals or disease models of
non-human mammals (e.g., mice, rats, rabbits, sheep, swine, bovine,
cats, dogs, monkeys, etc., more specifically, immunodeficiency
model rats, mice, rabbits, etc.) are administered with drugs (e.g.,
an immunomodulator, etc.) or physical stress (e.g., soaking stress,
electric shock, light and darkness, low temperature, etc.) or the
like, and blood or particular organ (e.g., brain, liver, kidney,
etc.), or the tissues or cells isolated from the organ are obtained
after a specified period of time. Tissue sections are prepared from
the thus obtained organs, tissues, cells, etc. in a conventional
manner followed by immunostaining with the antibody of the present
invention. The staining intensity of the receptor protein on the
cell surface is quantified to confirm the protein on the cell
membrane, whereby the amount of GPR39 on the cell membrane can be
confirmed quantitatively or qualitatively.
[0384] (iv) The confirmation can also be made by the similar
method, using transformants expressing GPR39.
[0385] Examples of the test compounds include peptides, proteins,
non-peptide compounds, synthetic compounds, fermentation products,
cell extracts, plant extracts, animal tissue extracts, blood
plasma, etc. These test compounds may be either novel or publicly
known compounds.
[0386] As salts of the test compound, salts with physiologically
acceptable acids (e.g., inorganic acids, etc.) or bases (e.g.,
organic acids, etc.) may be used and preferably used are
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.
[0387] The compound or its salts obtained by the screening method
of the present invention are the compound or its salts that have
the action of changing the amount of GPR39 in cell membranes.
Specifically, these compounds are: (a) a compound or its salts that
increase the amount of GPR39 in cell membranes thereby to
potentiate the G protein-coupled receptor-mediated cell-stimulating
activities; and (b) a compound or its salts that decrease the
amount of GPR39 in cell membranes thereby to attenuate the cell
stimulating-activity.
[0388] The compounds may be peptides, proteins, non-peptide
compounds, synthetic compounds, fermentation products, and these
compounds may be novel or publicly known compounds.
[0389] As salts of the compound, salts with physiologically
acceptable acids (e.g., inorganic acids, etc.) or bases (e.g.,
organic acids, etc.) may be used and preferably used are
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.
[0390] The compound or its salts that increase the amount of GPR39
in cell membranes thereby to potentiate the cell-stimulating
activity are useful as safe and low toxic preventive/therapeutic
agents for diseases associated with the dysfunction of GPR39.
[0391] The compound or its salt that decreases the expression level
of GPR39 in cell membranes thereby to attenuate the cell
stimulating activity is useful as a safe and low toxic agent for
preventing/treating diseases caused by the overexpression of
GPR39.
[0392] Specifically, the compound or its salt that increases the
amount of GPR39 in cell membranes can be used as a
prophylactic/therapeutic agent for diseases, e.g., metal deficiency
symptoms such as growth retardation, wounds, burn, cold
constitution, declining of learning ability, hypogonadism,
dysgeusia, anosmia, prostatic hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression,
menoxenia, hypotonic bladder induced by sensory decrease of the
bladder by diabetes mellitus, etc., hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs; etc., or an
agent for promoting the secretion of cytokines (e.g., IL-8).
[0393] On the other hand, the compound or its salt that decreases
the amount of GPR39 in cell membranes can be used as a
prophylactic/therapeutic agent for diseases, e.g., excess
metal-induced symptoms such as renal dysfunction, pulmonary
dysfunction, allergic dermatitis, sensory neuropathy, Wilson's
disease, overactive bladder-induced pollakiuria, nocturia,
cystitis-induced pollakiuria including interstitial cystitis,
prostatic hyperplasia induced-pollakiuria, urinary incontinence,
urinary urgency, pelvic visceral pain, coital pain, bladder
irritation symptoms, various disorders caused by urinary calculus,
etc., an agent for suppressing the secretion of cytokines (e.g.,
IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy, large
vascular disorders, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), etc.
[0394] Where the compound or its salt, which is obtained by using
the screening method of the present invention, is used as the
pharmaceutical composition above, the compound or its salt can be
prepared into a pharmaceutical preparation in a conventional
manner.
[0395] For example, the compound can be used orally in the form of
tablets which may be tablets, if necessary, 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
compound, with a physiologically acceptable known carrier,
flavoring agent, excipient, vehicle, antiseptic, stabilizer,
binder, 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.
[0396] Additives miscible with tablets, capsules, etc. include a
binder such as gelatin, corn starch, tragacanth or gum arabic, an
excipient such as crystalline cellulose, a puffiness agent such as
corn starch, gelatin, alginic acid, etc., a lubricant such as
magnesium stearate, a sweetening agent such as sucrose, lactose or
saccharin, a flavoring agent 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.
[0397] Furthermore, the preventive/therapeutic agent described
above may also be formulated with a buffer (e.g., phosphate buffer,
sodium acetate buffer), a soothing agent (e.g., benzalkonium
chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human
serum albumin, polyethylene glycol, etc.), a preservative (e.g.,
benzyl alcohol, phenol, etc.), an antioxidant, etc. The thus
prepared liquid for injection is normally filled in an appropriate
ampoule.
[0398] Since the thus obtained pharmaceutical preparation is safe
and low toxic, the preparation can be administered to human or
mammals (e.g., rats, mice, rabbits, sheep, swine, bovine, cats,
dogs, monkeys, etc.).
[0399] The dose of the compound or its salts varies depending on
subject to be administered, target organ, conditions, methods for
administration, etc.; in oral administration, the compound or its
salt that increases the amount of GPR39 in cell membranes is
administered to the patient (as 60 kg body weight) with, e.g.,
cancer normally in a dose of 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, target
organ, conditions, methods for administration, etc. but in the form
of, e.g., injectable preparation, the compound or its salt that
increases the amount of GPR39 in cell membranes is advantageously
administered intravenously to the patient (as 60 kg body weight)
with, e.g., cancer 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 60 kg can be administered.
(9) Pharmaceutical Comprising the Antibody to GPR39
[0400] The neutralizing activity of the antibody to GPR39 means the
activity of inactivating the signal transduction function in which
GPR39 takes part. Thus, when the antibody has the neutralizing
activity, the antibody can inactivate signal transduction in which
GPR39 takes part, for example, the GPR39-mediated cell stimulating
activities (e.g., the activities that promote or inhibit
arachidonic acid release, acetylcholine release, intracellular
Ca.sup.2+ release, intracellular cAMP production, intracellular
cGMP production, inositol phosphate production, changes in cell
membrane potential, phosphorylation of intracellular proteins,
activation of c-fos, pH reduction, etc.; an action of promoting the
secretion of cytokines (e.g., IL-8) etc., in particular,
intracellular Ca.sup.2+ level increasing activity (intracellular
Ca.sup.2+ release activity), an action of promoting the secretion
of cytokines (e.g., IL-8)).
[0401] Therefore, the antibody to GPR39 (e.g., a neutralizing
antibody) can be used as a prophylactic/therapeutic agent for
diseases caused by the excessive action of GPR39 or abundance of
the metal element, for example, excess metal-induced symptoms such
as renal dysfunction, pulmonary dysfunction, allergic dermatitis,
sensory neuropathy, Wilson's disease, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria including
interstitial cystitis, prostatic hyperplasia induced-pollakiuria,
urinary incontinence, urinary urgency, pelvic visceral pain, coital
pain, bladder irritation symptoms, various disorders caused by
urinary calculus, etc., an agent for suppressing the secretion of
cytokines (e.g., IL-8), or a prophylactic/therapeutic agent for
inflammatory diseases (e.g., diabetic complications such as
neuropathy, large vascular disorders, etc.; inflammatory bowel
diseases such as ulcerative colitis, etc.; cystitis; irritable
bowel syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis or chronic obstructive pulmonary disease (COPD)),
etc.
[0402] The preventive/therapeutic agent described above can be
prepared in the same manner as in the aforesaid pharmaceutical
composition comprising the GPR39 or the antagonist to the GPR39 and
provided for use.
(10) Pharmaceutical Comprising the Antisense DNA of the Present
Invention
[0403] The antisense DNA of the present invention can be used as a
prophylactic/therapeutic agent for diseases caused by the excessive
action of GPR39, abundance of the metal element or the like, for
example, excess metal-induced symptoms such as renal dysfunction,
pulmonary dysfunction, allergic dermatitis, sensory neuropathy,
Wilson's disease, overactive bladder-induced pollakiuria, nocturia,
cystitis-induced pollakiuria including interstitial cystitis,
prostatic hyperplasia induced-pollakiuria, urinary incontinence,
urinary urgency, pelvic visceral pain, coital pain, bladder
irritation symptoms, various disorders caused by urinary calculus,
etc., an agent for suppressing the secretion of cytokines (e.g.,
IL-8), or a prophylactic/therapeutic agent for inflammatory
diseases (e.g., diabetic complications such as neuropathy, large
vascular disorders, etc.; inflammatory bowel diseases such as
ulcerative colitis, etc.; cystitis; irritable bowel syndrome;
neuralgia), allergic diseases (e.g., asthma, atopic dermatitis or
chronic obstructive pulmonary disease (COPD)), etc.
[0404] For example, where the antisense DNA is used, the antisense
DNA itself is administered; alternatively, the antisense DNA is
inserted into an appropriate vector such as retrovirus vector,
adenovirus vector, adenovirus-associated virus vector, etc. and
then administered in a conventional manner. The antisense DNA may
also be administered as naked, or with adjuvants to assist its
uptake by gene gun or through a catheter such as a catheter with a
hydrogel.
[0405] In addition, the antisense DNA may also be used as an
oligonucleotide probe for diagnosis to investigate the presence of
the DNA of the present invention or the state of its expression in
tissues or cells.
(11) Preparation of Animal Bearing the DNA of the Present
Invention
[0406] The present invention provides a non-human mammal bearing
DNA which is exogenous (hereinafter briefly referred to as the
exogenous DNA of the present invention) or its variant DNA
(sometimes briefly referred to as the exogenous variant DNA of the
present invention).
[0407] That is, the present invention provides:
[1] A non-human mammal bearing the exogenous DNA of the present
invention or its variant DNA;
[2] The mammal according to [I], wherein the non-human mammal is a
rodent;
[3] The mammal according to [2], wherein the rodent is mouse or
rat; and,
[4] A recombinant vector containing the exogenous DNA of the
present invention or its variant DNA and capable of expressing in a
mammal; etc.
[0408] The non-human mammal bearing the exogenous DNA of the
present invention or its variant DNA (hereinafter simply referred
to as the DNA transgenic animal of the present invention) can be
produced by transferring a desired DNA into an unfertilized egg, a
fertilized egg, a spermatozoon, a germinal cell containing a
primordial germinal cell thereof, or the like, preferably in the
embryogenic stage in the development of a non-human mammal (more
preferably in the single cell or fertilized cell stage and
generally before the 8-cell phase), by standard means, such as the
calcium phosphate method, the electric pulse method, the
lipofection method, the agglutination method, the microinjection
method, the particle gun method, the DEAE-dextran method, etc.
Also, it is possible to transfer the exogenous DNA of the present
invention into a somatic cell, a living organ, a tissue cell, or
the like by the DNA transfer, and utilize the transformant for cell
culture, tissue culture, etc. In addition, these cells may be fused
with the above-described germinal cell by a publicly known cell
fusion method to prepare the DNA transgenic animal of the present
invention.
[0409] Examples of the non-human mammal that can be used include
bovine, swine, sheep, goat, rabbits, dogs, cats, guinea pigs,
hamsters, mice, rats, etc. Above all, preferred are rodents,
especially mice (e.g., C57Bl/16 strain, DBA2 strain, etc. for a
pure line and for a cross line, B6C3F.sub.1 strain, BDF.sub.1
strain B6D2F.sub.1 strain, BALB/c strain, ICR strain, etc.), rats
(Wistar, SD, etc.) or the like, since they are relatively short in
ontogeny and life cycle from a standpoint of producing model
animals for human disease.
[0410] "Mammal" in a recombinant vector that can be expressed in
the mammals includes the aforesaid non-human mammal and human.
[0411] The exogenous DNA of the present invention refers to the DNA
of the present invention that is once isolated and extracted from
mammal, not the DNA of the present invention inherently possessed
by the non-human mammal.
[0412] The mutant DNA of the present invention includes mutants
resulting from variation (e.g., mutation, etc.) in the base
sequence of the original DNA of the present invention, specifically
DNAs resulting from base addition, deletion, substitution with
other bases, etc. and further including abnormal DNA.
[0413] The abnormal DNA is intended to mean DNA that expresses the
abnormal GPR39 and exemplified by the DNA that expresses GPR39 for
suppressing the function of normal GPR39.
[0414] The exogenous DNA of the present invention may be any one of
those derived from a mammal of the same species as, or a different
species from, the mammal as the target animal. In transferring the
DNA of the present invention, it is generally advantageous to use
the DNA as a DNA construct in which the DNA is ligated downstream a
promoter capable of expressing the DNA in the target animal. For
example, in the case of transferring the human DNA of the present
invention, a DNA transgenic mammal that expresses the DNA of the
present invention to a high level, can be prepared by
microinjecting a DNA construct (e.g., vector, etc.) ligated with
the human DNA of the present invention into a fertilized egg of the
target non-human mammal downstream various promoters which are
capable of expressing the DNA derived from various mammals (e.g.,
rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.)
bearing the DNA of the present invention highly homologous to the
human DNA.
[0415] As expression vectors for GPR39, there are Escherichia
coli-derived plasmids, Bacillus subtilis-derived plasmids,
yeast-derived plasmids, bacteriophages such as .lamda. phage,
retroviruses such as Moloney leukemia virus, etc., and animal
viruses such as vaccinia virus, baculovirus, etc. Of these vectors,
Escherichia coli-derived plasmids, Bacillus subtilis-derived
plasmids, or yeast-derived plasmids, etc. are preferably used.
[0416] Examples of these promoters for regulating the DNA
expression include (i) promoters for DNA derived from viruses
(e.g., simian virus, cytomegalovirus, Moloney leukemia virus, JC
virus, breast cancer virus, poliovirus, etc.) and (ii) promoters
derived from various mammals (human, rabbits, dogs, cats, guinea
pigs, hamsters, rats, mice, etc.), for example, promoters of
albumin, insulin II, uroplakin II, elastase, erythropoietin,
endothelin, muscular creatine kinase, glial fibrillary acidic
protein, glutathione S-transferase, platelet-derived growth factor
.beta., keratins K1, K10 and K14, collagen types I and II, cyclic
AMP-dependent protein kinase .beta.I subunit, dystrophin,
tartarate-resistant alkaline phosphatase, atrial natriuretic
factor, endothelial receptor tyrosine kinase (generally abbreviated
as Tie2), sodium-potassium adenosine triphosphorylase
(Na,K-ATPase), neurofilament light chain, metallothioneins I and
IIA, metalloproteinase I tissue inhibitor. MHC class I antigen
(H-2L), H-ras, renin, dopamine .beta.-hydroxylase, thyroid
peroxidase (TPO), protein chain elongation factor 1.alpha.
(EF-1.alpha.), .beta. actin, .alpha. and .beta. myosin heavy
chains, myosin light chains 1 and 2, myelin base protein,
thyroglobulins., Thy-1, immunoglobulins, H-chain variable region
(VNP), serum amyloid component P, myoglobin, troponin C, smooth
muscle .alpha. actin, preproencephalin A, vasopressin, etc. Among
them, cytomegalovirus promoters, human protein elongation factor
1.alpha. (EF-1.alpha.) promoters, human and fowl .beta. actin
promoters, etc., which are capable of high expression in the whole
body are preferred.
[0417] Preferably, the vectors described above have a sequence that
terminates the transcription of the desired messenger RNA in the
DNA transgenic animal (generally termed a terminator); for example,
a sequence of each DNA derived from viruses and various mammals,
and SV40 terminator of the simian virus and the like are preferably
used.
[0418] In addition, for further enhancing the expression of a
foreign DNA of interest, a splicing signal, an enhancer region, a
portion of the intron of eukaryotic DNA, etc. for each DNA can be
ligated at the 5' upstream of a promoter region, between the
promoter region and a translation region, or at the 3' downstream
of the translation region, depending upon the purpose.
[0419] The translational region for normal GPR39 can be obtained
using as a starting material the entire genomic DNA or its portion
of liver, kidney, thyroid cell or fibroblast origin from human or
various mammals (e.g., rabbits, dogs, cats, guinea pigs, hamsters,
rats, mice, etc.) or various commercially available genomic DNA
libraries, or using cDNA prepared by a publicly known method from
RNA of liver, kidney, thyroid cell or fibroblast origin as a
starting material. Also, an exogenous abnormal DNA can produce the
translational region through variation of the translational region
of normal GPR39 obtained from the cells or tissues described above
by point mutagenesis.
[0420] The translational region can be prepared by a conventional
DNA engineering technique, in which the DNA is ligated downstream
the aforesaid promoter and if desired, upstream the translation
termination site, as a DNA construct capable of being expressed in
the transgenic animal.
[0421] The exogenous DNA of the present invention is transferred at
the fertilized egg cell stage in a manner such that the DNA is
certainly present in all the germinal cells and somatic cells of
the target mammal. The fact that the exogenous DNA of the present
invention is present in the germinal cells of the animal prepared
by DNA transfer means that all offspring of the prepared animal
will maintain the exogenous DNA of the present invention in all of
the germinal cells and somatic cells thereof. The offspring of the
animal that inherits the exogenous DNA of the present invention
also have the exogenous DNA of the present invention in all of the
germinal cells and somatic cells thereof.
[0422] The non-human mammal in which the normal exogenous DNA of
the present invention has been transferred can be passaged as the
DNA-bearing animal under ordinary rearing environment, by
confirming that the exogenous DNA is stably retained by
crossing.
[0423] By transfer of the exogenous DNA of the present invention at
the fertilized egg cell stage, the DNA is retained to be excess in
all of the germinal and somatic cells. The fact that the exogenous
DNA of the present invention is excessively present in the germinal
cells of the prepared animal after transfer means that the DNA of
the present invention is excessively present in all of the germinal
cells and somatic cells thereof. The offspring of the animal that
inherits the exogenous DNA of the present invention have
excessively the DNA of the present invention in all of the germinal
cells and somatic cells thereof.
[0424] It is possible to obtain homozygous animals having the
transferred DNA in both homologous chromosomes and breed male and
female of the animal so that all the progeny have this DNA in
excess.
[0425] In a non-human mammal bearing the normal DNA of the present
invention, the normal DNA of the present invention has expressed at
a high level, and may eventually develop hyperfunction symptoms of
GPR39 by accelerating the function of endogenous normal DNA.
Therefore, the animal can be utilized as a pathologic model animal
for such a disease. For example, using the normal DNA transgenic
animal of the present invention, it is possible to elucidate the
mechanism of hyperfunction symptoms of GPR39 and the pathological
mechanism of the disease associated with GPR39 and to investigate
how to treat these diseases.
[0426] Furthermore, since a mammal transferred with the exogenous
normal DNA of the present invention exhibits symptoms of increasing
GPR39, the animal is usable for screening of a drug for the
treatment of diseases associated with GPR39.
[0427] On the other hand, a non-human mammal having the exogenous
abnormal DNA of the present invention can be passaged under normal
breeding conditions as the DNA-bearing animal by confirming stable
retention of the exogenous DNA via crossing. Furthermore, the
exogenous DNA of interest can be utilized as a starting material by
inserting the DNA into the plasmid described above. The DNA
construct with a promoter can be prepared by conventional DNA
engineering techniques. The transfer of the abnormal DNA of the
present invention at the fertilized egg cell stage is preserved to
be present in all of the germinal and somatic cells of the target
mammal. The fact that the abnormal DNA of the present invention is
present in the germinal cells of the animal after DNA transfer
means that all of the offspring of the prepared animal have the
abnormal DNA of the present invention in all of the germinal and
somatic cells. Such an offspring that passaged the exogenous DNA of
the present invention will have the abnormal DNA of the present
invention in all of the germinal and somatic cells. A homozygous
animal having the introduced DNA on both of homologous chromosomes
can be acquired, and by crossing these male and female animals, all
the offspring can be bred to retain the DNA.
[0428] In a non-human mammal bearing the abnormal DNA of the
present invention, the abnormal DNA of the present invention has
expressed to a high level, and may eventually develop the function
inactive type inadaptability to GPR39 by inhibiting the functions
of endogenous normal DNA. Therefore, the animal can be utilized as
a pathologic model animal for such a disease. For example, using
the abnormal DNA transgenic animal of the present invention, it is
possible to elucidate the mechanism of the function inactive type
inadaptability to GPR39 and the pathological mechanism of the
disease and to investigate how to treat the disease.
[0429] More specifically, the transgenic animal of the present
invention expressing the abnormal DNA of the present invention at a
high level is expected to serve as an experimental model to
elucidate the mechanism of the functional inhibition (dominant
negative effect) of normal GPR39 by the abnormal GPR39 in the
function inactive type inadaptability to GPR39.
[0430] A mammal bearing the abnormal exogenous DNA of the present
invention is also expected to serve in a test for screening a drug
for the treatment of the function inactive type inadaptability to
GPR39, since the animal exerts symptoms of increasing GPR39.
[0431] Other potential applications of two kinds of the DNA
transgenic animals of the present invention described above further
include, for example:
(i) Use as a cell source for tissue culture;
(ii) Analysis of the relation to GPR39 that is specifically
expressed or activated by GPR39, by direct analysis of DNA or RNA
in tissues of the DNA transgenic animal of the present invention or
by analysis of GPR39 tissues expressed by the DNA;
(iii) Research on the function of cells derived from tissues that
are usually cultured only with difficulty, using cells in tissues
bearing the DNA cultured by a standard tissue culture
technique;
(iv) Screening a drug that enhances the functions of cells using
the cells described in (iii) above;
(v) Isolation and purification of the variant GPR39 of the present
invention and preparation of an antibody thereto; and so on.
[0432] Furthermore, clinical conditions of a disease associated
with GPR39 including the function inactive type inadaptability to
GPR39 can be determined by using the DNA transgenic animal of the
present invention. Also, pathological findings on each organ in a
disease model associated with GPR39 can be obtained in more detail,
leading to the development of a new method for treatment as well as
the research and therapy of any secondary diseases associated with
the disease.
[0433] It is also possible to obtain a free DNA-transferred cell by
withdrawing each organ from the DNA transgenic animal of the
present invention, mincing the organ and degrading with a
proteinase such as trypsin, etc., followed by establishing the line
of culturing or cultured cells. Furthermore, the DNA transgenic
animal of the present invention can serve to identify cells capable
of producing GPR39, and to study in association with apoptosis,
differentiation or propagation or on the mechanism of signal
transduction in them to inspect any abnormality therein. Thus, the
DNA transgenic animal can provide an effective research material
for GPR39 and for investigation of its function and effect.
[0434] To develop a drug for the treatment of diseases associated
with GPR39, including the function inactive type inadaptability to
GPR39, using the DNA transgenic animal of the present invention, an
effective and rapid method for screening can be provided by using
the method for inspection and the method for quantification, etc.
described above. It is also possible to investigate and develop a
method for DNA therapy for the treatment of diseases associated
with GPR39, using the DNA transgenic animal of the present
invention or a vector capable of expressing the exogenous DNA of
the present invention.
(12) Knockout Animal
[0435] The present invention provides a non-human mammalian
embryonic stem cell bearing the DNA of the present invention
inactivated and a non-human mammal deficient in expressing the DNA
of the present invention.
[0436] Thus, the present invention provides:
[1] A non-human mammalian embryonic stem cell in which the DNA of
the present invention is inactivated;
[2] The embryonic stem cell according to [1], wherein the DNA is
inactivated by introducing a reporter gene (e.g.,
.beta.-galactosidase gene derived from Escherichia coli);
[3] The embryonic stem cell according to [1], which is resistant to
neomycin;
[4] The embryonic stem cell according to [1], wherein the non-human
mammal is a rodent;
[5] The embryonic stem cell according to [4], wherein the rodent is
mouse;
[6] A non-human mammal deficient in expressing the DNA of the
present invention, wherein the DNA is inactivated;
[0437] [7] The non-human mammal according to [6], wherein the DNA
is inactivated by inserting a reporter gene (e.g.,
.beta.-galactosidase derived from Escherichia coli) therein and the
reporter gene is capable of being expressed under control of a
promoter for the DNA of the present invention;
[8] The non-human mammal according to [6], which is a rodent;
[9] The non-human mammal according to [8], wherein the rodent is
mouse; and,
[0438] [10] A method of screening a compound that promotes or
inhibits (preferably inhibits) the activity of a promoter for the
DNA of the present invention, which comprises administering a test
compound to the mammal of [7] and detecting expression of the
reporter gene.
[0439] The non-human mammal embryonic stem cell in which the DNA of
the present invention is inactivated refers to a non-human mammal
embryonic stem cell that suppresses the ability of the non-human
mammal to express the DNA by artificially mutating the DNA of the
present invention, or the DNA has no substantial ability to express
GPR39 (hereinafter sometimes referred to as the knockout DNA of the
present invention) by substantially inactivating the activities of
GPR39 encoded by the DNA (hereinafter merely referred to as ES
cell).
[0440] As the non-human mammal, the same examples as described
above apply.
[0441] Techniques for artificially mutating the DNA of the present
invention include deletion of a part or all of the DNA sequence and
insertion of or substitution with other DNA, by genetic
engineering. By these variations, the knockout DNA of the present
invention may be prepared, for example, by shifting the reading
frame of a codon or by disrupting the function of a promoter or
exon.
[0442] Specifically, the non-human mammal embryonic stem cell in
which the DNA of the present invention is inactivated (hereinafter
merely referred to as the ES cell with the DNA of the present
invention inactivated or the knockout ES cell of the present
invention) can be obtained by, for example, isolating the DNA of
the present invention that the desired non-human mammal possesses,
inserting a DNA fragment having a DNA sequence constructed by
inserting a drug resistant gene such as a neomycin resistant gene
or a hygromycin resistant gene, or a reporter gene such as lacZ
(.beta.-galactosidase gene) or cat (chloramphenicol
acetyltransferase gene), etc. into its exon site thereby to disable
the functions of exon, or integrating to a chromosome of the target
animal by, e.g., homologous recombination, a DNA sequence that
terminates gene transcription (e.g., polyA additional signal, etc.)
in the intron between exons, thus inhibiting the synthesis of
complete messenger RNA and eventually destroying the gene
(hereinafter simply referred to as a targeting vector). The
thus-obtained ES cells to the southern hybridization analysis with
a DNA sequence on or near the DNA of the present invention as a
probe, or to PCR analysis with a DNA sequence on the targeting
vector and another DNA sequence near the DNA of the present
invention which is not included in the targeting vector as primers,
to select the knockout ES cell of the present invention.
[0443] The parent ES cells to inactivate the DNA of the present
invention by homologous recombination, etc. may be of a strain
already established as described above, or may originally be
established in accordance with a modification of the known method
by Evans and Kaufman described above. For example, in the case of
mouse ES cells, currently it is common practice to use ES cells of
the 129 strain. However, since their immunological background is
obscure, the C57BL/6 mouse or the BDF.sub.1 mouse (F.sub.1 hybrid
between C57BL/6 and DBA/2), wherein the low ovum availability per
C57BL/6 in the C57BL/6 mouse has been improved by crossing with
DBA/2, may be preferably used, instead of obtaining a pure line of
ES cells with the clear immunological genetic background and for
other purposes. The BDF.sub.1 mouse is advantageous in that, when a
pathologic model mouse is generated using ES cells obtained
therefrom, the genetic background can be changed to that of the
C57BL/6 mouse by back-crossing with the C57BL/6 mouse, since its
background is of the C57BL/6 mouse, as well as being advantageous
in that ovum availability per animal is high and ova are
robust.
[0444] In establishing ES cells, blastocytes at 3.5 days after
fertilization are commonly used. In the present invention, embryos
are preferably collected at the 8-cell stage, after culturing until
the blastocyte stage, the embryos are used to efficiently obtain a
large number of early stage embryos.
[0445] Although the ES cells used may be of either sex, male ES
cells are generally more convenient for generation of a germ cell
line chimera. It is also desirable that sexes are identified as
soon as possible to save painstaking culture time.
[0446] Methods for sex identification of the ES cell include the
method in which a gene in the sex-determining region on the
Y-chromosome is amplified by the PCR process and detected. When
this method is used, one colony of ES cells (about 50 cells) is
sufficient for sex-determination analysis, though which convention
karyotype analysis required about 10.sup.6 cells; therefore, the
first selection of ES cells at the early stage of culture can be
based on sex identification, and male cells can be selected early,
which saves a significant amount of time at the early stage of
culture.
[0447] Also, second selection can be achieved by, for example,
confirmation of the number of chromosomes by the G-banding method.
It is usually desirable that the chromosome number of the obtained
ES cells be 100% of the normal number. However, when it is
difficult to obtain the cells having the normal number of
chromosomes due to physical operations, etc. in the cell
establishment, it is desirable that the ES cell is again cloned to
a normal cell (e.g., in a mouse cell having the number of
chromosomes being 2n=40) after knockout of the gene of the ES
cells.
[0448] Although the embryonic stem cell line thus obtained shows a
very high growth potential, it must be subcultured with great care,
since it tends to lose its ontogenic capability. For example, the
embryonic stem cell line is cultured at about 37.degree. C. in a
carbon dioxide incubator (preferably 5% carbon dioxide and 95% air,
or 5% oxygen, 5% carbon dioxide and 90% air) in the presence of LIF
(1 to 10000 U/ml) on appropriate feeder cells such as STO
fibroblasts, treated with a trypsin/EDTA solution (normally 0.001
to 0.5% trypsin/0.1 to about 5 mM EDTA, preferably about 0.1%
trypsin/1 mM EDTA) at the time of passage to obtain separate single
cells, which are then plated on freshly prepared feeder cells. This
passage is normally conducted every 1 to 3 days; it is desirable
that cells be observed at the passage and cells found to be
morphologically abnormal in culture, if any, be abandoned.
[0449] Where ES cells are allowed to reach a high density in
mono-layers or to form cell aggregates in suspension under
appropriate conditions, it is possible to differentiate the ES
cells to various cell types, for example, pariental and visceral
muscles, cardiac muscle or the like [M. J. Evans and M. H. Kaufman,
Nature, 292. 154, 1981; G. R. Martin, Proc. Natl. Acad. Sci.
U.S.A., 78, 7634, 1981; T. C. Doetschman et al., Journal of
Embryology Experimental Morphology, 87, 27, 1985]. The cells
deficient in expression of the DNA of the present invention, which
are obtained from the differentiated ES cells of the present
invention, are useful for studying GPR39 in vitro or GPR39
cytologically.
[0450] The non-human mammal deficient in expressing the DNA of the
present invention can be distinguished from normal animal by
assaying the amount of mRNA in the animal according to a publicly
known method and comparing the expression level indirectly.
[0451] As the non-human mammal, the same examples as described
above apply.
[0452] With respect to the non-human mammal deficient in expression
of the DNA of the present invention, the DNA of the present
invention can be made knockout by transferring a targeting vector,
prepared as described above, to mouse embryonic stem cells or mouse
oocytes, and conducting homologous recombination in which a
targeting vector DNA sequence, wherein the DNA of the present
invention is inactivated by the transfer, is replaced with the DNA
of the present invention on a chromosome of a mouse embryonic stem
cell or mouse oocytes.
[0453] The knockout cells with the disrupted DNA of the present
invention can be identified by the southern hybridization analysis
using as a probe a DNA fragment on or near the DNA of the present
invention, or by the PCR analysis using as primers a DNA sequence
on the targeting vector and another DNA sequence at the proximal
region of other than the DNA of the present invention derived from
mouse used in the targeting vector. When non-human mammal stem
cells are used, a cell line wherein the DNA of the present
invention is inactivated by homologous recombination is cloned; the
resulting clones are injected to, e.g., a non-human mammalian
embryo or blastocyst, at an appropriate stage such as the 8-cell
stage. The resulting chimeric embryos are transplanted to the
uterus of the pseudopregnant non-human mammal. The resulting animal
is a chimeric animal constructed with both cells having the normal
locus of the DNA of the present invention and those having an
artificially mutated locus of the DNA of the present invention.
[0454] When some germ cells of the chimeric animal have a mutated
locus of the DNA of the present invention, an individual, which
entire tissue is composed of cells having a mutated locus of the
DNA of the present invention can be selected from a series of
offspring obtained by crossing between such a chimeric animal and a
normal animal, e.g., by coat color identification, etc. The
individuals thus obtained are normally deficient in heterozygous
expression of GPR39. The individuals deficient in homozygous
expression of GPR39 can be obtained from offspring of the
intercross between those deficient in heterozygous expression of
GPR39.
[0455] When an oocyte is used, a DNA solution may be injected,
e.g., into the prenucleus by microinjection thereby to obtain a
transgenic non-human mammal having a targeting vector introduced in
its chromosome. From such transgenic non-human mammals, those
having a mutation at the locus of the DNA of the present invention
can be obtained by selection based on homologous recombination.
[0456] As described above, the individuals in which the DNA of the
present invention is knocked out permit passage rearing under
ordinary rearing conditions, after the individuals obtained by
their crossing have proven to have been knockout.
[0457] Furthermore, the genital system may be obtained and retained
by conventional methods. That is, by crossing male and female
animals each having the inactivated DNA, homozygous animals having
the inactivated DNA in both loci can be obtained. The homozygotes
thus obtained may be reared so that one normal animal and two or
more homozygotes are produced from a mother animal to efficiently
obtain such homozygotes. By crossing male and female heterozygotes,
homozygotes and heterozygotes having the inactivated DNA are
proliferated and passaged.
[0458] The non-human mammalian embryonic stem cell, in which the
DNA of the present invention is inactivated, is very useful for
preparing a non-human mammal deficient in expression of the DNA of
the present invention.
[0459] Moreover, since the non-human mammal deficient in expression
of the DNA of the present invention lacks various biological
activities induced by GPR39, such an animal can be used as an
animal model of diseases suspected of inactivated biological
activities of GPR39 and hence, is useful in the etiological studies
to investigate the causes for and therapy for these diseases.
(12a) Method of Screening a Compound or its Salt Having a
Therapeutic/Preventive Effect on Diseases Caused by Deficiency,
Damages, etc. of the DNA of the Present Invention
[0460] The non-human mammal deficient in expression of the DNA of
the present invention can be employed for screening a compound or
its salt having a therapeutic/preventive effect on diseases caused
by deficiency, damages, etc. of the DNA of the present invention
(e.g., metal deficiency symptoms such as growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc.), or a drug for promoting the secretion of
cytokines (e.g., IL-8).
[0461] That is, the present invention provides a method of
screening a compound or its salt having a therapeutic/preventive
effect on diseases caused by deficiency, damages, etc. of the DNA
of the present invention, which comprises administering a test
compound or test element to a non-human mammal deficient in
expression of the DNA of the present invention and, observing and
measuring a change occurred in the animal.
[0462] As the non-human mammal deficient in expression of the DNA
of the present invention, which can be employed for the screening
method, the same examples as given hereinabove apply.
[0463] Examples of the test compound include peptides, proteins,
non-peptide compounds, synthetic compounds, fermentation products,
cell extracts, plant extracts, animal tissue extracts, blood
plasma, etc. These compounds may be novel compounds or publicly
known compounds.
[0464] As salts of the test compound, salts with physiologically
acceptable acids (e.g., inorganic acids, etc.) or bases (e.g.,
organic acids, etc.) may be used and preferably used are
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.
[0465] As the test elements, for example, the elements described
in, e.g., the Periodic Table, are used. Among them, metal elements
are preferred.
[0466] Specifically, the non-human mammal deficient in expression
of the DNA of the present invention is treated with a test compound
or test element, comparison is made with an intact animal for
control and a change in each organ, tissue, disease conditions,
etc. of the animal is used as an indicator to assess the
therapeutic/preventive effects of the test compound or test
element.
[0467] For treating an animal to be tested with a test compound or
test element, for example, oral administration, intravenous
injection, etc. are applied, and the treatment can be appropriately
chosen depending on conditions of the test animal, property of the
test compound or test element, etc. Furthermore, a dose of the test
compound or test element to be administered can be appropriately
chosen depending on the administration methods, properties of the
test compound, etc.
[0468] When a test compound or test element is administered to a
test animal in the screening method and the diseases described
above of the test animal are cured by at least about 10%,
preferably at least 30%, more preferably at least about 50%, the
test compound can be selected to be a compound or its salt having a
therapeutic/preventive effect on the diseases described above.
[0469] The compound or its salt, which is obtained using the above
screening method, is a compound selected from the test compounds
described above and can be used as drugs, for example, a safe and
low toxic agent for treating/preventing diseases caused by
deficiencies, damages, etc. of GPR39 (e.g., metal deficiency
symptoms such as growth retardation, wounds, burn, cold
constitution, declining of learning ability, hypogonadism,
dysgeusia, anosmia, prostatic hyperplasia, arteriosclerosis,
myocardial infarction, apoplexy, cirrhosis, cholesterol
accumulation, lowered resistance to infection, gout, cancer, hard
labor, diabetes mellitus, brown spots, anemia, alopecia,
respiratory disturbances, indigestion, cardiac disturbances, gray
hair, puffiness, wrinkles, saggings, hypothyroidism, depression,
menoxenia, hypotonic bladder induced by sensory decrease of the
bladder by diabetes mellitus, etc., hypotonic bladder induced by
postsurgical bladder anesthesia of the pelvic organs; etc.), an
agent for promoting the secretion of cytokines (e.g., IL-8), etc.
In addition, compounds derived from the compound obtained by the
screening described above can be used as well.
[0470] As salts of the compound obtained by the screening method,
salts with physiologically acceptable acids (e.g., inorganic acids,
organic acids, etc.) or bases (e.g., alkali metal salts, etc.) may
be used and preferably used are 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.
[0471] The pharmaceutical comprising the compound or its salt
obtained by the screening method can be manufactured in the same
manner as the aforesaid pharmaceuticals comprising the compound
that changes the binding property or signal transduction of GPR39
to the metal element
[0472] Since the pharmaceutical preparation thus obtained is safe
and low toxic, it can be administered to human or mammal (e.g.,
rat, mouse, guinea pig, rabbit, sheep, swine, bovine, horse, cat,
dog, monkey, etc.).
[0473] The dose of the compound or its salt may vary depending upon
target disease, subject to be administered, route of
administration, etc. For example, when the compound or its salt is
orally administered, the compound or its salt is administered to
the patient (as 60 kg body weight) with, e.g., cancer generally in
a daily dose of about 0.1 to about 100 mg, preferably about 1.0 to
about 50 mg and, more preferably about 1.0 to about 20 mg. In
parenteral administration, a single dose of the compound or its
salt may vary depending upon target subject, target disease, etc.
When the compound or its salt is administered to the patient (as 60
kg body weight) with, e.g., cancer in the form of an injectable
preparation, the compound or its salt is administered intravenously
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 60 kg
weight can be administered.
(12b) Method of Screening a Compound or its Salt that Promotes or
Inhibits the Activity of a Promoter for the DNA of the Present
Invention
[0474] The present invention provides a method of screening a
compound or its salts that promote or inhibit the activity of a
promoter for the DNA of the present invention, which comprises
administering a test compound to a non-human mammal deficient in
expression of the DNA of the present invention and detecting the
expression of the reporter gene.
[0475] In the screening method described above, an animal in which
the DNA of the present invention is inactivated by introducing a
reporter gene and the reporter gene is expressed under control of a
promoter for the DNA of the present invention is used as the
non-human mammal deficient in expression of the DNA of the present
invention, which is selected from the aforesaid non-human mammals
deficient in expression of the DNA of the present invention.
[0476] The same examples of the test compound apply to the
compounds used for the screening.
[0477] As the reporter gene, the genes as described above may be
used. Preferably, .beta.-galactosidase (lacZ), soluble alkaline
phosphatase gene, luciferase gene and the like, are suitable for
use.
[0478] Since the reporter gene is present under control of a
promoter for the DNA of the present invention in the non-human
mammal deficient in expression of the DNA of the present invention
wherein the DNA of the present invention is substituted with the
reporter gene, the activity of the promoter can be detected by
tracing the expression of a substance encoded by the reporter
gene.
[0479] When a part of the DNA region encoding GPR39 is substituted
with, e.g., .beta.-galactosidase gene (lacZ) derived from
Escherichia coli, .beta.-galactosidase is expressed in a tissue
where GPR39 should originally be expressed, instead of GPR39. Thus,
the expression state of GPR39 can be readily observed in vivo of an
animal by staining with a reagent, e.g.,
5-bromo-4-chloro-3-indolyl-.beta.-galactopyranoside (X-gal) which
is substrate for .beta.-galactosidase. Specifically, a mouse
deficient in GPR39, or its tissue section is fixed with
glutaraldehyde, etc. After washing with phosphate buffered saline
(PBS), the system is reacted with a staining solution containing
X-gal at room temperature or about 37.degree. C. for approximately
30 minutes to an hour. After the .beta.-galactosidase reaction is
terminated by washing the tissue preparation with 1 mM EDTA/PBS
solution, the color formed is observed. Alternatively, mRNA
encoding lacZ may be detected in a conventional manner.
[0480] The compound or its salt obtained by using the screening
method described above is a compound or its salt that is selected
from the test compounds described above and that promotes or
inhibits the activity of a promoter for the DNA of the present
invention.
[0481] As salts of the compound obtained by the screening method,
salts with physiologically acceptable acids (e.g., inorganic acids,
etc.) or bases (e.g., organic acids, etc.) may be used and
preferably used are 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.
[0482] The compound or its salt that promotes the activity of a
promoter for the DNA of the present invention can promote the
expression of GPR39 to promote the function of GPR39 and is thus
useful as a pharmaceutical such as a prophylactic/therapeutic agent
for, e.g., diseases associated with the dysfunction of GPR39, or as
an agent for promoting the secretion of cytokines (e.g., IL-8),
etc.
[0483] The compound or its salt that inhibits the activity of a
promoter for the DNA of the present invention can inhibit the
expression of GPR39 to inhibit the function of GPR39 and is thus
useful as a pharmaceutical such as a prophylactic/therapeutic agent
for, e.g., diseases caused by the excessive function of GPR39, or
as an agent for suppressing the secretion of cytokines (e.g.,
IL-8), etc.
[0484] The diseases associated with the dysfunction of GPR39
include metal deficiency symptoms such as growth retardation,
wounds, burn, cold constitution, declining of learning ability,
hypogonadism, dysgeusia, anosmia, prostatic hyperplasia,
arteriosclerosis, myocardial infarction, apoplexy, cirrhosis,
cholesterol accumulation, lowered resistance to infection, gout,
cancer, hard labor, diabetes mellitus, brown spots, anemia,
alopecia, respiratory disturbances, indigestion, cardiac
disturbances, gray hair, puffiness, wrinkles, saggings,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc.
[0485] The diseases caused by the excessive function of GPR39
include, for example, excess metal-induced symptoms such as renal
dysfunction, pulmonary dysfunction, allergic dermatitis, sensory
neuropathy, Wilson's disease, overactive bladder-induced
pollakiuria, nocturia, cystitis-induced pollakiuria including
interstitial cystitis, prostatic hyperplasia induced-pollakiuria,
urinary incontinence, urinary urgency, pelvic visceral pain, coital
pain, bladder irritation symptoms, various disorders caused by
urinary calculus, etc., or inflammatory diseases (e.g., diabetic
complications such as neuropathy, large vascular disorders, etc.;
inflammatory bowel diseases such as ulcerative colitis, etc.;
cystitis; irritable bowel syndrome; neuralgia), allergic diseases
(e.g., asthma, atopic dermatitis or chronic obstructive pulmonary
disease (COPD)), etc.
[0486] In addition, compounds derived from the compound obtained by
the screening described above can be used as well.
[0487] The pharmaceutical comprising the compound or its salt,
which is obtained by the screening method above, can be
manufactured in a manner similar to the method for manufacturing
the pharmaceutical comprising the compound or its salt that changes
the binding property of GPR39 or its salt to the metal element
described above.
[0488] Since the pharmaceutical preparation thus obtained is safe
and low toxic, it can be administered to, for example, human or
mammal (e.g., rat, mouse, guinea pig, rabbit, sheep, swine, bovine,
horse, cat, dog, monkey, etc.).
[0489] The dose of the compound or its salt may vary depending upon
target disease, subject to be administered, route of
administration, etc. For example, when the compound or its salt
that promotes the activity of a promoter for the DNA of the present
invention is orally administered, the compound or its salt is
generally administered to the patient (as 60 kg body weight) with,
e.g., cancer in a daily dose of about 0.1 to about 100 mg,
preferably about 1.0 to about 50 mg and, more preferably about 1.0
to about 20 mg. In parenteral administration, a single dose of the
compound or its salt may vary depending upon target subject, target
disease, etc. When the compound or its salt that promotes the
activity of a promoter for the DNA of the present invention is
administered to the patient (as 60 kg body weight) with, e.g.,
cancer in the form of an injectable preparation, the compound or
its salt is advantageously administered intravenously 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 60 kg weight can
be administered.
[0490] As stated above, the non-human mammal deficient in
expression of the DNA of the present invention is extremely useful
for screening the compound or its salt that promotes or inhibits
the activity of a promoter for the DNA of the present invention and
can greatly contribute to elucidation of causes for various
diseases suspected of deficiency in expression of the DNA of the
present invention and for the development of preventive/therapeutic
drugs for these diseases.
[0491] Also, a so-called transgenic animal (gene transferred
animal) can be prepared by using a DNA containing the promoter
region of GPR39, ligating genes encoding various proteins at the
downstream and injecting the same into oocyte of an animal. It is
thus possible to synthesize GPR39 therein specifically and study
its activity in vivo. When an appropriate reporter gene is ligated
to the promoter site described above and a cell line that expresses
the gene is established, the resulting system can be utilized as
the search system for a low molecular compound having the action of
specifically promoting or inhibiting the in vivo productivity of
GPR39 itself.
[0492] When bases, amino acids, etc. are represented by
abbreviation in the present specification and drawings,
abbreviations are based on those according to IUPAC-IUB Commission
on Biochemical Nomenclature or by the conventional abbreviations in
the art, examples of which are shown below. For amino acids, which
may have an optical isomer, L form is presented unless otherwise
indicated.
[0493] DNA: deoxyribonucleic acid
[0494] cDNA: complementary deoxyribonucleic acid
[0495] A: adenine
[0496] T: thymine
[0497] G: guanine
[0498] C: cytosine
[0499] RNA: ribonucleic acid
[0500] mRNA: messenger ribonucleic acid
[0501] dATP: deoxyadenosine triphosphate
[0502] dTTP: deoxythymidine triphosphate
[0503] dGTP: deoxyguanosine triphosphate
[0504] dCTP: deoxycytidine triphosphate
[0505] ATP: adenosine triphosphate
[0506] EDTA: ethylenediaminetetraacetic acid
[0507] SDS: sodium dodecyl sulfate
[0508] Gly: glycine
[0509] Ala: alanine
[0510] Val: valine
[0511] Leu: leucine
[0512] Ile: isoleucine
[0513] Ser: serine
[0514] Thr: threonine
[0515] Cys: cysteine
[0516] Met: methionine
[0517] Glu: glutamic acid
[0518] Asp: aspartic acid
[0519] Lys: lysine
[0520] Arg: arginine
[0521] His: histidine
[0522] Phe: phenylalanine
[0523] Tyr: tyrosine
[0524] Trp: tryptophan
[0525] Pro: proline
[0526] Asn: asparagine
[0527] Gln: glutamine
[0528] pGlu: pyroglutamic acid
[0529] *: corresponding to termination codon
[0530] Me: methyl group
[0531] Et: ethyl group
[0532] Bu: butyl group
[0533] Ph: phenyl group
[0534] TC: thiazolidine-4(R)-carboxamido group
[0535] Substituents, protecting groups and reagents generally used
in this specification are presented as the codes below.
[0536] Tos: p-toluenesulfonyl
[0537] CHO: formyl
[0538] Bzl: benzyl
[0539] Cl.sub.2Bzl: 2,6-dichlorobenzyl
[0540] Bom: benzyloxymethyl
[0541] Z: benzyloxycarbonyl
[0542] Cl-Z: 2-chlorobenzyloxycarbonyl
[0543] Br-Z: 2-bromobenzyloxycarbonyl
[0544] Boc: t-butoxycarbonyl
[0545] DNP: dinitrophenol
[0546] Trt: trityl
[0547] Burn: t-butoxymethyl
[0548] Fmoc: N-9-fluorenylmethoxycarbonyl
[0549] HOBt: 1-hydroxybenztriazole
[0550] HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
[0551] HONB: 1-hydroxy-5-norbornene-2,3-dicarboxyimide
[0552] DCC: N,N'-dicyclohexylcarbodiimide
[0553] The sequence identification numbers in the sequence listing
of the specification indicates the following sequence,
respectively.
SEQ ID NO: 1
[0554] This shows the amino acid sequence of human-derived
GPR39.
SEQ ID NO: 2
[0555] This shows the base sequence of cDNA encoding human-derived
GPR39.
SEQ ID NO: 3
[0556] This shows the base sequence of the primer used for TaqMan
PCR in EXAMPLE 3 below.
SEQ ID NO: 4
[0557] This shows the base sequence of the primer used for TaqMan
PCR in EXAMPLE 3 below.
SEQ ID NO: 5
[0558] This shows the base sequence of the probe used for TaqMan
PCR in EXAMPLE 3 below.
SEQ ID NO: 6
[0559] This shows the amino acid sequence of mouse-derived
GPR39.
SEQ ID NO: 7
[0560] This shows the base sequence of cDNA encoding mouse-derived
GPR39.
SEQ ID NO: 8
[0561] This shows the amino acid sequence of rat-derived GPR39.
SEQ ID NO: 9
[0562] This shows the base sequence of cDNA encoding rat-derived
GPR39.
SEQ ID NO: 10
[0563] This shows the base sequence of the primer used for TaqMan
PCR in EXAMPLE 7 below.
SEQ ID NO: 11
[0564] This shows the base sequence of the primer used for TaqMan
PCR in EXAMPLE 7 below.
SEQ ID NO: 12
[0565] This shows the base sequence of the probe used for TaqMan
PCR in EXAMPLE 7 below.
SEQ ID NO: 13
[0566] This shows the base sequence of the primer used for TaqMan
PCR in EXAMPLE 9 below.
SEQ ID NO: 14
[0567] This shows the base sequence of the primer used for TaqMan
PCR in EXAMPLE 9 below.
SEQ ID NO: 15
[0568] This shows the base sequence of the probe used for TaqMan
PCR in EXAMPLE 9 below.
EXAMPLES
[0569] Hereinafter the present invention will be described in
detail with reference to EXAMPLES but the scope of the present
invention is not deemed to be limited thereto. Gene using
Escherichia coli was performed in accordance with the method
described in Molecular cloning.
Example 1
Acquisition of Human GPR39-GFP Fusion Protein-Expressing CHO
Cells
[0570] An expression plasmid was constructed to express a fusion
protein of Green Fluorescent Protein (GFP) cDNA isolated from jelly
fish Auquorea victoria, fused to human GPR39 at the C terminus, so
as to match the translation frame. In this case, a fragment excised
from the expression vector pQBI25 (Takara Shuzo) for GFP was used
as GFP cDNA. In the GPR39, its termination codon was corrected by
PCR to the recognition sequence with restriction enzyme NheI, and
the GFP fragment was ligated thereto, which was inserted into the
expression vector pAKKO-111H (the same plasmid vector as
pAKKO-1.11H described in Biochem. Biophys. Acta, Hinuma, S. et al.,
1219, 251-259, 1994). Using this vector, the GPR39-GFP fusion
protein-expressing CHO cells were acquired.
Example 2
Detection of the Agonist Activity of Metal Elements for GPR39
[0571] Agonists for GPR39 were surveyed by the method described
below and as a result, it was found that particular metal elements
were agonists for GPR39.
[0572] The human GPR39-GFP expression vector-transfected CHO cell
line produced in EXAMPLE 1 (CHO/hGPR39-GFP) was diluted in
3.times.10.sup.4 cells/100 .mu.l. The dilution was dispensed onto a
Black walled 96-well plate (Costar) in 100 .mu.l each per well,
followed by incubation overnight in a CO.sub.2 incubator. Changes
in intracellular calcium levels were determined by the following
method using FLIPR (Molecular Device). In 21 .mu.l of DMSO (DOJIN),
50 .mu.g of Fluo-3AM (DOJIN) was dissolved and an equal volume of
20% Pluronic acid (Molecular Probes) was added to the solution.
After mixing them, the resulting mixture was added to 10.6 ml of
assay buffer [prepared by adding 20 ml of 1M HEPES (pH 7.4) (DOJIN)
to HBSS (Invitrogen Corp.) and further adding to the solution 10 ml
of a solution mixture, which mixture was prepared by dissolving 710
mg of Probenecid (Sigma) in 5 ml of 1N NaOH and then adding thereto
5 ml of the HBSS/HEPES solution above] supplemented with 105 .mu.l
of fetal calf serum to prepare a fluorescent dye solution. The
medium in the cell plate was removed. Immediately thereafter, the
fluorescent dye solution was dispensed in 100 .mu.l each/well and
the cells were incubated in a CO.sub.2 incubator for an hour so
that the fluorescent dye was taken up into the cells. The cells
after the incubation were washed with the assay buffer described
above and set on FLIPR. Compounds of metal elements to be added to
the cells were diluted to the respective concentration with the
assay buffer. The dilutions were dispensed onto plates for ligands
and set on FLIPR. Following the pre-treatment above, changes in
intracellular calcium levels after addition of metal elements were
determined by FLIPR to study agonist activities. The results
indicate that the intracellular calcium levels of CHO/hGPR39-GFP
were dose-dependently increased when cadmium chloride (CdCl.sub.2),
zinc chloride (ZnCl.sub.2), copper chloride (CuCl.sub.2) and nickel
chloride (NiCl.sub.2) were added (FIGS. 1 through 4). In the CHO
cells where the receptor was not expressed or in the CHO cells
where GPCR other than GPR39 was expressed, no such response was
observed. Accordingly, it was found that these metal salts (metal
ions) were the agonists specific to GPR39.
Example 3
Expression of Human GPR39 mRNA
[0573] The expression level of mRNA was assayed on ABI PRISM 7900HT
SequenceDetector (Applied Biosystems, Inc.). Primers
[5'-TGTGACATTGGCCGTATGCT-3' (SEQ ID NO: 3) and
5'-CAGTCGTGCTTGGGTTTGG-3' (SEQ ID NO: 4)] and probe
[5'-TGCCCAACCAGATTCGGAGGATCA-3' (SEQ ID NO: 5)] used for
quantification of the expression level were designed based on the
base sequence of human GPR39 (SEQ ID NO: 2) using software
PrimerExpress (Applied Biosystems, Inc.) exclusively used for ABI
PRISM SequenceDetector. As the cDNA to be used as a template, cDNA
synthesized from 1 .mu.g of total RNA derived from various human
tissues and cells by reverse transcription using random primers.
Reverse transcription was carried out according to the protocol
attached, using SuperScriptII (GIBCO BRL) as reverse transcriptase.
A reaction solution for ABI PRISM 7900HT SequenceDetector was
prepared by mixing 7.5 .mu.l of TaqMan Universal PCR Master Mix
(Applied Biosystems, Inc.), 0.9 .mu.M of each primer, 0.25 .mu.M of
the probe and the cDNA solution. Distilled water was added to the
mixture to make the volume 15 .mu.l. The reaction was carried out
at 50.degree. C. for 2 minutes and 95.degree. C. for 10 minutes and
then repeating 40 times the cycle of 96.degree. C. for 15 seconds
and 60.degree. C. for 1 minute.
[0574] The expression of mRNA in various human tissues and cells is
shown in FIG. 5 through FIG. 7. The mRNA was highly expressed in
central nervous tissues, hypophysis, kidney, gastrointestinal tract
tissues, fetal brain, primary cultured cells from the vascular
system, primary cultured cells from the renal system, or colon
cancer cell lines.
Reference Example 1
Acquisition of Human GPR39, Rat GPR39 and Mouse GPR39-Expressing
Cells
[0575] The cDNAs of human GPR39, rat GPR39 and mouse GPR39 were
acquired from cDNAs including the intestinal tract by RT-PCR, based
on known sequences of AF034633.1 (human), XM.sub.--222578 (rat),
AK016817 and AK082941.1 (mouse), respectively. The resulting DNA
fragments were inserted into expression vector pAKKO-111H (the same
plasmid vector as pAKKO-1.11H described in Biochem. Biophys. Acta,
Hinuma, S. et al., 1219, 251-259, 1994) for animal cells. Using
this vector, the GPR39-expressing CHO cells were acquired by the
method per se known.
Example 4
Detection of Signal with Cytosensor
[0576] Activation of the intracellular signal transduction system
by ligands using a Cytosensor was determined in terms of the pH
changes by metabolism of cells as an indicator.
[0577] The procedures were as follows. On the previous day, the
human GPR39-expressing CHO cell line was cultured in a culture
flask to reach a confluent state. After washing with PBS
(Invitrogen Corp.), the cells were scraped off using trypsin
(Invitrogen Corp.) and centrifuged for recovery. The cells were
then suspended in medium and the suspension was adjusted to
3.times.10.sup.5 cells/ml. The capsule of Cytosensor capsule kit
(Molecular Device) was placed on a 12-well plate (Costar) and 1 ml
of the cell suspension was charged therein. After covering with the
lid, the cells were incubated at 37.degree. C. in an incubator
until next day. On the day when the assay was conducted, Cytosensor
was activated as instructed by the protocol. Then, RPMI1640 medium
(pH 7.4) (Invitrogen Corp.) for Cytosensor was prepared and
equilibrated. The spacer was then placed in the capsule where the
cells prepared on the previous day were seeded, and the capsule was
set on Cytosensor. After equilibration with the medium, a sample
was added to the cells by switching the flow path and pH changes
were determined.
[0578] The results indicate that when nickel chloride was added,
any response was not detected in mock cells where GPR39 was not
expressed and in other receptor TGR7, whereas in the
GPR39-expressed cells, a cell response specific to nickel was
detected (FIG. 8).
Example 5
Detection of the Agonist Activity of Metal Elements for Rat
GPR39
[0579] Using the CHO cell line where rat GPR39 was stably
expressed, the agonist activity of metal ions was detected using
FLIPR in terms of the intracellular calcium-increasing reaction as
an indicator. The CHO cell line used, wherein rat GPR39 was stably
expressed, was cultured in HAM's .alpha.MEM (Invitrogen Corp.)
containing 10% calf fetal serum (Invitrogen Corp.). On the day
before the assay was conducted, the human GPR39-expressing CHO cell
line was cultured in a culture flask to reach a confluent state.
After washing with PBS, the cells were scraped off using trypsin
and centrifuged for recovery. The cells were then suspended in
medium and the suspension was adjusted to 3.times.10.sup.5
cells/ml. Then, the suspension was dispensed onto a black walled
96-well plate (Costar) in 100 .mu.l each per well, followed by
incubation overnight in a CO.sub.2 incubator.
[0580] On the day when the assay was conducted, various test
samples were added to the cells and the changes of intracellular
calcium levels in this case were determined by FLIPR (Molecular
Device). In order to determine the changes in intracellular calcium
levels on FLIPR, the cells were pre-treated by the following
procedures. First, for the purpose of adding a fluorescent dye
Fluo-3AM (DOJIN) to the cells or washing the cells immediately
before the FLIPR assay, an assay buffer was prepared. A solution
was prepared by adding 20 ml of 1M HEPES (pH 7.4) (DOJIN) to 1000
ml of HBSS (Invitrogen Corp.) (hereinafter HBSS/HEPES solution), to
which solution was added 10 ml of a solution mixture obtained by
dissolving 710 mg of Probenecid (Sigma) in 5 ml of 1N NaOH and
further adding 5 ml of HBSS/HEPES solution thereto. The resulting
solution was used as the assay buffer. Next, 50 .mu.g of Fluo-3AM
was dissolved in 21 .mu.l of DMSO (DOJIN) and an equal volume of
20% Pluronic acid (Molecular Probes) was added to and mixed with
the solution. The mixture was then added to 10.6 ml of the assay
buffer supplemented with 105 .mu.l of fetal calf serum to prepare a
fluorescent dye solution. The medium for the CHO cells where human
GPR39 was stably expressed was removed. Immediately thereafter, the
fluorescent dye solution was dispensed in 100 .mu.l each/well and
the cells were incubated in a CO.sub.2 incubator for an hour so
that the fluorescent dye was taken up into the cells. The cells
after the incubation was washed with the assay buffer described
above and set on FLIPR. A test sample to be added to the CHO cells
where rat GPR39 was stably expressed was prepared using the assay
buffer and set on FLIPR at the same time. Following the
pre-treatment above, the changes in intracellular calcium levels
after the addition of each test sample were determined by FLIPR.
The results indicate that, when cobalt chloride (CoCl.sub.2),
nickel chloride (NiCl.sub.2), copper chloride (CuCl.sub.2), zinc
chloride (ZnCl.sub.2) or cadmium chloride (CdCl.sub.2) was added,
the intracellular calcium levels of CHO/rGPR39 was dose-dependently
increased and the agonist activity for GPR39 was detected (FIG. 9
through FIG. 13).
Example 6
Detection of the Agonist Activity of Metal Elements for Mouse
GPR39
[0581] Using the CHO cell line where mouse GPR39 was stably
expressed, the agonist activity of metal ions was detected on FLIPR
in accordance with the procedures described in EXAMPLE 5, in terms
of the intracellular calcium-increasing reaction as an indicator.
The results indicate that, when cobalt chloride (CoCl.sub.2),
nickel chloride (NiCl.sub.2), copper chloride (CuCl.sub.2), zinc
chloride (ZnCl.sub.2) or cadmium chloride (CdCl.sub.2) was added,
the intracellular calcium levels of CHO/mGPR39 was dose-dependently
increased and the agonist activity for GPR39 was detected (FIG. 14
through FIG. 18).
Example 7
Analysis of the Tissue Distribution of GPR39 mRNA in Rats by
RT-PCR
[0582] Various organs were removed from Wistar rats and the total
RNA was prepared using Isogen (Nippon Gene Co., Ltd.) in accordance
with its manual. Using a random primer and SuperScriptII reverse
transcriptase (GIBCO BRL) as a reverse transcriptase, cDNA was
synthesized from 1.mu. of the total RNA obtained, according to the
manual. The cDNA synthesized was made a solution of 25 ng/.mu.l
when calculated as total RNA and the solution was used as a
template for the following RT-PCR. In RT-PCR, Sequence Detection
System Prism 7900 (PE Biosystems) was used; as primers for
amplification and detection, 5'-CCCATGGAGTTCTACAGCATCAT-3' (SEQ ID
NO: 10) and 5'-TGTGGAGCTTGCAGGACAGA-3' (SEQ ID NO: 11) were used
and 5'-(Fam)-TGGAACCCCCTGACCACACCCA-(Tamra)-3' (SEQ ID NO: 12) as a
TaqMan probe. A solution for RT-PCR was prepared by adding 0.135
.mu.l each of the primer solutions each adjusted to 100 .mu.M, 0.75
.mu.l of the TaqMan probe adjusted to 5 .mu.M and 1 .mu.l of the
cDNA solution prepared above to 7.5 .mu.l of TaqMan Universal PCR
Master Mix (PE Biosystems) and then adding distilled water to make
the whole volume of the reaction solution 15 .mu.l. PCR was carried
out, after keeping at 50.degree. C. for 2 minutes and 95.degree. C.
for 10 minutes, by repeating 40 times the cycle of 95.degree. C.
for 15 seconds and 60.degree. C. for 1 minute. The expression
levels of GPR39 mRNA in various rat tissues obtained were
calculated as the number of copies per 25 ng of total RNA (FIG.
19).
Example 8
Analysis of the Expression Distribution of GPR39 mRNA in Bladder by
In Situ Hybridization
[0583] Male Wistar rats were sacrificed and the bladder was taken
out. After rinsing with PBS, the bladder was embedded in OCT
Compound and frozen in liquid nitrogen, which was stored at
-80.degree. C.
[0584] GPR39 antisense and sense probes were prepared by the
following procedures. First, rat GPR39 cDNA was inserted into
plasmid vector pCRII TOPO (Invitrogen Corp.) by the method per se
known. This cDNA was amplified by PCR using M13 primer (Invitrogen
Corp.)/Advantage 2 PCR kit (Clontech Corp.), denatured and then
purified by ethanol precipitation. The cDNA was subjected to in
vitro transcription (40 .mu.l scale) by SP6 or T7 using DIG RNA
Labeling KIT (SP6/T7) (Roche). After purification by ethanol
precipitation, the cDNA was dissolved in distilled water to make
the volume 100 .mu.l. Based on the inserted direction of cDNA, the
DIG-labeled riboprobe produced by SP6 and the DIG-labeled riboprobe
produced by T7 were identified to be an antisense probe and a sense
probe, respectively.
[0585] For in situ hybridization, fresh frozen sections were used.
First, the frozen bladder tissue described above was sliced in a
thickness of 8 .mu.m on a silane-coated slide using Cryostat CM3050
(Leica). The slice was fixed in 4% paraformaldehyde-containing PBS
for 10 minutes, then thoroughly washed with PBS and acetylated by
treating (room temperature, 10 minutes) with 0.1M triethanolamine
(pH 8.0) containing 0.25% acetic anhydride. For hybridization, the
antisense probe or sense probe was diluted to 200-fold with a
hybridization buffer (50% formaldehyde, 10 mM Tris-HCl, pH 7.5,
1.times. Denhardt's solution, 200 .mu.g/ml tRNA, 10% dextran
sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA), denatured at
85.degree. C. for 10 minutes, and then added to the slice, followed
by reacting at 50.degree. C. for at least 12 hours. Subsequently,
the following procedures were carried out to wash the probe
hybridized non-specifically. 1) A treatment with 2.times.SSC(SSC;
1.times.SSC=150 mM NaCl, 15 mM sodium citrate, pH 7.0)/50%
formamide (60.degree. C. for 30 minutes, once), 2) a treatment with
2.times.SSC (60.degree. C. for 15 minutes, once) and 3) a treatment
with 0.1.times.SSC (60.degree. C. for 15 minutes, twice). Following
the procedures above, immunohistochemistry was applied to detect
the DIG-labeled probes. First, after washing with DIG-1 (100 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), non-specific
reactions were blocked by a treatment (37.degree. C. for an hour)
with DIG-1 containing 1.5% Blocking reagent (Roche), and DIG-1
(1:1000) containing anti-DIG fab-fragment antibody conjugated with
alkaline phosphatase (Roche) was reacted at room temperature for an
hour. After thoroughly washing with DIG-1, the tissue was rinsed
with DIG-3 (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM
MgCl.sub.2), followed by color-forming reaction at room temperature
in solutions obtained by adding dimethylformamide containing 0.18
mg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), 70%
dimethylformamide containing 0.34 mg/ml 4-nitroblue tetrazolium
(NBT) and polyvinyl alcohol in amounts of 0.35 ml, 0.45 ml and 3%,
respectively, based on 10 ml of DIG-3. After the color formation
was appropriately terminated by washing with running water, the
tissue was sealed in PBS containing 90% glycerol and observed with
an optical microscope. The results indicate that a positive signal
was observed by the GPR39 antisense probe on the transitional
epithelium in the bladder. To the contrary, any signal with the
sense probe was not detected in any site. In the lamina propria
mucosa, muscular layer and vascular endothelial cells, any signal
with the antisense probe was not detected. The foregoing results
revealed that GPR39 mRNA was expressed specifically on the
transitional epithelium in the bladder.
Example 9
Selection of GPR39-Expressed Cell Lines
[0586] The cell lines used were purchased from Dainippon
Pharmaceutical and incubation procedures were performed according
to the protocol attached. 5637 (derived from human) was incubated
in RPMI 1640 (Invitrogen Corp.) supplemented with 10% FCS to reach
a pre-confluent state. HT-1197 (derived from human) and HT-1376
(derived from human) were incubated in MINIMUM ESSENTIAL MEDIUM
(WITH EARLE'S SALTS, WITH L-GLUTAMINE) supplemented with 10% FCS
and MEM NON-ESSENTIAL AMINO ACIDS to reach a pre-confluent state.
UM-UC-3 (derived from human) and NBT-II (derived from rat) were
incubated in MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH
L-GLUTAMINE) supplemented with 10% FCS, 1% MEM NON-ESSENTIAL AMINO
ACIDS and 1 mM Sodium pyruvate to reach a pre-confluent state. T24
(derived from human) and MBT-2 (derived from mouse) were incubated
in MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH L-GLUTAMINE)
supplemented with 10% FCS to reach a pre-confluent state. After
washing with PBS, the incubated cells were scraped off in PBS/EDTA,
centrifuged for recovery and stored at -80.degree. C. Extraction of
RNA and synthesis of cDNA were performed according to the manual of
ISOGEN (Nippon Gene). The total RNA was thus extracted and
purified. From 1 .mu.g of the extracted RNA, first strand cDNA was
synthesized using random in accordance with the manual for
SuperScript II (Invitrogen Corp.), precipitated in ethanol and
dissolved in 10 .mu.l of TE. TaqMan quantification was performed on
the cell line-derived cDNA (corresponding to 25 ng RNA), using
amplification reagent TaqMan (trademark), Universal PCR Master Mix
(Applied Biosystems JAPAN Ltd.) and TaqMan (trademark) primer-probe
set for GPR39 detection. The primer-probe set described in EXAMPLE
3 was used for human-derived samples. The primer-probe set
described in EXAMPLE 7 was used for rat-derived samples. For
mouse-derived samples, 5'-GTACCCACTCACAAGGGACTCAAC-3' (SEQ ID NO:
13) and 5'-TATTGGAGTTTCCAGGTTCATCGT-3' (SEQ ID NO: 14) were used as
primers and 5'-(Fam)-CAACCTCTCTCGCACCCGCCA-(Tamra)-3' (SEQ ID NO:
15) was used as a TaqMan probe. The reaction was carried out in 15
.mu.l in total of the reaction solution. The final concentrations
of the respective primers and probes were in accordance with the
manual.
[0587] TaqMan (trademark) PCR was carried out on ABI PRISM
(trademark) 7900HT sequence detection system (Applied Biosystems
JAPAN Ltd.). The temperature cycle used was complied with the
manual of TaqMan (trademark) Universal PCR Master Mix (Applied
Biosystems JAPAN Ltd.).
[0588] Quantitative TaqMan analysis of the amplification product
was performed using 7900HT SDS software (Applied Biosystems JAPAN
Ltd.). The calibration curve used to count the copy number was
prepared from CT values in six logarithms from 10.sup.7 copies/well
to 10.sup.2 copies/well using the cDNA fragment (human GPR39) or
plasmid DNA (mouse GPR39) of known concentrations, including the
full length amplification region.
[0589] The results indicate that the expression of GPR39 was noted
in all cell lines of 5637, HT-1197, HT-1376, UM-UC-3, NBT-II, T24
and MBT-2 (FIG. 20).
Example 10
Confirmation of the Reactivity with Metal Ions in the Human Bladder
Cancer Cell Lines
[0590] The cell lines used were purchased from Dainippon
Pharmaceutical and incubation procedures were performed according
to the protocol attached. 5637 was incubated in RPMI 1640
(Invitrogen Corp.) supplemented with 10% FCS to reach a
pre-confluent state. HT-1197 and HT-1376 were incubated in MINIMUM
ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH L-GLUTAMINE)
supplemented with 10% FCS and MEM NON-ESSENTIAL AMINO ACIDS to
reach a pre-confluent state. UM-UC-3 and NBT-II were incubated in
MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH L-GLUTAMINE)
supplemented with 10% FCS and MEM NON-ESSENTIAL AMINO ACIDS to
reach a pre-confluent state. T24 and MBT-2 were incubated in
MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH L-GLUTAMINE)
supplemented with 10% FCS to reach a pre-confluent state. Changes
in intracellular calcium levels were determined on FLIPR according
to the procedures described in EXAMPLE 5. The results indicate that
a reaction with nickel was detected in UM-UC-3 and HT-1197.
Example 11
Promoted Secretion of IL-8 from GPR39-Expressed UM-UC-3 Cells by
Metal Ions
[0591] Using GPR39-expressed UM-UC-3 cells, the effects of adding
metal ions as the agonist for GPR39 were examined.
[0592] On the previous day of assay, UM-UC-3 was cultured in
MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH L-GLUTAMINE)
supplemented with 110% FCS and MEM NON-ESSENTIAL AMINO ACIDS to
reach a pre-confluent state. After washing with PBS, the cells were
scraped off using trypsin and centrifuged for recovery. The cells
were then suspended in medium and the suspension was adjusted to
1.5.times.10.sup.5 cells/ml. Next, the suspension was dispensed in
a 6-well plate (Falcon) at 5 ml each per well, followed by
incubation overnight in a CO.sub.2 incubator.
[0593] On the day when the assay was conducted, the medium for the
cells prepared on the previous day was removed and the cells were
washed with MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH
L-GLUTAMINE) supplemented with MEM NON-ESSENTIAL AMINO ACIDS. Then,
5 ml each was added to each well, followed by incubation at
37.degree. C. in a CO.sub.2 incubator for an hour. Thereafter, 5 ml
each of MINIMUM ESSENTIAL MEDIUM (WITH EARLE'S SALTS, WITH
L-GLUTAMINE) supplemented with MEM NON-ESSENTIAL AMINO ACIDS, to
which various test samples were added, was charged in each well,
followed by incubation at 37.degree. C. in a CO.sub.2 incubator for
12 hours. After the culture supernatant was recovered, IL-8
contained in the samples was quantified by EIA (Amersham). It was
confirmed by the results that the amount of IL-8 accumulated was
approximately twice in the presence of 1 mM copper chloride, than
in the absence of copper chloride, which revealed that GPR39
promoted the secretion of cytokines such as IL-8, etc., as one of
its functions.
Example 12
Effects of the GPR39 Agonist on Conscious Rats
[0594] Female SD rats of 9-10 weeks old were anaesthetized by
intramuscular injection with 0.1 ml/100 g of a solution obtained by
mixing ketamine (50 mg/mL) and xylazine (20 mg/mL) in 5:1. The
bladder was then exposed via a midline incision and a hole was made
with a 20-G needle to insert a polyethylene tube (PE-50), which
tube was sutured to the bladder with cotton thread. The
polyethylene tube cannulated into the bladder was cut at a length
of about 1 cm, which was connected to a silicon tube (No. 00) and
exposed to the dorsum through the skin. The animals were housed in
individual cages until they were used for the experiments.
[0595] One day after the surgery, the animal was anesthetized with
halothane and placed in Ballman cages under restricted conditions.
After a three-way cock was connected to the tube cannulated into
the bladder, a saline infusion tube was connected to a cystometry
tube (resistance to infusion). An electronic scale was placed under
the Ballman cages and the micturition volume was measured at the
same time. When the anesthetic completely worn off, the infusion of
saline into the bladder commenced and the bladder pressure and
micturition volume were recorded on a personal computer via an A-D
converter (MP-100, BIOPAC Systems) and analyzed using a dedicated
software (AcqKnowledge, BIOPAC Systems).
[0596] Saline was infused into the bladder at the rate of 6 ml/hr
and the infusion was continued for 1 or 2 hours until stable
micturition was recorded. In order to enhance the permeability of a
drug into the bladder, protamine sulfate (10 mg/mL in PBS) was
infused into the bladder for an hour (Chuang Y, et. al., 2003,
Urology, 63, 664-670). Any substantial change in micturition
intervals was noted by the protamine infusion when compared to
those prior to the infusion. Then, saline was reinfused into the
bladder and micturition was recorded at least 2 hours until
protamine was completely washed off. At the point of time when
micturition intervals became constant, the saline was switched to
an aqueous solution of 1 mM cupric chloride (CuCl.sub.2) in saline
as the GPR39 agonist, followed by observation for about an hour.
Mean micturition intervals during from about 30 minutes to about 60
minutes after starting the infusion of CuCl.sub.2 were calculated
to find a ratio to mean micturition intervals for about 30 minutes
before the infusion of CuCl.sub.2 (% of the pre-value).
[0597] When the constant infusion solution was changed from saline
to the CuCl.sub.2 solution, the basal pressure was not affected but
micturition intervals were shortened. The ratio was shortened to
42.2.+-.6.1% (n=4) when the pre-value was made 100%. Accordingly,
CuCl.sub.2 infused into the bladder suggested to induce
GPR39-mediated pollakiuria, indicating that GPR39 was involved in
the regulation of micturition.
INDUSTRIAL APPLICABILITY
[0598] By using GPR39 and its ligand or an ionizable metal element
such as cadmium, zinc, copper, nickel, etc. or a salt thereof, a
compound or its salt that changes the binding property of the metal
element or a salt thereof to GPR39 can be efficiently screened. In
addition, an agonist having a more potent activity can be found by
using the screening method of the present invention.
[0599] The agonist for GPR39 is useful as a
prophylactic/therapeutic agent for metal deficiency symptoms, for
example, growth retardation, wounds, burn, cold constitution,
declining of learning ability, hypogonadism, dysgeusia, anosmia,
prostatic hyperplasia, arteriosclerosis, myocardial infarction,
apoplexy, cirrhosis, cholesterol accumulation, lowered resistance
to infection, gout, cancer, hard labor, diabetes mellitus, brown
spots, anemia, alopecia, respiratory disturbances, indigestion,
cardiac disturbances, gray hair, swelling, wrinkles, sagging,
hypothyroidism, depression, menoxenia, hypotonic bladder induced by
sensory decrease of the bladder by diabetes mellitus, etc.,
hypotonic bladder induced by postsurgical bladder anesthesia of the
pelvic organs; etc., or an agent for promoting the secretion of
cytokines (e.g., IL-8).
[0600] The antagonist for GPR39 is useful as a
prophylactic/therapeutic agent for excess metal-induced symptoms,
for example, renal dysfunction, pulmonary dysfunction, allergic
dermatitis, sensory neuropathy, Wilson's disease, overactive
bladder-induced pollakiuria, nocturia, cystitis-induced pollakiuria
including interstitial cystitis, prostatic hyperplasia
induced-pollakiuria, urinary incontinence, urinary urgency, pelvic
visceral pain, coital pain, bladder irritation symptoms, excess
metal-induced symptoms including various disorders caused by
urinary calculus, etc; an agent for suppressing the secretion of
cytokines (e.g., IL-8); or a prophylactic/therapeutic agent for
inflammatory diseases (e.g., diabetic complications such as
neuropathy, large vascular disorders, etc.; inflammatory bowel
diseases such as ulcerative colitis, etc.; cystitis; irritable
bowel syndrome; neuralgia), allergic diseases (e.g., asthma, atopic
dermatitis, chronic obstructive pulmonary disease (COPD)), etc.
Sequence CWU 1
1
15 1 453 PRT Human 1 Met Ala Ser Pro Ser Leu Pro Gly Ser Asp Cys
Ser Gln Ile Ile Asp 5 10 15 His Ser His Val Pro Glu Phe Glu Val Ala
Thr Trp Ile Lys Ile Thr 20 25 30 Leu Ile Leu Val Tyr Leu Ile Ile
Phe Val Met Gly Leu Leu Gly Asn 35 40 45 Ser Ala Thr Ile Arg Val
Thr Gln Val Leu Gln Lys Lys Gly Tyr Leu 50 55 60 Gln Lys Glu Val
Thr Asp His Met Val Ser Leu Ala Cys Ser Asp Ile 65 70 75 80 Leu Val
Phe Leu Ile Gly Met Pro Met Glu Phe Tyr Ser Ile Ile Trp 85 90 95
Asn Pro Leu Thr Thr Ser Ser Tyr Thr Leu Ser Cys Lys Leu His Thr 100
105 110 Phe Leu Phe Glu Ala Cys Ser Tyr Ala Thr Leu Leu His Val Leu
Thr 115 120 125 Leu Ser Phe Glu Arg Tyr Ile Ala Ile Cys His Pro Phe
Arg Tyr Lys 130 135 140 Ala Val Ser Gly Pro Cys Gln Val Lys Leu Leu
Ile Gly Phe Val Trp 145 150 155 160 Val Thr Ser Ala Leu Val Ala Leu
Pro Leu Leu Phe Ala Met Gly Thr 165 170 175 Glu Tyr Pro Leu Val Asn
Val Pro Ser His Arg Gly Leu Thr Cys Asn 180 185 190 Arg Ser Ser Thr
Arg His His Glu Gln Pro Glu Thr Ser Asn Met Ser 195 200 205 Ile Cys
Thr Asn Leu Ser Ser Arg Trp Thr Val Phe Gln Ser Ser Ile 210 215 220
Phe Gly Ala Phe Val Val Tyr Leu Val Val Leu Leu Ser Val Ala Phe 225
230 235 240 Met Cys Trp Asn Met Met Gln Val Leu Met Lys Ser Gln Lys
Gly Ser 245 250 255 Leu Ala Gly Gly Thr Arg Pro Pro Gln Leu Arg Lys
Ser Glu Ser Glu 260 265 270 Glu Ser Arg Thr Ala Arg Arg Gln Thr Ile
Ile Phe Leu Arg Leu Ile 275 280 285 Val Val Thr Leu Ala Val Cys Trp
Met Pro Asn Gln Ile Arg Arg Ile 290 295 300 Met Ala Ala Ala Lys Pro
Lys His Asp Trp Thr Arg Ser Tyr Phe Arg 305 310 315 320 Ala Tyr Met
Ile Leu Leu Pro Phe Ser Glu Thr Phe Phe Tyr Leu Ser 325 330 335 Ser
Val Ile Asn Pro Leu Leu Tyr Thr Val Ser Ser Gln Gln Phe Arg 340 345
350 Arg Val Phe Val Gln Val Leu Cys Cys Arg Leu Ser Leu Gln His Ala
355 360 365 Asn His Glu Lys Arg Leu Arg Val His Ala His Ser Thr Thr
Asp Ser 370 375 380 Ala Arg Phe Val Gln Arg Pro Leu Leu Phe Ala Ser
Arg Arg Gln Ser 385 390 395 400 Ser Ala Arg Arg Thr Glu Lys Ile Phe
Leu Ser Thr Phe Gln Ser Glu 405 410 415 Ala Glu Pro Gln Ser Lys Ser
Gln Ser Leu Ser Leu Glu Ser Leu Glu 420 425 430 Pro Asn Ser Gly Ala
Lys Pro Ala Asn Ser Ala Ala Glu Asn Gly Phe 435 440 445 Gln Glu His
Glu Val 450 2 1359 DNA Human 2 atggcttcac ccagcctccc gggcagtgac
tgctcccaaa tcattgatca cagtcatgtc 60 cccgagtttg aggtggccac
ctggatcaaa atcaccctta ttctggtgta cctgatcatc 120 ttcgtgatgg
gccttctggg gaacagcgcc accattcggg tcacccaggt gctgcagaag 180
aaaggatact tgcagaagga ggtgacagac cacatggtga gtttggcttg ctcggacatc
240 ttggtgttcc tcatcggcat gcccatggag ttctacagca tcatctggaa
tcccctgacc 300 acgtccagct acaccctgtc ctgcaagctg cacactttcc
tcttcgaggc ctgcagctac 360 gctacgctgc tgcacgtgct gacactcagc
tttgagcgct acatcgccat ctgtcacccc 420 ttcaggtaca aggctgtgtc
gggaccttgc caggtgaagc tgctgattgg cttcgtctgg 480 gtcacctccg
ccctggtggc actgcccttg ctgtttgcca tgggtactga gtaccccctg 540
gtgaacgtgc ccagccaccg gggtctcact tgcaaccgct ccagcacccg ccaccacgag
600 cagcccgaga cctccaatat gtccatctgt accaacctct ccagccgctg
gaccgtgttc 660 cagtccagca tcttcggcgc cttcgtggtc tacctcgtgg
tcctgctctc cgtagccttc 720 atgtgctgga acatgatgca ggtgctcatg
aaaagccaga agggctcgct ggccgggggc 780 acgcggcctc cgcagctgag
gaagtccgag agcgaagaga gcaggaccgc caggaggcag 840 accatcatct
tcctgaggct gattgttgtg acattggccg tatgctggat gcccaaccag 900
attcggagga tcatggctgc ggccaaaccc aagcacgact ggacgaggtc ctacttccgg
960 gcgtacatga tcctcctccc cttctcggag acgtttttct acctcagctc
ggtcatcaac 1020 ccgctcctgt acacggtgtc ctcgcagcag tttcggcggg
tgttcgtgca ggtgctgtgc 1080 tgccgcctgt cgctgcagca cgccaaccac
gagaagcgcc tgcgcgtaca tgcgcactcc 1140 accaccgaca gcgcccgctt
tgtgcagcgc ccgttgctct tcgcgtcccg gcgccagtcc 1200 tctgcaagga
gaactgagaa gattttctta agcacttttc agagcgaggc cgagccccag 1260
tctaagtccc agtcattgag tctcgagtca ctagagccca actcaggcgc gaaaccagcc
1320 aattctgctg cagagaatgg ttttcaggag catgaagtt 1359 3 20 DNA
Artificial Sequence primer 3 tgtgacattg gccgtatgct 20 4 19 DNA
Artificial Sequence primer 4 cagtcgtgct tgggtttgg 19 5 24 DNA
Artificial Sequence primer 5 tgcccaacca gattcggagg atca 24 6 456
PRT Mouse 6 Met Ala Ser Ser Ser Gly Ser Asn His Ile Cys Ser Arg Val
Ile Asp 5 10 15 His Ser His Val Pro Glu Phe Glu Val Ala Thr Trp Ile
Lys Ile Thr 20 25 30 Leu Ile Leu Val Tyr Leu Ile Ile Phe Val Val
Gly Ile Leu Gly Asn 35 40 45 Ser Val Thr Ile Arg Val Thr Gln Val
Leu Gln Lys Lys Gly Tyr Leu 50 55 60 Gln Lys Glu Val Thr Asp His
Met Val Ser Leu Ala Cys Ser Asp Ile 65 70 75 80 Leu Val Phe Leu Ile
Gly Met Pro Met Glu Phe Tyr Ser Ile Ile Trp 85 90 95 Asn Pro Leu
Thr Thr Pro Ser Tyr Ala Leu Ser Cys Lys Leu His Thr 100 105 110 Phe
Leu Phe Glu Thr Cys Ser Tyr Ala Thr Leu Leu His Val Leu Thr 115 120
125 Leu Ser Phe Glu Arg Tyr Ile Ala Ile Cys His Pro Phe Lys Tyr Lys
130 135 140 Ala Val Ser Gly Pro Arg Gln Val Lys Leu Leu Ile Gly Phe
Val Trp 145 150 155 160 Val Thr Ser Ala Leu Val Ala Leu Pro Leu Leu
Phe Ala Met Gly Ile 165 170 175 Glu Tyr Pro Leu Val Asn Val Pro Thr
His Lys Gly Leu Asn Cys Asn 180 185 190 Leu Ser Arg Thr Arg His His
Asp Glu Pro Gly Asn Ser Asn Met Ser 195 200 205 Ile Cys Thr Asn Leu
Ser Asn Arg Trp Glu Val Phe Gln Ser Ser Ile 210 215 220 Phe Gly Ala
Phe Ala Val Tyr Leu Val Val Leu Ala Ser Val Ala Phe 225 230 235 240
Met Cys Trp Asn Met Met Lys Val Leu Met Lys Ser Lys Gln Gly Thr 245
250 255 Leu Ala Gly Thr Gly Pro Gln Leu Gln Leu Arg Lys Ser Glu Ser
Glu 260 265 270 Glu Ser Arg Thr Ala Arg Arg Gln Thr Ile Ile Phe Leu
Arg Leu Ile 275 280 285 Val Val Thr Leu Ala Val Cys Trp Met Pro Asn
Gln Ile Arg Arg Ile 290 295 300 Met Ala Ala Ala Lys Pro Lys His Asp
Trp Thr Arg Thr Tyr Phe Arg 305 310 315 320 Ala Tyr Met Ile Leu Leu
Pro Phe Ser Asp Thr Phe Phe Tyr Leu Ser 325 330 335 Ser Val Val Asn
Pro Leu Leu Tyr Asn Val Ser Ser Gln Gln Phe Arg 340 345 350 Lys Val
Phe Trp Gln Val Leu Cys Cys Arg Leu Thr Leu Gln His Ala 355 360 365
Asn Gln Glu Lys Arg Gln Arg Ala Arg Phe Ile Ser Thr Lys Asp Ser 370
375 380 Thr Ser Ser Ala Arg Ser Pro Leu Ile Phe Leu Ala Ser Arg Arg
Ser 385 390 395 400 Asn Ser Ser Ser Arg Arg Thr Asn Lys Val Phe Leu
Ser Thr Phe Gln 405 410 415 Thr Glu Ala Lys Pro Gly Glu Ala Lys Pro
Gln Pro Leu Ser Pro Glu 420 425 430 Ser Pro Gln Thr Gly Ser Glu Thr
Lys Pro Ala Gly Ser Thr Pro Glu 435 440 445 Asn Ser Leu Gln Glu Gln
Glu Val 450 455 7 1368 DNA Mouse 7 atggcttcat ccagtggctc caaccacatc
tgctcccgtg tcatcgatca tagccatgtt 60 cctgaatttg aggtggccac
ttggatcaaa atcaccctca tcttggtgta cctgatcatt 120 tttgtggtag
gcatcttggg caacagcgtc accatcaggg ttacgcaggt attgcagaag 180
aagggctatt tgcagaagga ggtgacagat cacatggtca gtttggcttg ttcagatatc
240 ttggtctttt tgattggcat gcccatggag ttctacagca tcatttggaa
ccccctgacc 300 acacccagct atgctctgtc ctgtaagctc cacacgttcc
tctttgagac gtgcagctac 360 gccacactgc tgcacgtgct gaccctcagc
tttgagcgct acattgccat ttgtcatccc 420 ttcaagtata aagcagtgtc
tggacctcgc caggtgaaac tgctgattgg ctttgtatgg 480 gtcacctccg
ccctggtggc actgcctttg ctctttgcca tgggtatcga gtaccctctg 540
gtaaacgtac ccactcacaa gggactcaac tgcaacctct ctcgcacccg ccaccacgat
600 gaacctggaa actccaatat gtccatctgc acgaacctct ccaaccgttg
ggaggtcttc 660 cagtccagca tctttggggc ctttgctgtt tacctggtgg
tcctggcgtc tgtggctttc 720 atgtgttgga atatgatgaa agtgctaatg
aagagcaagc agggcactct tgcagggacc 780 gggccacagc tccagctgag
gaagtcagag agtgaggaga gccggacagc aagaagacag 840 accatcatat
tcctgagact gattgtggtg acgttggccg tgtgttggat gcccaatcag 900
atccgacgga tcatggctgc agcaaaaccc aaacatgact ggaccagaac gtacttcagg
960 gcatacatga tcctcctgcc cttctctgat accttcttct acctcagctc
tgtggtcaac 1020 cctctcctct acaacgtgtc ctctcagcag ttccggaagg
tgttctggca ggtgctctgc 1080 tgccgcctga ctctgcagca tgccaaccaa
gagaaacgcc agcgtgcccg cttcatctcc 1140 accaaggaca gcaccagctc
agcccgcagc cccctcatct tcctagcctc ccggcgcagt 1200 aactcttcct
ccaggagaac taacaaggtt ttcttaagca cttttcagac tgaggccaag 1260
cctggagagg ctaagcccca gcccttgagt cctgagtcac cacagactgg ctcagagacc
1320 aaaccagctg ggtccacccc agaaaatagt ttacaggagc aggaagta 1368 8
456 PRT Rat 8 Met Ala Ser Ser Ser Gly Ser Ser Asn Ile Cys Ser Arg
Val Ile Asp 5 10 15 His Ser His Val Pro Glu Phe Glu Val Ala Thr Trp
Ile Lys Ile Thr 20 25 30 Leu Thr Leu Val Tyr Leu Ile Val Phe Val
Val Gly Ile Leu Gly Asn 35 40 45 Ser Val Thr Ile Arg Val Thr Gln
Val Leu Gln Lys Lys Gly Tyr Leu 50 55 60 Gln Lys Glu Val Thr Asp
His Met Ile Ser Leu Ala Cys Ser Asp Ile 65 70 75 80 Leu Val Phe Leu
Ile Gly Met Pro Met Glu Phe Tyr Ser Ile Ile Trp 85 90 95 Asn Pro
Leu Thr Thr Pro Ser Tyr Ala Leu Ser Cys Lys Leu His Thr 100 105 110
Phe Leu Phe Glu Thr Cys Ser Tyr Ala Thr Leu Leu His Val Leu Thr 115
120 125 Leu Ser Phe Glu Arg Tyr Ile Ala Ile Cys His Pro Phe Arg Tyr
Lys 130 135 140 Asp Val Ser Gly Pro Cys Gln Val Lys Leu Leu Ile Gly
Phe Val Trp 145 150 155 160 Val Thr Ser Ala Leu Val Ala Leu Pro Leu
Leu Phe Ala Met Gly Ile 165 170 175 Glu Tyr Pro Leu Ala Asn Val Pro
Thr His Lys Gly Leu Asn Cys Asn 180 185 190 Leu Ser Arg Thr Arg His
His Asp His Pro Gly Asp Ser Asn Met Ser 195 200 205 Ile Cys Thr Asn
Leu Ser Ser Arg Trp Glu Val Phe Gln Ser Ser Ile 210 215 220 Phe Gly
Ala Phe Ala Val Tyr Leu Val Val Leu Val Ser Val Ala Phe 225 230 235
240 Met Cys Trp Asn Met Met Lys Val Leu Met Lys Ser Lys Arg Gly Thr
245 250 255 Leu Ala Gly Thr Gly Pro Gln Leu Gln Leu Arg Lys Ser Glu
Ser Glu 260 265 270 Glu Ser Arg Thr Ala Arg Arg Gln Thr Ile Ile Phe
Leu Arg Leu Ile 275 280 285 Val Val Thr Leu Ala Val Cys Trp Met Pro
Asn Gln Ile Arg Arg Ile 290 295 300 Met Ala Ala Ala Lys Pro Lys His
Asp Trp Thr Lys Ser Tyr Phe Lys 305 310 315 320 Ala Tyr Met Ile Leu
Leu Pro Phe Ser Asp Thr Phe Phe Tyr Leu Ser 325 330 335 Ser Val Val
Asn Pro Leu Leu Tyr Asn Val Ser Ser Gln Gln Phe Arg 340 345 350 Lys
Val Phe Trp Gln Val Leu Cys Cys Arg Leu Thr Leu Gln His Ala 355 360
365 Asn Gln Glu Lys Gln Gln Arg Ala Tyr Phe Ser Ser Thr Lys Asn Ser
370 375 380 Ser Arg Ser Ala Arg Ser Pro Leu Ile Phe Leu Ala Ser Arg
Arg Ser 385 390 395 400 Asn Ser Ser Ser Arg Arg Thr Asn Lys Val Phe
Leu Ser Thr Phe Gln 405 410 415 Ala Glu Ala Lys Pro Leu Glu Gly Glu
His Gln Pro Leu Ser Pro Glu 420 425 430 Ser Pro Gln Thr Gly Ser Glu
Thr Lys Pro Ala Gly Ser Ala Thr Glu 435 440 445 Asn Ser Leu Gln Glu
Gln Glu Val 450 455 9 1368 DNA Rat 9 atggcttcat ccagtggctc
cagcaacatc tgctcccgag tcatcgatca cagccatgtc 60 cctgagttcg
aagtggccac ttggatcaaa atcaccctca ccttggtgta cctgatcgtc 120
ttcgtggtag gcatcttggg caatagcgtc accatccggg ttacgcaggt attgcagaaa
180 aagggctatt tgcagaagga ggtgacagat cacatgatca gtttggcttg
ttcagatatc 240 ttggtctttt tgattggcat gcccatggag ttctacagca
tcatctggaa ccccctgacc 300 acacccagct atgctctgtc ctgcaagctc
cacacgttcc tctttgagac gtgtagctac 360 gccacattgc tgcatgtgct
gaccctcagc tttgagcgct acattgccat ttgtcatccc 420 ttcagatata
aggacgtgtc tggaccttgc caggtgaaac tgctgatcgg ctttgtatgg 480
gtcacctccg ctctggtggc actgcccttg ctctttgcca tgggtattga gtaccctctg
540 gcgaacgtcc ccactcacaa gggactcaac tgtaacctct ctcgtacccg
ccaccacgat 600 catcctggag actccaatat gtccatctgc acgaacctct
ccagccgttg ggaggtcttc 660 cagtccagca tctttggggc cttcgctgtt
tacctggtgg tcctggtgtc tgtggctttc 720 atgtgttgga acatgatgaa
agtgctaatg aagagcaagc ggggtactct ggcagggacc 780 ggaccacagc
tgcagctgcg gaagtcagag agtgaggaga gccggacagc gagaagacag 840
accatcatat tcctgagact gatcgtggtg acactggccg tgtgttggat gccaaatcag
900 atccgacgga tcatggccgc agcaaaaccc aaacatgact ggaccaagtc
gtacttcaag 960 gcgtacatga tcctcctccc cttctccgac accttcttct
acctcagctc cgtggtcaac 1020 cctctcctct acaacgtgtc ttctcagcag
ttccggaagg ttttctggca ggttctctgc 1080 tgccggctga ctctgcagca
tgccaaccag gagaaacagc agcgtgccta cttcagctct 1140 accaaaaaca
gcagccgctc agcccgaagc ccgctcatct tcctagcctc ccggcgtagt 1200
aactcttcct cccggagaac taacaaggtt ttcttaagca cttttcaggc ggaggctaag
1260 cctctagagg gcgagcacca gcccttgagt cctgagtcac cacagaccgg
ctcagagacc 1320 aaacctgctg gttccgccac agaaaatagt ttacaggagc
aggaagtg 1368 10 23 DNA Artificial Sequence primer 10 cccatggagt
tctacagcat cat 23 11 20 DNA Artificial Sequence primer 11
tgtggagctt gcaggacaga 20 12 22 DNA Artificial Sequence primer 12
tggaaccccc tgaccacacc ca 22 13 24 DNA Artificial Sequence primer 13
gtacccactc acaagggact caac 24 14 24 DNA Artificial Sequence primer
14 tattggagtt tccaggttca tcgt 24 15 21 DNA Artificial Sequence
primer 15 caacctctct cgcacccgcc a 21
* * * * *