U.S. patent application number 11/476481 was filed with the patent office on 2007-07-12 for drug for inhibiting myometrial contraction.
This patent application is currently assigned to Shionogi & Co., Ltd.. Invention is credited to Toshihiko Yanagita.
Application Number | 20070161555 11/476481 |
Document ID | / |
Family ID | 26498061 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070161555 |
Kind Code |
A1 |
Yanagita; Toshihiko |
July 12, 2007 |
Drug for inhibiting myometrial contraction
Abstract
A composition for inhibiting spontaneous myometrial contraction
or bradykinin-induced contraction, comprising adrenomedullin. The
composition of the present invention may be used to selectively
inhibit spontaneous myometrial contraction or bradykinin-induced
contraction to prevent premature labor, prevent miscarriage, arrest
parturition prior to cesarean section, or to treat
dysmenorrhea.
Inventors: |
Yanagita; Toshihiko;
(Miyazaki-shi, JP) |
Correspondence
Address: |
FAY SHARPE LLP
1100 SUPERIOR AVENUE, SEVENTH FLOOR
CLEVELAND
OH
44114
US
|
Assignee: |
Shionogi & Co., Ltd.
Osaka
JP
|
Family ID: |
26498061 |
Appl. No.: |
11/476481 |
Filed: |
June 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10030298 |
Dec 21, 2001 |
|
|
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11476481 |
Jun 28, 2006 |
|
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Current U.S.
Class: |
514/12.1 ;
514/12.5 |
Current CPC
Class: |
A61K 38/22 20130101;
A61P 15/06 20180101; A61P 15/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/22 20060101
A61K038/22 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2000 |
JP |
2000-079171 |
Jun 23, 1999 |
JP |
11-177548 |
Jun 23, 2000 |
JP |
PCT/JP00/04167 |
Claims
1-9. (canceled)
10. A method for inhibiting abnormal bradykinin-induced myometrial
contraction, comprising administering to the patient in need of
treatment, a therapeutically effective amount of adrenomedullin,
wherein the adrenomedullin is: (d.) a peptide comprising an amino
acid sequence from Met in position -94 to Leu in position 91 of SEQ
ID NO: 2.
11-18. (canceled)
19. A method according to claim 10, wherein the adrenomedullin is a
peptide having an amino acid sequence which has about 60% homology
with the amino acid sequence of SEQ ID NO: 2, and having an action
of inhibiting myometrial contraction.
20. A method according to claim 10, wherein the adrenomedullin is a
peptide having an amino acid sequence which has about 70% homology
with the amino acid sequence of SEQ ID NO: 2, and having an action
of inhibiting myometrial contraction.
21. A method according to claim 10, wherein the adrenomedullin is a
peptide having an amino acid sequence which has about 80% homology
with the amino acid sequence of SEQ ID NO: 2, and having an action
of inhibiting myometrial contraction.
22. A method according to claim 10, wherein the adrenomedullin is a
peptide having an amino acid sequence which has about 90% homology
with the amino acid sequence of SEQ ID NO: 2, and having an action
of inhibiting myometrial contraction.
23. A method for inhibiting bradykinin-induced myometrial
contraction comprising administering to the patient in need of
treatment, a therapeutically effective amount of adrenomedullin
wherein the adrenomedullin is a peptide having an amino acid
sequence which has about 60% homology with the amino acid sequence
of SEQ ID NO: 2, and having an action of inhibiting myometrial
contraction.
24. A method for inhibiting abnormal bradykinin-induced myometrial
contraction comprising administering to the patient in need of
treatment, a therapeutically effective amount of adrenomedullin
wherein the adrenomedullin is a peptide having an amino acid
sequence which has about 70% with the amino acid sequence of SEQ ID
NO: 2, and having an action of inhibiting myometrial
contraction.
25. A method for inhibiting abnormal bradykinin-induced myometrial
contraction comprising administering to the patient in need of
treatment, a therapeutically effective amount of adrenomedullin
wherein the adrenomedullin is a peptide having an amino acid
sequence which has about 80% homology with the amino acid sequence
of SEQ ID NO: 2, and having an action of inhibiting myometrial
contraction.
26. A method for inhibiting abnormal bradykinin-induced myometrial
contraction comprising administering to the patient in need of
treatment, a therapeutically effective amount of adrenomedullin
wherein the adrenomedullin is a peptide having an amino acid
sequence which has about 90% homology with the amino acid sequence
of SEQ ID NO: 2, and having an action of inhibiting myometrial
contraction.
27. A method according to any of claims 1 to 4, wherein the
C-terminus of the adrenomedullin is amidated.
28. A method according to any of claims 1 to 4, wherein Gly is
added to the C-terminus of the adrenomedullin.
29. A method according to any of claims 1 to 4, wherein the
adrenomedullin, Cys in position 16 and Cys in position 21 of SEQ ID
NO: 2 are crosslinked.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for
inhibiting spontaneous myometrial contraction or bradykinin-induced
contraction, comprising adrenomedullin. More specifically, the
present invention relates to a composition for selectively
inhibiting spontaneous myometrial contraction or bradykinin-induced
contraction, comprising adrenomedullin.
BACKGROUND ART
[0002] The management of premature labor is one of the most
important issues in the field of obsterics. Premature labor refers
to parturition on or after 22 weeks and before 37 weeks of
pregnancy, which accounts for 5 to 10% of the total number of
childbirths. A neonate delivered by premature labor is called a
pronatus, which is frequently a low birth weight infant. Recently,
although the care of neonates has progressed significantly, the
morbidity and mortality of pronatuses are higher compared to
normally delivered neonates. Therefore, it is desired that
premature labor is avoided to continue pregnancy as long as
possible.
[0003] At present, widely used drugs for preventing premature labor
include .beta..sub.2-adrenergic sympathetic agonist, magnesium
sulfate, and indomethacin (a prostaglandin synthesis
inhibitor).
[0004] Ritodrine, which is a representative .beta..sub.2-adrenergic
agonist, causes various cardiovascular and metabolic side effects,
including tachycardia, elevation of renin secretion, and
hyperglycemia in mothers (and hypoglycemia in neonates). Other
.beta..sub.2-adrenergic agents, such as for example terbutaline and
albuterol, cause side effects similar to those of ritodrine.
[0005] Magnesium sulfate having a plasma concentration of 4 to 8
mg/dL, which exceeds the therapeutically acceptable range, causes
inhibition of cardiac conduction and neuromascular transmission,
hypopnea, and cardiac arrest. Therefore, when renal function is
impaired, this agent is not preferable.
[0006] Indomethacin causes side effects in fetuses, such as for
example pulmonary artery hypertension and persistent truncus
arteriosus, which are contraindications to large dosage and
long-term use of indomethacin.
[0007] As described above, currently known drugs for preventing
premature labor have various drawbacks. Therefore, there is a
demand for a novel drug for preventing premature labor without such
drawbacks.
[0008] The mechanism of the onset of parturition, i.e., the
beginning of pains, has not been yet fully revealed, but it has
been suggested that oxytocin, prostaglandin, and the like having a
uterine contracting action are involved. Bradykinin has a uterine
contracting action similar to oxytocin and prostaglandin, but the
physiological or pathophysiological meaning is still unknown.
However, bradykinin is inherently an inflammatory mediator, and it
has been suggested that there is a possibility that premature labor
and miscarriage is caused by abnormally increased bradykinin in the
gravid uterus, (Reference 1; List of references are described at
the end of this specification). Therefore, if a drug capable of
inhibiting spontaneous myometrial contraction or bradykinin-induced
myometrial contraction action, or a drug capable of selectively
inhibiting spontaneous myometrial contraction or bradykinin-induced
contraction action, was especially found, the drug would be
expected to be useful not only for preventing premature labor but
also for preventing miscarriage and arresting of parturition prior
to cesarean section.
[0009] Further, such an agent would be expected to be useful in the
treatment of dysmenorrhea. This is because dysmenorrhea is
characterized by a periodic pain in association with menses during
a ovulocycle, and the pain is believed to be derived from uterine
contraction and ischemia.
[0010] Adrenomedullin (AM), a member of the calcitonin gene-related
peptide (CGRP) family, was originally isolated from human
pheochromocytoma as a peptide having a hypotensive action
(Reference 2). It is known that AM plays multiple roles in a
variety of tissues (Reference 3). This suggests that AM has a
nonuniform mechanism of action in an organism.
[0011] Levels of AM protein or AM mRNA in the female reproductive
system (e.g., pituitary posterior lobe [Reference 3] and the uterus
[Reference 4]), are as high as those in adrenal medulla. Also,
level of circulating AM in maternal blood (Reference 5), and AM and
AM mRNA abundance in fetal-placental tissues (Reference 6) and the
uterus (Reference 7) were both elevated during normal pregnancy. In
gestosis, a complication of pregnancy, the maternal plasma AM level
did not alter (Reference 5), or lowered (Reference 8), whereas the
AM content in amniotic fluid and umbilical vein were higher,
compared to normal pregnancy (Reference 9). However, physiological
roles of AM and details of AM function in these fetal and maternal
tissues remain elusive.
[0012] As to the effect of AM on uterine contraction, it has only
been reported that AM inhibited galanin-induced tonic contraction
of the uterus at high concentrations of 5 .mu.M or more, and the
action of AM was eliminated by CGRP[8-37] (Reference 7). Galanin is
a neuropeptide contained in a neuron (CGRP). However, the
importance of galanin-induced uterine contraction is not understood
at all. AM action is not found at concentrations of the order of
nanomolar (nM) or less (which are the concentrations reported in a
number of papers as concentrations at which AM can function). It
has been only confirmed that AM can function at high concentrations
of the order of micromolar (.mu.M) or more. Therefore, it is hardly
believed that these effects reflect a physiological function of
AM.
[0013] The motility of the uterus (contraction/relaxation) is not
only regulated by nerves, such as sympathetic nerves and
parasympathetic nerves, but also is coordinately regulated by
various substances, such as for example CGRP (Reference 10), nitric
oxide (NO), oxytocin, and prostaglandin F.sub.2.alpha.
(PGF.sub.2.alpha.: a representative prostaglandin having various
actions: elevation of blood pressure; vasoconstriction; promotion
of intestine movement; uterine contraction; promotion of regression
of corpus luteum; and bronchoconstriction, and used as a
parturifacient). Similar to bradykinin described above, substances
which can cause abnormal contraction leading to premature labor
also influence the motility of the uterus. However, what effect is
obtained by AM on spontaneous uterine contraction; the
contractility evoked by regulatory factors, such as oxytocin,
PGF.sub.2, and the like; or bradykinin-induced contraction, is not
known at all.
[0014] The present invention is intended to solve the
above-described problems. The objective of the present invention is
to provide a novel agent for inhibiting, preferably selectively
inhibiting, spontaneous myometrial contraction or
bradykinin-induced contraction.
DISCLOSURE OF THE INVENTION
[0015] The inventor found that adrenomedullin, originally
identified as a peptide having a hypotentive action, has an action
of selectively inhibiting spontaneous myometrial contraction and
bradykinin-induced contraction and that the inhibitor action is
selective, and based on this finding, completed the present
invention.
[0016] A composition of the present invention for inhibiting
spontaneous myometrial contraction or bradykinin-induced
contraction comprises AM. The composition of the present invention
may be used for selectively inhibiting spontaneous myometrial
contraction or bradykinin-induced contraction, preventing premature
labor, preventing miscarriage, arresting parturition prior to
cesarean section, and treating dysmenorrhea.
[0017] In one embodiment, the AM includes: (a) a peptide comprising
an amino acid sequence from Ser in position 13 to Tyr in position
52 of SEQ ID NO: 2 in SEQUENCE LISTING;
[0018] (b) a peptide comprising an amino acid sequence having one
or several amino acid deleted, substituted, or added in the amino
acid sequence (a), and having an action of inhibiting myometrial
contraction; (c) a peptide comprising an amino acid sequence from
Tyr in position 1 to Tyr in position 52 of SEQ ID NO: 2 in SEQUENCE
LISTING; (d) a peptide comprising an amino acid sequence having one
or several amino acid deleted, substituted, or added in the amino
acid sequence (c), and having an action of inhibiting myometrial
contraction; (e) a peptide comprising an amino acid sequence from
Ala in position -73 to Tyr in position 52 of SEQ ID NO: 2 in
SEQUENCE LISTING; (f) a peptide comprising an amino acid sequence
having one or several amino acid deleted, substituted, or added in
the amino acid sequence (e), and having an action of inhibiting
myometrial contraction; (g) a peptide comprising an amino acid
sequence from Met in position -94 to Leu in position 91 of SEQ ID
NO: 2 in SEQUENCE LISTING; and (h) a peptide comprising an amino
acid sequence having one or several amino acid deleted,
substituted, or added in the amino acid sequence (g), and having an
action of inhibiting myometrial contraction.
[0019] In another embodiment, the C-terminus of the AM may be
amidated. Gly may be added to the C-terminus of the AM.
[0020] In another embodiment, in the AM, Cys in position 16 and Cys
in position 21 of SEQ ID NO: 2 in SEQUENCE LISTING may be
crosslinked. The crosslink may be a disulfide bond or a
--CH.sub.2--CH.sub.2-- bond.
[0021] A method of the present invention for preventing premature
labor or miscarriage uses a composition comprising AM.
[0022] The present invention also provides use of AM in production
of a drug for preventing premature labor or miscarriage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1(A) is a schematic diagram showing a uterus site from
which uterine strips were isolated in examples. FIG. 1(B) is a
schematic diagram showing the shape of the prepared uterine
strips.
[0024] FIG. 2(a) is a graph showing the result of measuring
myometrial contraction when distilled water was added to the
uterine strip a in FIG. 1(A). FIG. 2(b) is a graph showing the
result of measuring myometrial contraction when distilled water was
added to the uterine strip b in FIG. 1(A). FIG. 2(c) is a graph
showing the result of measuring myometrial contraction when 100 nM
AM was added to the uterine strip a in FIG. 1(A). FIG. 2(d) is a
graph showing the result of measuring myometrial contraction when
100 nM AM was added to the uterine strip d in FIG. 1(A).
[0025] FIG. 3(a) is a graph showing the result of measuring
myometrial contraction when 1 to 100 nM AM was added to the uterine
strip b in FIG. 1(A). FIG. 3(b) is a graph showing the result of
measuring myometrial contraction when 1 to 100 nM AM was added to
the uterine strip a in FIG. 1(A). FIG. 3(a) is a graph showing the
result of measuring myometrial contraction when distilled water was
added to the uterine strip d in FIG. 1(A).
[0026] FIG. 4(a) is a graph showing the result of measuring
myometrial contraction when bradykinin and then 100 nM AM were
added to the uterine strip b in FIG. 1(A). FIG. 4(b) is a graph
showing the result of measuring myometrial contraction when
bradykinin and then distilled water were added to the uterine strip
b in FIG. 1(A).
[0027] FIG. 5 is a diagram showing the amino acid sequence of AM
derived from human pheochromocytoma. RE1 to RE6 indicate fragments
produced by digesting the amino acid sequence with
arginylendopeptidase.
[0028] FIG. 6 is a diagram showing concentration-dependent
inhibition of spontaneous myometrial contraction of the uterus by
AM; and prevention by AM[22-52] or CGRP[8-37]. FIGS. 6(a) to 6(e)
are representative records from five separate experiments having
similar results. *p<0.05, comparison with responses without a
drug (one-way analysis of variance); *p<0.05, comparison with AM
alone (two-way analysis of variance).
[0029] FIG. 7 is a diagram showing inhibition of bradykinin-induced
uterine contraction which was caused by AM but not by prevention of
oxytocin or PGF.sub.2.alpha. by AM[22-52] or CGRP[8-37]. FIGS. 7(a)
to 7(e) show representative records from five separate experiments
having similar results.
BEST MODE FOR CARRYING OUT THE INVENTION
[0030] In embodiments of the present invention, protein
purification and analysis methods, recombinant DNA techniques, and
assays, which are known in the art, are employed unless otherwise
specified.
[0031] I. Definition
[0032] Hereinafter, the terms used herein to explain the present
invention will be described.
[0033] As described above, "adrenomedullin" is a peptide having a
hypotensive action, originally isolated from human
pheochromocytoma. The term "adrenomedullin" as used herein is not
limited to the particular peptide, but includes peptides having
substantial homology with the amino acid sequence of that peptide.
Examples of the homologous peptides include species mutants and
allelic mutants. Human-derived AM comprises an amino acid sequence
from Tyr in position 1 to Tyr in position 52 of SEQ ID NO: 2 in
SEQUENCE LISTING. (The peptide consisting of an amino acid sequence
from Met in position -94 to Leu in position 91 of SEQ ID NO: 2 in
SEQUENCE LISTING is believed to be preproadrenomedullin. The
peptide obtained by processing of a signal peptide and consisting
of an amino acid sequence from Ala in position -73 to Leu in
position 91 of SEQ ID NO: 2 in SEQUENCE LISTING is, believed to be
proadrenomedullin. The peptide consisting of an amino acid sequence
from Ser in position 13 to Tyr in position 52 of SEQ ID NO: 2 in
SEQUENCE LISTING is an AM fragment which has been confirmed to have
a hypotensive action. AM in any of the above-described forms may be
employed in the present invention.) Human-derived AM may be encoded
by a polynucleotide sequence from T in position 447 to C in
position 602 of SEQ ID NO: 1 in SEQUENCE LISTING. Porcine-derived
AM comprises an amino acid sequence from Tyr in position 1 to Tyr
in position 52 of SEQ ID NO: 4 in SEQUENCE LISTING. Porcine-derived
AM may be encoded by a polynucleotide sequence from T in position
430 to C in position 585 of SEQ ID NO: 3 in SEQUENCE LISTING.
Rat-derived AM comprises an amino acid sequence from Tyr in
position 1 to Tyr in position 50 of SEQ ID NO: 6 in SEQUENCE
LISTING. Rat-derived AM may be encoded by a polynucleotide sequence
from T in position 433 to T in position 582 of SEQ ID NO: 5 in
SEQUENCE LISTING.
[0034] Clearly, human-derived peptides are preferable for human
diseases or treatment of a human. However, homologous peptides
derived from other mammals may also be employed for some purposes.
Further, comparison of human-derived peptides with peptide derived
from other mammals is important when an attempt is made to obtain a
variant maintaining a desired activity of a human-derived
peptide.
[0035] AM used in the present invention is not necessarily limited
to the above-described sequences, but includes, as subjects,
homologous peptides having an amino acid sequence having one or
several amino acid deleted, substituted, or added in the
above-described sequences and maintaining a desired activity.
[0036] Amino acid conservative substitution is one preferable means
for obtaining homologous peptides. Conservative substitution
representatively includes substitutions conducted within the
following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid; asparagine, glutamine; serine,
threonine; lysine, arginine; and phenylalanine, tyrosine.
[0037] The homology between two amino acid sequences is determined
by optionally introducing a gap to optimize residue matching. A
peptide having an amino acid sequence, which has substantially
homology with the amino acid sequence of human AM, has
representatively about 60% homology with the amino acid sequence of
human AM, preferably at least about 70%, more preferably at least
about 80%, and in an especially preferable embodiment, at least
about 90% or more. Software for determining homology is easily
available.
[0038] In the present invention, a peptide is by definition
referred to "having an action of inhibiting myometrial contraction"
if the degree of spontaneous contraction of uterine muscle is about
90% or less and preferably about 80% or less of the value indicated
in the Control sample of Example 1 or if the degree of
bradykinin-induced contraction is about 90% or less and preferably
about 80% or less of the value indicated in the Control sample of
Example 2, when both are measured under substantially the same
conditions as those of Example 1 below (the concentration of AM
added was 100 nM).
[0039] In the present invention, a peptide is by definition
referred to as "not inhibiting contraction" if the degree of
spontaneous contraction of uterine muscle is about 90% or more and
preferably about 95% or more of the value indicated in Control
sample of in Example 1; if the degree of bradykinin-induced
contraction is about 90% and preferably about 95% or more of the
value indicated in the Control sample shown in Example 2; or if
degree of contraction induced by oxytocin or prostaglandin
F.sub.2.alpha. is higher about 90% and preferably about 95% or more
of a value indicated before the addition of AM in Example 4, when
measured respectively under substantially the same conditions as
those of Example 1 (the concentration of AM added was 100 nM).
[0040] In the present invention, the peptide is referred to as
having an action of selectively inhibiting myometrial contraction
when although spontaneous myometrial contraction or
bradykinin-induced contraction is inhibited, myometrial contraction
induced by oxytocin and prostaglandin F.sub.2.alpha. is not
inhibited.
[0041] The C-terminus of a peptide used in the present invention
may or may not be amidated. "Amidation of C-terminus" refers to one
of modification reactions of a peptide, in which the COOH group of
the C-terminal amino acid of a peptide is changed to the form of
CONH.sub.2. A number of biologically active peptides functioning in
vivo are first biosynthesized as a precursor protein having a
larger molecular weight. The precursor protein is then matured by a
modification reaction such as for example the amidation of the
C-terminus. The amidation is conducted by a C-terminal amidating
enzyme acting on the precursor protein. The precursor protein
always includes a Gly residue on the C-terminal side of a residue
to be amidated, which is frequently followed by a basic amino acid
sequence pair, such as for example Lys-Arg or Arg-Arg, on the
C-terminal side (Reference 11).
[0042] II. AM Having an Action of Inhibiting Myometrial
Contraction
[0043] In the present invention, AM is used as an effective
component of a composition for inhibiting, preferably selectively
inhibiting, spontaneous myometrial condition or bradykinin-induced
contraction. AM is also used as an effective component for
producing a drug for preventing premature labor or miscarriage. AM
may be those isolated from naturally-occurring sources, those
produced using recombinant DNA techniques, or those chemically
synthesized.
[0044] When AM is isolated from naturally-occurring sources,
purification may be conducted, for example, in the following way.
For example, firstly human pheochromocytoma is pulverized to obtain
a crude extract, followed by various chromatography techniques so
that adenomodullin may be purified. In this case, by monitoring an
increase in the cAMP activity of platelets, a fraction containing
AM of interest can be obtained. Methods for isolation and
purification of AM are described in Japanese Laid-Open Publication
No. 7-196693.
[0045] When AM is produced using recombinant DNA techniques, the
DNA sequence encoding a peptide of interest is expressed using
various recombinant systems. Construction of expression vectors and
preparation of transformants having appropriate DNA sequences are
conducted by methods known in the art. Expression may be conducted
using prokaryote systems or eukaryote systems.
[0046] Prokaryote hosts used include E. coli, bacillus, and other
bacteria. For such prokaryote hosts, plasmid vectors having
replication sites and control sequences compatible with the hosts
are used. For example, E. coli is typically transformed with a
derivative of pBR322 which is a plasmid derived from E. coli. In
this case, the control sequence herein includes a promoter for
initiation of transcription, an operator if necessary, and a
ribosome binding site. Such a control sequence includes generally
used promoters such as for example .beta.-lactamase and lactose
promoter systems (Reference 12), tryptophan promoters (Reference
13), and P.sub.L promoters derived from .lamda. and N-gene ribosome
binding sites (Reference 14).
[0047] As an eukaryote host, yeast and mammalian cells may be used,
for example. For such an eukaryote host, a plasmid vector having a
replication site and a control sequence compatible with the host is
used. For example, yeast is transformed with pYEUra3 (Clontech).
Other promoter classes useful in a eukaryote host include, for
example, promoters for synthesizing a glycolytic enzyme, which
include a promoter for 3-phosphoglycerate kinase (Reference 15); a
promoter derived from an enolase gene; a promoter derived from a
Leu2 gene obtained from YEp13; a promoter derived from
metallothionein; an early or late promoter derived from SV40; and
other virus promoters such as for example those derived from
polyoma virus, adenovirus II, bovine papilloma virus and avian
sarcoma virus. A combination of a host cell and an appropriate
promoter is known to those skilled in the art and may be
appropriately selected if necessary.
[0048] A transformant can be obtained by introducing an expression
vector into an appropriate host cell. A desired AM can be obtained
by culturing the transformant under appropriate conditions.
[0049] Chemical synthesis of AM may be conducted within a method
known in the art. For example, AM may be synthesized by such a
method is a solid phase method on a peptide synthesizer. A
C-terminal amidated peptide can be synthesized using a peptide
synthesizer by condensing amino acids sequentially from the
C-terminal amino acid to the N-terminal amino acid using a
benzhydryl amine resin and a standard DCC/HOBt, and cutting out an
intended peptide from the resultant peptide resin by a standard
cleavage method (trifluoromethanesulfonic acid method).
[0050] A C-terminal amidated AM may be obtained by one of the
following: a carboxyl group at the C-terminus of the peptide
obtained by expression in a host is chemically amidated; or a
peptide is prepared so as to have Gly added to the C-terminus of an
intended amino acid sequence, and is then allowed to react with the
above-mentioned C-terminal amidating enzyme for amidation.
[0051] Alternatively, the peptide obtained by adding Gly to the
C-terminus of AM may be amidated due to an action of a C-terminal
amidating enzyme in vivo as described above.
[0052] A disulfide bond can be formed, for example, by oxidizing a
peptide by air oxidization or with an appropriate oxidant. The
substitution of the disulfide bond can be conducted with a
--CH.sub.2--CH.sub.2-- bond by a well-known method (Reference 16).
Generally, cleavage in the disulfide bond is avoided by
substituting a --CH.sub.2--CH.sub.2-- bond for the disulfide bond,
resulting in stabilization of the protein.
[0053] Assay methods for action of selectively inhibiting
myometrial contraction, which are known in the art, may be used to
confirm that the thus-obtained AM has an action of inhibiting,
preferably selectively inhibiting, myometrial contraction. Examples
of such assay methods include: a method using the uterus of a
female rat previously treated with estrogen; a method using the
uterus of a virgin female rat in proestrus or estrus; a method
using the uterus of a female rat during pregnancy or during or
after parturition; and the like. When the uterus of a female rat
previously treated with estrogen is used, action of inhibiting
myometrial contraction may be assayed, for example, under the
following conditions: the uterus is isolated from a female rat to
which estrogen (for example, 17.beta.-estradiol) has been
administered, and is cut into several parts to obtain uterus
fragments. Portions of the uterine strips to which blood vessels
are attached are removed to obtain uterine strips. While the
resultant uterine strips are immersed in a buffer solution such as
for example Ringer's solution, a measurement apparatus, such as for
example an isometric transducer and an isotonic transducer, is used
to continuously examine myometrial contraction. When a rhythm of
uterine muscle in spontaneous contraction become constant, or after
bradykinin, oxytocin, or prostaglandin F.sub.2.alpha. is added to
the solution and thereafter a subject peptide is added to the
solution, a change in myometrial contraction is examined. Uterine
muscle is caused to contract in the presence or absence of the
subject peptide to compare levels of contraction, thereby judging
the myometrial contraction inhibiting action of the peptide. In
this manner, an action of the subject peptide on spontaneous
myometrial contraction, or myometrial contraction induced by
bradykinin, oxytocin, or prostaglandin F.sub.2.alpha. is
determined. If a subject peptide inhibits spontaneous myometrial
contraction and bradykinin-induced contraction, but not oxytocin-
or prostaglandin F.sub.2.alpha.-induced myometrial contraction, it
is judged that the peptide has a selective myometrial contraction
inhibiting action.
[0054] III. Preparation of a Composition for Inhibiting Myometrial
Contraction
[0055] A composition of the present invention comprises, in
addition to an effective amount of AM, any excipient known to those
skilled in the art. Examples of the excipients include lactose,
cornstarch, magnesium stearate, and alum.
[0056] The composition of the present invention is prepared in
accordance with methods known in the art.
[0057] The composition of the present invention may be in any form.
The composition of the present invention may be a solid, such as
for example a tablet, a pill, a capsule, and a granule; or a
liquid, such as for example an aqueous solution and a suspension.
When the composition of the present invention is orally
administered as a tablet, an excipient, such as for example
lactose, cornstarch, and magnesium stearate, may be typically used.
When the composition of the present invention is orally
administered as a capsule, an excipient, such as for example
lactose and dried cornstarch, may be typically used. In order to
orally administer AM as an aqueous suspension, the AM may be used
in combination with an emulsion or a suspension. The aqueous
suspension may optionally contain a sweetner and an aroma chemical.
When the composition of the present invention is intramuscularly,
intraperitoneally, subcutaneously, or intravenously injected, AM is
dissolved in a sterilized solution to prepare a buffer solution
which is in turn adjusted into an appropriate pH. When the
composition of the present invention is intravenously administered,
the composition is preferably isotonic.
[0058] The composition of the present invention may be used as an
drug for preventing premature labor or miscarriage.
[0059] IV. Administration of a Composition for Inhibiting
Myometrial Contraction
[0060] The composition of the present invention may be administered
in the form of a conventional peptide formulation as described in
Remington's Pharmaceutical Sciences, Mack Publishing, Easton, Pa.
For example, the composition of the present invention may be
administered orally, or alternatively parenterally, such as for
example intravenous administration, intramuscular injection,
intraperitoneal injection, and subcutaneous injection. These
peptides may be supplemented into the amniotic fluid. Preferably,
these peptides may also be administered by injection.
[0061] When the composition of the present invention is
administered into a human, typically, the dose per day can be
appropriately determined by those skilled in the art by taking into
consideration a patient's symptoms, severity, individual
differences in sensitivity, weight, age, and the like. The
composition of the present invention may be administered once a day
or several times a day.
[0062] Premature labor or miscarriage would be prevented by
administration of the composition of the present invention.
EXAMPLES
[0063] Hereinafter, action of AM as an drug of the present
invention for inhibiting, or preferably selectively inhibiting,
spontaneous myometrial contraction or bradykinin-induced
contraction will be more specifically described. The present
invention is not limited to the following examples. AM used in the
examples is a synthesized peptide consisting of an amino acid
sequence from Tyr in position 1 to Tyr in position 50 of SEQ ID NO:
6 (available from Peptide Institute, Inc.).
Example 1
Effect of AM on Spontaneous Contraction of the Uterus of a Female
Rat)
[0064] 1 .mu.g of 17.beta.-estradiol in 0.2 ml of 30% ethanol was
subcutaneously injected to 10 to 12 week old female rats.
[0065] On the following day, the rats were sacrified by hammering
their heads. Thereafter, the rats were decapitated, followed by
exsanguination. The uteri were isolated from the rats. Each
isolated uterus was cut into four fragments a to d (FIG. 1(A)).
Portions of each strip to which blood vessels were attached were
cut off, thereby obtaining uterine strips (FIG. 1(B)).
[0066] The effect of AM on the rat uterus was examined by measuring
contractions of the uterine strips using an isotonic transducer
TD-112S (manufactured by Nippon Kohden Corporation) where the
tension was 1 g.
[0067] While the uterine strip was immersed in 30 ml of a modified
Krebs-Ringer bicarbonate (KRB) solution with glucose) (hereinafter
simply referred to as "modified KRB solution"), the uterine strip
was attached to the isotonic transducer. The composition of the
modified KRB solution is as follows: 122 mM NaCl, 26 mM
NaHCO.sub.3, 5 mM KCl, 1 mM MgSO.sub.4.7H.sub.2O, 0.03 mM EDTA-2Na,
2.4 mM CaCl.sub.2, and 11 mM glucose; pH 7.4).
[0068] Myometrial contraction was continuously measured. After
spontaneous rhythm of the uterine muscle was confirmed to be
constant, 30 .mu.l of 10.sup.-4 M AM (experimental sample) or
distilled water (control sample) was added to the respective
modified KRB solution with glucose, where the concentration of AM
was 100 nM. 30 minutes after the addition of AM or distilled water,
300 .mu.l of 4.5 M KCl was added to make a KCl concentration of 45
mM.
[0069] The results are shown in FIGS. 2(a) to 2(d). Here, FIGS.
2(a) to 2(d) correspond to the results obtained by using the
uterine strips for the portions a to d in FIG. 1(A). FIGS. 2(a) and
2(b) show controls. FIGS. 2(c) and 2(d) show the results of the
addition of 100 nM AM. Arrows on the left side of each Figure
indicate times at which distilled water or AM was added. Arrows on
the right side of each Figure indicate the times at which 45 mM KCl
was added.
[0070] As shown in FIGS. 2(a) and 2(b), spontaneous myometrial
contraction was not affected by the addition of distilled water. In
the case of the addition of AM, spontaneous myometrial contraction
was significantly inhibited (FIGS. 2(c) and 2(d)). Further, since
the addition of 45 mM KCl caused strong contradiction both in the
control added sample and the AM added sample), it was found that
the addition of AM does not affect muscle contraction due to
activation of voltage-dependent Ca channels caused by
depolarization of smooth muscle cells in the uterus.
[0071] Note that when the isometric transducer was used to conduct
the same experiment, the same results as above were obtained (data
not shown).
Example 2
Concentration-Dependent Effect of AM on the Uterus of a Female
Rat
[0072] In a manner similar to that of Example 1, uterine strips
were prepared, and attached to an isotonic transducer in a modified
KRB solution. Myometrial contraction was continuously measured.
After it was confirmed that the spontaneous rhythm of uterine
muscle became constant, 30 .mu.l of 1.times.10.sup.-6,
2.times.10.sup.-6, 7.times.10.sup.-6, 2.times.10.sup.-5, or
7.times.10.sup.-5 M AM (experimental samples), or distilled water
(control sample) was added to the modified KRB solution at time 0
min (initial addition), 5 min later, 12 min later, 22 min later,
and 32 min later, where AM concentrations were 1, 3, 10, 30, and
100 nM, respectively. Thereafter, after 45 minutes from the initial
addition of AM or distilled water, 300 .mu.l of 4.5 M KCl was added
to the solution to be a KCl concentration of 45 mM.
[0073] The results are shown in FIGS. 3(a) to 3(c). Here, FIGS.
3(a) to 3(c) correspond to the results obtained by using the
uterine strips for the portions b to d in FIG. 1(A), respectively.
FIGS. 3(a) and 3(b) show the results of AM addition having various
concentrations from 1 to 100 nM. FIG. 3(a) shows the results of the
controls. Arrows in each figure indicate times at which AM,
distilled water, or KCl was added.
[0074] As shown in FIGS. 3(a) and (b), it was found that
spontaneous myometrial contraction was inhibited by the addition of
AM in a concentration-dependent manner.
Example 3
Inhibition of Bradykinin-Induced Contraction by AM
[0075] In a manner similar to that of Example 1, uterine strips
were prepared and attached to an isotonic transducer in a modified
KRB solution. At the time when 10 nM bradykinin (Peptide Institute,
Inc.) was added to the modified KRB solution, myometrial
contraction was continuously measured. 20 minutes after the
addition of bradykinin, 100 nM AM or distilled water was further
added.
[0076] The results are shown in FIGS. 4(a) and 4(b). Here, FIGS.
4(a) and 4(b) correspond to the results obtained by using the
uterine strips for the portions a and c in FIG. 1(A), respectively.
FIG. 4(a) shows the results of addition of 100 nM AM. FIG. 4(b)
shows the results of addition of distilled water. Arrows in each
figure indicate times at which bradykinin, AM, or distilled water
were added.
[0077] As shown in FIGS. 4(a) and 4(b), bradykinin-induced
contraction was inhibited by the addition of AM.
Example 4
Effect of AM on Contraction Induced by Oxytocin or Prostaglandin
F.sub.2.alpha.
[0078] 8 to 12 weeks old female rats were used to prepare uterine
strips in a manner similar to that of Example 1.
[0079] Thereafter, the uterine strip was placed in a tissue chamber
filled with 30 ml of a modified KRB solution with aeration of 95%
O.sub.2/5% CO.sub.2 at 37.degree. C. to measure contraction of the
uterine strip in a manner similar to that of Example 1. After 40
minutes of equilibrium, the uterine strip was preincubated for 15
minutes in the presence or absence of 1 .mu.M AM[22-52] or 1 .mu.M
CGRP[8-37]. Thereafter, AM was added to the modified KRB solution
in the tissue chamber to gradually increase the AM concentration
from 1 nM to 100 nM, exposing the uterine strip to AM.
[0080] In another experiment, rats without injection of
17.beta.-estradiol were used. The effect of 100 nM AM on uterine
contraction induced by 10 nM bradykinin, 1 nM oxytocin, or 1 .mu.M
PGF.sub.2.alpha. was tested in the presence or absence of 1 .mu.M
AM[22-52] or 1 .mu.M CGRP[8-37].
[0081] In all measurements, the uterine strips were eventually
contracted due to 45 mM KCl depolarization to confirm responses of
the myometrium. The results are shown in FIGS. 6(a) to 6(f) and
FIGS. 7(a) to 7(e).
[0082] The uterine strips isolated from the rats treated with
17.beta.-estradiol, a type of estrogen, contracted spontaneously in
a rhythmical manner (FIG. 6(a); in this case, distilled water was
added instead of an AM solution). AM was added to the chamber so as
to gradually increase the concentration (1 to 100 nM). Spontaneous
contraction was inhibited in a concentration-dependent manner
(IC.sub.50=23 nM) (FIGS. 6(b) and 6(c)). The inhibitory effect of
AM could be reversed by removal of AM by washing out and exchanging
the modified KRB solution even when uterine muscle was completely
relaxed with 100 nM AM (FIG. 6(c)). The preliminary addition of 1
.mu.M AM[22-52] or 1 .mu.M CGRP[8-37] per se had no effect, but it
substantially completely prevents the contraction inhibitory effect
of the addition of 1 to 100 nM AM (FIGS. 6(d) and 6(e)). FIG. 6(f)
is a graph showing comparison of the results shown in FIGS. 6(b),
6(d), and 6(e).
[0083] When uterine strips were prepared from rats which were not
treated with estrogen, these were spontaneously contracted with
various intervals and amplitudes. As shown in FIGS. 7(a), 7(b) and
7(c), either 1 nM oxytocin, 1 .mu.M PGF.sub.2.alpha., or 10 nM
bradykinin all significantly stimulated contraction. 100 nM AM
substantially had almost no effect on contraction induced by
oxytocin (FIG. 7(a)) or PGF.sub.2.alpha. (FIG. 7(b)). In the other
hand, bradykinin-induced contraction was completely blocked by 100
nM AM which is a concentration where spontaneous contraction can be
completely inhibited (FIG. 7(c)). The inhibitory effect of AM on
bradykinin-induced contraction was eliminated by the preliminary
addition of AM[22-52] or CGRP[8-37] (FIGS. 7(d) and 7(e)).
[0084] (Discussion of Examples)
[0085] To the inventor's knowledge, the above-described examples
are the first to demonstrate that AM inhibits autonomous and
spontaneous contraction of the uterus of a rat in
concentration-dependent and reversible manners. Further, AM
inhibited bradykinin-induced contraction, but had no effect on
contraction induced by oxytocin or PGF.sub.2.alpha., or contraction
caused by a high K stimulus. This suggests that the AM action does
not directly relax smooth muscle of the uterus, and selectively
inhibits mechanisms generating spontaneous contraction or
bradykinin-induced contraction.
[0086] In the above-described examples, the AM uterine contraction
inhibiting action was blocked by both AM[22-52] which is an
antagonist for an AM receptor and CGRP[8-37] which is an antagonist
for a CGRP receptor. Therefore, the AM action is considered to be
expressed through the AM receptor and the CGRP receptor. As to that
the AM action is blocked by CGRP[8-37], besides the examples, it
has been reported that the vasodilatory action of AM was blocked by
CGRP[8-37] in the isolated rat mesentery vasculature (Reference
17), that elevation of heart rate and blood pressure due to AM
administration into a rat cerebral ventricle was blocked by
AM[22-52] or CGRP[8-37] (Reference 18), and the like. Further, it
has been reported that in a binding test using the uterus of a rat,
AM could display both .sup.125I-AM binding and .sup.125I-CGRP
binding, i.e., AM could bind to not only a binding site for AM but
also a binding site for CGRP (Reference 7). These findings support
the results obtained in the examples.
[0087] Expression of the AM protein or the AM gene in the uterus is
as abundant as the expression level in the suprarenal medulla at
which AM was discovered (Reference 7 and 3). It has been reported
that in rat and human uteruses, AM is expressed in endometrial
tissues rather than smooth muscle tissues of the uterus. Therefore,
it is inferred that AM produced in a endometrium acts on uterine
smooth muscle as a paracrine factor.
[0088] Further, the clinical importance of the uterine contraction
inhibiting action of AM will be discussed.
[0089] In the pregnant uterus, an expression amount of AM is
increased by a factor of about 1.8 to about 4.5 compared to the
non-pregnant uterus. An amount of .sup.125I-AM binding is increased
by a factor of about 10, and an amount of .sup.125I-CGRP binding is
increased by a factor of about 4 (Reference 7; Reference 4; and
Reference 19). However, it has been reported that the expression of
CGRP is reduced to the limit of detection or less (Reference 7).
Based on these findings and the results of the examples, i.e., "AM
inhibits uterine contraction through the AM receptor and the CGRP
receptor", a possibility is suggested that in the pregnant uterus,
an increased expression amount of AM inhibits uterine contraction,
thereby playing an important role in maintaining pregnancy.
[0090] Furthermore, in the examples, AM inhibited
bradykinin-induced contraction, but not contraction induced by
oxytocin and PGF.sub.2.alpha.. Generally, it is believed that
contractions induced by oxytocin and PGF.sub.2.alpha. play an
important role in parturition. While the physiological or
pathophysiological importance of bradykinin-induced contractions
has not yet been revealed, since bradykinin is inherently an
inflammatory mediator locally produced by an inflammation reaction
(Reference 20), the possibility has been suggested that an abnormal
increase in bradykinin in the pregnant uterus leads to premature
labor and miscarriage (Reference 1). Therefore, a possibility is
suggested that AM selectively inhibits abnormal contractions due to
bradykinin but not contractions during normal parturition due to
oxytocin and PGF.sub.2.alpha., to prevent premature labor and
miscarriage and maintain pregnancy.
[0091] The above-described results will be briefly described below.
In uterine strips isolated from a nonpregnant rat, AM inhibited
spontaneous rhythmic contraction in a concentration-dependent
manner (IC.sub.50=23 nM). The inhibitory effect of AM was perfectly
blocked by either calcitonin gene-related peptide[8-37]
(CGRP[8-37]) which is a putative antagonist for the CGRP receptor
or AM[22-52] which is a putative antagonist for the AM receptor. AM
also attenuated uterine contraction induced by bradykinin.
Bradykinin-induced uterine contraction is blocked by either
CGRP[8-37] or AM[22-52]. AM do not have an inhibitory effect or
contractile responses induced by oxytocin or prostaglandin
F.sub.2.alpha.. These results show that AM selectively inhibits
spontaneous myometrial contraction and bradykinin-induced
contraction.
REFERENCES
[0092] 1. Schrey et al., Prostaglandins, Leukotrienes, and
Essential Fatty Acids (1992) 45:137-142 [0093] 2. Kitamura et al.,
Biochem. Biophys. Res. Commun. (1993) 192:553-560 [0094] 3. Cameron
et al., Endocrinology (1998) 139:2253-2264 [0095] 4. Makino et al.,
Eur. J. Pharmacol. (1999) 371:159-167 [0096] 5. Minegishi et al.,
Mol. Hum. Reprod. (1999) 5:767-770 [0097] 6. Dilorio et al., Eur.
J. Endocrinol. (1999) 140:201-206 [0098] 7. Upton et al.,
Endocrinology (1997) 138:2508-2514 [0099] 8. Hata et al., Lancet
(1997) 350:1600 [0100] 9. Marinoni et al., Obstet. Gynecol. (1999)
93:964-967 [0101] 10. Anouar et al., Arch. Pharmacol. (1998)
357:446-453 [0102] 11. Mizuno, Seikagaku, Vol. 61, No. 12, pp.
1435-1461 (1989) [0103] 12. Chang et al., Nature (1977) 198, 1056
[0104] 13. Goeddel et al., Nucleic Acids Res. (1980) 8:4057 [0105]
14. Shimatake, Nature (1981) 292:128 [0106] 15. Hitzeman et al., J.
Biol. Chem. (1980) 255:2073 [0107] 16. O. Keller et al., Helv.
Chim. Acta (1974) 57:1253 [0108] 17. Nuki et al., Biochem. Biophys.
Res. Commun. (1993) 196:245-251 [0109] 18. Saita et al., Am. J.
Physiol. (1998) 274:R979-R984 [0110] 19. Dong et al., Am. J.
Obstet. Gynecol. (1998) 179:497-506 [0111] 20. DeLa et al., Am. J.
Physiol. (1991) 260:G213-219
INDUSTRIAL APPLICABILITY
[0112] The present invention provides a composition for inhibiting,
preferably selectively inhibiting, spontaneous myometrial
contraction and bradykinin-induced contraction, comprising AM. This
composition is useful in prevention of premature labor and
miscarriage, arresting of parturition during cesarean section, and
treatment of dysmenorrhea.
Sequence CWU 1
1
6 1 1457 DNA Homo sapiens CDS (165)..(719) mat peptide (447)..(602)
1 ggcacgagct ggatagaaca gctcaagcct tgccacttcg ggcttctcac tgcagctggg
60 cttggacttc ggagttttgc cattgccagt gggacgtctg agactttctc
cttcaagtac 120 ttggcagatc actctcttag cagggtctgc gcttcgcagc cggg atg
aag ctg gtt 176 Met Lys Leu Val tcc gtc gcc ctg atg tac ctg ggt tcg
ctc gcc ttc cta ggc gct gac 224 Ser Val Ala Leu Met Tyr Leu Gly Ser
Leu Ala Phe Leu Gly Ala Asp -90 -85 -80 -75 acc gct cgg ttg gat gtc
gcg tcg gag ttt cga aag aag tgg aat aag 272 Thr Ala Arg Leu Asp Val
Ala Ser Glu Phe Arg Lys Lys Trp Asn Lys -70 -65 -60 tgg gct ctg agt
cgt ggg aag agg gaa ctg cgg atg tcc agc agc tac 320 Trp Ala Leu Ser
Arg Gly Lys Arg Glu Leu Arg Met Ser Ser Ser Tyr -55 -50 -45 ccc acc
ggg ctc gct gac gtg aag gcc ggg cct gcc cag acc ctt att 368 Pro Thr
Gly Leu Ala Asp Val Lys Ala Gly Pro Ala Gln Thr Leu Ile -40 -35 -30
cgg ccc cag gac atg aag ggt gcc tct cga agc ccc gaa gac agc agt 416
Arg Pro Gln Asp Met Lys Gly Ala Ser Arg Ser Pro Glu Asp Ser Ser -25
-20 -15 ccg gat gcc gcc cgc atc cga gtc aag cgc tac cgc cag agc atg
aac 464 Pro Asp Ala Ala Arg Ile Arg Val Lys Arg Tyr Arg Gln Ser Met
Asn -10 -5 -1 1 5 aac ttc cag ggc ctc cgg agc ttt ggc tgc cgc ttc
ggg acg tgc acg 512 Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys Arg Phe
Gly Thr Cys Thr 10 15 20 gtg cag aag ctg gca cac cag atc tac cag
ttc aca gat aag gac aag 560 Val Gln Lys Leu Ala His Gln Ile Tyr Gln
Phe Thr Asp Lys Asp Lys 25 30 35 gac aac gtc gcc ccc agg agc aag
atc agc ccc cag ggc tac ggc cgc 608 Asp Asn Val Ala Pro Arg Ser Lys
Ile Ser Pro Gln Gly Tyr Gly Arg 40 45 50 cgg cgc cgg cgc tcc ctg
ccc gag gcc ggc ccg ggt cgg act ctg gtg 656 Arg Arg Arg Arg Ser Leu
Pro Glu Ala Gly Pro Gly Arg Thr Leu Val 55 60 65 70 tct tct aag cca
caa gca cac ggg gct cca gcc ccc ccg agt gga agt 704 Ser Ser Lys Pro
Gln Ala His Gly Ala Pro Ala Pro Pro Ser Gly Ser 75 80 85 gct ccc
cac ttt ctt taggatttag gcgcccatgg tacaaggaat agtcgcgcaa 759 Ala Pro
His Phe Leu 90 gcatcccgct ggtgcctccc gggacgaagg acttcccgag
cggtgtgggg accgggctct 819 gacagccctg cggagaccct gagtccggga
ggcaccgtcc ggcggcgagc tctggctttg 879 caagggcccc tccttctggg
ggcttcgctt ccttagcctt gctcaggtgc aagtgcccca 939 gggggcgggg
tgcagaagaa tccgagtgtt tgccaggctt aaggagagga gaaactgaga 999
aatgaatgct gagacccccg gagcaggggt ctgagccaca gccgtgctcg cccacaaact
1059 gatttctcac ggcgtgtcac cccaccaggg cgcaagcctc actattactt
gaactttcca 1119 aaacctaaag aggaaaagtg caatgcgtgt tgtacataca
gaggtaacta tcaatattta 1179 agtttgttgc tgtcaagatt ttttttgtaa
cttcaaatat agagatattt ttgtacgtta 1239 tatattgtat taagggcatt
ttaaaagcaa ttatattgtc ctcccctatt ttaagacgtg 1299 aatgtctcag
cgaggtgtaa agttgttcgc cgcgtggaat gtgagtgtgt ttgtgtgcat 1359
gaaagagaaa gactgattac ctcctgtgtg gaagaaggaa acaccgagtc tctgtataat
1419 ctatttacat aaaatgggtg atatgcgaac agcaaacc 1457 2 185 PRT Homo
sapiens 2 Met Lys Leu Val Ser Val Ala Leu Met Tyr Leu Gly Ser Leu
Ala Phe -90 -85 -80 Leu Gly Ala Asp Thr Ala Arg Leu Asp Val Ala Ser
Glu Phe Arg Lys -75 -70 -65 Lys Trp Asn Lys Trp Ala Leu Ser Arg Gly
Lys Arg Glu Leu Arg Met -60 -55 -50 Ser Ser Ser Tyr Pro Thr Gly Leu
Ala Asp Val Lys Ala Gly Pro Ala -45 -40 -35 Gln Thr Leu Ile Arg Pro
Gln Asp Met Lys Gly Ala Ser Arg Ser Pro -30 -25 -20 -15 Glu Asp Ser
Ser Pro Asp Ala Ala Arg Ile Arg Val Lys Arg Tyr Arg -10 -5 -1 1 Gln
Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys Arg Phe 5 10 15
Gly Thr Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln Phe Thr 20
25 30 Asp Lys Asp Lys Asp Asn Val Ala Pro Arg Ser Lys Ile Ser Pro
Gln 35 40 45 50 Gly Tyr Gly Arg Arg Arg Arg Arg Ser Leu Pro Glu Ala
Gly Pro Gly 55 60 65 Arg Thr Leu Val Ser Ser Lys Pro Gln Ala His
Gly Ala Pro Ala Pro 70 75 80 Pro Ser Gly Ser Ala Pro His Phe Leu 85
90 3 1493 DNA Sus scrofa CDS (148)..(711) mat peptide (430)..(585)
3 gcggaacagc tcgagccttg ccacctctag tttcttacca cagcttggac gtcggggttt
60 tgccactgcc agagggacgt ctcagacttc atcttcccaa atcttggcag
atcaccccct 120 tagcagggtc tgcacatctc agccggg atg aag ctg gtt ccc
gta gcc ctc atg 174 Met Lys Leu Val Pro Val Ala Leu Met -90 tac ctg
ggc tcg ctc gcc ttc ctg ggc gct gac aca gct cgg ctc gac 222 Tyr Leu
Gly Ser Leu Ala Phe Leu Gly Ala Asp Thr Ala Arg Leu Asp -85 -80 -75
-70 gtg gcg gca gag ttc cga aag aaa tgg aat aag tgg gct cta agt cgt
270 Val Ala Ala Glu Phe Arg Lys Lys Trp Asn Lys Trp Ala Leu Ser Arg
-65 -60 -55 gga aaa aga gaa ctt cgg ctg tcc agc agc tac ccc acc ggg
atc gcc 318 Gly Lys Arg Glu Leu Arg Leu Ser Ser Ser Tyr Pro Thr Gly
Ile Ala -50 -45 -40 gac ttg aag gcc ggg cct gcc cag act gtc att cgg
ccc cag gat gtg 366 Asp Leu Lys Ala Gly Pro Ala Gln Thr Val Ile Arg
Pro Gln Asp Val -35 -30 -25 aag ggc tcc tct cgc agc ccc cag gcc agc
att ccg gat gca gcc cgc 414 Lys Gly Ser Ser Arg Ser Pro Gln Ala Ser
Ile Pro Asp Ala Ala Arg -20 -15 -10 atc cga gtc aag cgc tac cgc cag
agt atg aac aac ttc cag ggc ctg 462 Ile Arg Val Lys Arg Tyr Arg Gln
Ser Met Asn Asn Phe Gln Gly Leu -5 -1 1 5 10 cgg agc ttc ggc tgt
cgc ttt ggg acg tgc acc gtg cag aag ctg gcg 510 Arg Ser Phe Gly Cys
Arg Phe Gly Thr Cys Thr Val Gln Lys Leu Ala 15 20 25 cac cag atc
tac cag ttc acg gac aaa gac aag gac ggc gtc gcc ccc 558 His Gln Ile
Tyr Gln Phe Thr Asp Lys Asp Lys Asp Gly Val Ala Pro 30 35 40 cgg
agc aag atc agc ccc cag ggc tac ggc cgc cgg cgc cga cgc tct 606 Arg
Ser Lys Ile Ser Pro Gln Gly Tyr Gly Arg Arg Arg Arg Arg Ser 45 50
55 ctg ccc gaa gcc agc ctg ggc cgg act ctg agg tcc cag gag cca cag
654 Leu Pro Glu Ala Ser Leu Gly Arg Thr Leu Arg Ser Gln Glu Pro Gln
60 65 70 75 gcg cac ggg gcc ccg gcc tcc ccg gcg cat caa gtg ctc gcc
act ctc 702 Ala His Gly Ala Pro Ala Ser Pro Ala His Gln Val Leu Ala
Thr Leu 80 85 90 ttt agg att taggcgccta ctgtggcagc agcgaacagt
cgcgcatgca 751 Phe Arg Ile tcatgccggc gcttcctggg gcggggggct
tcccggagcc gagcccctca gcggctgggg 811 cccgggcaga gacagcattg
agagaccgag agtccgggag gcacagacca gcggcgagcc 871 ctgcattttc
aggaacccgt cctgcttgga ggcagtgttc tcttcggctt aatccagccc 931
gggtccccgg gtgggggtgg agggtgcaga ggaatccaaa ggagtgtcat ctgccaggct
991 cacggagagg agaaactgcg aagtaaatgc ttagaccccc aggggcaagg
gtctgagcca 1051 ctgccgtgcc gcccacaaac tgatttctga aggggaataa
ccccaacagg gcgcaagcct 1111 cactattact tgaactttcc aaaacctaga
gaggaaaagt gcaatgtatg ttgtatataa 1171 agaggtaact atcaatattt
aagtttgttg ctgtcaagat ttttttttgt aacttcaaat 1231 atagagatat
ttttgtacgt tatatattgt attaagggca ttttaaaaca attgtattgt 1291
tcccctcccc tctattttaa tatgtgaatg tctcagcgag gtgtaacatt gtttgctgcg
1351 cgaaatgtga gagtgtgtgt gtgtgtgtgc gtgaaagaga gtctggatgc
ctcttgggga 1411 agaagaaaac accatatctg tataatctat ttacataaaa
tgggtgatat gcgaagtagc 1471 aaaccaataa actgtctcaa tg 1493 4 188 PRT
Sus scrofa 4 Met Lys Leu Val Pro Val Ala Leu Met Tyr Leu Gly Ser
Leu Ala Phe -90 -85 -80 Leu Gly Ala Asp Thr Ala Arg Leu Asp Val Ala
Ala Glu Phe Arg Lys -75 -70 -65 Lys Trp Asn Lys Trp Ala Leu Ser Arg
Gly Lys Arg Glu Leu Arg Leu -60 -55 -50 Ser Ser Ser Tyr Pro Thr Gly
Ile Ala Asp Leu Lys Ala Gly Pro Ala -45 -40 -35 Gln Thr Val Ile Arg
Pro Gln Asp Val Lys Gly Ser Ser Arg Ser Pro -30 -25 -20 -15 Gln Ala
Ser Ile Pro Asp Ala Ala Arg Ile Arg Val Lys Arg Tyr Arg -10 -5 -1 1
Gln Ser Met Asn Asn Phe Gln Gly Leu Arg Ser Phe Gly Cys Arg Phe 5
10 15 Gly Thr Cys Thr Val Gln Lys Leu Ala His Gln Ile Tyr Gln Phe
Thr 20 25 30 Asp Lys Asp Lys Asp Gly Val Ala Pro Arg Ser Lys Ile
Ser Pro Gln 35 40 45 50 Gly Tyr Gly Arg Arg Arg Arg Arg Ser Leu Pro
Glu Ala Ser Leu Gly 55 60 65 Arg Thr Leu Arg Ser Gln Glu Pro Gln
Ala His Gly Ala Pro Ala Ser 70 75 80 Pro Ala His Gln Val Leu Ala
Thr Leu Phe Arg Ile 85 90 5 1376 DNA Rattus norvegicus CDS
(154)..(708) mat peptide (433)..(582) 5 tccagccttt accgctcctg
gtttctcggc ttctcatcgc agtcagtctt ggactttgcg 60 ggttttgccg
ctgtcagaag gacgtctcgg actttctgct tcaagtgctt gacaactcac 120
cctttcagca gggtatcgga gcatcgctac aga atg aag ctg gtt tcc atc gcc
174 Met Lys Leu Val Ser Ile Ala -90 ctg atg tta ttg ggt tcg ctc gcc
gtt ctc ggc gcg gac acc gca cgg 222 Leu Met Leu Leu Gly Ser Leu Ala
Val Leu Gly Ala Asp Thr Ala Arg -85 -80 -75 ctc gac act tcc tcg cag
ttc cga aag aag tgg aat aag tgg gcg cta 270 Leu Asp Thr Ser Ser Gln
Phe Arg Lys Lys Trp Asn Lys Trp Ala Leu -70 -65 -60 -55 agt cgt ggg
aag agg gaa cta caa gcg tcc agc agc tac cct acg ggg 318 Ser Arg Gly
Lys Arg Glu Leu Gln Ala Ser Ser Ser Tyr Pro Thr Gly -50 -45 -40 ctc
gtt gat gag aag aca gtc ccg acc cag act ctt ggg ctc cag gac 366 Leu
Val Asp Glu Lys Thr Val Pro Thr Gln Thr Leu Gly Leu Gln Asp -35 -30
-25 aag cag agc acg tct agc acc cca caa gcc agc act cag agc aca gcc
414 Lys Gln Ser Thr Ser Ser Thr Pro Gln Ala Ser Thr Gln Ser Thr Ala
-20 -15 -10 cac att cga gtc aaa cgc tac cgc cag agc atg aac cag ggg
tcc cgc 462 His Ile Arg Val Lys Arg Tyr Arg Gln Ser Met Asn Gln Gly
Ser Arg -5 -1 1 5 10 agc act gga tgc cgc ttt ggg acc tgc aca atg
cag aaa ctg gct cac 510 Ser Thr Gly Cys Arg Phe Gly Thr Cys Thr Met
Gln Lys Leu Ala His 15 20 25 cag atc tac cag ttt aca gac aaa gac
aag gac ggc atg gcc ccc aga 558 Gln Ile Tyr Gln Phe Thr Asp Lys Asp
Lys Asp Gly Met Ala Pro Arg 30 35 40 aac aag atc agc cct caa ggc
tat ggc cgc cgg cgc cgg cgt tcc ctg 606 Asn Lys Ile Ser Pro Gln Gly
Tyr Gly Arg Arg Arg Arg Arg Ser Leu 45 50 55 cca gag gtc ctc cga
gcc cgg act gtg gag tcc tcc cag gag cag aca 654 Pro Glu Val Leu Arg
Ala Arg Thr Val Glu Ser Ser Gln Glu Gln Thr 60 65 70 cac tca gct
cca gcc tcc ccg gcg cac caa gac atc tcc aga gtc tct 702 His Ser Ala
Pro Ala Ser Pro Ala His Gln Asp Ile Ser Arg Val Ser 75 80 85 90 agg
tta taggtgcggg tggcagcatt gaacagtcgg gcgagtatcc cattggcgcc 758 Arg
Leu tgcggaatca gagagcttcg caccctgagc ggactgagac aatcttgcag
agatctgcct 818 ggctgcccct aggggaggca gaggaaccca agatcaagcc
aggctcacgt cagaaaccga 878 gaattacagg ctgatactct ctccgggcag
gggtctgagc cactgccttg cccgctcata 938 aactggtttt ctcacggggc
atacggctca ttacttactt gaactttcca aaacctagcg 998 aggaaaagtg
caatgcttgt tatacagcca aaggtaacta tcatatttaa gtttgttgat 1058
gtcaagaggt tttttttttt gtaacttcaa atatatagaa atatttttgt acgttatata
1118 ttgtattaag ggcattttaa agcgattata ttgtcacctt cccctatttt
aagaagtgaa 1178 tgtctcagca aggtgtaagg ttgtttggtt ccgtgtgtgt
gtgtgtgtgt gtgtgtgtgt 1238 gtgtgtgtgt gtgtgtgtaa ggtggagagc
gcctgattac cgcctgtgga tgaagaaaaa 1298 acattgtgtc ttctataatc
tatttacata aaatatgtga tctgggaaaa agcaaaccaa 1358 taaactgtct
caatgctg 1376 6 185 PRT Rattus norvegicus 6 Met Lys Leu Val Ser Ile
Ala Leu Met Leu Leu Gly Ser Leu Ala Val -90 -85 -80 Leu Gly Ala Asp
Thr Ala Arg Leu Asp Thr Ser Ser Gln Phe Arg Lys -75 -70 -65 Lys Trp
Asn Lys Trp Ala Leu Ser Arg Gly Lys Arg Glu Leu Gln Ala -60 -55 -50
Ser Ser Ser Tyr Pro Thr Gly Leu Val Asp Glu Lys Thr Val Pro Thr -45
-40 -35 -30 Gln Thr Leu Gly Leu Gln Asp Lys Gln Ser Thr Ser Ser Thr
Pro Gln -25 -20 -15 Ala Ser Thr Gln Ser Thr Ala His Ile Arg Val Lys
Arg Tyr Arg Gln -10 -5 -1 1 Ser Met Asn Gln Gly Ser Arg Ser Thr Gly
Cys Arg Phe Gly Thr Cys 5 10 15 Thr Met Gln Lys Leu Ala His Gln Ile
Tyr Gln Phe Thr Asp Lys Asp 20 25 30 35 Lys Asp Gly Met Ala Pro Arg
Asn Lys Ile Ser Pro Gln Gly Tyr Gly 40 45 50 Arg Arg Arg Arg Arg
Ser Leu Pro Glu Val Leu Arg Ala Arg Thr Val 55 60 65 Glu Ser Ser
Gln Glu Gln Thr His Ser Ala Pro Ala Ser Pro Ala His 70 75 80 Gln
Asp Ile Ser Arg Val Ser Arg Leu 85 90
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