U.S. patent application number 10/189493 was filed with the patent office on 2003-09-04 for smooth muscle growth inhibitory composition, diagnostic method for arteriosclerosis, and kit therefor.
Invention is credited to Matsuzawa, Yuji, Ohmoto, Yasukazu.
Application Number | 20030166551 10/189493 |
Document ID | / |
Family ID | 17848257 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166551 |
Kind Code |
A1 |
Matsuzawa, Yuji ; et
al. |
September 4, 2003 |
Smooth muscle growth inhibitory composition, diagnostic method for
arteriosclerosis, and kit therefor
Abstract
The invention provides not only a smooth muscle growth
inhibitory composition and a composition for inhibiting expression
of adhesion molecules in vascular endothelial cells, each
comprising the adipose tissue-specific secretory factor apM1 as an
active ingredient, but also a method for diagnosis of
arteriosclerosis which comprises assaying apM1 in a sample, an
antibody against apM1, and a diagnostic kit for arteriosclerosis
which comprises the antibody as an active component, all of which
contribute to the elucidation of obesity-related genes and
corresponding expression products which are useful for the
etiologic exploration and establishment of therapeutic modalities
for various obesity-related diseases, particularly arteriosclerosis
inclusive of angina pectoris and myocardial infarction. By
utilizing the information thus obtained, therapeutic and diagnostic
methods for various diseases can be established.
Inventors: |
Matsuzawa, Yuji;
(Takarazuka-shi, JP) ; Ohmoto, Yasukazu;
(Itano-gun, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 Pennsylvania Avenue, NW
Washington
DC
20037-3213
US
|
Family ID: |
17848257 |
Appl. No.: |
10/189493 |
Filed: |
July 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10189493 |
Jul 8, 2002 |
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09530423 |
May 1, 2000 |
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6461821 |
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09530423 |
May 1, 2000 |
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PCT/JP98/04862 |
Oct 27, 1998 |
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Current U.S.
Class: |
435/7.1 ;
435/338; 435/7.21; 514/1.9; 514/16.5; 514/7.4 |
Current CPC
Class: |
A61P 9/10 20180101; G01N
33/6893 20130101; A61P 11/00 20180101; A61P 43/00 20180101; Y10S
530/809 20130101; A61K 38/00 20130101; C07K 14/5759 20130101; A61P
29/00 20180101; G01N 2333/4704 20130101; C07K 16/26 20130101; G01N
2800/323 20130101; A61P 19/02 20180101; C07K 16/18 20130101; G01N
33/5091 20130101; A61P 11/06 20180101 |
Class at
Publication: |
514/12 ;
435/7.21; 435/338 |
International
Class: |
A61K 038/17; G01N
033/567; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 1997 |
JP |
297569/1997 |
Claims
1. A smooth muscle growth inhibitory composition comprising a
pharmacologically effective amount of at least one member selected
from the group consisting of the adipose tissue-specific secretory
factor apM1 and a pharmacologically acceptable salt thereof in
combination with a pharmaceutically acceptable carrier.
2. A prophylactic and therapeutic composition for arteriosclerosis
which comprises a pharmacologically effective amount of at least
one member selected from the group consisting of the adipose
tissue-specific secretory factor apM1 and a pharmacologically
acceptable salt thereof in combination with a pharmaceutically
acceptable carrier.
3. A prophylactic and therapeutic composition for post-angioplasty
restenoses which comprises a pharmacologically effective amount of
at least one member selected from the group consisting of the
adipose tissue-specific secretory factor apM1 and a
pharmacologically acceptable salt thereof in combination with a
pharmaceutically acceptable carrier.
4. A diagnostic agent for arteriosclerosis which comprises an
antibody against the adipose tissue-specific secretory factor
apM1.
5. Hybridoma KOC09108 having an ability to produce a monoclonal
antibody against the adipose tissue-specific secretory factor apM1
as deposited under the accession number of FERM BP-6542.
6. The monoclonal antibody against the adipose tissue-specific
secretory factor apM1 as produced by Hybridoma KOCO9108.
7. A diagnostic kit for arteriosclerosis which comprises an
antibody against the adipose tissue-specific factor apM1 as an
active component.
8. A method of inhibiting growth of smooth muscle which comprises
administering a pharmacologically effective amount of at least one
member selected from the group consisting of the adipose
tissue-specific secretory factor apM1 and a pharmacologically
acceptable salt thereof to a patient in whom a treatment for
inhibiting growth of smooth muscle is required.
9. A method for the prophylaxis and therapy of arteriosclerosis
which comprises administering a pharmacologically effective amount
of at least one member selected from the group consisting of the
adipose tissue-specific secretory factor apM1 and a
pharmacologically acceptable salt thereof to a patient in whom a
treatment for inhibiting growth of smooth muscle is required.
10. A method for the prophylaxis and therapy of post-angioplasty
restenoses which comprises administering a pharmacologically
effective amount of at least one member selected from the group
consisting of the adipose tissue-specific secretory factor apM1 and
a pharmacologically acceptable salt thereof to a patient in whom a
prophylactic or therapeutic treatment for post-angioplasty
restenoses is required.
11. A method for diagnosis of arteriosclerosis which comprises
quantitating the adipose tissue-specific secretory factor apM1 in a
sample with an antibody against the adipose tissue-specific
secretory factor apM1 and comparing the result of the assay with
the measured values in healthy subjects and patients with
arteriosclerosis to establish a diagnosis of arteriosclerosis.
12. Use of at least one member selected from the group consisting
of the adipose tissue-specific secretory factor apM1 and a
pharmacologically acceptable salt thereof for the manufacture of a
smooth muscle growth inhibitory composition.
13. Use of at least one member selected from the group consisting
of the adipose tissue-specific secretory factor apM1 and a
pharmacologically acceptable salt thereof for the manufacture of a
prophylactic and therapeutic composition for arteriosclerosis.
14. Use of at least one member selected from the group consisting
of the adipose tissue-specific secretory factor apM1 and a
pharmacologically acceptable salt thereof for the manufacture of a
prophylactic and therapeutic composition for post-angioplasty
restenoses.
15. Use of an antibody against the adipose tissue-specific
secretory factor apM1 for the manufacture of a diagnostic agent for
arteriosclerosis.
Description
TECHNICAL FIELD
[0001] This invention relates to a smooth muscle growth inhibitory
composition comprising apM1 (adipose most abundant gene transcript
1) as an active ingredient, a method for diagnosis of
arteriosclerosis (atherosclerosis) which comprises assaying said
apM1 in a sample, and a diagnostic kit for arteriosclerosis which
comprises an antibody against said apM1 as an active component.
BACKGROUND ART
[0002] It is well known that, in modern society, obesity or an
excessive accumulation of body fat is involved in the development
of diabetes mellitus, hyperlipidemia, hypertension, and
atherosclerotic diseases inclusive of angina pectoris and
myocardial infarction. With obesity, not only genetic factors but
also environmental factors are associated.
[0003] Recently, leptin and many other obesity-related genes have
been isolated from animal models. While the group of these genes
thus isolated is suspected to be involved in the establishment of
obesity in man, various environmental factors such as the excessive
food intake and insufficient physical exercise by contemporary man
are also considered to be playing a crucial role in the development
of diabetes mellitus and atherosclerosis via fat storage.
[0004] Not only the search for obesity-related genes but also the
approach toward elucidation of the specific genes expressed in
adipose tissues under overnutrition and of the influences of such
gene transcripts on the individual seem to be of remarkable
significance for expatiation of the etiologies of said diseases and
establishment of relevant therapeutic modalities.
[0005] The object of this invention is to cast light on the
obesity-related genes and their expression products which should be
useful for elucidation of the pathogenesis of various
obesity-related diseases, particularly atherosclerotic diseases
such as angina pectoris, myocardial infarction, etc., and
establishment of pertinent therapeutic modalities and to establish
therapeutic and diagnostic methods for the diseases by utilizing
such genes and expression products.
[0006] The inventors have conducted intensive studies for
accomplishing the above object and made it clear previously that
accumulation of fat, particularly visceral fat in the abdominal
cavity, is closely associated with abnormal glucose tolerance,
hyperlipidemia and hypertension. Furthermore, through large-scale
sequencing analyses of the genes expressed in adipose tissue, they
elucidated that many secretory protein genes have been expressed in
adipose tissue and that, particularly in visceral fat, the
expression of various bioactive substance genes can be observed. In
addition to the cloning of those known genes, the inventors
succeeded in cloning an adipose tissue-specific collagen-like
protein apM1 gene [Biochem. Biophys. Res. Commun., 221, 286-289
(1996)].
[0007] This apM1 gene was found to be coding for the secretory
protein (apM1) consisting of 244 amino acid residues, contain a
66-residue collagen-like motif (G-X-Y), and have homology with the
C1q subcomponent of the complement system and collagen X and VII.
However, the physiological function of this gene and its expression
product apM1 remained to be known.
[0008] In the ensuring research, the inventors made a series of
investigations in regard to the expression of said apM1 gene by the
genetic engineering technique, preparation of an antibody against
the expression product apM1, establishment of an apM1 assay system
utilizing said antibody, and relationship of the blood apM1
concentration determined by using said assay system to the body fat
distribution or various diseases. The research led to the novel
finding, inter alia, that apM1 has smooth muscle growth inhibitory
activity and that the blood apM1 concentration faithfully reflects
the atherosclerotic change.
[0009] Furthermore, the inventors obtained the novel finding that
apM1 is effective in the prevention and treatment of
post-angioplasty restenoses, such as restenosis after percutaneous
transluminal coronary angioplasty (PTCA) using a stent, and for
that matter, in the prophylaxis and therapy of atherosclerotic
diseases accompanied by angiopathy, such as angina pectoris and
myocardial infarction. This invention has been developed on the
basis of the above finding.
DISCLOSURE OF INVENTION
[0010] In accordance with the invention, there is provided a smooth
muscle growth inhibitory composition comprising a pharmacologically
effective amount of at least one member selected from the group
consisting of apM1 and its salt in combination with a
pharmaceutically acceptable carrier.
[0011] Furthermore, in accordance with the invention, there is
provided a therapeutic and prophylactic composition for
post-angioplasty restenoses which comprises a pharmacologically
effective amount of at least one member selected from the group
consisting of apM1 and its salt in combination with a
pharmaceutically acceptable carrier.
[0012] There is also provided in accordance with the invention a
prophylactic and therapeutic composition for arteriosclerosis which
comprises a pharmacologically effective amount of at least one
member selected from the group consisting of apM1 and its salt in
combination with a pharmaceutically acceptable carrier.
[0013] There is also provided in accordance with the invention a
method for diagnosis of arteriosclerosis which comprises
quantitating apM1 in a sample with an anti-apM1 antibody and
comparing the value thus found with the values measured in healthy
subjects and in patients with arteriosclerosis.
[0014] In addition, the present invention provides a diagnostic kit
for arteriosclerosis, which comprises an anti-apM1 antibody as an
active component, and a monoclonal anti-apM1 antibody which is
effective in diagnosing the arteriosclerosis.
[0015] The smooth muscle growth inhibitory composition according to
the invention is effective, through its smooth muscle growth
inhibitory activity, in the prophylaxis and therapy of
atherosclerotic diseases accompanied by angiopathy, such as angina
pectoris, myocardial infarction inclusive of thrombosis, brain
infarction, etc. and in the arrest of progression of such
atherosclerotic diseases. In fact, apM1 as the active ingredient of
the composition of the invention has an ability to inhibit
expression of the cell adhesion molecules governing the onset of
arteriosclerosis, namely VCAM-1 (vascular cell adhesion
molecule-1), ELAM (endothelial leukocyte adhesion molecule), ICAM-1
(intercellular adhesion molecule-1), and so on. It is because of
this action that the composition of the invention antagonizes the
onset of various atherosclerotic diseases.
[0016] Consequently, the invention further provides a
pharmaceutical composition for inhibiting the expression of
adhesion molecules in vascular endothelial cells.
[0017] The fact that apM1 inhibits expression of said cell adhesion
molecules indicates that the composition of the invention can be
indicated for the prophylaxis and therapy of bronchial asthma which
is a disease related to type I allergy accompanying eosinophilic
infiltration and also known to be a disease associated with an
enhanced expression of VCAM-1, for instance.
[0018] Furthermore, in view of the fact that said ICAM-1 and ELAM
are known to be inflammation-related adhesion molecules, the
composition of the invention comprising apM1 as an active
ingredient may be indicated as an antiinflammatory agent or a
therapeutic drug for rheumatoid arthritis, for instance, by taking
advantage of said inhibitory effect on the expression of adhesion
molecules.
[0019] Furthermore, the composition of the invention is effective
in the prevention and treatment of post-angioplasty restenoses, for
example in stent PTCA cases. Thus, after an operation for
neovascularization against the coronary artery stenosis in angina
pectoris or myocardial infarction, the post-ischemic reperfusion
and injury of vascular endothelial cells evoke expression of
adhesion molecules in vascular endothelial cells and consequent
proliferation of smooth muscle cells to induce a restenosis. The
composition of the invention inhibits such expression of adhesion
molecules and growth of smooth muscle cells to thereby contribute
to the prevention of ischemic restenoses after angioplasty.
[0020] The method for diagnosis of arteriosclerosis according to
the invention utilizes a new marker, that is smooth muscle growth
potency (DNA-synthesizing ability of smooth muscle cells). The
diagnosis method of the invention is carried out by determining and
quantitating the apM1 level in a sample with a specific antibody
against apM1.
[0021] The production of apM1 for use as the active ingredient of
the composition of the invention, preparation of the composition
using apM1 as an active ingredient, production of an antibody
against apM1, and assay of apM1 are now described in sequence.
[0022] The designation of amino acids, peptides, nucleotide
sequences, nucleic acids, etc. by abbreviations in this
specification is in conformity with the rules of nomenclature
recommended by IUPAC-IUB (IUPAC-IUB Communication on Biological
Nomenclature, Eur. J. Biochem., 138, 9(1984)), "The Guidelines for
Drafting of Specifications Etc. Containing Nucleotide Sequence or
Amino Acid Sequence Information" (Edited by the Japanese Patent
Office, June, 1998) and the conventions in the relevant field of
art.
[0023] apM1 can be provided in the form of a recombinant protein by
the established genetic engineering techniques [e.g. Science, 224,
1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc.
Natl. Acad. Sci., USA., 80, 5990 (1983)]. In this case, as the apM1
gene, the gene which was previously established by the present
inventors can be used [Biochem. Biophys. Res. Commun., 221, 286-289
(1996)].
[0024] As an alternative, apM1 can be produced by the conventional
method for chemical synthesis in accordance with the information on
the amino acid sequence encoded by said gene.
[0025] More particularly, the production of apM1 by a genetic
engineering technique comprises constructing a recombinant DNA with
which the gene coding for the objective protein may be expressed in
a host cell, introducing the DNA into the host cell to obtain a
transformant and culturing the transformant.
[0026] As the host cell mentioned above, cells derived from
eucaryotes and prokaryotes can be employed. The eucaryotic cell
includes cells of vertebrates and cells of eucaryotic
microorganisms. As the cell of a vertebrate, the monkey cell line
COS [Cell, 23, 175 (1981)], the Chinese hamster ovarian cell line
and the corresponding dihydrofolate reductase-deficient cell line
[Proc. Natl. Acad. Sci., USA., 77, 4216 (1980)] and the like are
frequently used but these are not exclusive choices.
[0027] As the expression vector of a vertebrate origin, a vector
having a promoter sequence located upstream of the gene to be
expressed, RNA (precursor) splice site, a polyadenylation site and
a transcription terminating sequence, among others, can be
generally used. Where necessary, the vector may further have a
replication origin. As an example of such expression vector,
pSV2dhfr harboring an early promoter of SV40 can be mentioned (Mol.
Cell. Biol., 1, 854 (1981)).
[0028] As the eucaryotic microorganisms, yeasts are generally used
and, among them, yeasts of the genus Saccharomyces can be used with
advantage. As the expression vector derived from a eucaryotic
microorganism such as a yeast, pAM82 having a promoter for the acid
phosphatase gene [Proc. Natl. Acad. Sci., USA., 80, 1 (1983)],
among others, can be utilized.
[0029] As the prokaryotic host, Escherichia coli and Bacillus
subtilis are generally used most frequently. When they are used as
hosts, it is advantageous to select a plasmid vector which can be
replicated in the host microorganism and in order that the
objective gene may be expressed in the vector, use an expression
plasmid provided with a promoter region and an SD (Shine-Dalgano)
sequence upstream of the gene and, further, with an initiation
codon (e.g. ATG) required for the start of protein synthesis. As
said Escherichia coli as the host, E. coli K12 is generally used,
and as the vector, pBR322 or its modification product is generally
used. However, these are not exclusive choices but the various
known bacterial strains and vectors can likewise be employed.
Examples of the promoter that can be used are tryptophan (trp)
promoter, 1pp promoter, 1ac promoter, and PL/PR promoter.
[0030] Introduction of the resulting recombinant DNA into the host
cell for transformation can be carried out in the routine
manner.
[0031] The transformant obtained can be cultivated by the
conventional manner, whereby the objective recombinant protein is
expressed, produced, and accumulated or secreted intracellularly,
extracellularly or on the cell membrane. The medium for the
cultivation can suitably be selected from among various media in
routine use according to the host cell selected. The cultivation of
the transformant can also be carried out under conditions suited to
the particular host cell.
[0032] Where necessary, the apM1 obtained in the above manner can
be isolated and purified by various separation procedures utilizing
the physical, chemical and other characteristics thereof
[Biochemical Data Book II, 1175-1259, First Edition, 1st
impression, Jun. 23, 1980, published by Tokyo Kagaku Dojin, K.K.;
Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313
(1987), etc.]. More particularly, said isolation and purification
can be achieved by the conventional reconstitution treatment,
treatment with a protein-precipitating (salting-out) agent,
centrifugation, osmotic pressure shock method, sonication,
ultrafiltration, various types of liquid chromatography such as
molecular sieve chromatography (gel filtration), adsorption
chromatography, ion-exchange chromatography, affinity
chromatography, high-performance liquid chromatography (HPLC),
etc., dialysis, and soon, as used either singly or in
combination.
[0033] Alternatively, the apM1 mentioned above can also be produced
by the general method for chemical synthesis based on the amino
acid sequence information. The method includes the conventional
liquid-phase and solid-phase methods for peptide synthesis. In more
detail, each of these methods includes the so-called stepwise
elongation technique which comprises condensing component amino
acids one after another for chain extension according to the amino
acid sequence information, and the fragment condensation technique
which comprises synthesizing fragment peptides each consisting of
several amino acid residues in advance and coupling them together
one after another according to said information.
[0034] The condensation reaction for use in the above method of
peptide synthesis can also be carried out in the conventional
manner. For example, the method which can be used includes the
azide method, mixed acid anhydride method, DCC method, activated
ester method, redox method, DPPA (diphenylphosphoryl azide) method,
DCC+additive (e.g. 1-hydroxybenzotriazole, N-hydroxysuccinimide, or
N-hydroxy-5-norbornene-2- ,3-dicarboximide) method, and Woodward's
method, among others.
[0035] The solvent that can be used in those methods can also
suitably be selected from among those solvents which are well known
to be of use in peptide-forming condensation reactions. As specific
examples, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate,
etc., inclusive of mixtures thereof, can be mentioned.
[0036] In conducting the peptide synthesis reactions, the carboxyl
groups of amino acids or peptides which are not to be involved in
the respective reactions can be protected generally by
esterification, e.g. in the form of a lower alkyl ester, e.g.
methyl ester, ethyl ester or tert-butyl ester, or an aralkyl ester,
e.g. benzyl ester, p-methoxybenzyl ester or p-nitrobenzyl
ester.
[0037] Amino acids having a functional group in the side chain, for
example the hydroxyl group of a tyrosine residue, may be protected
with acetyl, benzyl, benzyloxycarbonyl, tert-butyl or the like,
although such protection is not necessarily indispensable.
[0038] Moreover, the guanidino group of an arginine residue, for
instance, can be protected with a suitable protective group such as
nitro, tosyl, p-methoxybenzenesulfonyl, methylene-2-sulfonyl,
benzyloxycarbonyl, isobornyloxycarbonyl or
adaman-tyloxycarbonyl.
[0039] The reactions for elimination of such protective groups from
the protected amino acids or peptides, or from the final protein,
can also be carried out by the conventional procedures, for example
by the catalytic reduction method or by the method using liquid
ammonia/sodium metal, hydrogen fluoride, hydrogen bromide, hydrogen
chloride, trifluoroacetic acid, acetic acid, formic acid, or
methanesulfonic acid.
[0040] The apM1 thus obtained can be purified by the various
procedures mentioned hereinbefore, for example the procedures in
routine use in peptide chemistry, such as ion exchange
chromatography, partition chromatography, gel permeation
chromatography, counter-current distribution, and so forth.
[0041] The composition of the invention comprises apM1 or a
pharmacologically acceptable salt thereof as an active ingredient.
The salt includes those with alkali metals, alkaline earth metals
and ammonium, such as the sodium, potassium, lithium, calcium,
magnesium, barium and ammonium salts. These salts can be produced
by the methods well known in the art. The above-mentioned salt
further includes acid addition salts which can be prepared by
reacting apM1 with a suitable organic or inorganic acid in the per
se known manner. Examples of the acid addition salts are
hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,
valerate, oleate, laurate, borate, benzoate, lactate, phosphate,
p-toluenesulfonate (tosylate), citrate, maleate, fumarate,
succinate, tartrate, sulfonate, glycolate, ascorbate,
benzenesulfonate, napsylate and like salts.
[0042] The composition according to the invention is generally
provided and put to use in the form of a pharmaceutical preparation
containing a pharmacologically effective amount of said active
ingredient together with a suitable pharmaceutical carrier.
[0043] The carrier that can be utilized in such a pharmaceutical
preparation includes various diluents and/or excipients, such as
fillers, volume builders, binders, humectants, disintegrators,
surfactants, lubricants, and the like. These carriers are
conventionally used according to the desired unit dosage form.
[0044] The unit dosage forms of the pharmaceutical preparation can
be selected from a broad variety according to the therapeutic
objectives. Typical examples include solid forms such as tablets,
pills, powders, fine powders, granules and capsules, and liquid
forms such as a solution, a suspension, an emulsion, syrup and an
elixir. These preparations are classified, by route of
administration, into oral preparations, parenteral preparations,
transnasal preparations, vaginal preparations, rectal
suppositories, sublingual tablets, ointments, and the like, and
each can be formulated and molded or otherwise processed by the
established pharmaceutical procedure. Furthermore, such
pharmaceutical preparations may be supplemented with various
additives which can be formulated in ordinary pharmaceutical
preparations, such as the stabilizer, antibacterial agent, buffer,
isotonizing agent, chelating agent, pH control agent and
surfactant, each at a suitable level.
[0045] The stabilizer includes human serum albumin and those
L-amino acids, carbohydrates and cellulose derivatives which are
conventionally used. These can be used each alone or in combination
with a surfactant or the like. Particularly, such a combination may
contribute to an enhanced stability of the active ingredient.
[0046] The L-amino acids are not particularly restricted but
include glycine, cysteine, glutamic acid and so on.
[0047] The carbohydrates are not particularly restricted but
include monosaccharides such as glucose, mannose, galactose and
fructose; sugar alcohols such as mannitol, inositol and xylitol;
disaccharides such as sucrose, maltose and lactose; and
polysaccharides such as dextran, hydroxypropyl-starch, chondroitin
sulfate and hyalluronic acid; inclusive of derivatives thereof.
[0048] The surfactants are not particularly restricted but ionic
and nonionic surfactants can be employed. Examples of the
surfactants are polyoxyethylene glycol sorbitan alkyl esters,
polyoxyethylene alkyl ethers, sorbitan monoacyl esters, and fatty
acid glycerides.
[0049] The cellulose derivatives are not particularly restricted
but include methylcellulose, ethylcellulose, hdroxyethylcellulose,
hydroxypropylcellulose, hydroxy-propylmethylcellulose,
carboxymethylcellulose sodium and so on.
[0050] The carbohydrates can be used at least about 0.0001 mg,
preferably within the range of about 0.01-10 mg per 1 .mu.g of the
active ingredient. The surfactants can be used at least about
0.00001 mg, preferably within the range of about 0.0001-0.01 mg per
1 .mu.g of the active ingredient. The human serum albumin can be
used at least about 0.0001 mg, preferably within the range of about
0.001-0.1 mg per 1 .mu.g of the active ingredient. The amino acids
can be used within the range of about 0.001-10 mg per 1 .mu.g of
the active ingredient. The cellulose derivatives can be used at
least about 0.00001 mg, preferably within the range of about
0.001-0.1 mg per 1 .mu.g of the active ingredient.
[0051] The proportion of the active ingredient in the
pharmaceutical preparation of the invention can be liberally
selected from a broad range. Generally, the active ingredient
accounts for the range of about 0.00001-70 weight %, preferably
about 0.0001-5 weight % of the final preparation.
[0052] The buffer which may be optionally incorporated in the
pharmaceutical preparation includes boric acid, phosphoric acid,
acetic acid, citric acid, .epsilon.-aminocaproic acid, glutamic
acid and the corresponding salts (e.g. salts with alkali metals or
alkaline earth metals such as the sodium, potassium, calcium and
magnesium salts). The isotonizing agent includes sodium chloride,
potassium chloride, sugars and glycerine, among others. The
chelating agent includes sodium edetate and citric acid.
[0053] The pharmaceutical preparation of the invention encompasses
not only liquid preparations but also lyophilized preparations for
extemporaneous reconstitution as prepared by freeze-drying liquid
preparations for extended shelf lives. The lyophilized preparations
are to be administered after dissolution in water or a buffer
solution inclusive of physiological saline.
[0054] In molding the pharmaceutical composition of the invention
into the tablet form, there can be used, as the carrier, various
excipients such as lactose, sucrose, sodium chloride, glucose,
urea, starch, calcium carbonate, kaolin, crystalline cellulose,
silicic acid and potassium phosphate; binders such as water,
ethanol, propanol, simple syrup, glucose solution, starch solution,
gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose,
methyl cellulose and polyvinyl pyrrolidone; disintegrators such as
sodium carboxymethyl cellulose, calcium carboxymethyl cellulose,
low-substituted hydroxypropyl cellulose, dry starch, sodium
alginate, agar powder, laminaran powder, sodium hydrogen carbonate
and calcium carbonate; surfactants such as polyoxyethylene sorbitan
fatty acid ester, sodium lauryl sulfate and stearyl monoglyceride;
disintegration inhibitors such as sucrose, stearin, cacao butter
and hydrogenated oil; absorption promoters such as quaternary
ammonium base and sodium lauryl sulfate; humectants such as
glycerin and starch; adsorbents such as starch, lactose, kaolin,
bentonite and colloidal silicic acid; and lubricants such as
purified talc, stearic acid salt, boric acid powder and
polyethylene glycol.
[0055] Furthermore, where necessary, the tablets obtained can be
coated with the conventional coating materials to provide
sugar-coated tablets, gelatin-coated tablets, enteric tablets,
film-coated tablets or double- or multi-layer tablets.
[0056] The pills can be prepared using, as the carrier, excipients
such as glucose, lactose, starch, cacao butter, hydrogenated
vegetable oil, kaolin and talc; binders such as gum arabic powder,
gum tragacanth powder, gelatin and ethanol, and disintegrators such
as laminaran and agar.
[0057] Capsules are generally manufactured by formulating the
active ingredient with a carrier or carriers such as those
mentioned above by way of example and encapsulating the composition
using hard gelatin or soft capsule shells, for instance.
[0058] The liquid preparation for oral administration includes
solutions, emulsions, suspensions, syrup and elixirs. Each can be
prepared using a conventional inert diluent, for example a
pharmacologically acceptable vehicle inclusive of water. The liquid
preparation may further be supplemented with various auxiliary
agents such as a wetting agent, an emulsifier and/or a suspending
agent and can be prepared by the established procedure.
[0059] The liquid preparation for parenteral administration, for
example a sterile aqueous or nonaqueous solution, emulsion or
suspension, can be prepared by using a diluent such as water, ethyl
alcohol, propylene glycol, polyethylene glycol, ethoxylated
isostearyl alcohol, polyoxylated isostearyl alcohol,
polyethoxylated sorbitan fatty acid esters and vegetable oils such
as olive oil. The liquid preparation may be supplemented with an
organic ester which can be injected or infused, such as ethyl
oleate. Such preparations may be further supplemented with the
solubilizer, buffer, wetting agent, emulsifier, suspending agent,
preservative, dispersant and other additives.
[0060] Such pharmaceutical preparations can be sterilized by
filtration through a bacterial filter, incorporation of a
bactericide, irradiation treatment, heat treatment, or the like.
Moreover, such pharmaceutical preparations may each be provided in
the form of a sterile solid composition, which can be dissolved in
sterilized water or a suitable sterilizable medium for
extemporaneous sterilization.
[0061] Rectal suppositories and vaginal preparations can be
prepared by using polyethylene glycol, cacao butter, a higher
alcohol, a higher alcohol ester, gelatin or a semi-synthetic
glyceride as the carrier or base.
[0062] Ointments such as pastes, creams and gels can be prepared by
using such a diluent or diluents as white petrolatum, paraffin,
glycerin, cellulose derivatives, propylene glycol, polyethylene
glycol, silicones, bentonite, and vegetable oils such as olive
oil.
[0063] Pharmaceutical preparations for transnasal or sublingual
administration can be prepared using the well-known standard
excipient or excipients in the conventional manner.
[0064] Furthermore, where necessary, the pharmaceutical preparation
of the invention can be supplemented with coloring agents,
preservatives, perfumes, flavors, sweeteners or other
pharmaceutical compositions.
[0065] The method for administration of said pharmaceutical
preparation is not particularly restricted but can be judiciously
selected with reference to the dosage form, patient background
inclusive of age and sex, severity of illness, and other
conditions. For example, the tablets, pills, solutions,
suspensions, emulsions, granules and capsules are orally
administered. The injections are intravenously administered either
alone or in admixture with an ordinary infusion such as glucose and
amino acid, and if necessary, administered alone intramuscularly,
intradermally, subcutaneously or intraperitoneally. The
suppositories are inserted into the rectum, while vaginal
preparations are administered into the vagina. Transnasal
preparations are administered into the nostrils, sublingual
preparations into the oral cavity, and ointments for transdermal
absorption.
[0066] The amount of the active ingredient to be contained in said
pharmaceutical preparation and the dosage are not particularly
restricted but each can be judiciously selected from a broad range
according to the expected therapeutic effect, method of
administration, treatment time, patient factors such as age and
sex. The dosage is usually selected so that the blood concentration
of the active ingredient will be preferably about 1-200 .mu.g/ml,
more preferably about 10-20 .mu.g/ml. This preparation can be
administered once or in a few divided doses a day.
[0067] Production of a specific antibody against apM1 is now
described in detail. The specific antibody against apM1 can be
produced as an antiserum (polyclonal antibody) or a monoclonal
antibody by utilizing apM1, a fragment thereof or a complex protein
containing it as a hapten as an immunogen.
[0068] The technology for producing such antibodies are well
understood by those skilled in the art. The antibody according to
the invention can also be produced in accordance with the known
method [e.g. Biochemical Experiments Series: "Methods for
Immunobiochemical Research", Japanese Biochemical Society (ed.)
(1986)].
[0069] More particularly, the monoclonal anti-apM1 antibody can be
produced by, for example, a process which comprises constructing a
fusion cell (hybridoma) between a plasmocyte (immunocyte) from a
mammal immunized with said immunogen and a plasmacytoma cell of
mammal origin, selecting a clone producing the desired
apM1-recognizing antibody, and cultivating the clone.
[0070] The apM1 that can be used as an immunogen in the above
procedure is not particularly restricted but may be any of the
known recombinant apM1 species prepared by the recombinant DNA
technology, a peptide having a partial amino acid sequence thereof,
or the corresponding conjugated protein containing a prosthetic
group. The apM1 mentioned above is known to be an adipose
tissue-specific secretory factor and any protein having the
equivalent activity or action, for example GBP 28 (gelatin-binding
protein of 28 kDa), can also be used likewise as said
immunogen.
[0071] The mammal to be immunized with said immunogen in the above
procedure is not particularly restricted but is preferably selected
from the standpoint of compatibility with the plasmacytoma cell to
be used in cell fusion. Generally, the mouse, rat or rabbit is used
with advantage.
[0072] The immunization of said mammal is carried out by the
routine method, for example by injecting said immunogen by the
intravenous, intradermal, subcutaneous, or intraperitoneal route.
Preferably, the immunogen is administered alone or optionally in
combination with an ordinary adjuvant to a laboratory animal, such
as the mouse, several times at 2- to 14-day intervals, in a total
dose of about 100-500 .mu.g/mouse. As the immunogen, it is
preferable to use splenocytes isolated about 3 days after the last
immunization.
[0073] As regards the mammalian plasmacytoma cell as the other
parent cell to be fused with the immunocyte, any of the various
cells already known, for example p3 (p3/.times.63-Ag8) [Nature,
256, 495-497 (1975)], p3-U1 [Current Topics in Microbiology and
Immunology, 81, 1-7 (1978)], NS-1 [Eur. J. Immunol., 6, 511-519
(1976)], MPC-11 [Cell, 8, 405-415 (1976)], SP2/0 [Nature, 276,
269-270 (1978)], FO [J. Immunol. Meth., 35, 1-21 (1980)],
.times.63.6.5.3. [J. Immunol., 123, 1548-1550 (1979)], S194 [J.
Exp. Med., 148, 313-323 (1978)], etc. and myeloma cells such as rat
R210 [Nature, 277, 131-133 (1979)], can be employed.
[0074] The cell fusion reaction between said immunocyte and
plasmacytoma cell can be carried out generally in accordance with
the method of Milstein et al. [Methods in Enzymology, Vol. 73, p. 3
(1981)], for instance. More particularly, the above fusion reaction
can be conducted in a usual medium in the presence of an ordinary
fusion inducer, such as polyethylene glycol (PEG), Sendai virus
(HVJ) or the like. To achieve an improved fusion efficiency, the
medium may be optionally supplemented with an auxiliary agent such
as dimethyl sulfoxide.
[0075] The ratio of the immunocyte to the plasmacytoma cell for use
is not different from the ratio commonly used in this type of
procedure. Thus, for example, the immunocyte is generally used in a
proportion of about 1- to 10-fold as large as the amount of the
plasmacytoma cell. Examples of the medium useful for the cell
fusion are RPMI-1640 and MEM, which are generally used for
proliferation of plasmacytoma cells, and other media which are used
for cell culture of this kind. It is usually preferable that the
serum component such as fetal calf serum (FCS) be omitted from the
medium formulations.
[0076] Fusion is achieved by a procedure which comprises mixing
predetermined amounts of said immunocyte and plasmacytoma cell
thoroughly in said medium and adding a solution of PEG, for example
a PEG with an average molecular weight of about 1000-6000, which
has been prewarmed to about 37.degree. C., to the medium usually in
a concentration of about 30-60 w/v %, followed by stirring.
Thereafter, serial addition of a suitable medium, centrifugation
and removal of the supernatant are repeated until the desired
hybridoma has been obtained.
[0077] The hybridoma thus produced can be isolated by cultivating
it in a usual selection medium, such as HAT (a medium containing
hypoxanthine, aminopterin and thymidine). This culture using HAT
medium is carried out for a sufficient time to kill the cells
(unfused cells etc.) other than the desired hybridoma, usually for
several days to a few weeks. The resulting hybridoma is subjected
to a search for the clone producing the desired antibody by the
usual limiting dilution method, followed by the production of
monoclonal antibody.
[0078] The search for the desired antibody-producing clone can be
carried out by the various methods in routine use for detection of
antibodies, such as ELISA [Engvall, E., Meth. Enzymol., 70, 419-439
(1980)], plaque method, spot method, agglutination method,
Ouchterlony method and radioimmunoassay (RIA) [Hybridoma Techniques
and Monoclonal Antibodies, published by R&D Planning, K.K., pp.
30-53, Mar. 5, 1982]. The immunogen mentioned hereinbefore can be
used for the purpose of this search.
[0079] The thus-obtained hybridoma producing the desired monoclonal
antibody which recognizes apM1 can be subcultured in ordinary media
and can also be stored for a long time in liquefied nitrogen.
[0080] Harvesting of the desired antibody from the above hybridoma
can be effected by the method which comprises cultivating the
hybridoma in the routine manner and harvesting the antibody as a
culture supernatant or the method which comprises inoculating a
compatible mammal with the hybridoma to cause it to multiply and
harvesting the antibody as an ascites fluid. The former method is
suitable for preparation of the antibody of high purity and the
latter method is suitable for mass production of the antibody.
[0081] The antibody thus obtained can be purified by the
conventional procedure such as precipitation, gel filtration,
affinity chromatography, and so forth. In this manner, the desired
anti-apM1 monoclonal antibody which specifically binds apM1 can be
obtained.
[0082] The invention further provides a technique for the assay of
apM1 in a sample and an associated method for diagnosis of
arteriosclerosis.
[0083] The diagnosis of arteriosclerosis according to the invention
comprises determining the amount of apM1 in a patient's blood or
urine sample with the anti-apM1 antibody by a liquid-phase or
solid-phase immunoassay technique and comparing the measured value
with the corresponding value in patients with arteriosclerosis and
the corresponding value in healthy persons to see whether the apM1
level in the sample is higher or lower than the level in healthy
persons.
[0084] The preferred immunoassay method is ELISA by the sandwich
technique.
[0085] The above method is now described in detail. Its principle
is based on the enzyme antibody method. Thus, this method typically
comprises sowing an anti-human apM1 monoclonal antibody (the first
antibody) on a 96-well plate and, to prevent nonspecific
adsorption, carrying out blocking (immobilization). To this
monoclonal antibody-immobilized plate, the human apM1 standard
solution or the test sample is added and reacted (the first
reaction). After the plate is washed, the anti-human apM1 antibody
(the second antibody) is added and reacted (the second reaction).
The plate is washed and the HRP-labeled anti-rabbit IgG antibody
(the third antibody) is added and reacted (the third reaction).
After the plate is washed, the substrate is added and the enzymatic
reaction is carried out (the fourth reaction). Then, the activity
is read as the absorbance at the wavelength of 492 nm.
[0086] In the above procedure, it is also possible to use the
polyclonal antibody as the first antibody and the monoclonal
antibody as the second antibody.
[0087] In the above method, the higher the concentration of apM1 in
the standard solution or test sample used is, the higher is the
enzyme activity (absorbance) detected. By constructing a standard
curve (calibration curve) by plotting the absorbance values of
standard solutions and comparing the absorbance of the test sample
with the curve, the amount of apM1 in the test sample can be
expediently found to diagnose whether the patient has
arteriosclerosis or not.
[0088] Furthermore, the above diagnosis of arteriosclerosis
according to this invention can be carried out more expediently by
means of a kit and this invention further provides such a kit.
[0089] The kit of the invention and the method of detecting the
amount of apM1 in a test sample are now described in detail.
[0090] In the assay procedure using the kit of the invention, the
test sample is preferably a urine sample or a blood sample
(particularly a serum or plasma sample from fasted blood), and such
a sample can be obtained and prepared from the test subject in the
routine manner.
[0091] The kit according to the invention comprises an anti-apM1
monoclonal or polyclonal antibody (anti-apM1 antibody) as an
essential component, preferably a combination thereof with the
anti-apM1 polyclonal antibody or monoclonal antibody, respectively,
as the case may be, or a combination of said monoclonal
antibodies.
[0092] The monoclonal antibody mentioned above is preferably used
as immobilized by coupling it to a carrier on a plate in advance.
As an alternative, said antibody can be directly used in an
affinity gel form or used as the gel prepared in a vessel or test
tube which can be shaken and centrifuged and lends itself to
extraction of the non-affinity gel-bound fraction.
[0093] It is also preferable to equilibrate said
antibody-immobilized plate or gel in advance with a suitable buffer
solution, for example 0.01M Tris-HCl buffer (pH 7.4)+0.15M NaCl.
Furthermore, said antibody-immobilized plate or gel may be
supplemented with a usual preservative such as sodium azide.
[0094] More preferably, as an additional component of the kit of
the invention, a labeled anti-human apM1 polyclonal antibody is
supplied as the second antibody for ELISA. Where necessary, the kit
of the invention may further comprise a stabilizer such as
saccharose or bovine serum protein and/or a preservative. The
preservative is selected from among substances which do not affect
the test result with the kit and includes a diluted solution of
sodium azide as a representative example.
[0095] Moreover, the kit of the invention may optionally comprise a
water-soluble or -miscible substance such as glycerin, alcohol,
glycol, glycoether or the like and, for degreasing purposes, a
mixed organic solvent such as ethanol-diethyl ether,
methanol-diethyl ether, or chloroform-methanol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] FIG. 1 shows a restriction enzyme map of a plasmid
containing the nucleotide sequence of the apM1 gene.
[0097] FIG. 2 shows a standard curve constructed by using a
recombinant apM1.
[0098] FIG. 3 is a graph showing the smooth muscle antagonizing
effect of apM1 as measured in accordance with Example 3.
[0099] FIG. 4 show diagrammatically the blood apM1 levels in
patients with coronary artery disease and healthy subjects as
determined in accordance with Example 4.
[0100] FIG. 5 is a graph showing the concentration-dependent
inhibitory effect of apM1 on the expression of an adhesion molecule
on the cell surface as determined in accordance with Example 5.
BEST MODE FOR CARRYING OUT THE INVENTION
[0101] The following examples illustrate the invention in further
detail and should not be limitative of the scope of the
invention.
EXAMPLE 1
[0102] Production of a Recombinant apM1
[0103] (1) Expression of apM1 in Escherichia coli
[0104] 1) apM1 PCR
[0105] The nucleotide sequence of the apM1 gene and the amino acid
sequence encoded thereby have been deposited with the GenBank under
the accession number of D45371. The coding region (CDS) is shown as
the sequence from 27th to 761st of the nucleotide sequence. Its
deduced amino acid sequence is shown in SEQ ID NO:1. In this
sequence, the 1st to 14th residues constitute a signal peptide and
the 15th to 244th residues represent the mature apM1.
[0106] The apM1 gene was amplified by PCR using the plasmid donated
by Dr. Funahashi, the Second Department of Internal medicine, Osaka
University School of Medicine as a template. The restriction enzyme
map of the plasmid containing the nucleotide sequence of the apM1
gene is shown in FIG. 1.
[0107] Designing so that the 693 bp sequence from 69th to 761st of
the nucleotide sequence of the apM1 would be amplified with an NdeI
site at the 5'-end and a BamHI site at the 3'-end, PCR primers were
produced using an automatic DNA synthesizer. The PCR primer
sequences are shown in SEQ ID NO:3 (forward) and NO:4
(reverse).
[0108] 2) Subcloning of the apM1 Gene
[0109] The PCR product obtained in the above step 1) was subcloned
into pT7 Blue T-Vector (Novagen) and it was confirmed that there
was no mutation in its nucleotide sequence (pT7-apM1).
[0110] 3) Construction of an Expression Vector
[0111] The expression vector pET3c (Novagen) was digested with NdeI
and BamHI to recover a fragment of approximately 4600 bp. On the
other hand, the pT7-apM1 obtained in the above step 1) was digested
with NdeI and BamHI to recover a fragment of approximately 700 bp.
These fragments were ligated and the expression vector thus
obtained was named pET3c-apM1.
[0112] 4) Expression in Escherichia coli
[0113] The host E. coli strain BL21(DE3)pLysS was transformed with
the pET3c-apM1 constructed in the above step 3) and cultured in
2xT.Y.Amp. (tryptone 16 g, yeast extract 10 g, and NaCl 5 g). When
the organism had entered into the logarithmic growth phase, IPTG
(isopropyl .beta.-D-thiogalactopyranoside) was added for inducing
the production of a recombinant apM1. The E. coli cells before and
after this IPTG induction and the inclusion body (the insoluble
fraction of E. coli) after said IPTG induction were sampled and
subjected to SDS-PAGE and Western blotting to confirm the
expression of apM1.
[0114] 5) Results and Discussion
[0115] The expression product in E. coli, obtained in the above
manner, was a 230-residue protein corresponding to the .sup.15Gly
to .sup.244Asn, exclusive of the signal sequence, of the amino acid
sequence of apM1, with the addition of Met derived from the
initiation codon at the N-terminus. This amino acid sequence is
shown in SEQ ID NO:2.
[0116] The E. coli obtained by the above procedure was analyzed by
SDS-PAGE. As a result, an approximately 30 kD band could be
confirmed in the E. coli cell and inclusion body after IPTG
induction.
[0117] Then, Western blotting was performed using two kinds of
antibodies [polyclonal antibodies (synthetic peptides)]. Both
antibodies reacted with said approximately 30 kD band, whereas no
reaction was detected at all with the host E. coli.
[0118] The above approximately 30 kD band was excised to
investigate the sequence of its 10 amino acid residues at
N-terminal. The sequence was the same as the expected sequence,
with the deletion of the N-terminal Met having been found in a
minor population.
[0119] It became clear from the above results that the recombinant
apM1 had been expressed as an approximately 30 kD protein. Most of
the recombinant apM1 expressed had been intracellularly accumulated
as an inclusion body.
[0120] (2) Purification of the Recombinant apM1 from E. coli
[0121] Purification of the recombinant apM1 from E. coli was
carried out by the following 5-step procedure.
[0122] 1) Culture of E. coli
[0123] The E. coli BL21(DE3)pLysS (Novagen) transformed with the
expression vector pET3c-apM1 was precultured in 2xT.Y.Amp.Cm.
(tryptone 16 g, yeast extract 10 g, chloramphenicol 25 .mu.g/ml,
and NaCl 5 g) (37.degree. C., shake culture). On the following day,
the culture was diluted with 100 volumes of 2xT.Y.Amp. and further
incubated. After 2-3 hours of incubation when the OD550 of the
culture fluid had become 0.3-0.5, IPTG was added at a final
concentration of 0.4 mM for inducing the production of recombinant
apM1. About 3-5 hours after addition of IPTG, the culture fluid was
centrifuged (5000 rpm, 20 min., 4.degree. C.) and the E. coli
pellet thus obtained was stored frozen.
[0124] 2) Preparation of an Inclusion Body from E. coli
[0125] The E. coli pellet was suspended in 50 mM Tris-HCl (pH 8.0)
and treated with lysozyme at 37.degree. C. for 1 hour. Then, Triton
X-100 (Katayama Kagaku) was added at a final concentration of 0.2%.
This solution was sonicated (Branson Sonifier, output control 5, 30
sec.) and centrifuged (12000 rpm, 30 min, 4.degree. C.) and the
pellet was recovered. This pellet was suspended in 25 ml of 0.2%
Triton X-100-supplemented 50 mM Tris-HCl (pH 8.0) and the
suspension was sonicated (under the same conditions as above).
[0126] The resulting solution was centrifuged and the pellet was
washed by the same procedure as above. The pellet thus obtained was
taken as the inclusion body.
[0127] 3) Refolding of the Inclusion Body
[0128] The inclusion body was solubilized with a small quantity of
7 M guanidine HCl, 100 mM Tris-HCl (pH 8.0) and 1% 2ME. This
solution was dropped into 200-fold volume of 2 M urea, 20 mM
Tris-HCl (pH 8.0) for dilution and allowed to stand at 4.degree. C.
for 3 nights.
[0129] 4) Concentration of the Refolded Solution
[0130] The solution after the above refolding was centrifuged (9000
rpm, 30 min, 4.degree. C.) and the supernatant was concentrated to
about {fraction (1/100)} by ultrafiltration using an Amicon YM-10
membrane. This concentrate was dialyzed against 20 mM Tris-HCl (pH
8.0) and the dialysate was filtered through a 0.45 .mu.m
filter.
[0131] 5) DEAE-5PW Anion-Exchange HPLC
[0132] The sample obtained in the above step 4) was fractionally
purified by anion-exchange high performance liquid chromatography
(HPLC) with DEAE-5PW (Tosoh Corporation). As the starting buffer,
20 mM Tris-HCl (pH 7.2) was used, and elution was carried out on a
NaCl gradient (0.fwdarw.1M NaCl/60 ml) under absorbance monitoring
at 280 nm. The eluate was collected in 1 ml fractions and each
fraction was analyzed by SDS-PAGE.
[0133] 6) Results and Discussion
[0134] Because the recombinant apM1 had been expressed as an
inclusion body in E. coli, its purification was carried out by
solubilization and refolding of the inclusion body. As a result,
the recombinant apM1 was solubilized and separated on the
anion-exchange column. The peak fractions (fraction Nos. 30-37)
were pooled and analyzed by SDS-PAGE. As a result, an approximately
30 kD band was observed. In this analysis, a faint smear band was
detected on the background but as most of the protein was
considered to be the recombinant apM1. This approximately 30 kD
band (recombinant apM1) was used as the antigen in the subsequent
immunization of rabbits and mice.
[0135] (3) Preparation of Anti-apM1 Polyclonal and Monoclonal
Antibodies
[0136] 1) Preparation of the Polyclonal Antibody
[0137] The recombinant apM1, 100 .mu.g/body, was mixed with
complete adjuvant in a 1:1 ratio and 5 rabbits were immunized with
the mixture 8 times at 2-week intervals to obtain an anti-apM1
polyclonal antibody (Identification codes: OCT9101-OCT9105).
[0138] 2) Preparation of the Monoclonal Antibody
[0139] The recombinant apM1, 20 .mu.g/body, was mixed with complete
adjuvant in a 1:1 ratio and mice were immunized with the mixture 3
times at 2-week intervals. Then, the final immunization was carried
out without the adjuvant 3 days before cell fusion. Cell fusion
between the mouse spleen cell and myeloma cell was carried out by
the PEG method and the hybridoma was selected in HAT medium.
[0140] Screening for an apM1 antibody-producing cell line was
carried out by ELISA using the antigen (recombinant apM1)-coated
immunoplate, and the hybridoma was cloned by the limiting dilution
method.
[0141] In the above manner, 11 anti-apM1 antibody producing
hybridoma lines named KOCO9101-KOCO9111 were obtained. One
hybridoma, among them, was deposited with the National Institute of
Bioscience and Human Technology, the Ministry of International
Trade and Industry, Japan (NIBH, Higashi 1-1-3, Tsukuba-shi,
Ibaraki, Japan) as of Jun. 8, 1998 (original deposit date) (the
identification code assigned by the depositor: KOC09108) and the
demand for conversion to the deposit under Budapest Treaty was
filed as of Oct. 7, 1998. The accession number of the final deposit
is FERM BP-6542.
[0142] The hybridomas as single clones were respectively
administered intraperitoneally to mice treated with pristane in
advance and the ascites fluid was harvested (Identification codes:
ANOC9101-9111).
[0143] 3) Purification of Antibodies
[0144] The rabbit antiserum (polyclonal antibody) and mouse ascites
fluid (monoclonal antibody) were respectively purified using a
protein A column.
[0145] 4) Expression of apM1 in Animal Cells
[0146] The cDNA of the apM1 was excised with EcoRI and inserted
into the EcoRI site of the expression vector pCIneo (Promega
Corp.). The COS-1 cell (ATCC CRL1650) was transfected with the
above pCIneo-apM1 using LlipofectAMINE, GIBCO BRL, and the culture
supernatant and the cells were respectively harvested after 72
hours.
[0147] 5) Western Blotting of apM1
[0148] First, the adipose tissue extract, COS-1 cells, COS-1 cell
culture supernatant, healthy human plasma, and recombinant apM1
were subjected to 2 ME(+) SDS-PAGE and transferred to a
nitrocellulose membrane.
[0149] This membrane was reacted with the anti-apM1 monoclonal
antibody (ANOC9104) and, then, with the HRP-labeled antibody, and
detection was carried out with ECL (Western blot detecting reagent,
Amersham).
[0150] As a result, an approximately 35 kD band was detected for
the adipose tissue extract, pCIneo-apM1/COS-1 cell, and healthy
human plasma but was not observed for pCIneo/COS-1 cell or
pCIneo/COS-1 cell culture supernatant.
[0151] With the culture supernatant of pCIneo-apM1/COS-1 cells, a
35 kD band could be confirmed although it was too weak in intensity
to be readily discernible.
EXAMPLE 2
[0152] Determination of apM1 in Samples
[0153] 1) Western Blotting of apM1
[0154] Healthy human plasma was diluted 1:10 with PBS and 5 .mu.l
of the dilution was subjected to 2ME(+), (-)SDS-PAGE and
transferred to a nitrocellulose filter. After blocking, the filter
was reacted with a 1000-fold dilution of the anti-apM1 polyclonal
antibody (OCT9101-9105) or 5 .mu.g/ml of the anti-apM1 monoclonal
antibody (ANOC9101-1) and further with HRP-labeled anti-rabbit IgG
antibody or HRP-labeled anti-mouse IgG antibody, and detection was
carried out with ECL.
[0155] As a result, with all the polyclonal antibodies, an apM1
band of approximately 35 kD in the case of 2ME(+) and that of
approximately 70 kD in the case of 2ME(-) could be confirmed. Among
the monoclonal antibodies, ANOC9104 and ANOC9108 reacted intensely
with the apM1 band. The above results suggested that as the
monoclonal antibody for use in ELISA, ANOC9104 and ANOC9108 are
suitable.
[0156] 2) Construction of the apM1 ELISA System
[0157] The anti-apM1 monoclonal antibody (ANOC9108) was coated on
an immunoplate and, after each well was blocked, the apM1 standard
and sample were added and incubated. After each well was washed, a
dilution of anti-apM1 polyclonal antibody (OCT9104) was added and
incubated. After each well of the plate was washed, a dilution of
HRP-labeled anti-rabbit IgG antibody was added and incubated. After
each well was washed, OPD was added to each well for staining and
the absorbance at 492 nm was measured. As the apM1 standard, the
recombinant apM1 expressed in E. coli, purified and quantitated for
protein by a protein assay using BSA as reference was used.
[0158] As a result, the detection range with the above combination
of ANOC9108 and OCT9104 was 0.1 ng/ml-20 ng/ml.
[0159] 3) Gel Filtration and Western Blotting of Healthy Human
Plasma
[0160] When healthy human plasma was measured by the above apM1
ELISA, the concentration expected from the result of Western
blotting analysis could not be obtained. Therefore, the healthy
human plasma was subjected to Superose 12 (Pharmacia)
gel-filtration and each fraction was subjected to SDS-PAGE and
analyzed by Western blotting with ANOC9104. The molecular weight
markers were also subjected to gel filtration under the same
conditions and their positions were compared with the elution
position of apM1.
[0161] As a result, apM1 was found to have been broadly eluted in
the fractions corresponding to not less than 290 KD in molecular
mass. The above findings suggested that apM1 in blood is associated
with the other plasma components to form large molecules not less
than 290 kD in molecular mass so that the antibody recognition site
is masked. With the thought, therefore, that if the plasma be
treated with an SDS-containing buffer, the antibody might be
enabled to react with apM1, the following study was undertaken.
[0162] 4) Treatment of a Plasma Sample
[0163] The plasma was boiled in an SDS-containing buffer and the
conditions of treatment (boiling time, mixing ratio of plasma to
SDS buffer) were studied. As to boiling time, the healthy human
plasma was diluted 10-fold with SDS buffer and boiled for 10 sec.,
30 sec., 1 min., 3 min., 5 min. or 10 min. and after final dilution
to 5000-fold from the original plasma, the assay of apM1 was
carried out. As regards the mixing ratio of plasma to SDS buffer, a
2, 3, 5, 10 or 20-fold dilution of healthy human plasma in SDS
buffer was boiled for 5 minutes and, after final dilution to
10000-fold, the assay of apM1 was carried out.
[0164] As a result, apM1 could be detected at levels of the same
order as the results of Western blotting of healthy human plasma.
Comparison of the above different conditions of treatment suggested
that it was appropriate to dilute plasma 10-fold in SDS buffer and
boil the dilution for 5 minutes.
[0165] 5) Dilution of the Plasma Sample
[0166] In view of the finding that the detection range of apM1
ELISA is 70 .mu.g/ml-20 ng/ml and that the blood apM1 concentration
is of the .mu.g order, it was considered that serum (plasma) must
be diluted prior to assay. Therefore, in order to determine the
proper dilution factor, the SDS-treated plasma was serially diluted
and apM1 in each dilution was assayed. Thus, healthy human serum
was treated with SDS and, then, diluted 200-10.times.10.sup.4-fold
serially and apM1 was assayed.
[0167] As a result, within the measurement range of this ELISA,
apM1 was detected in proportion to dilution factor, and in
consideration of absorbance, among other conditions, it was
considered appropriate to perform assays at an about 5000-fold
final dilution.
[0168] 6) Study of Blood Sampling Conditions
[0169] Sampling of blood from healthy subjects was carried out
under various conditions: serum, heparin and EDTA, and the
differences in apM1 titer according to differences in blood
sampling conditions were investigated. Thus, blood sampling was
carried out in 10 healthy subjects and each serum (plasma) sample
was treated with SDS and apM1 was assayed in a final 5000-fold
dilution.
[0170] As a result, blood apM1 levels in the 10 healthy subjects
showed substantially no difference due to the difference in blood
sampling method.
[0171] 7) Study of Sample Storage Conditions
[0172] The conditions of storage of samples for apM1 assays were
studied with plasma and SDS-treated plasma samples. Regarding
plasma storage conditions, samples left standing at 4.degree. C.,
room temperature, or 37.degree. C. for 1, 2, 3 and 6 days,
respectively, were treated with SDS and, to investigate the
influence of the freeze-thaw procedure on plasma, samples subjected
to 1, 2, 4 and 8 freeze-thaw cycles, respectively, were treated
with SDS, and apM1 was assayed in final 5000-fold dilutions. As to
SDS-treated samples, the plasma was diluted 10-fold with SDS
buffer, boiled for 5 minutes, diluted 10-fold, and subjected to the
same experiment as above.
[0173] As a result, under the above conditions, little influence
was noted on the measured values of apM1. While the influence of
the freeze-thaw procedure was also investigated using plasma and
SDS-treated plasma samples, little influence was observed.
[0174] 8) Study of Diurnal Variation and Circadian Variation
[0175] The diurnal and circadian variations in the measured values
of apM1 in the sera (plasmas) of healthy subjects were studied. As
regards the diurnal variation, the same sample was assayed 8 times
and CV was calculated. As to the circadian variation, the same
sample was assayed 4 times on different days and CV was
calculated.
[0176] As a result, the CV of diurnal variation was within 5% and
the CV of circadian variation was within 10%.
[0177] 9) Specificity Study
[0178] Swine, bovine, equine, goat, rat and mouse sera were
subjected to apM1 ELISA and the specificity of the assay system was
studied. The serum from each animal species was treated with SDS
buffer and apM1 was assayed in a final series of 100-8100-fold
dilutions.
[0179] As a result, whereas a cross sensitivity of about 10% was
found between bovine and equine sera, substantially no cross
sensitivity was observed among other animal species.
EXAMPLE 3
[0180] The Smooth Muscle Growth Inhibitory Effect of apM1
[0181] A plastic plate was sown with human arterial smooth muscle
cells (Clontech) at a concentration of 1.times.10.sup.4/cm.sup.2
and allowed to stand overnight in DMEM (Gibco) supplemented with
10% FBS (Gibco), 100 IU/ml penicillin and 100 .mu.g/ml streptomycin
and, then, incubated in 5% CO.sub.2+95% air at 37.degree. C.
[0182] The DNA synthesis into the human arterial smooth muscle
cells was quantitated according to [methyl .sup.3H]-thymidine
uptake (4 replicates).
[0183] Thus, the cells sown on a 96-well plate were treated with 10
.mu.g/ml of apM1 and/or, as control, 10 ng/ml of HB-EGF
(recombinant human heparin-binding EGF-like growth factor, R&D
Systems) in 2% FBS-DMEM for 24 hours. Then, [methyl
.sup.3H]-thymidine was added, 1 .mu.Ci/well, over 5 hours. The
cells were then treated with trypsin and, using an automatic cell
harvester, taken out onto a glass fiber filter. Then, the amount of
[methyl .sup.3H]-thymidine uptake was directly measured with a
.beta.-counter.
[0184] The results are presented in FIG. 3. This bar graph was
constructed by plotting the relative ratio of DNA synthesis on the
ordinate against control (no test drug added), addition of apM1
(this invention), addition of HB-EGF (control), and addition of
apM1+HB-EGF (this invention) on the abscissa.
[0185] It is apparent from the graph that apM1 antagonizes the DNA
synthesis of human smooth muscle cells (growth of smooth muscle).
Thus, by the addition of apM1, the control DNA synthesis can be
significantly (p<0.001) inhibited and even the DNA synthesis
augmented by HB-EGF can also be significantly (p<0.001)
inhibited.
[0186] These results indicate that apM1 is an effective smooth
muscle growth inhibitor.
EXAMPLE 4
[0187] Determination of Blood apM1 Levels in Patients With Coronary
Artery Disease
[0188] In 24 male and 10 female patients verified to have a
stenosis of not less than 75% by coronary angiography (indicated as
CAD(+)) and 66 male and 39 female patients with a stenosis of less
than 75% (indicated as CAD(-)), the blood was collected and the
plasma apM1 concentration was determined by the method of the
invention as described in Example 2.
[0189] At the same time, a CT scan was taken at the umbilical level
of each patient and the visceral fat area (VFA, cm.sup.2) was
calculated from CT findings.
[0190] With the plasma apM1 level (.mu.g/ml) being plotted on the
ordinate and VFA on the abscissa, the correlation between the
measured value and calculated value was analyzed by the unpaired
t-test for the values other than CAD(+) and CAD(+). The results are
presented in FIG. 4.
[0191] It is apparent from the graph that the apM1 levels in the
patients with coronary artery disease [CAD(+) group] were
invariably low regardless of the magnitude of visceral fat area
(VFA), indicating that apM1 is effective in diagnosing an advanced
coronary artery stenosis.
[0192] It was also found that apM1 is an important marker of the
onset and progression of arteriosclerosis, suggesting its
usefulness in the therapy of the disease.
EXAMPLE 5
[0193] The Inhibitory Effect of apM1 on the Onset of
Arteriosclerosis
[0194] Human aortic vascular endothelial cells (HAEC; purchased
from Clontech) sown on a 96-well plate were cultured (5% CO.sub.2,
37.degree. C.) in the vascular endothelial cell culture medium.
Using Becton Dickinson's "Biocoat" (trade name), culture was
continued until the growth had become confluent, after which time
the medium was changed to TCM 199 [Tissue culture medium 199; Osaka
University Microbial Research-Nakalai Tesque]+0.5% FCS (fetal calf
serum; Japan Biomaterials)+3% BSA (bovine serum albumin;
Sigma).
[0195] Then, the recombinant apM1 obtained in Example 1 was added
in varying amounts, i.e. 1, 5, 10, 25 and 50 .mu.g/ml, and the
system was further incubated for 18 hours. Thereafter, human
recombinant TNF-.alpha. (tumor necrosis factor-.alpha., R & D;
10 U/ml) was added and the system was further incubated for 6 hours
(the apM1-added experimental group).
[0196] As a control, the group without addition of said recombinant
apM1 (TNF-.alpha. stimulation only) was provided.
[0197] Using the cell-ELISA method (Takami, S., et al.,
Circulation, 97 (8), 721-728 (1998)), it was analyzed whether apM1
would suppress the expression of adhesion molecule proteins, namely
VCAM-1 (vascular cell adhesion molecule-1), ELAM (endothelial
leukocyte adhesion molecule) and ICAM-1 (intercellular adhesion
molecule-1), on the HAEC surface by the above TNF-.alpha.
stimulation. As the anti-ICAM-1 antibody, DAKO's 6.5B5 was
used.
[0198] The apM1-added group was compared with the apM1-free group
and the difference was tested for statistical significance by
Student's test.
[0199] The results for VCAM-1 are presented in FIG. 5.
[0200] In FIG. 5, the abscissa represents the level of addition of
apM1 (.mu.g/ml;--indicates no addition) while the ordinate
represents the relative expression level of the adhesion molecule
protein (VCAM-1) in each experimental group or control group, with
the expression level of VCAM-1 on the cell surface of HAEC in the
absence of TNF-.alpha. stimulation being taken as unity (1).
[0201] The following can be deduced from FIG. 5. Thus, beginning at
the concentration of 1 .mu.g/ml, apM1 inhibited the
TNF-.alpha.-augmented expression of the adhesion molecule VCAM-1 in
HAEC significantly (p<0.05) and dose-dependently.
[0202] It was also found that the expression of the other major
adhesion molecules ELAMI and CAM-1 was similarly inhibited by
apM1.
[0203] It has been reported that injuries to vascular endothelial
cells and the resultant monocyte adhesion are cardinal factors in
the pathogenesis of arteriosclerosis [Ross, R., Nature, 362 (6423),
801-804 (1993)]. The finding that apM1 inhibited expression of the
adhesion molecules decisive of the onset of arteriosclerosis
(VCAM-1, ELAM, ICAM-1, etc.) indicates that apM1 acts in an
inhibitory way on the onset of arteriosclerosis, thus being
effective as a prophylactic agent for arteriosclerosis.
INDUSTRIAL APPLICABILITY
[0204] The invention provides a smooth muscle growth inhibitory
composition and a composition for inhibiting the expression of
adhesion molecules in vascular endothelial cells, both of which
comprise apM1 as an active ingredient. These compositions of the
invention are useful in the pharmaceutical field. Furthermore, the
invention provides a method for assay of apM1 which comprises using
an antibody specific to apM1 and by which a novel method for
diagnosis of arteriosclerosis can be established.
Sequence CWU 1
1
4 1 244 PRT Abdominal fat tissue from myoma uteri 1 Met Leu Leu Leu
Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His 1 5 10 15 Asp Gln
Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 20 25 30
Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35
40 45 His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly
Glu 50 55 60 Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys
Gly Asp Ile 65 70 75 80 Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro
Arg Gly Phe Pro Gly 85 90 95 Ile Gln Gly Arg Lys Gly Glu Pro Gly
Glu Gly Ala Tyr Val Tyr Arg 100 105 110 Ser Ala Phe Ser Val Gly Leu
Glu Thr Tyr Val Thr Ile Pro Asn Met 115 120 125 Pro Ile Arg Phe Thr
Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp 130 135 140 Gly Ser Thr
Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe 145 150 155 160
Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe 165
170 175 Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu
Asn 180 185 190 Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu
Glu Val Gly 195 200 205 Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly
Glu Arg Asn Gly Leu 210 215 220 Tyr Ala Asp Asn Asp Asn Asp Ser Thr
Phe Thr Gly Phe Leu Leu Tyr 225 230 235 240 His Asp Thr Asn 2 231
PRT Abdominal fat tissue from myoma uteri 2 Met Gly His Asp Gln Glu
Thr Thr Thr Gln Gly Pro Gly Val Leu Leu 1 5 10 15 Pro Leu Pro Lys
Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly 20 25 30 His Pro
Gly His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr 35 40 45
Pro Gly Glu Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys 50
55 60 Gly Asp Ile Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg
Gly 65 70 75 80 Phe Pro Gly Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu
Gly Ala Tyr 85 90 95 Val Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu
Thr Tyr Val Thr Ile 100 105 110 Pro Asn Met Pro Ile Arg Phe Thr Lys
Ile Phe Tyr Asn Gln Gln Asn 115 120 125 His Tyr Asp Gly Ser Thr Gly
Lys Phe His Cys Asn Ile Pro Gly Leu 130 135 140 Tyr Tyr Phe Ala Tyr
His Ile Thr Val Tyr Met Lys Asp Val Lys Val 145 150 155 160 Ser Leu
Phe Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr 165 170 175
Gln Glu Asn Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu 180
185 190 Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu
Arg 195 200 205 Asn Gly Leu Tyr Ala Asp Asn Asp Asn Asp Ser Thr Phe
Thr Gly Phe 210 215 220 Leu Leu Tyr His Asp Thr Asn 225 230 3 29
DNA PCR primer(forward) sequence for apM1 3 aacatatggg gcatgaccag
gaaaccacg 29 4 29 DNA PCR primer(reverse) sequence for apM1 4
aaggatcctc agttggtgtc atggtagag 29
* * * * *