U.S. patent application number 09/030061 was filed with the patent office on 2003-05-22 for il-18 as an osteoclastgenic inhibitor.
Invention is credited to GILLSPIE, MATTHEW TODD, HORWOOD, NICOLE JOY, KURIMOTO, MASASHI, UDAGAWA, NOBUYUKI.
Application Number | 20030095946 09/030061 |
Document ID | / |
Family ID | 12999446 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030095946 |
Kind Code |
A1 |
GILLSPIE, MATTHEW TODD ; et
al. |
May 22, 2003 |
IL-18 AS AN OSTEOCLASTGENIC INHIBITOR
Abstract
An osteoclastgenic inhibitory agent which comprises an
interleukin-18 and/or its functional equivalent. The agent can be
arbitrarily used as an ingredient for cell culture and agents for
regulating bone resorption and for osteoclast-related diseases,
directed to treat and/or prevent hypercalcemia, osteoclastoma,
osteoporosis, etc.
Inventors: |
GILLSPIE, MATTHEW TODD;
(VICTORIA, AU) ; HORWOOD, NICOLE JOY; (VICTORIA,
AU) ; UDAGAWA, NOBUYUKI; (CHIBA, JP) ;
KURIMOTO, MASASHI; (OKAYAMA, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Family ID: |
12999446 |
Appl. No.: |
09/030061 |
Filed: |
February 25, 1998 |
Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61P 19/08 20180101;
A61P 19/00 20180101; A61K 38/00 20130101; C07K 14/54 20130101; A61P
43/00 20180101 |
Class at
Publication: |
424/85.2 |
International
Class: |
A61K 038/20 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 25, 1997 |
JP |
55468/1997 |
Claims
We claim:
1. An osteoclastgenic inhibitory agent, which comprises an
interleukin-18 or its functional equivalent.
2. The inhibitory agent of claim 1, wherein said interleukin-18
includes the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3 as partial amino acid sequences.
3. The inhibitory agent of claim 1, wherein said interleukin-18
includes the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 5
as partial amino acid sequences.
4. The inhibitory agent of claim 1, wherein said interleukin-18
includes the amino acid sequence of SEQ ID NO: 6.
5. The inhibitory agent of claim 1, wherein said interleukin-18 is
human origin.
6. The inhibitory agent of claim 1, wherein said interleukin-18
includes the amino acid sequence of SEQ ID NO: 7.
7. The inhibitory agent of claim 1, which is a therapeutic agent
for osteoclast-related diseases.
8. The inhibitory agent of claim 1, which contains a protein,
buffer, or saccharide as a stabilizer.
9. The inhibitory agent of claim 1, which is in the form of a
liquid, paste, or solid.
10. The inhibitory agent of claim 1, which contains 0.000002-100
w/w % of said interleukin-18.
11. A method for treating and/or preventing osteoclast-related
diseases, which comprising administering said inhibitory agent of
claim 1 to patients suffering from said diseases at a dose of about
0.5 .mu.g to 100 mg per shot, 2 to 6 fold a day or 2 to 10 fold a
week for one day to one year.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to an osteoclastgenic
inhibitory agent comprising an interleukin-18 (hereinafter
abbreviated as "IL-18") or its functional equivalent.
[0003] 2. Description of the Prior Art
[0004] Osteoblasts' bone formation and osteoclasts' bone resorption
are well balanced in healthy living bodies, and this keeps the bone
tissues in normal conditions while old bone tissues are being
replaced with fresh ones without altering the original bone shape.
The phenomenon plays an important role in keeping living bodies'
homeostasis such as the controlling of blood calcium concentration
within a desired range. Once the balance is lost, especially when
the bone resorption level exceeds the bone formation level,
bone-related diseases and other diseases may be induced. Therefore,
elucidation of the whole mechanism of bone resorption in living
bodies, particularly, elucidation of osteoclasts is greatly
highlighted due to scientific and clinical significance
thereof.
[0005] However, the mechanism of osteoclast formation has not yet
been completely elucidated even though interleukin 1 as a promoter
and interleukin 4 as an inhibitor were found. This is because,
similarly as various phenomena in living bodies, osteoclast
formation in living bodies is controlled by the close and
complicated relationship between promoters and inhibitors. Based on
these, it is greatly expected to establish an effective
osteoclastgenic inhibitory agent from the viewpoint of scientific
and clinical aspects.
SUMMARY OF THE INVENTION
[0006] The object of the present invention is to provide a novel
and effective osteoclastgenic inhibitory agent. To solve the object
the present inventors energetically studied for IL-18, i.e., one of
cytokines as communication transferring substances in immune
systems, which induces production of interferon-.gamma.
(hereinafter abbreviated as "IFN-.gamma."), an important
biologically active substance for immunocompetent cells, and
granulocyte/macrophage colony-stimulating factor (hereinafter
abbreviated as "GM-CSF"), and augments cytotoxicity and induces
formation of killer cells. At the finding, IL-18 was described as
an interferon-.gamma.-inducing factor as reported by Haruki OKAMURA
in Japanese Patent Kokai Nos. 27,189/96 and 193,098/96, and in
Nature, Vol. 378, No. 6,552, pp. 88-91 (1995), and then called
IL-18 according to the proposal of Shimpei USHIO et al., in The
Journal of Immunology, Vol. 156, pp. 4,274-4,279 (1996).
[0007] The present inventors found that a particular gene, capable
of inhibiting osteoclast formation from osteoclastic precursor
cells in vitro, is specifically expressed in quantities in stroma
cells derived from mouse myeloma. Their further detailed analysis
revealed that (i) the gene encodes IL-18 that includes SEQ ID NO: 7
as a core sequence, (ii) IL-18 and functional equivalents thereof
effectively inhibit osteoclast formation, and (iii) the inhibition
is mainly due to the action of GM-CSF induced and produced by
IL-18.
[0008] Based on these, the present inventors solved the present
object by an osteoclastgenic inhibitory agent comprising IL-18 or
its functional equivalent as an effective ingredient.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0009] FIG. 1 shows the structure of the recombinant DNA
pKGFHH2.
[0010] FIG. 2 shows the structure of the recombinant DNA
pCSHIGIF/MUT35.
[0011] FIG. 3 shows the structure of the recombinant DNA
pCSHIGIF/MUT42.
[0012] FIG. 4 shows the structure of the recombinant DNA
pBGHuGF.
[0013] FIG. 5 shows the structure of the recombinant DNA
pKGFMH2.
[0014] In these figures, KGFHH2 cDNA means a cDNA encoding the
IL-18 according to the present invention: IGIF/MUT35; a DNA
encoding the IL-18 according to the present invention: IGIF/MUT42;
a DNA encoding the IL-18 according to the present invention:
HuIGIF; a chromosomal DNA encoding the IL-18 according to the
present invention: KGFMH2 cDNA; a CDNA encoding the IL-18 according
to the present invention: 5S; a gene for 5S ribosomal RNA: Ptac; a
tac promoter: rrnBT1T2; a termination region of a ribosomal RNA
operon: AmpR; an ampicillin resistent gene: pBR322ori; a
replication origin of Escherichia coli: CMV; a cytomegalovirus
promoter: IFNss; a nucleotide sequence encoding a signal peptide
for subtype .alpha.2b of human interferon-.alpha..
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention relates to an osteoclastgenic
inhibitory agent comprising IL-18 or its functional equivalent as
an effective ingredient. The wording "IL-18" as referred to in the
invention includes polypeptides with the above property
independently of their sources and origins. For example, the IL-18
used in the present invention includes, as internal partial amino
acid sequences, the amino acid sequences of SEQ ID NO: 1, SEQ ID
NO: 2, and SEQ ID NO: 3, as well as SEQ ID NO: 4 and SEQ ID NO: 5,
and includes the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO:
7 as a whole. The wording "functional equivalent(s)" as referred to
in the present invention includes (i) those wherein one or more
amino acids in the amino acid sequence of IL-18 are replaced with
different amino acids, (ii) those wherein one or more amino acids
are added to the N- and/or C-termini of the amino acid sequence of
IL-18, (iii) those wherein one or more amino acids are inserted
into the internal sites of the amino acid sequence of IL-18, (iv)
those wherein one or more amino acids in the N- and/or C-terminal
regions of the amino acid sequence of IL-18 are deleted, and (v)
those wherein one or more amino acids in the internal regions of
the amino acid sequence of IL-18 are deleted; all of these
modifications should be made within the range that does not
substantially lose the property of osteoclast formation by IL-18
among the inherent property of IL-18. Examples of such functional
equivalents are described along with their detailed amino acid
sequences in Japanese Patent Application No. 20,906/97 by the same
applicant of the present applicant, i.e., polypeptides which are
capable of inducing production of interferon-gamma by
immunocompetent cells, wherein said polypeptides contain either
amino acid sequence wherein one or more cysteines are replaced with
different amino acid(s) while leaving respective consensus
sequences as shown in SEQ ID NOs: 1, 2 and 4 intact, or that
wherein one or more amino acids are added, removed and/or replaced
at one or more sites including those in the consensus sequences but
excluding those of the replaced cysteine. The different amino acids
to replace the cysteine(s) are not restricted to any types, as far
as the resulting polypeptide, containing an amino acid sequence
replaced with the different amino acid(s), exhibits an activity of
inducing production of IFN-.gamma. by immunocompetent cells in the
presence or absence of an appropriate cofactor, as the wild-type
polypeptides containing SEQ ID NOs: 1, 2 and 4 as consensus partial
amino acid sequences, and a stability significantly higher than
that of the wild-type polypeptides. The different amino acids
include serine, threonine, alanine, valine, leucine, isoleucine,
histidine, tyrosine, phenylalanine, tryptophan, and methionine,
among which the most preferable amino acid is serine or alanine.
Embodiments of the amino acid sequences, containing SEQ ID NOs: 1,
2 and 4 as consensus partial amino acid sequences, in which one or
more cysteines are to be replaced with different amino acid(s) are
the wild-type polypeptides containing SEQ ID NO: 6 or 7. SEQ ID NO:
6 contains cysteines at the 38th, 68th, 76th, and 127th positions
from the N-terminus. SEQ ID NO: 7 contains cysteines at the 7th,
75th, and 125th positions. The polypeptides include those
containing the amino acid sequence of any one of SEQ ID NOs: 20-26,
which are derived from the wild-type polypeptide containing SEQ ID
NO: 6, those containing the amino acid sequence of SEQ ID NO: 27 or
28, which are derived from the wild-type polypeptide containing the
amino acid sequence of SEQ ID NO: 7, and those containing an amino
acid sequence derived from any one of SEQ ID NOs: 20-28 by adding,
removing, and/or replacing one or more amino acids to and/or at
position(s) excepting the positions where the cysteine(s) have been
replaced while retaining the desired biological activities and
stability. The wording "one or more amino acids" means the number
of amino acids which conventional methods such as site-directed
mutagenesis can usually add, remove or replace. The polypeptides
containing any one of SEQ ID NOs: 20-28 possess both stability and
biological activities significantly higher than those of the
wild-type polypeptides.
[0016] The functional equivalents as referred to in the present
invention further include glycosylated polypeptides of IL-18 and
the above polypeptides. Any of these IL-18 and functional
equivalents thereof, both of which are included to and referred to
as "IL-18" in the present invention, unless specified otherwise,
can be used in the present invention independently of their
origins; those prepared by separating from natural sources such as
cell cultures and from artificially synthesized ones using
recombinant DNA technology and peptide synthesis.
[0017] With economical viewpoint, methods of recombinant DNA
technology are advantageously used; generally, desired IL-18 can be
obtained by introducing DNAs encoding IL-18 into appropriate hosts
derived from microorganisms, plants, and animals to form
transformants, culturing the transformants in nutrient culture
media in a conventional manner, and purifying the cultures by
conventional methods used for purifying cytokines. Any DNAs can be
used as the above DNAs as long as they contain a DNA encoding
IL-18, and can be suitably selected depending on the purpose of the
use of the present osteoclastgenic inhibitory agent or on the
recombinant DNA technology used. For example, Japanese Patent Kokai
Nos. 193,098/96, 231,598/96, and 27,189/96 by the same applicant of
the present invention disclose in detail methods for producing
IL-18 by culturing transformed microorganisms into which DNAs
including a cDNA encoding mouse or human IL-18 are introduced; and
Japanese Patent Application No. 185,305/96 by the same applicant of
the present invention discloses in detail a method for producing
IL-18 encoding human IL-18 by culturing transformed animal cells
which have an introduced DNA that includes a chromosomal DNA
encodes human IL-18. Japanese Patent Application No. 20,906/97 by
the same applicant of the present invention discloses in detail a
method for producing IL-18 by culturing transformed animal cells
having an introduced DNA which includes a DNA encoding a functional
equivalent of human IL-18.
[0018] The aforesaid recombinant DNA technology has an economical
advantage, but depending on the hosts and DNA sequences used, the
IL-18 thus obtained may have somewhat different physicochemical
property from those of IL-18 produced and functions in vivo.
Japanese Patent Application No. 67,434/96 by the same applicant of
the present invention discloses in detail a preparation of IL-18
using established human cell lines as natural sources, and Japanese
Patent Application No. 213,267/96 by the same applicant also
discloses in detail the preparation using an
interleukin-1.beta.-converting enzyme. The IL-18 obtained by those
preparations can be estimated to have substantially the same or
equal physicochemical property to that of IL-18 that is produced
and functions in vivo, and the yield can be estimated to be
slightly lower. However, such IL-18 has an advantage that it has a
fewer side effects when used as pharmaceuticals directed to
administering to warm-blooded animals in general and including
humans. When applying purification methods using monoclonal
antibodies specific to IL-18, as disclosed in Japanese Patent
Application No. 231,598/96 by the same applicant of the present
invention, a relatively-high purity IL-18 can be obtained in a
minimum labor and cost.
[0019] The present osteoclastgenic inhibitory agent comprising the
aforesaid IL-18 includes any types and forms usable to inhibit
osteoclast formation both in vivo and in vitro. The present agent
can be advantageously used as ingredients for cell culture media
for animal cells, which satisfactorily inhibit osteoclast
formation, maintain, proliferate, and/or differentiate the desired
cells; components of screening kits for bone-related therapeutic
agents; bone-resorption regulatory agents; and agents for
osteoclast-related diseases. The bone-resorption regulatory agents
include medicaments and health foods that exert an osteoclastgenic
inhibitory activity in vivo, control bone resorption to normal
conditions, and improve unfavorable physical conditions such as a
relatively-insignificant arthralgia. The agents for
osteoclast-related diseases include medicaments used to prevent
and/or treat diseases caused by an excessive osteoclast formation
and/or its function. Examples of such diseases are hypercalcemia,
osteoclastoma, Behcet's syndrome, osteosarcoma, arthropathy,
chronic rheumatoid arthritis, deformity ostitis, primary
hyperthyroidism, osteopenia, and osteoporosis. Varying depending on
the types of agents and diseases to be treated, the present agent
is usually formulated into a liquid, paste, or solid form which
contains 0.000002-100 w/w %, preferably, 0.0002-0.5 w/w % of
IL-18.
[0020] The present osteoclastgenic inhibitory agent can be IL-18
alone or compositions comprising IL-18 and one or more other
ingredients such as carriers, excipients, diluents, adjuvants,
antibiotics, and proteins such as serum albumin and gelatin as
stabilizers; saccharides such as glucose, maltose, maltotriose,
maltotetraose, trehalose, sucrose, isomaltose, lactose, panose,
erlose, palatinose, lactosucrose, raffinose, fructooligosaccharide,
galactooligosaccharide, lentinan, dextrin, pullulan, and sugar
alcohols including sorbitol, maltitol, lactitol, and maltotriitol;
buffers comprising phosphates or citrates mainly; and reductants
such as 2-mercaptoethanol, dithiothreitol, and reduced glutathione;
and optionally biologically active substances such as
interferon-.alpha., interferon-.beta., interferon-.gamma.,
interleukin-2, interleukin-3, interleukin-6, interleukin-12,
TNF-.alpha., TNF-.beta., GM-CSF, estrogen, progesterone,
chlormadinone acetate, calcitonin, somatokine, somatomedin,
insulin-like growth factor, ipriflavone, parathyroid hormone (PTH),
norethisterone, busulfan, ancitabine, cytarabine, fluorouracil,
tetrahydrofurfuryl fluorouracil, methotrexate, vitamin D.sub.2,
active vitamin D, Krestin.RTM. or polysaccharide K, L-asparaginase,
and OK-432 or Picibanil.RTM.; and calcium salts such as calcium
lactate, calcium chloride, calcium monohydrogenphosphate, and
L-calcium L-aspartate. When used as agents for administering to
warm-blooded animals in general and including humans, i.e., agents
for osteoclast-related diseases, the present agent can be
preferably formulated into compositions by appropriately combining
with one or more of the above physiologically-acceptable
substances.
[0021] The present osteoclastgenic inhibitory agent includes
medicaments in a unit dose form used for administering to
warm-blooded animals in general and including humans. The wording
"unit dose form" means those which contain IL-18 in an amount
suitable for a daily dose or in an amount up to four fold by
integers or up to {fraction (1/40)} fold of the dose, and those in
a physically separated and formulated form suitable for prescribed
administrations. Examples of such formulations are injections,
liquids, powders, granules, tablets, capsules, troches, collyriums,
nebulas, and suppositories.
[0022] The present agent as an osteoclastgenic inhibitory agent
effectively treat and prevent osteoclast-related diseases
independently of oral and parenteral administrations. Varying
depending on the types and symptoms of patients' diseases, the
present agent can be administered to the patients orally,
intradermally, subcutaneously, muscularly, or intravenously at a
dose of about 0.5 .mu.g to 100 mg per shot, preferably, at a dose
of about 2 .mu.g to 10 mg per shot of IL-18, 2-6 fold a day or 2-10
fold a week for one day to one year.
[0023] In the below, with reference to experiments, the
preparation, physicochemical property, and biological activity of
the IL-18 according to the present invention are described:
[0024] Experiment 1
[0025] Preparation of Human IL-18
[0026] According to the method in Japanese Patent Kokai No.
231,598/96 by the same applicant of the present invention, an
autonomously-replicable recombinant DNA, pKGFHH2, linked to a cDNA
encoding human IL-18, was prepared. Dideoxyribonucleotide
sequencing analyzed that, as shown in FIG. 1, in the recombinant
DNA, KGFHH2 cDNA containing the base sequence of SEQ ID NO: 8 was
linked to the downstream of Ptac, a Tac promoter. The recombinant
DNA pKGFHH2 contained the amino acid sequences of SEQ ID NOs: 1 to
5; these amino acid sequences were respectively encoded by
nucleotides 46-63, 88-105, 400-420, 151-165, and 214-228 in SEQ ID
NO: 8.
[0027] According to the method in Japanese Patent Kokai No.
231,598/96, the recombinant DNA pKGFHH2 was introduced into an
Escherichia coli Y1090 strain, ATCC 37197, and the strain was
cultured. The produced polypeptide was purified by immunoaffinity
chromatography to obtain a purified human IL-18 with a purity of at
least 95% in a yield of about 25 mg/l culture. According to the
method in Japanese Patent Kokai No. 193,098/96 by the same
applicant of the present invention, the purified human IL-18 was
analyzed for biological activity and physicochemical property as
indicated below: When culturing human lymphocytes, collected by a
conventional manner from a healthy donor, in the presence of the
purified human IL-18, IFN-.gamma. production was observed depending
on the concentration of IL-18, resulting in a confirmation that
IL-18 has an activity of inducing IFN-.gamma. production by
lymphocytes as an immunocompetent cell. In accordance with the
method as reported by U. K. Laemmli in Nature, Vol. 227, pp.
680-685 (1970), the purified IL-18 was subjected to SDS-PAGE,
resulting in a major band with an IFN-.gamma. inducing activity at
a position corresponding to 18,500.+-.3,000 daltons. The IL-18 gave
a pI of 4.9.+-.1.0 as determined by conventional chromatofocusing.
Conventional analysis using "PROTEIN SEQUENCER MODEL 473A", an
apparatus of Applied Biosystems, Inc., Foster City, USA, revealed
that the IL-18 had the amino acid sequence of SEQ ID NO: 9, i.e.,
the amino acid sequence of SEQ ID NO: 8 where a methionine residue
was linked to the N-terminus.
[0028] Experiment 2
[0029] Preparation of Human IL-18
[0030] According to the method in Japanese Patent Application No.
67,434/96 by the same applicant of the present invention, THP-1
cells, ATCC TIB 202, a human monocyte cell line derived from a male
with acute monocytic leukemia, were inoculated to the dorsum
subcutaneous tissues of new born hamsters, followed by feeding the
hamsters for three weeks. Tumor masses, about 15 g weight each,
formed in the subcutaneous tissues of each hamster, were extracted,
dispersed in media, and disrupted. The polypeptide obtained from
the disrupted cells was purified by immunoaffinity chromatography
to obtain a purified human IL-18 in a yield of an about 50
ng/head.
[0031] Similarly, according to the method in Japanese Patent
Application No. 67,434/96, the purified human IL-18 was analyzed
and determined for biological activity and physicochemical property
as indicated below: It was confirmed that culturing human
lymphocytes, collected from healthy donors in a conventional
manner, in the presence of different concentrations of the human
IL-18, resulted in an IL-18 dose-dependent IFN-.gamma. production.
This revealed that the human IL-18 has a biological activity of
inducing IFN-.gamma. production by lymphocytes as an
immunocompetent cell. In accordance with the method as reported by
U. K. Laemmli in Nature, Vol. 227, pp. 680-685 (1970), the purified
human IL-18 was subjected to SDS-PAGE using 2 w/v % dithiothreitol
as a reductant, resulting in a major band with an IFN-.gamma.
production inducing activity at a position corresponding to
18,000-19,500 daltons. According to the peptide map disclosed in
Japanese Patent Application No. 67,434/96, the human IL-18 was
treated with clostripain commercialized by Sigma Chemical Company,
Missouri, USA, to obtain polypeptide fragments, followed by
subjecting the fragments for fractionation to high-performance
liquid chromatography (HPLC) using "ODS-120T", a column
commercialized by Tosoh Corporation, Tokyo, Japan, and analyzing
the amino acid sequences of the fragments from the N-terminus to
reveal the following amino acid sequences of SEQ ID NOs: 10 to 13.
These amino acid sequences were completely coincided with amino
acids 148-157, 1-13, 45-58, and 80-96 in SEQ ID NO: 6. The data
shows that the human IL-18 obtained in Experiment 2 has the amino
acid sequence of SEQ ID NO: 6 and all the partial amino acid
sequences of SEQ ID NOs: 1 to 5.
[0032] Experiment 3
[0033] Preparation of Functional Equivalents
[0034] According to the method in Japanese Patent Application No.
20,906/97 by the same applicant of the present invention, it was
prepared an autonomously-replicable recombinant DNA,
pCSHIGIF/MUT35, was linked to a DNA encoding a functional
equivalent of human IL-18 where cysteines 38, 68, and 76 in SEQ ID
NO: 6 were respectively replaced with serine, serine, and alanine.
Dideoxyribonucleotide sequence analysis revealed that as shown in
FIG. 2, in the recombinant DNA, DNA IGIF/MUT35 with SEQ ID NO: 14
linked to the downstream of a base sequence encoding a signal
peptide of subtype .alpha.2b in human interferon-.alpha. in the
same reading-frame, as reported by K. Henco et al., in Journal of
Molecular Biology, Vol. 185, pp. 227-260 (1985), and had a stop
codon for protein synthesis at further downstream. As shown in
parallel in SEQ ID NO: 14, the amino acid sequence encoded by the
recombinant DNA corresponded to SEQ ID NO: 6 where cysteines 38,
68, and 76 in SEQ ID NO: 6 were respectively replaced with serine,
serine, and alanine. The recombinant DNA contained a nucleotide
which encodes all the amino acid sequences of SEQ ID NOs: 1 to 4
and the one of SEQ ID NO: 5 where cysteine at amino acid 5 in SEQ
ID NO: 5 was replaced with alanine. These amino acid sequences were
respectively encoded by nucleotides 46-63, 88-105, 400-420,
151-165, and 214-228 in SEQ ID NO: 14.
[0035] According to the method in Japanese Patent Application No.
20,906/97 by the same applicant of the present invention, the
recombinant DNA pCSHIGIF/MUT35 was introduced into COS-1 cells,
ATCC CRL 1650, an established cell line derived from SV40
transformed African Green monkey kidney, followed by culturing the
transformed cells. The produced polypeptide in the culture was
purified by immunoaffinity chromatography to obtain a purified
functional equivalent of human IL-18 in a yield of about 40 ng/ml
culture. According to the method in Japanese Patent Application No.
20,906/97, the purified functional equivalent was analyzed and
determined for biological activity and physicochemical property as
indicated below: When culturing KG-1 cells, ATCC CCL 246, an
established cell line derived from human acute myelogenous
leukemia, in the presence of different concentrations of the
purified functional equivalent of human IL-18, IFN-.gamma.
production was observed depending on the concentration of the
IL-18, revealing that the IL-18 has a biological activity of
inducing IFN-.gamma. production by KG-1 cells as an immunocompetent
cell. In accordance with the method as reported by U. K. Laemmli in
Nature, Vol. 227, pp. 680-685 (1970), the purified functional
equivalent was subjected to SDS-PAGE in the presence of 2 w/v %
dithiothreitol as a reductant, resulting in a major band with an
IFN-.gamma. production inducing activity at a position
corresponding to 18,000-19,500 daltons. Conventional analysis using
"PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems,
Inc., Foster City, USA, revealed that the N-terminal region of the
functional equivalent had the amino acid sequence of SEQ ID NO: 15
which corresponded to the amino acid sequence in the N-terminal
region as shown in parallel in SEQ ID NO: 14.
[0036] Experiment 4
[0037] Preparation of Functional Equivalent
[0038] According to the method in Japanese Patent Application No.
20,906/97 by the same applicant of the present invention, it was
prepared an autonomously-replicable recombinant DNA,
pCSHIGIF/MUT42, which was linked to a DNA encoding for a functional
equivalent of human IL-18 where cysteines 38, 68, 76, and 127 in
SEQ ID NO: 6 were respectively replaced with serine, serine,
alanine, and serine. Dideoxyribonucleotide sequencing revealed
that, as shown in FIG. 3, in the recombinant DNA, DNA IGIF/MUT42
with SEQ ID NO: 16 linked to the downstream of a base sequence
encoding a signal peptide for subtype .alpha.2b of human
interferon-.alpha. in the same reading frame, as reported by K.
Henco et al., in Journal of Molecular Biology, Vol. 185, pp.
227-260 (1985), and had a stop codon for protein synthesis at
further downstream. As shown in parallel in SEQ ID NO: 16, the
amino acid sequence encoded by the recombinant DNA corresponded to
SEQ ID NO: 6 where cysteines 38, 68, 76, and 127 in SEQ ID NO: 6
were respectively replaced with serine, serine, alanine, and
serine. The recombinant DNA contained a nucleotide sequence which
encodes all the amino acid sequences of SEQ ID NOs: 1 to 4 and the
one of SEQ ID NO: 5 where cysteine 5 in SEQ ID NO: 5 was replaced
with alanine. These amino acid sequences were respectively encoded
by nucleotides 46-63, 88-105, 400-420, 151-165, and 214-228 in SEQ
ID NO: 16.
[0039] According to the method in Japanese Patent Application No.
20,906/97 by the same applicant of the present invention, the
recombinant DNA pCSHIGIF/MUT42 was introduced into COS-1 cells,
followed by culturing the cells. The produced polypeptide in the
culture was purified by immunoaffinity chromatography to obtain a
purified functional equivalent of human IL-18 in a yield of about
20 ng/ml culture. According to the method in Japanese Patent
Application No. 20,906/97, the purified functional equivalent was
analyzed and determined for biological activity and physicochemical
property as indicated below: When cultured KG-1 cells in the
presence of different concentrations of the purified functional
equivalent, a dose-dependent IFN-.gamma. production was observed,
and this revealed that the functional equivalent has a biological
activity of inducing IFN-.gamma. production by KG-1 cells as an
immunocompetent cell. In accordance with the method as reported by
U. K. Laemmli in Nature, Vol. 227, pp. 680-685 (1970), the purified
functional equivalent was subjected to SDS-PAGE in the presence of
2 w/v % dithiothreitol as a reductant, resulting in a major band
with an IFN-.gamma. inducing activity at a position corresponding
to 18,000-19,500 daltons. Conventional analysis using "PROTEIN
SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc.,
Foster City, USA, revealed that the N-terminal region of the
functional equivalent had the amino acid sequence of SEQ ID NO: 15
which completely corresponded to the amino acid sequence in the
N-terminal region as shown in parallel in SEQ ID NO: 16.
[0040] Experiment 5
[0041] Preparation of Human IL-18
[0042] According to the method in Japanese Patent Application No.
185,305/96 by the same applicant of the present invention, an
autonomously-replicable recombinant DNA, pBGHuGF, linked to a
chromosomal DNA encoding human IL-18, was obtained.
Dideoxyribonucleotide sequencing analysis revealed that as shown in
FIG. 4, in the recombinant DNA, a chromosomal DNA, which encodes
human IL-18, i.e., DNA HuIGIF with SEQ ID NO: 17, was linked to the
downstream of a restriction site by a restriction enzyme, Hind III.
As shown in SEQ ID NO: 17, the chromosomal DNA HuIGIF consists of
11,464 bp where the exon was fragmented by four introns positioning
at nucleotides 83-1,453, 1,466-4,848, 4,984-6,317, and
6,452-11,224. Among the resting nucleotide sequence excluding these
introns, nucleotides 3-11,443 from the 5'-terminus are the part
that encodes a precursor of human IL-18, and nucleotides
4,866-4,983 are the part that encodes an active human IL-18. The
chromosomal DNA contained nucleotides sequences encoding SEQ ID
NOs: 1 to 5; these amino acid sequences were respectively encoded
by nucleotides 4,911-4,928, 4,953-4,970, 11,372-11,392,
6,350-6,364, and 6,413-6,427 in SEQ ID NO: 17.
[0043] According to the method in Japanese Patent Application No.
185,305/96, the recombinant DNA pBGHuGF was introduced into CHO-K1
cells, ATCC CCL 61, an established cell line derived from Chinese
hamster ovary, followed by culturing the cells. The culture
supernatant was contacted with a supernatant of cell disruptant
prepared from a THP-1 cell culture to produce a polypeptide which
was then purified by immunoaffinity chromatography to obtain a
purified human IL-18 in a yield of about 15 mg/l culture. According
to the method in Japanese Patent Application No. 185,305/96, the
polypeptide was analyzed and determined for biological activity and
physicochemical property as indicated below: It was confirmed that
human lymphocytes, which were collected from a healthy donor,
produced IFN-.gamma. depending on the purified human IL-18
concentration when cultured at different concentrations of the
human IL-18, revealing that the human IL-18 has a biological
activity of inducing IFN-.gamma. production by lymphocytes as an
immunocompetent cell. In accordance with the method as reported by
U. K. Laemmli in Nature, Vol. 227, pp. 680-685 (1970), the purified
human IL-18 was subjected to SDS-PAGE in the presence of 2 w/v %
dithiothreitol as a reductant, resulting in a major band with an
IFN-.gamma. inducing activity at a position corresponding to
18,000-19,500 daltons. The N-terminal region of the human IL-18
contained the amino acid sequence of SEQ ID NO: 15 which completely
corresponded to the amino acid sequence in the N-terminal region of
SEQ ID NO: 17 for an active IL-18.
[0044] Experiment 6
[0045] Preparation of Mouse IL-18
[0046] To a 0.5-ml reaction tube were added 8 .mu.l of 25 mM
magnesium chloride, 10 .mu.l of 10.times.PCR buffer, one .mu.l of
25 mM dNTP mix, one .mu.l of 2.5 units/.mu.l of amplitaq DNA
polymerase, one ng of a recombinant DNA, which encodes mouse IL-18
having the nucleotide sequence of SEQ ID NO: 18 and the amino acid
sequence of SEQ ID NO: 7, prepared from a phage DNA clone according
to the method in Japanese Patent Kokai No. 27,189/96, and adequate
amounts of a sense and antisense primers having nucleotide
sequences represented by 5'-ATAGAATTCAAATGAACTTTGGCCGAC- TTCACTG-3'
and 5'-ATAAAGCTTCTAACTTTGATGTAAGTT-3', respectively, which were
chemically synthesized based on the amino acid sequences nearness
to the N- and C-termini of SEQ ID NO: 7, and the mixture solution
was brought up to a volume of 100 .mu.l with sterilized distilled
water. The solution thus obtained was subjected in a usual manner
to PCR reaction of the following three cycles of successive
incubations at 94.degree. C. for one minute, 43.degree. C. for one
minute, and 72.degree. C. for one minute, and further 40 cycles of
successive incubations at 94.degree. C. for one minute, 60.degree.
C. for one minute, and 72.degree. C. for one minute.
[0047] The product obtained by the PCR reaction and "pCR-Script SK
(+)", a plasmid vector commercialized by Stratagene Cloning
Systems, California, USA, were in a conventional manner ligated
together using a DNA ligase into a recombinant DNA which was then
introduced into "XL-1 Blue MRF'Kan", an Escherichia coli strain
commercialized by Stratagene Cloning Systems, California, USA., to
obtain a transformant. The transformant was inoculated to L-broth
(pH 7.2) containing 50 .mu.g/ml ampicillin, followed by the
incubation at 37.degree. C. for 18 hours under shaking conditions.
The culture was centrifuged to obtain the proliferated
transformants which were then treated with a conventional
alkali-SDS method to isolate a recombinant DNA. A portion of the
recombinant DNA isolated was analyzed by dideoxyribonucleotide
sequencing, revealing that the recombinant DNA contained
restriction sites of Eco RI and Hind III at the 5'- and 3'-termini
of SEQ ID NO: 18, respectively, and a DNA containing a methionine
codon for initiating polypeptide synthesis and a TAG codon for
terminating polypeptide synthesis, which were located in just
before and after the N- and C-termini of the amino acid sequence as
shown in parallel in SEQ ID NO: 18. The recombinant DNA contained
the nucleotide sequences of SEQ ID NOs: 1 to 5. These amino acid
sequences were encoded by nucleotides 46-63, 85-102, 394-414,
148-162, and 211-225 in SEQ ID NO: 18.
[0048] The remaining portion of the recombinant DNA was in a
conventional manner cleaved with restriction enzymes of Eco RI and
Hind II, and the resulting 0.1 .mu.g of an Eco RI-Hind III DNA
fragments, obtained by using "DNA LIGATION KIT VER 2", a DNA
ligation kit commercialized by Takara Shuzo Co., Ltd., Tokyo,
Japan, and 10 ng of pKK223-3, a plasmid vector commercialized by
Pharmacia LKB Biotechnology AB, Uppsala, Sweden, which had been
cleaved with a restriction enzyme were linked together, by
incubating at 16.degree. C. for 30 min to obtain an
autonomously-replicable recombinant DNA, pKGFMH2. Using competent
cell method, an Escherichia coli Y1090 strain, ATCC 37197, was
transformed using the recombinant DNA pKGFMH2, and the resulting
transformant, KGFMH2, was inoculated to L-broth (pH 7.2) containing
50 .mu.g/ml ampicillin, and cultured at 37.degree. C. for 18 hours
under shaking conditions. The culture was centrifuged to collect
the proliferated transformants, followed by applying a conventional
SDS-alkali method to a portion of the transformants to extract the
recombinant DNA pKGFMH2. Dideoxyribonucleotide sequencing analysis
revealed that, as shown in FIG. 5, KGFMH2 cDNA containing the
nucleotide sequence of SEQ ID NO: 18 was linked to the downstream
of the Tac promoter in the recombinant DNA pKGFMH2.
[0049] Ampicillin was added to L-broth (pH 7.2), which had been
sterilized by autoclaving, to give a concentration of 50 .mu.g/ml,
cooled to 37.degree. C., and inoculated with the transformant
KGFMH2, followed by the culture at 37.degree. C. for 18 hours.
Eighteen liters of a fresh preparation of the same culture medium
was placed in a 20-l jar fermenter, similarly sterilized as above,
admixed with ampicillin, cooled to 37.degree. C., and inoculated
with one v/v % of the seed culture obtained in the above, followed
by the culture at 37.degree. C. for 8 hours under
aeration-agitation conditions. The resulting culture was
centrifuged to collect the cultured cells which were then suspended
in a mixture solution (pH 7.3) containing 150 mM sodium chloride,
16 mM disodium hydrogenphosphate, and 4 mM sodium
dihydrogenphosphate, disrupted by ultrasonication, and centrifuged
to remove cell disruptant, and this yielded an about two liters of
a supernatant.
[0050] To an about two liters of the supernatant was added 10 mM
phosphate buffer (pH 7.3) containing ammonium sulfate to give a 40%
ammonium saturation. The resulting sediment was removed by
centrifugation, and the supernatant was mixed with ammonium sulfate
to give an 85% ammonium saturation, allowed to stand at 4.degree.
C. for 18 hours, and centrifuged at about 8,000 rpm for 30 min to
obtain a newly formed sediment. The sediment thus obtained was
dissolved in 10 mM phosphate buffer (pH 6.6) containing 1.5 M
ammonium sulfate to give a total volume of about 1,300 ml, and the
solution was filtered, and fed to a column packed with about 800 ml
of "PHENYL SEPHAROSE CL-6B", a gel commercialized by Pharmacia LKB
Biotechnology AB, Uppsala, Sweden, followed by washing the column
with a fresh preparation of the same buffer and feeding to the
column a linear gradient buffer of ammonium sulfate decreasing from
1.5 M to 0 M in 10 mM phosphate buffer (pH 6.6) at an SV (space
velocity) 1.5. Fractions eluted at around 1 M ammonium sulfate were
pooled, concentrated using a membrane filter, and dialyzed against
10 mM phosphate buffer (pH 6.5) at 4.degree. C. for 18 hours. The
dialyzed solution was fed to a column packed with about 55 ml of
"DEAE-5PW", a gel commercialized by Pharmacia LKB Biotechnology AB,
Uppsala, Sweden, which had been equilibrated with 10 mM phosphate
buffer (pH 6.5). The column was washed with a fresh preparation of
the same buffer, and fed with a linear gradient buffer of sodium
chloride increasing from 0 M to 0.5 M in 10 mM phosphate buffer (pH
6.5) at SV 5.5, followed by collecting fractions eluted at around
0.2 M sodium chloride. Thereafter, the fractions were pooled and
concentrated similarly as above up to give an about nine
milliliters, followed by dialyzing the concentrate against PBS
(phosphate buffered saline) at 4.degree. C. for 18 hours, and
feeding the dialyzed solution to a column packed with "SUPERDEX
75", a gel commercialized by Pharmacia LKB Biotechnology AB,
Uppsala, Sweden, which had been equilibrated with a fresh
preparation of the same PBS. The column was fed with a fresh
preparation of the same PBS to collect fractions with an
IFN-.gamma. inducing activity, and the fractions were pooled and
concentrated with a membrane filter to obtain a purified mouse
IL-18 in a yield of about 350 .mu.g/l culture.
[0051] According to the method in Japanese Patent Kokai No.
27,189/96, the purified mouse IL-18 was analyzed and determined for
biological activity and physicochemical property as indicated
below: Culturing mouse spleen cells, collected by a conventional
manner, under different concentrations of the mouse IL-18 resulted
in an IFN-.gamma. production depending on the concentrations of the
mouse IL-18, and this revealed that the mouse IL-18 has an activity
of inducing IFN-y production by spleen cells as an immunocompetent
cell. In accordance with the method as reported by U. K. Laemmli in
Nature, Vol. 227, pp. 680-685 (1970), the purified human IL-18 was
subjected to SDS-PAGE under non-reducing conditions, resulting in a
major band with an IFN-.gamma. inducing activity at a position
corresponding to 19,000.+-.5,000 daltons. The N-terminal region of
the mouse IL-18 contained the amino acid sequence of SEQ ID NO: 19
which corresponded to the N-terminal region of SEQ ID NO: 18.
[0052] With reference to Experiment 7, the biological activity of
the IL-18 according to the present invention will be described in
more detail, and Experiment 8 describes the cytotoxicity of the
IL-18:
[0053] Experiment 7
[0054] Biological Activity
[0055] Experiment 7-1
[0056] Induction of GM-CSF Production
[0057] Using a heparinized syringe, blood was collected from a
healthy volunteer and diluted two fold with serum-free RPMI 1640
medium (pH 7.4). The diluent was overlaid on a ficoll and
centrifuged, and the collected lymphocytes were washed with RPMI
1640 medium (pH 7.4) supplemented with 10 v/v % fetal calf serum,
and suspended in a fresh preparation of the same medium to give a
cell density of 1.times.10.sup.6 cells/ml, followed by distributing
the cell suspension to a 12-well microplate by two ml/well.
[0058] Using RPMI 1640 medium (pH 7.4) supplemented with 10 v/v %
fetal calf serum, an IL-18 preparation obtained by the method in
Experiment 1 was prepared into a one .mu.g/ml solution which was
then distributed to the above microplate by 20-200 .mu.l/well. To
the microplate was further added a fresh preparation of the same
buffer, supplemented with 500 .mu.l/ml of Concanavalin A, by 10
.mu.l/well, followed by the incubation at 37.degree. C. for 48
hours in a 5 v/v % CO.sub.2 incubator. After completion of the
culture, supernatants in each well were sampled by 0.1 ml/well, and
determined for GM-CSF content using a conventional enzyme
immunoassay. In parallel, a culture system free of IL-18 as a
control was provided and treated similarly as above. The data is in
Table 1:
1 TABLE 1 IL-18* GM-CSF yield (nM) (pg/ml) 0 510 0.7 2,150 2.8
3,050 5.6 3,950 Note: The symbol "*" means that IL-18 was added to
the culture system in the presence of 2.5 .mu.g/ml of Concanavalin
A.
[0059] The results in Table 1 indicate that lymphocytes as an
immunocompetent cell produced GM-CSF depending on the concentration
of IL-18 when contacted with IL-18 in the presence of Concanavalin
A as a cofactor. It was also confirmed that all of the IL-18
preparations and functional equivalents thereof, which were
obtained by the methods in Experiments 2 to 5, induced GM-CSF
production even when used alone similarly as above. An IL-18
preparation obtained by the method in Experiment 6 was tested in
accordance with Experiment 7-1 except that the human lymphocytes
used in the experiment were replaced with spleen cells prepared
from mouse by a conventional manner, revealing that the IL-18
preparation also induced GM-CSF production.
[0060] Experiment 7-2
[0061] Inhibition of Osteoclast Formation
[0062] Experiment 7-2(a)
[0063] As reported by T. J. Martin and K. W. Ng in Journal of
Cellular Biochemistry, Vol. 56, pp. 357-366 (1994), it is
considered requisite for contacting osteoclastic precursor cells,
derived from hematopoietic stem cells, with osteoblasts or bone
marrow stromas to generally differentiate osteoclastic precursor
cells into mature osteoclasts. As described by G. D. Roodman in
Endocrine Reviews, Vol. 17, No. 4, pp. 308-332 (1996), it is
generally recognized that osteoclasts have characters of
multinucleated cells, tartaric acid-resistant acid phosphatase
(hereinafter abbreviated as "TRAP") activity, and a calcitonin
receptor. In a co-culture system of osteoblasts and bone marrow
cells as reported by Nobuyuki UDAGAWA et al., in Journal of
Experimental Medicine, Vol. 182, pp. 1,461-1,468 (1995), these
cells respond to factors such as 1.alpha.,25-dihydroxyvitamin
D.sub.3, prostaglandin E.sub.2, adrenocortical hormone, interleukin
1, interleukin 6, and interleukin 11, to form osteoclast-like cells
(hereinafter may be abbreviated as "OCL"). The formed OCL has
characters of osteoclasts in vivo. Therefore, the co-culture system
well reflects in vitro the processes of osteoclast formation in
vivo. Using this system, experiments for osteoclast formation and
osteoclastgenic inhibitory agents can be carried out.
[0064] The osteoclastgenic inhibitory activity of the IL-18
according to the present invention was studied using the above
co-culture system. The osteoblasts used in this experiment were
prepared in a conventional manner by treating a newborn mouse
calvaria with 0.1 w/v % collagenase commercialized by Worthington
Biochemical Co., Freefold, Australia, and 0.2 w/v % dispase
commercialized by Godo Shusei Co., Ltd., Tokyo, Japan. The bone
marrow cells were prepared from a mature mouse in a conventional
manner. As a negative control, 2.times.10.sup.4 cells of a primary
cell culture of osteoblasts and 5.times.10.sup.5 cells of bone
marrow cells were co-cultured in each well of a 48-well microplate
containing 0.4 ml/well of .alpha.-MEM medium supplemented with 10
v/v % fetal calf serum (hereinafter designated as "Medium"
throughout Experiment 4-2) at 37.degree. C. for seven days in a 5
v/v % CO.sub.2 incubator. As a positive control, the above
two-types of cells were co-cultured similarly as in the negative
control except that they were cultured in other wells containing
10.sup.-8M of 1.alpha.,25-dihydroxyvitamin D.sub.3 commercialized
by Wako Pure Chemicals, Tokyo, Japan, and 10.sup.-7M of
prostaglandin E.sub.2 commercialized by Sigma Chemical Company,
Missouri, USA. The aforesaid two-types of cells were co-cultured
similarly as in the positive control except that they were cultured
in other wells containing 1.alpha.,25-dihydroxyvitamin D.sub.3
commercialized by Wako Pure Chemicals, Tokyo, Japan, and
prostaglandin E.sub.2 commercialized by Sigma Chemical Company,
Missouri, USA., in the same concentrations as used in the positive
control, and a concentration of 0.01-10 ng/ml of an IL-18
preparation prepared by the method in Experiment 6. In every
co-culture system, the media in each well were replaced with fresh
preparations of the same media used in the co-culture systems on
the 3rd day after the initiation of each culture. According to the
method by Nobuyuki UDAGAWA in Journal of Experimental Medicine,
Vol. 182, pp. 1,461-1,468 (1995), the cells on the 6th day after
the initiation of each culture were fixed and stained based on TRAP
activity, followed by counting the stained cells (hereinafter
called "TRAP-positive cells") per well. Throughout Experiment 4-2,
quadruplet wells under the same conditions were provided for each
co-culture system, and the mean value for the TRAP-positive cells
per well in each system was calculated. The results are in Table
2:
2TABLE 2 IL-18 Osteoclastgenic (ng/ml) formation factor*1 Number of
TRAP-positive cells per well*2 0 - 2 0 + 110 0.01 + 114 0.1 + 111
0.5 + 106 1 + 63 2 + 29 4 + 12 8 + 2 10 + 2 Note: *1: The symbols
of "+" and "-" show co-culture systems with and without 10.sup.-8M
1.alpha.,25-dihydroxyvitam- in D.sub.3 and 10.sup.-7M prostaglandin
E.sub.2, respectively. *2: It shows a mean value of the data from
quadruplet wells cultured under the same conditions.
[0065] As shown in Table 2, the formation of TRAP-positive cells
was not substantially observed in the negative control, but the
distinct formation was observed in the positive control. In the
co-culture systems, i.e., the positive control supplemented
additionally with IL-18, the formation of TRAP-positive cells was
inhibited depending on the concentration of IL-18, and the maximum
inhibition, i.e., a level equal to that in the negative control,
was found at eight ng/ml or more of IL-18. These data strongly
indicates that IL-18 has a concrete activity of inhibiting OCL
formation in vitro and also inhibits osteoclast formation.
[0066] Experiment 7-2(b)
[0067] As described hereinbefore, it was confirmed that there exist
factors that induce the formation of osteoclast-like cells in the
co-culture systems used throughout Experiment 7-2. Therefore, in
this Experiment 7-2(b), it was studied whether the inhibitory
activity of IL-18 on osteoclast formation observed in Experiment
7-2(a) was specific to some factors or not; the osteoclast-like
cells were cultured by the same method as used in the negative
control in Experiment 7-2(a) except for using a medium supplemented
with 10.sup.-8M 1.alpha.,25-dihydroxyvita- min D.sub.3, 10.sup.-7M
prostaglandin E.sub.2, 200 ng/ml parathyroid hormone, 100 ng/ml
interleukin 1, or 20 ng/ml interleukin 11. These culture systems
were for positive controls. In parallel, the cells were cultured in
other wells by the same method used in the positive controls except
for using a medium containing 10 ng/ml of an IL-18 preparation
obtained by the method in Experiment 6, in addition to any one of
the above factors at the same concentration. After completion of
the cultures, TRAP-positive cells in each well were counted, and
the numbers were compared similarly as in Experiment 7-2(a). The
results are in Table 3:
3TABLE 3 Osteoclast formation factor*1 Number of (concentration)
IL-18*2 TRAP-positive cells per well*3 D.sub.3 (10.sup.-8M) - 94 +
3 PGE.sub.2 (10.sup.-7M) - 77 + 3 PTH (200 ng/ml) - 63 + 3 IL-11
(100 ng/ml) - 84 + 3 IL-1 (20 ng/ml) - 71 + 3 Note: *1: D.sub.3,
PGE.sub.2, PTH, IL-11, and IL-1 are respectively
1.alpha.,25-dihydroxyvitamin D.sub.3, prostaglandin E.sub.2,
parathyroid hormone, interleukin-11, and interleukin-1 which were
added to wells to give the concentrations as indicated in
parentheses. *2: The symbol "+" means that IL-18 was added to a
well to give a concentration of 10 ng/ml, and the symbol "-" means
that IL-18 was not added to. *3: It shows a mean value of the data
from quadruplet wells cultured under the same conditions.
[0068] As shown in Table 3, a distinct formation of TRAP-positive
cells was observed in every positive control, but the formation was
almost completely inhibited in the presence of IL-18. This strongly
indicates that IL-18 has a wide and general activity of inhibiting
osteoclast formation independently of osteoclast-formation-related
factors.
[0069] Experiment 7-2(c)
[0070] It was studied whether the osteoclastgenic inhibition by
IL-18, confirmed in Experiments 7-2(a) and 7-2(b), was caused by
the action of the IL-18-induced GM-CSF. For positive and negative
controls, the same co-culture systems employed in Experiment 7-2(a)
were used. Using other wells, the co-culture of osteoblasts and
bone marrow cells was carried out similarly as the method used for
the positive controls except for using a medium supplemented with
1.alpha.,25-dihydroxyvitamin D.sub.3 and prostaglandin E.sub.2 at
the same concentrations used in the positive control, and with (i)
10 .mu.g/ml of an anti-mouse GM-CSF polyclonal antibody
commercialized by R&D Systems, Minnesota, USA, (ii) 10 ng/ml of
an IL-18 preparation obtained by the method in Experiment 6, (iii)
(ii) plus 10 .mu.g/ml of an anti-mouse polyclonal antibody, (iv)
0.1 ng/ml of a mouse GM-CSF commercialized by R&D Systems,
Minnesota, USA, or (v) (iv) plus 10 .mu.g/ml of an anti-mouse
GM-CSF polyclonal antibody. After completion of the culture,
TRAP-positive cells in each well were counted, and the numbers were
compared similarly as in Experiment 7-2(a). The data is shown in
Table 4 where the symbols "i" to "v" coincide with those used in
the co-culture systems other than the control systems.
4TABLE 4 Culture Osteoclastgenic Anti-GM-CSF Number of
TRAP-positive system*1 factor*2 IL-18*3 GM-CSF*4 antibody*5 cells
per well*6 N - - - - 3 p + - - - 122 i + - - + 112 ii + + - - 3 iii
+ + - + 111 iv + - + - 4 v + - + + 106 Note: *1; where the symbols
"N" and "P" mean negative and positive controls, respectively, and
the symbols "i" to "v" correspond to those in the five types
co-culture systems used. *2; where the symbol "+" means that
1.alpha.,25-dihydroxyvitamin D.sub.3 and prostaglandin E.sub.2 were
respectively added to a well to give respective concentrations of
10.sup.-8M and 10.sup.-7M, and the symbol "-" means that these
compounds were not added to. *3; The symbol "+" means that IL-18
was added to a well to give a concentration of 10 ng/ml, and the
symbol "-" means that IL-18 was not added to. *4; The symbol "+"
means that GM-CSF was added to a well to give a concentration of
0.1 ng/ml, and the symbol "-" means that GM-CSF was not added to.
*5; The symbol "+" means that an anti-GM-CSF polyclonal antibody
was added to a well to give a concentration of 10 .mu.g/ml, and the
symbol "-" means that the polyclonal antibody was not added to.
[0071] As shown in Table 4, the formation of TRAP-positive cells
was almost completely inhibited by IL-18, cf., the co-culture
system (ii), but the inhibition was almost completely inhibited by
the addition of the anti-mouse polyclonal antibody, cf., the
co-culture system (iii). Mouse GM-CSF exhibited an activity of
inhibiting the formation of TRAP-positive cells similar to IL-18,
cf., the co-culture system (iv), and the inhibition was almost
completely inhibited by the addition of the anti-mouse GM-CSF
polyclonal antibody, cf., the co-culture system (v). The sole use
of the anti-mouse GM-CSF polyclonal antibody gave no influence on
the formation of TRAP-positive cells, cf., the co-culture system
(i). These data strongly indicates that the osteoclastgenic
inhibition by IL-18 was due to the action of the IL-18-induced
GM-CSF.
[0072] Experiment 8
[0073] Acute Toxicity Test
[0074] Eight-week-old mice were in a conventional manner injected
percutaneously, orally, or intraperitoneally with either of IL-18
preparations obtained by the methods in Experiments 1 to 6. The
results showed that these IL-18 preparations had an LD.sub.50 of
about one mg/kg or more in mice independent of the route of
administration. The data evidences that IL-18 can be incorporated
into pharmaceuticals for warm-blooded animals in general and
including humans without causing no serious side effects.
[0075] As described in Nikkei Biotechnology Annual Report 1996, pp.
498-499 (1995), published by Nikkei BP Publisher, Tokyo, Japan
(1995), the IL-18-induced GM-CSF has not yet been clinically used
in Japan, but applied clinically in USA and Europe. The fact would
show that IL-18 has substantially no serious side effects. These
facts indicate that the osteoclastgenic inhibitory agent according
to the present invention can be successively administered to
warm-blooded animals in general and including humans to induce
osteoclast formation and exert a satisfactory therapeutic and/or
prophylactic effect on osteoclast-related diseases without causing
serious side effects.
[0076] The following Examples describe the present osteoclastgenic
inhibitory agent according to the present invention:
EXAMPLE 1
[0077] Liquid
[0078] Either of IL-18 preparations, obtained by the methods in
Experiments 1 to 6, was dissolved in physiological saline
containing one w/v % human serum albumin as a stabilizer to give a
concentration of two mg/ml of the IL-18 preparation. The resulting
solutions were in a conventional manner membrane filtered for
sterilization into liquids.
[0079] The liquids have a satisfactory stability and can be
arbitrarily used as ingredients for cell culture and agents in the
form of an injection, ophthalmic solution, or collunarium for
regulating bone resorption and for osteoclast-related diseases,
directed to treat and/or prevent hypercalcemia, osteoclastoma,
osteoporosis, etc.
EXAMPLE 2
[0080] Dry Agent
[0081] Fifty milligrams of either of IL-18 preparations, obtained
by the methods in Experiments 1 to 6, was dissolved in 100 ml of
physiological saline containing one w/v % purified gelatin as a
stabilizer. The solutions thus obtained were in a conventional
manner membrane filtered for sterilization, distributed to vials by
one milliliter, lyophilized, and sealed with caps.
[0082] The products have a satisfactory stability and can be
arbitrarily used as ingredients for cell culture and agents in the
form of a dry injection for regulating bone resorption and for
osteoclast-related diseases, directed to treat and/or prevent
hypercalcemia, osteoclastoma, osteoporosis, etc.
EXAMPLE 3
[0083] Dry Agent
[0084] Fifty milligrams of either of IL-18 preparations, obtained
by the methods in Experiments 1 to 6, was dissolved in 100 ml of
physiological saline containing one w/v % trehalose as a
stabilizer. The solutions were in a conventional manner membrane
filtered for sterilization, distributed to vials by one milliliter,
lyophilized, and sealed with caps.
[0085] The products have a satisfactory stability and can be
arbitrarily used as ingredients for cell culture and agents in the
form of a dry injection for regulating bone resorption and for
osteoclast-related diseases, directed to treat and/or prevent
hypercalcemia, osteoclastoma, osteoporosis, etc.
EXAMPLE 4
[0086] Ointment
[0087] "HIVIS WAKO GEL.RTM. 104", a carboxyvinylpolymer
commercialized by Wako Pure Chemical Industries, Ltd., Tokyo,
Japan, and a high-purity trehalose were dissolved in a sterilized
distilled water to give respective concentrations of 1.4 w/w % and
2.0 w/w %, and the solution was mixed to homogeneity with either of
IL-18 preparations obtained by the methods in Experiments 1 to 6,
and adjusted to pH 7.2 to obtain a paste containing about one mg of
an IL-18 preparation per g of the product.
[0088] Each product thus obtained has a satisfactory spreadability
and stability and can be arbitrarily used as an agent in the form
of an ointment for regulating bone resorption and for
osteoclast-related diseases, directed to treat and/or prevent
hypercalcemia, osteoclastoma, osteoporosis, etc.
EXAMPLE 5
[0089] Tablet
[0090] "FINETOSE.RTM.", an anhydrous crystalline a-maltose powder
commercialized by Hayashibara Biochemical Laboratories, Inc.,
Okayama, Japan, was mixed to homogeneity with either of IL-18
preparations, obtained by the methods in Experiments 1 to 6, and
"LUMIN" or
1-1'-1"-trihepthyl-11-chinolyl(4).multidot.4.multidot.4'-penthamethinchyn-
ocyanine-1,1"-dijodide. The mixtures were in a conventional manner
tabletted to obtain tablets, about 200 mg weight each, containing
an about two milligrams of either of the IL-18 preparations and an
about two milligrams of LUMIN per tablet.
[0091] The products have a satisfactory swallowability, stability,
and cell-activating activity and can be arbitrarily used as agents
in the form of a tablet for regulating bone resorption and for
osteoclast-related diseases, directed to treat and/or prevent
hypercalcemia, osteoclastoma, osteoporosis, etc.
[0092] As described above, the osteoclastgenic inhibitory agent
according to the present invention effectively inhibits osteoclast
formation. Therefore, the agent can be arbitrarily used as an
ingredient for cell culture and agents for regulating bone
resorption and for osteoclast-related diseases, directed to treat
and/or prevent hypercalcemia, osteoclastoma, osteoporosis, etc.
[0093] Thus the present invention with these useful activities and
functions is a significant invention that would greatly contribute
to this field.
[0094] While there has been described what is at present considered
to be the preferred embodiments of the invention, it will be
understood the various modifications may be made therein, and it is
intended to cover in the appended claims all such modifications as
fall within the true spirits and scope of the invention.
Sequence CWU 1
1
* * * * *