Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFICATION
THERAPEUTIC AGENT FOR CARTILAGINOUS DISEASES
Technical Field
The present invention relates to a medicine useful for
the prevention and treatment of cartilaginous diseases. More
particularly, the present invention relates to a therapeutic
agent for cartilaginous diseases, an accelerator for
chondrocyte proliferation and an accelerator for proteoglycan
production comprising HGF (Hepatocyte Growth Factor) as an
active component.
Background Art
Cartilage is a connective tissue composed of chondrocytes
and matrices surrounding them, and exists in articulatio>
intervertebral disk of spina, costal cartilage, auricle,
external acoustic meatus, pubic symphysis, throat tectum and
the like. Cartilage is composed of chondrocytes and cartilage
matrices produced by the chondrocytes, and the cartilage
matrix consists mainly of a fiber component such as a collagen
fiber, proteoglycan and water. Cartilage can be classified
into hyaline cartilage (costal cartilage, throat cartilage,
articular cartilage and the like), elastic cartilage (auricle
cartilage and the like) and fibrocartilage (intervertebral
disk cartilage, pubic bone cartilage, articular cartilage and
the like) depending on the mixing condition of a cartilage
a
:,
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matrix. In the aforesaid cartilage matrix, it is said that
the collagen fiber concerns the stiffness and strength of
cartilage against tension and shear force, proteoglycan
concerns the strength of cartilage against compression force,
and water manifests a feature as a viscoelastic material for
an organism tissue, and for example, in the articular
cartilage, 78.6% of the weight of cartilage is occupied by
water, 20% is occupied by collagen and 7% is occupied by
proteoglycan. The effect of cartilage includes the reduction
1 0 in abrasion of epiphysis (cartilage between bones),
maintenance of. elasticity (auricle cartilage and the like),
and motion function (rib cartilage, pubic bone cartilage and
the like).
As described above, cartilage has an important effect for
1 5 maintaining the function of an organism, and conventionally,
there are known various disorders caused by cartilage
diseases, including, for example, chondrodystrophy,
osteoarthritis, diastrophic discopathy, failure in restoration
and cure of fracture and the like. Particularly. thPrP hae
20 been a remarkable increase in the number of the patient
suffering from anthropathy because of the arrival an aging
society, the increase of injury due to sports and the
appearance of occupational diseases represented by keypuncher
disease and the like, and there is desired the development of
2 5 medical care in this field.
Various therapeutic methods have been conventionally
tried for the treatment of cartilage diseases. They were not
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intended for direct solution of causes, but were only nosotropic methods, for
example, a method in which disorder such as pain due to the disease is
inhibited by
administering an antiphlogistic; a method in which the motion of an
articulatio is
made smooth by injecting a hyaluronic acid preparation into the articulatio,
and the
S like.
As described above, radical therapeutics for articular disorders has not been
found, and there are many patients suffering from osteoarthritis, therefore,
an
effective therapeutic method for articular disorders is eagerly desired.
The present inventors have been studied intensely to solve the
above-mentioned problems. As a result, it has been found that HGF promotes the
proliferation of a chondrocyte, has an effect to promote the production of
proteoglycan, and is effective for the treatment of various disorders caused
by
cartilaginous diseases, and the present invention has been completed.
The above-described HGF is a protein which was found as a factor to
IS proliferate liver parenchyma cells in vitro (Biochem Biophys Res Commun,
122,
1450, 1984, T. Nakamura et al.; Proc. Natl. Acad. Sci. USA, 83, 6489, 1986, T.
Nakamura et al.; FEBS Letter, 224, 311, 1987, T. Nakamura et al.; Nature, 342,
440, 1989, T. Nakamura et al.; Proc. Natl. Acad. Sci. USA, 87, 3200, 1990, K.
Tashiro et al.). It has become apparent as a result of the recent studies by
many
researchers including the present inventors that HGF, which was found as a
factor
to specifically proliferate liver parenchyma cells, manifests various
activities such
as the cure of tissue injury in vivo, and HGF is expected to be not
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only a research object but also therapeutic agents applied to
human and animals.
It is known that HGF is produced mainly by mesenchymal
cells, and it has become clear that so-called paracrine
mechanism exists in which HGF is optionally supplied from
adjacent cells. However, it is believed that HGF is supplied
also by so-called endocrine mechanism since when liver or
kidney is injured, the production of HGF is increased also in
the organs which are not injured, for example lung and the
1 0 1 ike.
Regarding the receptor of HGF, it has been identified
form the recent studies that c-Met proto-oncogene codes the
HGF receptor (Bottaro et al., Science 251, 802-804, 1991;
Naldini et al., Oncogene 6, 501-504, 1991).
As described above, though various information are known
regarding HGF, the chondrocyte growth accelerating effect and
proteoglycan production accelerating effect are novel
knowledge which are conventionally not known, and the present
invention accomplished based on the knowledge provides a
medicine useful for the treatment of various disorders caused
by cartilaginous diseases.
Disclosure of the Invention
The present invention is a therapeutic agent for
2 5 cartilaginous diseases comprising HGF as an active component.
Further, the present invention relates to an accelerator
for chondrocyte proliferation comprising HGF as an active
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component; an accelerator for proteoglycan production comprising HGF as an
active
component; and a therapeutic method for cartilaginous diseases of human or
mammals comprising administering an effective amount of HGF.
The above-described HGF may be one derived from the tissue or blood
component of human or animals, or one produced by gene recombination.
The active component HGF is effective for the treatment and prevention of
various disorders caused by cartilaginous diseases, since HGF promotes the
growth
of chondrocytes and promotes the production of proteoglycan.
In another aspect, the present invention provides a therapeutic composition
for a cartilaginous disease comprising Hepatocyte Growth Factor (HGF) and a
pharmaceutically acceptable carrier, wherein said cartilaginous disease is
osteoarthritis, cure and restoration of fracture, restoration of articular
cartilage and
articular disk due to injury, rheumatoid arthritis, or restoration of
cartilage after bone
transplantation.
In another aspect, the present invention provides Hepatocyte Growth Factor
(HGF) for treating a cartilaginous disease, wherein said cartilaginous disease
is
osteoarthritis, cure and restoration of fracture, restoration of articular
cartilage and
articular disc due to injury, rheumatoid arthritis, or restoration of
cartilage after bone
transplantation.
Brief Explanation of the Drawings
Fig. 1 is a microphotograph showing the expression of HGF mRNA in limb
buds of mice at the early developmental stage (the left part is bright field,
the right
part is corresponding dark field). In Fig. 1, each A to D is the longitudinal
section of
a 10.5 days p. c. embryo, each E to H is the longitudinal section of a 11 days
p. c.
embryo.
Fig. 2 is a microphotograph showing the expression of HGF mRNA in
limb buds of mice at the digitus forming stage (the left part is bright field,
the
right part is corresponding dark field). In Fig. 2, each A and B is the
section of a
12. 5 days p. c. embryo, each C to F is the section of a 13 days p. c. embryo,
each
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Sa
G to J is the section of a 14 days p. c. embryo. Further, Fe indicates a
femur,
Fi indicates a fibula, Ta indicates a tarsal bone, and each I to V indicates
digit
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numb a r.
Fig. 3 is a microphotograph showing the expression of HGF
mRNA in limb buds and thoraxes of mice at the developmental
stage (the left part is bright field, the right part is
corresponding dark field). In Fig.3, each A and B is the
horizontal section of the hind limb of a 16 days p.c. embryo,
each C and D is the longitudinal section of the thorax of a 13
days p.c. embryo, each E and F is the longitudinal section of
the thorax of a 14 days p.c. embryo. Further, Ta indicates a
tarsal bone, Ti indicates a tibia and Rib indicates the
precartilaginous condensation of rib cartilage.
Fig. 4 is a microphotograph showing the scatter activity
of HGF on chondrocytes. In Fig.4, A indicates control (no-HGF-
treatment), B indicates HGF-treatment.
Fig. 5 is a drawing showing the effect of HGF on
chondrocyte proliferation. In Fig. S, A indicates the effect
on DNA synthesis of articular chondrocytes, B indicates the
effect on DNA synthesis of synovial cells, and C indicates the
effect on proliferation (cell number) of articular
2 0 chondrocytes.
Fig. 6 is a drawing showing the effect of HGF on
proteoglycan production.
Fig. 7 is a drawing showing the effect of HGF on DNA
synthesis (Fig. 7A) and proteoglycan production (Fig. 7B) in
the presence of an anti-HGF antibody.
Fig. 8 is an electrophoretic photo showing the expression
of HGF receptor mRNA in chondrocytes.
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The Best Mode for carrying out the Invention
As HGF used in the present invention, there can be used one which
prepared by various methods if it is purified to an extent that it can be used
as a
medicine. Various methods are known for preparing HGF. For example, HGF can
be obtained by extraction and purification from organs (e. g. liver, spleen,
lung,
bone marrow, brain, kidney, placenta and the like), serum, plasma and blood
cells
(e. g. platelet, leucocyte and the like) of mammals such as rat, cow, horse,
sheep
and the like (see FEBS Letter, 224, 311, 1987, T. Nakamura et al.; Proc. Natl.
Acad. Sci. USA, 86, 5844, 1989, E. Gherardi et al., and the like).
Also, it is possible to obtain HGF by cultivation of primary culture cells or
cell lines producing HGF, followed by separation and purification from the
culture
product (e. g. culture supernatant, cultured cell, etc.). Further, HGF can be
obtained by gene engineering method which comprises cloning the gene coding
HGF with a proper vector, inserting it into a proper host cell to give a
transformant,
and separating the desired recombinant HGF from the culture supernatant of the
transformant (e. g. Nature, 342, 440, 1989, T. Nakamura et al.; Japanese
Patent
Kokai No. 111383/1993, T. Nakamura et al.; Biochem. Biophys. Res. Commun.,
163, 967, 1989, K. Miyazawa et al.). The host cell is not specifically
limited, and
various host cells conventionally used in gene engineering methods can be
used,
which are, for example, Escherichia coli, Bacillus subtilis, yeast,
filamentous
fungi, and plant or
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animal cells.
More specifically, the method of extracting and purifying HGF from live
tissues is, for example, to administer carbon tetrachloride to a rat
intraperitoneally,
remove a liver from the rat with hepatitis, grind it, and purify by the
ordinary
protein purifying technique such as gel column chromatography using
S-Sepharose* and heparin Sepharose*, HPLC and the like.
Further, by the gene engineering method, the gene coding the amino acid
sequence of human HGF is cloned into a vector such as bovine papilloma virus
DNA and the like to obtain an expression vector, and by using this expression
vector, animals cells such as Chinese hamster ovary (CHO) cells, mouse C127
cells, monkey COS cells and the like are transformed, and HGF can be obtained
from the culture supernatant of the transformants.
In thus obtained HGF, a part of the amino acid sequence of HGF may be
deleted or substituted by other amino acid(s), another amino acid sequence may
be
inserted, one or more amino acids may be bonded to the N-terminal and/or
C-terminal, or saccharide chains) may likewise be deleted or substituted,
providing it has substantially the same effect as HGF.
The therapeutic agent and accelerator of the present invention comprise the
above-mentioned HGF as an active component, and HGF promotes the
proliferation of chondrocytes and has an effect to promote the production of
proteoglycan, as shown in Test Examples mentioned later. Further, HGF has a
feature that there is little tendency to cause side effect,
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since it shows no effect on the chondrocytes not injured and
shows an effect only on the chondrocytes injured. Therefore,
the therapeutic agent and accelerator of the present invention
are effective for the treatment and prevention of various
disorders caused by cartilaginous diseases, including, for
example, the following disorders.
Osteoarthritis
Chondrodystrophy
Cure and restoration of fracture
Restoration of articular cartilage and articular disk due to
injury
Acute suppurative arthritis
Tuberculous arthritis
Syphilitic arthritis
Rheumatoid arthritis
Rheumatic fever
Systemic lupus erythematosus
Spondylitis deformans
Herniated disk
Restoration due to bone transplantation
The therapeutic agent and accelerator of the present
invention may be applied to the treatment and prevention of
various disorders caused by cartilaginous diseases in human
and the other animals (for example, cow, horse, pig, sheep,
dog, cat and the like).
The therapeutic agent and accelerator of the present
invention can be prepared in various preparation forms (e. g.,
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1 0
liquid preparation, solid preparation, capsule preparation and
the like), and in general, it is prepared in the form of
injection preparation, inhalation preparation, suppository
preparation or oral preparation containing HGF as the active
component alone or together with common carrier. The afore-
said injection preparation can be prepared by a conventional
method, for example, it can be prepared by dissolving HGF in a
suitable solvent (e. g. , sterilized water, buffer solution,
physiological saline and the like), sterilizing the solution
by filtration through a filter or the like, and then filling
the sterilized solution in aseptic vessels. The HGF content
in the injection preparation may be usually from about 0.0002
to 0.2 (W/V%), preferably from about 0.001 to 0. 1 (W/V%).
Further, the oral preparation may be formed as a preparation
such as a tablet, granule, grain, powder, soft or hard
capsule, liquid preparation, emulsion, suspension, syrup and
the like, and these preparations can be prepared by a
conventional pharmaceutical method. The suppository
preparation can also be prepared by a conventional
pharmaceutical method using a common base (for example, cacao
oil, laurin oil, glycero-gelatin, macrogol, witepsol and the
like). Further, the inhalation preparation can be prepared by
a conventional pharmaceutical method.
The HGF content in the preparation may be suitably
adjusted depend on the preparation form, disease to be applied
and the like.
In production of the preparation, a stabilizer is
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preferably added, and the stabilizer includes, for example, albumin, globulin,
gelatin, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and
the like.
Further, the preparation of the present invention may contain additives
necessary
for pharmaceutical preparation, for example, an excipient, a dissolving aid,
an
antioxidant, a pain-alleviating agent, an agent for isotonicity and the like.
In the
liquid preparation, it is preferable to store it under frozen conditions or
after the
removal of water by a process such as freeze-drying. The freeze-dried
preparation
is used by dissolving again in distilled water for injection and the like
before use.
The therapeutic agent and accelerator of the present invention can be
administered via a suitable administration route depend on the preparation
form.
For example, the injection preparation can 'be administered by intravenous,
intraarterial, subcutaneous, intramuscular and the like. The dose is suitably
adjusted depend on symptoms, age, body weight and the like of a patient, and
usually from 0.05 mg to 500 mg, preferably from 1 mg to 100 mg of HGF is
administered once or several times per day.
In another aspect, the present invention provides a therapeutic composition
for cartilaginous diseases comprising Hepatocyte Growth Factor (HGF) wherein
said cartilaginous disease is osteoarthritis, cure and restoration of
fracture,
restoration of orticulor cartilage and articular disk due to injury,
rheumatoid
arthritis, or restoration of cartilage after bone transplantation.
In another aspect, the present invention provides use of an effective amount
of Hepatocyte Growth Factor (HGF) for treating cartilaginous diseases of
humans
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lz
or mammals, wherein said cartilaginous disease is osteoarthritis, cure and
restoration of fracture, restoration of acticular cartilage and articular disk
due to
injury, rheumatoid arthritis, or restoration of cartilage after bone
transplantation.
In another aspect, the present invention provides use of a wild-type full
length Hepatocyte Growth Factor (HGF) for treating cartilaginous diseases and
disorders in mammals, wherein said cartilaginous diseases and disorders are
osteoarthritis, failure in restoration and cure of fracture, articular
cartilage and
articular disk injury, rheumatoid arthritis, or restoration of cartilage after
bone
transplantation.
Industrial Applicability
In the present invention, an active component HGF has an effect to promote
the proliferation of chondrocytes and to promote the production of
proteoglycan.
Therefore, the therapeutic agent and accelerator of the present invention are
useful
for the treatment and prevention of various disorders caused by cartilaginous
diseases. Further, according to the invention, there can be obtained a
medicine
having little side effect, since HGF acts only on cartilaginous tissue
injured.
Example
The following Examples and Production Examples further illustrate the present
invention in detail but are not to be construed to limit the scope thereof.
The
materials and methods used in the following experiments are shown below.
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Materials and methods
In situ h~rbridization
An EcoRI fragment (1.4 kb) of a rat HGF-cDNA (RBC1 clone)
(Proc. Natl. Acad. Sci. USA, 87, 3200, 1990, K. Tashiro et al.) was
subcloned to a pGEM7'~ vector to produce sense and antisense RNA
probes labeled with [a-35S]UTP (400 Ci/mmol, manufactured by
Amersham Ltd.). The labeled transcripts were hydrolyzed with an alkali
to a SO to 150 bases substance as a ribo-probe.
In situ hybridization was carried out by the method described in a literature
(Biochem. Biophys. Res. Commun., 173, 42, 1990, S. Noji et al.). The sample
was
fixed in 4% paraformaldehyde in phosphate-physiological saline solution,
dehydrated with ethanol, washed with toluene, and then embedded in paraffin. 5
pm sections were cut and mounted on slide glasses coated with poly-L-lysine.
The
sections were deparaffinized with glycine and acetic anhydride, and hybridized
with the probes at SO°C for 16 hours. Then, the sections were washed
with 0. xSSC
solution at 50°C for 1 hour, treated with RNAase A (20 ,pg/ml) at
37°C for 30
minutes, washed twice with 2xSSC solution at 37°C for 10 minutes. The
sections
were immersed in an emulsion (1:1 dilution of Kodak NBT-2*), and exposed to
light for 2 weeks. The sections were developed with Kodak D-19*, fixed and
stained with hematoxylin-eosin.
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Cells and cell incubation
Chondrocytes were isolated from 23 day-old embryo and 4 week-old
postnatal New Zealand white rabbit by the method described in a literature (J.
Cell.
Physiol., 133, 491, 1987, Y. Kato et al.). Articular cartiLages were isolated
from
the knee joint femoral articular cartilage, and costal cartilages were
isolated from
the hyaline cartilage of rib (Dev. Biol., 136, 500, 1989, M. Iwamoto et al.).
Synovial fibroblasts were isolated from the synovial tissue of knee joint.
Minced
fragments of the synovial tissue were cultured for 10 days in DMEM containing
10% FBS, and there were collected the proliferated cells by trypsin treatment.
Embryonic mesenchymal cells were isolated from the limb muscular tissue of 20
day-old rat embryo by the method described in a literature (Exp. Cell Res.,
157,
483, 1985, Thompson AY. et al.). Limb bud mesenchymal cells were isolated
from 10.5 day-old rat embryo. The limb buds were dissected under a surgical
microscope, treated with 0.25% trypsin for 30 minutes, then pipetted and
passed
through a nylon sieve to isolate single cells. The all cells except limb bud
cells
were maintained in DMEM containing 10% FBS and 60 4~,g/ml kanamycin
(hereinafter, referred to as medium A) at 37°C under 5% C02 /95% air.
~ Measurement of DNA synthesis
The DNA synthesis rate was evaluated by measuring the incorporation of
[3H]-thymidine ([6-3H]-thymidine, manufactured by Amersham Ltd., 20 Ci/mmol)
into a 10% TCA insoluble-cell precipitate (J. Clin. Invest., 85, 626, 1990, T.
Koike
et al.). The cells were seeded at a density of l.Sx104/6 mm well (96 wells
plate),
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and incubated to be confluent. For the growth arrest, the cells were
preincubated
with 0.1 ml of DMEM containing 0.3% FBS. Various concentrations of HGF were
added to the media. The incubation continued for 24 hours. One pCi/ml of
[3H]-thymidine was added 3 hours before the termination of the incubation.
After
5 the labelling, the cells were washed three times with ice-cooled PBS, twice
with
5% TCA containing 3 mM thymidine, once with ethanolldimethyl ether (3:1). The
residues in the well were solubilized with 100 pl of 0.1 N NaOH, transferred
to a
liquid scintillation vial, neutralized with 1 N HCI, and radioactivity was
measured
by a scintillation counter (Rack-beta, manufactured by Pharmacia Ltd.).
10 ~ Measurement of proteoglycan synthesis
Chondrocytes were seeded at a density of I.Sx104/6 mm well, and
maintained in 0.1 ml of the medium A. When the cells became confluent, they
were preincubated for 24 hours in 0. I ml of DMEM containing 0.3% FBS. Then,
they were preincubated for 24 hours in 0. 1 ml of DMEM containing 0. 3% FBS
15 and HGF. One p,Ci/ml [35S]-sulfate was added 20 hours before the
termination of
incubation. The proteoglycan synthesis was evaluated by measuring the
incorporation of [35S]-sulfate into a precipitate with cetylpyridinium
chloride after
protease digestion (Exp. CeII Res., I30, 73, 1980, Y. Kato et al.).
~ Total RNA preparation and reverse transcribed PCR
Total RNA from cartilage was prepared by a modified method of the
method described in a literature (Anal. Biochem., 203, 352, 1992, Georgeann
Smale and Joachim Sasse). Freshly isolated tissue fragments (wet weight 0.1 g)
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were quickly homogenized in 2 ml of a 4 M GITC solution of 1
2-mercaptoethanol, 0. 1 M Tris-HCl (pH 7.5) and 4 M guanidine thiocyanate. The
homogenate was mixed with 100 p,l of 10% SDS, and the mixture was centrifuged
by a microcentrifuge for 5 minutes. The supernatant (2 ml) was layed on the
same
volume of 1.6 g cesium trifluoroacetate and 1 mM EDTA (pH 8.0) in a Beckmann
polyalloma centrifuge tube (13x51 mm). The samples were centrifuged at 35,000
rpm (147,OOOxg) for 20 hours at 18°C. The supernatant was removed under
suction, then the precipitate was dissolved in 200 p,l of 4 M GITC solution
and
extracted with phenol: chloroform: isoamyl alcohol (25:24:1), then the
extracted
material was mixed with 20 ~l of 3 M sodium acetate (pH 4. 8), and
precipitated
with 2-fold volume (440 pl) of ethanol. The precipitate was dissolved in
DEPC-treated water.
First, a first-strand cDNA was synthesized from 0. 5 ~.g of the total RNA
using a SuperScript* reverse transcriptase (Gibco-BRL) and downstream
anti-sense primers. Subsequently, PCR amplification was carried out. The
amplification was conducted in 35 cycles (in the case of chondrocytes) or 40
cycles
(in the case of cartilaginous tissue), each cycle being conducted 30 seconds
at
94°C, 1 minute at 58°C and 1.5 minutes at 72°C. As a
primer base sequence for
PCR amplification, a 725 by fragment was prepared with
5-CAGT(A/G)ATGATCTCAATGGGCAAT-3' and
5'-AATGCCCTCTTCCTATGACTTC-3' for c-Met of a rat and mouse (Oncogene,
2, 593, 1988, Chan AM-L. et al.)
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16a
Example 1
HGF mRlelA expression in developmental limb
The expression of HGF mRNA in the limb bud of a developmental mouse
was tested by in situ hybridization method. The results are shown in Fig. l,
Fig. 2
S and Fig. 3.
Fig. 1 shows the expression of HGF mRNA in limb buds of early
developmental stage mice, and is a microphotograph of longitudinal sections of
the hind-limb. The bright field (left part) and the corresponding dark field
(right
part) were photographed after in situ hybridization, autoradiography and
staining.
In this figure, each A to D indicates the section of a 0. 5 days p. c. embryo,
and
each E to H indicates the section of a 11 days p. c. embryo.
Fig. 2 shows the expression of HGF mRNA in limb buds of digitus forming
stage mice, and is a microphotograph of longitudinal sections of the hind-
limb. The
bright field (left part) and the corresponding dark field (right part) were
photographed after in situ hybridization, autoradiography and staining. In
this
figure, each A and B indicates the section
1 7 2191869
of a 12. 5 days p. c. embryo, each C to F indicates the section
of a 13 days p.c. embryo, and each G to J indicates the
section of a 14 days p.c. embryo. Further, Fe indicates a
femur; Fi indicates a fibula, Ta indicates a tarsal bone, and
each I to V indicates digit number.
Fig. 3 is a microphotograph showing the expression of HGF
mRNA in limb buds and thoraxes of mice at developmental stage,
and the bright field (left part) and the corresponding dark
field (right part) were photographed after in situ
hybridization, autoradiography and staining. In this figure,
each A and B indicates the horizontal section of the hind limb
of a 16 days p. c. embryo, each C and D indicates the
longitudinal section of the thorax of a 13 days p.c. embryo,
each E and F is the longitudinal section of the thorax of a 14
1 5 days p. c. embryo. Further, Ta indicates a tarsal bone, Ti
indicates a tibia and Rib indicates the precartilaginous
condensation of rib cartilage.
As shown in Fig. 1, there was detected the diffuse
expression of HGF mRNA around the bottom region of the limb on
11th day. At this stage, the cartilaginous condensation was
not formed in the limb. With the progress of the
cartilaginous condensation, the expression site of HGF mRNA
became more restricted. When stylopodium, zygopodium and
autopodium were formed on 12.5th day, the expression of HGF
mRNA was observed at wrist/heel and elbow/knee joint regions
(see Fig. 2A and B). For convenience, knee and heel are
shown). In the later stage (13 to 14 days), HGF mRNA was
1 s 2197869
expressed in the restricted mesenchymal cells adjacent to the
cartilaginous condensation at wrist/heel and elbow/knee joint
regions (see Fig. 2C to J). On 16th day, HGF mRNA was
localized in the restricted mesenchymal cells adjacent to the
cartilage of tarsal bone (see Fig. 3A and B). The expression
level in the limb of HGF mRNA was decreased with the
differentiation. Throughout the tests, HGF mRNA was not
detected in the growing disk of hand and foot.
1 0 Example 2
HGF mRNA expression in developing thorax
The expression of HGF mRNA in the thorax of developing
mouse was tested by in situ hybridization method. The results
are shown in Fig. 3C to F.
1 5 As shown in Figs. 3C to F, HGF mRNA was expressed in an
ambient intercostal mesenchymal tissue at the end point of a
precartilaginous condensation formed by extension of the
intercostal bone. In the precartilaginous~condensation, the
signal of hybridization was not detected.
Example 3
Scatter activity test of HGF on chondrocytes
To determine which is the target cell of HGF around the
differentiating cartilage tissue, there were prepared the
2 5 cultured cells of chondrocytes derived from articular
cartilage of knee joint and costal cartilage, synovial cells
derived from knee joint and fibroblasts proliferated from limb
1 9 2197869
muscular tissue, and the effects of HGF exogenously added to
these cells were examined.
Namely, articular chondrocytes of rabbit were seeded at a
density of 3x103/16 mm well, and maintained in medium A for 2
days. Then, the chondrocytes were treated with HGF for 2
days. When the incubation was over, phase-contrast
photomicrographs were taken. The results are shown in Fig. 4.
As shown in Fig. 4, in no-HGF-treated test (control),
polyhedron chondrocytes proliferated and formed islands (Fig.
4A). On the other hand, in the culture treated with HGF (3
ng/ml), each chondrocytes existed a single cell and did not
form an island (Fig. 4B). Therefore, it became clear that HGF
stimulates the migration of chondrocytes. In contrast, HGF
scattered neither fibroblasts nor synovial cells.
Example 4
Effect of HGF on chondrocyte roliferation
The effect of HGF on chondrocytes proliferation was
examined.
Namely, articular chondrocytes isolated from 4 week-old
rabbit were incubated. The cells which had become confluent
was subjected to serum deleting treatment for 24 hours, then
treated with various concentrations of HGF, and the
incorporation amount of [3H]-thymidine was measured by the
method described in the aforesaid Materials and Methods.
Further, the same test was conducted with respect to the
rabbit synovial fibroblasts. The results are shown in Fig. 5A
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(articular chondrocytes) and 5B (synovial fibroblasts). Here,
the result is shown in terms of average ~ standard deviation
of three tests (likewise also in Fig. 5C, Fig. 6 and Fig. 7A
and B) .
As shown in Fig. 5A, HGF increased the incorporation of
[3H]-thymidine into rabbit articular chondrocytes depending on
the dose, and had an effect to promote DNA synthesis, namely
an effect to promote proliferation of the articular
chondrocytes. With 1 ng/ml HGF, DNA synthesis was recognized
to increase three times against the control. On the other
hand, as shown in Fig. 5B, the synovial fibroblasts did not
react to HGF.
Further, in the other experiment, the effect of HGF on
the cell number of articular cartilage was examined. Namely,
1 5 rabbit articular chondrocytes were seeded at a density of
1x104/16 mm well, and maintained in a DMEM medium containing
10% FBS. Then, 10 ng/ml HGF was added and the mixture was
incubated for 48 hours, and after the incubation, the cell
number was measured. The results were shown in Fig. 5C.
As shown in Fig. 5C, 10 ng/ml HGF increased the cell
number 1.8 times as compared with the control.
Example 5
Effect of HGF on proteoglycan production
2 5 Since HGF promoted the proliferation of chondrocytes as
described above, the effect of articular chondrocytes on
proteoglycan production was examines by the method described
CA 02197869 2004-07-16
21
in the aforesaid Materials and Methods. The proteoglycan synthesis was
evaluated
by measuring the incorporation of [35S]-sulfate into macromolecules
(glycosaminoglycans) precipitated with cetylpyridiniurn chloride after
protease
digestion (Exp. Cell Res., 130, 73, 1980, Y. Kato et al.). Further, there were
conducted tests on the following factors instead of HGF. Insulin-like growth
factor (IGF)-I: concentration 100 ng/ml IGF-II: concentration 100 ng/ml
Parathyroid hormone (PTH): concentration 10-~ M TGF-(3 : concentration 3
ng/ml.
The results are shown in Fig. 6. As shown in Fig. 6, HGF increased the
incorporation of [35S]-sulfate depending on the dose. The maximum increase was
recognized at 1 ng/ml HGF. This effect is approximately the same as those of
IGF-I and II (Exp. Cell Res., 130, 73, 1980, Y. Kato et al.), though weaker
than
those of TGF-(3 J3 (J. Cell Physiol., 138, 329, 1989, H, moue et al.) and PTH
(J.
Clin. Invest., 85, 626, 1990, T. Koike et al.).
Example 6
Effect of HGF on DNA synthesis and proteo~lycan production in the presence of
anti-HGF antibody
As described above, HGF is usually thought to act on target cells by
paracrine mechanism, and it is considered that the result of the aforesaid in
situ
hybridization supports this idea. Then, to recognize this point, it was
examined
whether or not the anti-HGF polyclonal antibody modulates the functions of
chondrocytes.
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22
Namely, rabbit articular chondrocytes which had become
confluent were treated with 25 ~cg/ml anti-HGF polyclonal
antibody (IgG fraction purified by affinity) or not treated in
the presence or absence of 3 ng/ml HGF. Then, the
chondrocytes were labeled with [3H]-thymidine or [35S]-sulfate
by the method described in the aforesaid Materials and Methods
to measure the DNA synthesis or proteoglycan production. The
results are shown in Fig. 7A (DNA synthesis) and B
(proteoglycan production). In these figures, Ab indicates an
anti-HGF polyclonal antibody.
As shown in Fig. 7, the addition of the anti-HGF
polyclonal antibody alone did not change the DNA synthesis and
proteoglycan production in the articular chondrocytes.
However, the anti-HGF polyclonal antibody completely blocked
1 5 the effect of the exogenously added HGF. This means that
chondrocytes do not produce sufficient HGF to modulate the
functions of themselves.
Example 7
Expression of HGF receptor mRNA in chondrocytes
The.expression of HGF receptor (c-Met) in chondrocytes in
vivo and in vitro was examined by reverse transcription PCR.
Various parts of articular tissue 'and costal cartilage were
dissected out from 4 week-old rat under a surgical microscope,
and the total RNA was extracted as described in Materials and
Methods. The extracted RNA (0.5 ug) was subjected to reverse
transcription, amplified using c-Met primers, and then
CA 02197869 2004-07-16
23
analyzed by 1.5 % agarose gel electrophoresis. The results
are shown in Fig, 8.
As shown in Fig. 8, after the amplification was repeated
40 cycles with respect to the articuiar chondrocytes and
costal chondrocytes, slight c-Met expression was detected, and
after the amplification of the cultured chondrocytes was
repeated 35 cycles, remarkable c-Met expression was detected.
Production Example 1
Production example of HGF preparation
(1) HGF 20 ug
human serum albumin 100 mg
The above-mentioned substances were dissolved in 0.01 M
PBS of pH '7.0, and the total amount was adjusted to 20 ml and
sterilized. The solution was poured into vials by 2 ml,
freeze-dried and sealed.
(2) HGF 40 ~.cg
Tween 80 ~ 1 mg
human serum albumin 100 mg
2 0 The above-mentioned substances were dissolved in
physiological saline for injection, and the total amount was
adjusted to 20 ml and sterilized. The solution was poured
into vials by 2 ml, freeze-dried and sealed.
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