WO1996005855A1

WO1996005855A1 - Cartilage disease remedy

Info

Publication number: WO1996005855A1
Application number: PCT/JP1995/000121
Authority: WO
Inventors: Masahiro Iwamoto; Sumihare Noji; Toshikazu Nakamura
Original assignee: Sumitomo Pharmaceuticals Co., Ltd.
Priority date: 1994-08-19
Filing date: 1995-01-30
Publication date: 1996-02-29
Also published as: JPH0859502A; JP3737532B2

Abstract

A cartilage disease remedy, cartilage cell growth accelerator and proteoglycan formation accelerator, each containing a hepatocyte growth factor (HGF) as the active ingredient, and a method of treating human and mammalian cartilage diseases by administering an effective dose of HGF. The HGF as the active ingredient accelerates cartilage cell growth and proteoglycan formation. Therefore the remedy and accelerators are useful for preventing and treating various cartilage diseases.

Description

Description Treatment agent for cartilage disorders Technical field

The present invention relates to an agent useful for treating and preventing cartilage disease. More specifically, the present invention relates to a therapeutic agent for cartilage disorders, a chondrocyte proliferation promoter and a proteoglycan production promoter containing HGF (Hepatocyte Growth Factor) as an active ingredient. Background art

Cartilage is connective tissue consisting of chondrocytes and the matrix surrounding it, and is found in joints, spinal discs, costal cartilage, auricles, ear canals, pubic connections, and epiglottis. Cartilage is composed of chondrocytes and cartilage matrix produced by chondrocytes. The cartilage matrix is mainly composed of fibrous components such as collagen fibers, proteoglycans and water. The cartilage is mixed with the cartilage matrix to produce hyaline cartilage (costum). Cartilage, throat cartilage, articular cartilage, etc.), elastic cartilage (auricular cartilage, etc.) and fibrocartilage

(Disc cartilage, pubic cartilage, articular cartilage, etc.). In the above-mentioned cartilage matrix, collagen fibers are involved in rigidity and strength of cartilage against tension and shear force, proteoglycans are involved in strength against compressive force, and water has never before been characterized as a viscoelastic body of living tissue. For example, in the case of articular cartilage, water accounts for 78.6% of cartilage mass, collagen accounts for 20%, and proteoglycan accounts for 7%. The effects of cartilage include reduction of epiphyseal friction (cartilage between bones), retention of elasticity (such as auricular cartilage), and motor function (such as costal cartilage and pubic cartilage). As described above, cartilage has an important effect in maintaining the function of a living body, and various diseases caused by cartilage disorders have been known, for example, dyschondrodysplasia, osteoarthritis, deformation Examples include intervertebral disc disease, fracture repair, and poor healing. In particular, with the advent of an aging society, an increase in trauma due to sports, and the emergence of occupational diseases represented by Kipanchia disease, etc. The number of health care professionals has increased significantly, and medical progress in this area is demanded.

Conventionally, various treatments have been tried to treat cartilage disorders, but they are not aimed at directly resolving the cause. For example, by administering an anti-inflammatory agent, etc. It was only a symptomatic treatment, such as a method of suppressing pain and other injuries caused by the disease; a method of injecting a hyaluronic acid preparation or the like into a joint to lubricate the movement of the joint.

As described above, no curative treatment for joint disorders has been found. Particularly, osteoarthritis has a large number of patients, and there is an eager need for effective treatments.

The present inventors have conducted intensive studies to solve the above-mentioned problems.As a result, HGF has an effect of promoting the growth of chondrocytes and the production of proteoglycan, and has been shown to inhibit various diseases caused by cartilage disorders. The present inventors have found that they are effective for treatment and completed the present invention.

The above-mentioned HGF is a protein found as a factor for growing hepatocytes in vitro (Biochem Biophys Res Commun, 122, 1450, 1984, Proc. Natl. Acad. Sci. USA, 83, 6489, 1986, FEBS Letter, 224, 311, 1987, Nature, 342, 440, 1989, Proc. Natl. Acad. Sci. USA, 87, 3200, 1990). HGF, which was discovered as a factor that specifically promotes hepatocyte proliferation, has been shown to have various activities such as tissue injury healing and the like in vivo by recent research results by many researchers including the present inventors. The results are clear, and expectations are high for its application not only as a research target but also as a therapeutic drug for humans and animals.

It has been elucidated that HGF is produced by cells of the mesenchymal system in life, and it has been clarified that a so-called paracrine mechanism has been established in which HGF is supplied from neighboring cells as needed. I have. However, when liver and spine are injured, HGF production is also increased in uninjured organs, such as lungs, so that it is considered that HGF is also supplied by the so-called endocrine mechanism.

Recent studies on the receptor for HGF have confirmed that the c-Met primordial tumor fist gene encodes the HGF receptor (Bot taro et al., Science 251_, 802-804, 1991; Naldini et al., Oncogen e 6, 501-504, 1991).

Although a great deal of knowledge has been obtained on HGF as described above, the action of HGF to promote chondrocyte proliferation and proteoglycan production is a novel finding that has not been known so far, and the present invention based on such knowledge has been made. Is to provide an agent useful for treating various diseases caused by cartilage disorders. Disclosure of the invention

The present invention is a remedy for cartilage disorders comprising HGF as an active ingredient.

Another aspect of the present invention provides a chondrocyte proliferation promoting agent comprising HGF as an active ingredient; a proteoglycan production promoting agent comprising HGF as an active ingredient; and administering an effective amount of HGF. And a method for treating cartilage disorders in a human or mammal.

The above HGF may be derived from human or animal tissues or blood components, or may be produced by genetic recombination.

HGF, which is an active ingredient, has the effect of promoting the proliferation of chondrocytes and the production of proteoglycan, and is therefore effective for the treatment and prevention of various diseases caused by cartilage disorders. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a micrograph showing the expression of HG FmRNA in the limb buds of early nascent mice (bright field on the left and corresponding dark field on the right). In the figure, A to D are 10.5-day-old fetuses, and E to H are longitudinal section sections of the 11-day-old fetus.

Figure 2 is a photomicrograph showing the expression of HG FmRNA in the limb buds of the mouse during finger formation (bright field on the left, corresponding 喑 field on the right). In the figure, A and B are sections of a 12.5 day old fetus, C to F are sections of a 13 day old fetus, and G to J are sections of a 14 day old fetus. Fe is the femur, Fi is the fibula, Ta indicates a tarsal bone, and I to V indicate finger numbers.

FIG. 3 is a micrograph showing the expression of HGF mRNA in the limb buds and thorax of a developing mouse (the bright field is on the left, and the corresponding dark field is on the right). In the same figure, A and B show cross sections of hind limbs of a 6-day-old fetus; C and D show 13-day- 鹼 fetuses, and E and F show longitudinal-section sections of the thorax of 14-day-old fetuses. Ta indicates tarsal bone, Ti indicates tibia, and Rib indicates pre-cartilaginous accumulation of rib cartilage.

FIG. 4 is a micrograph showing the scatter activity of HGF on chondrocytes. In the figure, A indicates control (non-HGF processing) and B indicates HGF processing.

FIG. 5 shows the effect of HGF on chondrocyte proliferation. In the figure, A shows the effect on DNA synthesis of articular chondrocytes, B shows the effect on DNA synthesis of peritoneal cells, and C shows the effect on proliferation (cell number) of articular chondrocytes.

FIG. 6 is a diagram showing the effect of HGF on proteoglycan production. FIG. 7 is a graph showing the effects of HGF on DNA synthesis (FIG. 7A) and production of open-ended theoglycans (FIG. 7B) in the presence of an anti-HGF antibody. FIG. 8 is an electrophoretic photograph showing the expression of HGF receptor mRNA in chondrocytes. BEST MODE FOR CARRYING OUT THE INVENTION

As the HGF used in the present invention, those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals.

Various methods are known for preparing HGF. For example, from liver, spleen, lung, bone marrow, organs such as bone marrow, brain, back, placenta, etc., blood cells such as platelets, leukocytes, plasma, serum, etc. It can be obtained by extraction and purification (see FEBS Letter, 224, 312, 1987, Proc. Natl. Acad. Sci. USA, 86, 5844, 1989, etc.).

In addition, primary cultured cells and cell lines that produce HGF are cultured and cultured. HGF can be isolated and purified from culture supernatants, cultured cells, etc., or HGF-encoding genes can be incorporated into an appropriate vector by genetic engineering techniques and inserted into an appropriate host. To obtain the desired recombinant HGF from the culture supernatant of this transformant (for example, Nature, 342, 440, 1989, Japanese Patent Application Laid-Open No. 5-111383). Gazette, Biochem. Biophys. Res. Commun., 163, 967, 1989). The above host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques, for example, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plants, or animal cells can be used.

More specifically, as a method for extracting and purifying HGF from living tissue, for example, a method of intraperitoneally administering carbon tetrachloride to a rat, extracting and crushing the liver of the rat in a hepatitis state, and crushing the S-sepharose The protein can be purified by conventional protein purification methods such as gel column chromatography such as heparin sepharose and HPLC.

In addition, animal cells, such as Chinese hamster ovaries (eg, Chinese hamster ovaries), are obtained by using an expression vector in which a gene encoding the amino acid sequence of human HGF has been incorporated into a vector such as sipapilloma virus DNA using a genetic recombination method. CHO) cells, mouse C127 cells, monkey COS cells, etc. can be transformed and obtained from the culture supernatant.

In the HGF thus obtained, as long as it is substantially equivalent to HGF, a part of the amino acid sequence is deleted or replaced by another amino acid, or another amino acid sequence is partially inserted. Or one or more amino acids may be bound to the N-terminus and / or C-terminus, or the sugar chain may be similarly deleted or substituted.

The therapeutic agent and accelerator of the present invention contain the above-mentioned HGF as an active ingredient, and HGF promotes the growth of chondrocytes and the production of proteoglycan, as shown in the test examples described later. Furthermore, since HGF does not act on undisturbed cartilage tissue and acts only on impaired cartilage tissue, HGF has a feature that it is less likely to cause side effects. Therefore, the therapeutic agent and the accelerator of the present invention are useful for treating various diseases caused by cartilage disorders. It is effective for prevention, and these include, for example, the following diseases.

Osteoarthritis

Chondrodysplasia

Fracture healing and repair

Repair of articular cartilage and articular disc due to trauma

Acute purulent arthritis

Tuberculous arthritis

Syphilitic arthritis

Rheumatoid arthritis

Rheumatic fever, systemic lupus erythematosus

Degenerative spondylosis

Herniated disc

Bone graft repair

The therapeutic agent and enhancer of the present invention are useful for the treatment and prevention of various diseases caused by cartilage disorders in mammals (for example, horses, horses, pigs, sheep, dogs, cats, etc.) in addition to humans. Applied.

The therapeutic agent and enhancer of the present invention can be used in various forms (for example, liquids, solids, capsules, etc.). Generally, the active ingredient HGF alone or an injection thereof together with a conventional carrier, Inhalant, suppository or oral. The injection can be prepared by a conventional method. For example, after dissolving HGF in an appropriate solvent (for example, sterilized water, buffer, physiological saline, etc.), sterilize by passing through a filter or the like. Then, it can be prepared by filling in a sterile container. The HGF content in the injection is usually adjusted to about 0.002 to 0.2 (W / V5, preferably about 0.001 to 0.1 (W / V%). Are formulated into tablets, granules, fine granules, powders, soft or hard capsules, solutions, emulsions, suspensions, syrups, etc. Suppositories can also be prepared by a conventional method using a conventional base (for example, cacao butter, laurin butter, glycemic gelatin, macrocrogol, witepsol, etc.). Inhalants should also be prepared in accordance with the usual pharmaceutical procedures. Can be.

The HGF content in the preparation can be appropriately adjusted according to the dosage form, the disease to be applied and the like.

At the time of formulation, a stabilizer is preferably added. Examples of the stabilizer include 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 preparation, for example, excipients, solubilizers, antioxidants, soothing agents, isotonic agents and the like. When a liquid preparation is used, it is desirable to store it after freezing or freezing it to remove water. The lyophilized preparation is reconstituted with distilled water for injection and used before use.

The therapeutic agent and enhancer of the present invention can be administered by an appropriate route depending on the form of the preparation. For example, it can be administered in the form of an injection into a vein, artery, subcutaneous, intramuscular, or the like. The dose is adjusted appropriately depending on the patient's condition, age, weight, etc., but is usually 0.05 mg to 500 ing, preferably lmg to 100 mg as HGF, and is divided into once or several times a day. It is appropriate to administer. Industrial applicability

In the present invention, HGF, which is an active ingredient, has the effect of promoting the growth of chondrocytes and promoting the production of priteoglycan. Therefore, the therapeutic agent and the promoting agent of the present invention are useful for the treatment and prevention of the above-mentioned various diseases caused by cartilage disorders. Furthermore, since HGF acts only on damaged cartilage tissue, it is possible to obtain a drug with few side effects. Example

Hereinafter, the present invention will be described in more detail based on Examples and Production Examples, but the present invention is not limited to these Examples. The materials and methods used in the following experiments are as follows. Materials and methods

① In situ hybridization

Rat HG F-cDNA (RBC1 clone) (Proc. Natl. Acad.

The 1.4 kb EcoRI fragment of Sci. USA, 87, 3200, 1990) was subcloned into the pGEM7 vector and [a— ³ 'S] UTP (400 Ci / mmo).

1, Amersham) to prepare antisense and sense RNA probes. The labeled transcript was hydrolyzed to 50-150 base as a riboprobe.

The in situ hybridization was carried out by the method described in the literature (Biochem. Biophys. Res. Commun., 73, 42, 1990). Samples were fixed in 4% paraformaldehyde monophosphate saline solution, dehydrated with ethanol, washed with toluene, and embedded in paraffin. A 5 μπι section was cut out and mounted on a slide glass coated with poly L-lysine. Sections were deparaffinized with glycine and acetic anhydride and hybridized with a probe at 50 ° C for 16 hours. Thereafter, the sections were washed with 0.1 XSSC solution at 50 ° C for 1 hour, treated with RNAase A (20 g / m]) at 37 ° C for 30 minutes, and then treated with 2XSSC solution. Washing was performed twice at 37T for 10 minutes. The sections were immersed in the emulsion (11 Kodak NBT-2 diluted solution) and exposed for 2 weeks. The sections were developed and fixed on Kodak D-19 and stained with hematoxylin and eosin.

② Cells and cell culture

Chondrocytes were isolated from 23-day-old fetuses and 4-week-old newborns of New Zealand white herons according to the method described in the literature (J. Cell. Physiol., 133, 491, 1987). Articular cartilage was isolated from femoral articular cartilage of the knee, and costal cartilage was isolated from hyaline cartilage of the rib (Dev. Biol., 136, 500, 1989). Meningeal fibroblasts were isolated from the meningeal tissue of the knee joint. The minced synovial tissue fragments were cultured in DMEM containing 10% FBS for 10 days, and cells grown by trypsin treatment were collected. According to the method described in the literature (Exp. Cell Res., 157, 483, 1985), fetal mesenchymal cells were isolated from leg muscle tissues of fetal rats on day 20. Leg bud mesenchymal cells were isolated from rat embryos at 10.5 days. Leg buds are surgical The cells were cut out under a microscope for use, treated with 0.25% tribcine for 30 minutes, and then pitted to obtain isolated cells with nylon gauze. All cells except Asymmetric cells, 1 0% FBS, 6 0 μ DM EM ( hereinafter, referred to as medium A) containing g / 1 of kanamycin 3 7 ° at _{C, 5% C 0 2/} 9 5% air Maintained below.

③ Measurement of DNA synthesis

DNA synthesis rate is 10 ° /. Of ^[3 H] to T CA insoluble cell sedimentation - ^{thymidine.. ([6 - 3 H} ] - thymidine, Amersham, 2 0 C immo 1) of was evaluated by measuring the incorporation (J. Clin Invest, 85, 626, 1990). Cells were seeded at a density of 1.5 11 per 96-mm plate in a 96-well plate (Γ density) and cultured until confluent. To stop the growth, cells were incubated with 0.3% FBS. .. containing preincubation with 0. 1 ml of DM EM various concentrations of HGF was added to the medium the incubation was continued for 24 hours ^{1 C i / ml [a H} ] -. thymidine Inkyubeshi Yung stop 3 After labeling, cells were washed three times with ice-cold PBS, twice with 5% TCA containing 3 mM thymidine, and once with ethanol: dimethyl ether (3: 1). After solubilization with 100 µl of 0.1 NaOH, the solution was transferred to a liquid scintillation vial, neutralized with INHC1, and radioactivity was measured with a scintillation counter (Rack-beta, Pharmacia).

測定 Measurement of proteoglycan synthesis

Chondrocytes were seeded at a density of 1.5 x 1 (Γ) per 6 mm well and maintained in 0.1 ml medium A. When cells reached confluence, they contained 0.3% FBS. Preincubation was performed for 24 hours in 0.1 ml DMEM, followed by incubation for 24 hours in 0.1 ml DMEM containing 0.3% FBS and HGF. ^ae S] The monosulfate group was added 20 hours before the end of the incubation.The proteoglycan synthesis was based on [ ³ 'S] of the precipitate on cetylpyridinium chloride after protease digestion. —Evaluated by measuring sulfate uptake (Exp. Cell Res., 130, 73, 1980).

⑤Total RNA preparation and reverse transcription PCR

Total RNA from cartilage was prepared by a modification of the method described in the literature (Anal. Biochem., 203, 352, 1992). Freshly isolated tissue fragments (0.1 g wet weight) were prepared using 4 M guanidine thiosinate, 0.1 MT ris hydrochloric acid (pH 7.5), 1% 2-mercaptoethanol in 4 MGITC solution 2 The homogenization was performed quickly at m 1. The homogenate was mixed with 10% SDS 1001 and centrifuged for 5 minutes in a microcentrifuge. 2 ml of the supernatant is layered on a Beckman polyaroma-centrifuge tube (13 x 51 mm) over an equal volume of 6 g cesium trifluoroacetate and 1 mM EDTA (pH 8.0). did. The sample was centrifuged at 35 ° C, OOOrpm (147, OOOXg) at 18 ° C for 20 hours. After removing the supernatant by suction, dissolve the precipitate in 200 μl of 4 MGITC solution, extract with phenol: chloroform: isoamylalcosol (25: 24: 1), then add 20 μl of 3-acetic acid. Sodium (ρΗ4.8) was mixed and precipitated with 2 volumes (440 μl) of ethanol. The precipitate was dissolved in DEPC-treated water.

First, first-strand cDNA was synthesized from 0.5 μg of total RNA using Supersrcript revertase (Gibco-BRL) and an antisense primer in the downstream region. Subsequently, PCR expansion was performed. Proliferation is for 30 cycles at 94 ° C for 30 seconds, 58 for 1 minute, and 72 ° C for 5 minutes for 35 cycles (for chondrocytes) or 40 cycles (for cartilage tissue). I went in. The primer base sequence for PCR amplification is 5 'for rat and mouse c-Met (Oncogene, 2, 593, 1988). — CA GT (A / G) ATGATCT CAA T GGG CAA T-3' And 5'-AA TGCCCTCTTCCTAT GA CTTC-3 'to generate a 72 bp fragment. Example 1

HG F mRNA expression in developing limbs

Expression of HGF mRNA in limb buds of developing mice Tested by the reduction method. The results are shown in Fig. 1, Fig. 2 and Fig. 3. Fig. 1 shows the expression of HG FmRNA in the limb buds of mice in early development, and is a photomicrograph of a longitudinal section of the hind limb. The corresponding dark field (right) was taken after in situ hybridization, autoradiography and staining. In the figure, A to D are sections of a 10.5 day old fetus, and E to H are sections of an 11 day old fetus.

Figure 2 shows the expression of HG FmRNA in the limb buds of the mouse during finger formation. Photomicrographs of longitudinal sections of the hind limb. The bright field (left) and the corresponding dark field (right) are in situ hybridized. Photographs were taken after zession, autoradiography and staining. In the figure, A and B are sections of a 12.5 day old fetus, CF are sections of a 13 day old fetus, and GJ are sections of a 14 day old fetus. Fe represents a femur, Fi represents a fibula, Ta represents a tarsal bone, and I to V represent finger numbers.

Figure 3 is a micrograph showing the expression of HG FmRNA in the limb buds and rib cage of the developing mouse. The bright field (left) and the corresponding dark field (right) are in situ hybridization and autoradio. It was taken after the graphic and dyeing. In the same figure, A and B show cross-sections of hind limbs of 16-day-old fetuses; C and D show 13-day-old fetuses, and E and F show longitudinal cross-sections of thorax of 14-day-old fetuses. Ta indicates a tarsal bone, Ti indicates a tibia, and R ib indicates pre-cartilaginous accumulation of rib cartilage.

As shown in FIG. 1, on day 11, diffuse expression of HG FmRNA was detected around the bottom region of the limb. At this stage, cartilaginous accumulation had not occurred in the limbs. As cartilage accumulation progresses, the expression site of HG FmRNA has been more restricted. HGF mRNA expression was observed in the joint area of the wrist malleolus and elbow Z knee when the base leg, the joint leg, and the self-leg portion were formed on Day 2.5 (see FIGS. 2A and B. For convenience). , Knees and ankles). Late (days 13 to 14), HGF mRNA was expressed in adjacent and restricted mesenchymal cells of the cartilage accumulation in the wrist Z ankle and elbow / knee joint regions (Fig. 2C-J See). On day 16, HG FmRNA was localized to limited mesenchymal cells adjacent to tarsal cartilage (see FIGS. 3A and B). HG Limb expression levels of FmRNA decreased with differentiation. No HG FmRNA was detected in the growth plates of the limbs throughout the study. Example 2

HG F mRNA expression in the developing rib cage

The expression of HG F mRNA in the thorax of developing mice was tested by the in situ hybridization method. The results are shown in FIGS.

As shown in FIGS. 3C-F, HG FmRNA was expressed in the intercostal mesenchymal tissue around the tip of the intercostal elongated pre-cartilaginous accumulation. No signal of hybridization was detected in pre-cartilaginous accumulation. Example 3

Scatter activity test of HGF for chondrocytes

To determine which cells are the target cells for HGF around the differentiating cartilage tissue, chondrocytes from knee joint cartilage and costal cartilage, synovial cells from knee joint, and fibroblasts grown from limb muscle tissue Cultured cells were prepared, and the effect of exogenously added HGF on these cells was examined.

That is, perforated articular chondrocytes were seeded at a density of ³ × 10 ³ cells in 16-mm wells and maintained in medium A for 2 days. Thereafter, the cells were treated with HGF for 2 days. At the end of the incubation, phase contrast micrographs were taken. Fig. 4 shows the results.

As shown in Fig. 4, when HGF was not treated (control), polygonal chondrocytes proliferated and became insular (Fig. 4A). On the other hand, in the culture treated with HGF (3 ng Zml), chondrocytes did not form islets in a single cell state (FIG. 4B). Therefore, HGF was found to stimulate chondrocyte migration. HGF was not dispersed in fibroblasts and synovial cells. Example 4

Effect of HG F on The effect of HGF on chondrocyte proliferation was examined.

That is, articular chondrocytes collected from 4 weeks old egrets were cultured. After the configurator Ruen Bok cells became 24 hours serum withdrawal treatment, were treated with HG F at various concentrations, by the method described in Materials and Methods ^[a H] - uptake of thymidine was measured . In addition, a similar test was performed on the heron membrane fibroblasts. The results are shown in FIGS. 5A (articular chondrocytes) and B (synovial fibroblasts). The results show the average soil standard deviation of three tests (the same applies to Figs. 5C, 6 and Figs. 7A and B).

As shown in FIG. 5 A, HG F is Usagi articular cartilage into cells ^[3 H] - increased thymidine incorporation in a dose-dependent manner, enhancement of DNA synthesis, a growth promoting effect on immediate Chi articular chondrocytes It was shown to have. DNA synthesis showed a 3-fold increase in HGF at 1 ng / ml over control. On the other hand, as shown in FIG. 5B, meningeal fibroblasts did not respond to HGF.

In another experiment, the effect of HGF on the number of articular cartilage cells was examined. That is, 1 × 1 (Γ cells were seeded in 16 mm wells and maintained in a DMEM medium containing 10% FBS. Then, 10 ng / m 1 of HGF was added thereto. After incubation for 8 hours, the number of cells was counted after the incubation, and the results are shown in Figure 5C.

As shown in FIG. 5C, ^ 〇 in 101 8 111] increased the cell number by about 1.8 times compared to the control. Example 5

Effect of HGF on proteoglycan formation

As described above, HGF promoted the proliferation of chondrocytes. Next, the effect of articular chondrocytes on proteoglycan production was examined by the method described in the above-mentioned Materials and Methods. Proteoglycan synthesis was studied by measuring the incorporation of [ ³ 'S] -sulfate groups into macromolecules (glycosaminoglycans) that precipitate on cetylpyridinium chloride after protease digestion (Exp. Cell Res., 130, 73, 1980). Instead of HG F, the following factors Was also tested.

Insulin-like growth factor (IGF) — I: concentration 100 n gZm 1

I G F-I I: concentration 100 ng / m 1

Parathyroid hormone (PTH): Concentration 1 0- ⁷ M

TG F-β: concentration 3 ng / m 1

Figure 6 shows the results. As shown in FIG. 6, HGF dose-dependently increased ⁶ S] -sulfate uptake. The maximum increase was obtained with 1 ng / ml of HGF. This effect is due to TG F— / 3 (J. Cell Physiol.,

138, 329, 1989) and PTH (J. Clin. Invest., 85, 626, 1990), but comparable to IGF-I and II (Exp. Cell Res.,

130, 73, 1980). Example 6

Effect of HGF on DNA synthesis and proteoglycan production in the presence of anti-HGF antibody

As described above, HGF is generally considered to act on target cells by a paracrine mechanism, and the results of the in situ hybridization described above are considered to support this idea. Therefore, in order to confirm this point, it was examined whether the HGF polyclonal antibody changes the function of chondrocytes.

That is, the confluent Egret articular chondrocytes were cultured with 25 μg / ml anti-HGF polyclonal antibody (IgG fraction purified by affinity) in the presence or absence of 3 ng Zml of HGF. ) Treated or non-treated. Thereafter, the materials and methods described in the Methods Nyori, ^[a H] - thymidine or ^[36 S] - labeled with sulfate group was measured DN A synthesis or proteoglycans generation. The results are shown in FIGS. 7A (DNA synthesis) and B (proteoglycan generation). In the figure, Ab indicates an anti-HGF polyclonal antibody.

As shown in Figure 7, addition of anti-HGF polyclonal antibody alone did not alter DNA synthesis or proteoglycan production in articular chondrocytes. Was. However, the anti-HGF polyclonal antibody completely inhibited the effect of exogenously added HGF. This indicates that chondrocytes do not produce enough HGF to regulate the function of cartilage itself. Example 7

Expression of HGF receptor mRNA in chondrocytes

The expression of HGF receptor 1 (c-Met) in chondrocytes in vivo and in vitro was examined by reverse transcription PCR. Joint tissue and costal cartilage were excised from a 4-week-old rat under a surgical microscope, and total RNA was extracted as described in Methods and Materials. The extracted RNA (0.5 μg) was reverse transcribed, amplified using c-Met primer, and analyzed by 1.5% agarose gel electrophoresis. Figure 8 shows the results.

As shown in Fig. 8, after 40 times of amplification in articular and costal cartilage tissues, trace amounts of c-Met expression were detected, and cultured chondrocytes showed significant c after 35 times of amplification. One met expression was observed. Production Example 1

Production example of HGF preparation

(1) HGF 20 g

Human serum albumin 100 mg

The above substance was dissolved in 0.01 M PBS having a pH of 7.0, the total amount was adjusted to 20 ml, and after sterilization, 2 ml was dispensed into vials and sealed by freeze-drying.

(2) HGF 40 g

Tween 8 0 1 mg

Human serum albumin 100 mg

The above substance was dissolved in physiological saline for injection, the total volume was adjusted to 2 Oml, and after sterilization, 2 ml was dispensed into vials and sealed by freeze-drying.

Claims

Billing scope

1. A therapeutic agent for cartilage disorders comprising HGF as an active ingredient.

2. The therapeutic agent for cartilage disorders according to claim 1, wherein the HGF is produced by genetic recombination.

3. Cartilage disorders include osteoarthritis, achondroplasia, healing and repair of fractures, repair of articular cartilage and articular disc due to trauma, acute suppurative arthritis, tuberculous arthritis, syphilitic arthritis, rheumatoid arthritis 3. The therapeutic agent for cartilage disorders according to claim 1 or 2, which is repaired by rheumatic fever, systemic lupus erythematosus, osteoporosis, herniated disc or bone graft.

4. A chondrocyte proliferation promoter comprising HGF as an active ingredient.

5. A proteoglycan production promoter comprising HGF as an active ingredient.

6. The agent according to claim 4 or 5, wherein the HGF is produced by recombination of the arrested child.

7. A method for treating cartilage disorders in a human or mammal, comprising administering an effective amount of HGF.

8. The method for treating a cartilage disorder according to claim 6, wherein the HGF is produced by genetic recombination.

9. Cartilage disorders include osteoarthritis, chondrodysplasia, healing and repair of fractures, repair of articular cartilage and discs due to trauma, acute suppurative arthritis, tuberculosis arthritis, syphilitic arthritis, rheumatoid arthritis, 9. The method for treating a cartilage disorder according to claim 7 or 8, which is repaired by rheumatic fever, systemic lupus erythematosus, osteoporosis, herniated disc or bone graft.