Title:  Therapeutic agent for cartilaginous diseases

United States Patent:  6,756,358

Issued:  June 29, 2004

Inventors:  Iwamoto; Masahiro (Minoo, JP); Noji; Sumihare (Tokushima, JP); Nakamura; Toshikazu (Takatsuki, JP)

Assignee:  Sumitomo Pharmaceuticals Co., Ltd. (Osaka, JP)

Appl. No.:  921874

Filed:  August 6, 2001

Abstract

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, and a treatment method for cartilaginous diseases of human or mammals comprising administering an effective amount of HGF. The 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 prevention and treatment of various disorders caused by 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 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 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 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, there has 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 medical care in this field.

Various therapeutic methods have been conventionally tried for the treatment of cartilage diseases. They were not 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 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 proliferate liver parenchyma cells 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). 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 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 like.

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 cartilaginous diseases comprising HGF as an active component.

Further, the present invention relates to an accelerator for chondrocyte proliferation comprising HGF as an active 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.

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, 312, 1987; Proc. Natl. Acad. Sci. USA, 86, 5844, 1989, 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, Japanese Patent Kokai No. 111383/1993, Biochem. Biophys. Res. Commun., 163, 967, 1989). 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 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 chain(s) 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, 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., 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 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.

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.

Claim 1 of 14 Claims

What is claimed is:

1. A method of treating cartilaginous diseases and disorders in mammals comprising administering a wild-type full length Hepatocyte Growth Factor (HGF) in an amount which is effective in treating said cartilaginous diseases and disorders, wherein said cartilaginous diseases and disorders are osteoarthritis, chondrodystrophy, failure in restoration and cure of fracture, articular cartilage and articular disk injury, acute suppurative arthritis, tuberculous arthritis, syphilitic arthritis, rheumatoid arthritis, rheumatic fever, systemic lupus erythematosus, spondylitis deformans, or herniated disk.

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