HK1101351B

HK1101351B - Remedy or preventive for arthritis

Info

Publication number: HK1101351B
Application number: HK07106190.8A
Authority: HK
Inventors: 中尾一和; 北村秀智
Original assignee: 中外制药株式会社; 中尾一和
Priority date: 2004-03-31
Filing date: 2005-03-31
Publication date: 2015-02-18

Description

Therapeutic or prophylactic agent for arthritis

Technical Field

The present invention relates to a therapeutic or prophylactic agent for arthritis, particularly osteoarthritis and similar types of arthritis, or to an articular chondrocyte growth promoter, or to a method for inhibiting arthritis or a method for promoting articular chondrocyte growth using a guanylate cyclase B (hereinafter referred to as "GC-B") activator. The present invention further relates to a method for screening an arthritis therapeutic agent or an articular chondrocyte growth promoter using GC-B activity as an index.

Background

Arthritis is an inflammatory disease of the joints, and rheumatoid arthritis and osteoarthritis (or osteoarthrosis) are common joint diseases.

Rheumatoid arthritis is considered to be an autoimmune disease accompanied by joint pain, stiffness and swelling, and as the disease progresses, may often cause degeneration of the articular cartilage surface similar to osteoarthritis, which leads to severe destruction of the bones and cartilage of the joint.

Osteoarthritis is a degenerative disease of the articular cartilage that often occurs in the elderly. Osteoarthritis (OA) involves destruction of cartilage and proliferative changes of bone and cartilage resulting from degeneration of components of the joint, wherein the changes result in secondary arthritis (e.g., synovitis). Osteoarthritis occurs primarily in weight-bearing joints, such as the knee, elbow, and hip joints (Virchows Arch 1996; 260: 521-. In addition, temporomandibular arthritis with similar morbidity has been identified to occur in the temporomandibular joint (J Orofac Pain 1999; 13 (4): 295-.

It is known that cartilage matrix protein, which is a functional entity of cartilage in osteoarthritis, decreases and the number of chondrocytes decreases (Arth Rheum 2002; 46 (8): 1986-. However, due to the lack of vascularity, the low number of highly differentiated chondrocytes, the low number of chondrocyte precursor cells and the slow renewal of the cartilage matrix in cartilage tissue, said cartilage has a self-renewal capacity that is too low to guarantee a natural cure in osteoarthritis from diseases associated with a reduced number of articular cartilage matrix and chondrocytes (Novartis focus. Sypm. 2003; 249: 2-16). In addition, in osteoarthritis, arthritis occurs simultaneously with cartilage degeneration, which is the cause of joint pain (JRheumatotol 2001; 28 (6): 1330-1337).

Examples of therapeutic/prophylactic agents for arthritis such as rheumatoid arthritis and osteoarthritis which have been reported include, for example, protein tyrosine kinase inhibitors (Japanese patent laid-open (Kohyo)11-512708A (1999)), N-acyl-2-glucosamine derivatives (Japanese patent laid-open (Kohyo)2004-507490A) and quinoline/quinazoline derivatives (Japanese patent laid-open (Kokai)9-169646A (1997)). In addition, standard therapeutic agents for osteoarthritis that have been widely used at present are oral anti-inflammatory analgesics or intra-articular injections of hyaluronic acid and adrenocortical steroid formulations. All these drugs can only relieve joint pain, which means that there is a need for drugs having inhibitory effects on the degeneration of articular cartilage (precision Base 7, 2002).

Guanylate Cyclase (GC) is a membrane protein belonging to a family of enzymes that catalyse the synthesis of the second messenger, cGMP, from GTP, examples of which include GC-A, GC-B, … …, and GC-F. GC-B is found primarily in vascular endothelial cells and is thought to be involved in smooth muscle relaxation. Natriuretic Peptide (NP) is known as active GC. NPs are classified into ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and CNP (C-type natriuretic peptide), which are thought to exhibit biological activity by increasing intracellular cGMP levels through two guanylate cyclase binding receptors (NPR-A for ANP and BNP, and NPR-B for CNP) (Ann Rev Biochem 1991; 60: 229-K255).

NPR-C is not a guanylate cyclase binding receptor and is considered to be a scavenger receptor for NP not involved in signal transduction (Science, 1987; 238: 675-678). However, in a system whereby mouse bone marrow macrophages are stimulated with Lipopolysaccharide (LPS), prostaglandin E₂(PGE₂) Is induced by cyclooxygenase 2(COX-2), ANP and CNP have been reported to have a PGE response by reducing intracellular cAMP levels via NPR-C₂Suggesting that NPR-C is involved in signal transduction of NPs (Endocrinology 2002; 143 (3): 846-852). This report describes that ANP appears to be on PEG by stimulation of mouse Bone Marrow Macrophages (BMM) with LPS₂The enhancement of production is about 70% inhibition at the most, however CNP shows only about 20% inhibition at the most, and thus CNP has a weak effect. Since it is known that the control of COX-2 production by cyclic nucleotides such as cAMP and cGMP shows a response that is either promoted or inhibited depending on the cell type and the type of stimulation, it is unclear whether the inhibition of the production of PGE2 induced by LPS in BMM cells by CNP can be applied to other cells and stimulation. In addition in Endocrinology 2002; 143(3): 846-852 it was reported that ANP exhibited inhibition in a system in which administration of LPS increased blood thromboxane B in mice₂(TXB₂) On the contrary, the same mechanism of cANF is enhanced. In addition, although the report describes the reference of ANP to immune-related diseases such as arthritis and sepsis, there is no mention of the use of CNP for these related diseases. Therefore, no findings have been obtained regarding the effect of CNP on arthritis.

NPs have been reported to play important roles in controlling fluid constancy and regulating blood pressure (JClin Invest 1987; 93: 1911-1921, J Clin Invest 1984; 87: 1402-1412), and their expression and expression of biological activity in various tissues outside the cardiovascular system are known (Endocrinol 1991; 129: 1104-1106, Ann Rev Biochem 1991; 60: 553-575). As for cartilage, the use of CNP for the treatment of elongation of growing cartilage (autotonic gristle) and cartilage insufficiency in transgenic mice overexpressing BNP (Proc. Natl. Acad. Sci. U.S.A.1998; 95: 2337-2342) or CNP has been reported (Nat Med 2004; 10 (1): 80-86, Japanese patent application (Kokai) 2003-113116A). However, the growing cartilage is a temporary cartilage which eventually disappears with calcification and is replaced by bone, and is known to have biological properties different from those of permanent cartilage, such as articular cartilage and tracheal cartilage, which exist during life (Dev Biol 1989; 136 (2): 500-. In addition, although it has been reported that the in vitro activity of CNP enhances the overgrowth of articular chondrocytes, which are permanent cartilages, there has been no finding about the in vivo effect on articular cartilage in normal animals, or on articular cartilage degeneration or arthritis in osteoarthritis.

In osteoarthritis, swelling of articular cartilage at the initial stage of the disease results in a temporary increase in the volume of cartilage tissue (J Rheum 1991; 18 (3): 1905) 1915), and as the disease progresses, degeneration/destruction of the cartilage matrix increases, resulting in a decrease in the volume (Arthritis Rheum 2004; 50 (2): 476) 487). Due to apoptosis, the number of articular chondrocytes is reduced (Arthritis Rheum 2004; 50 (2): 507-515). On the other hand, the remaining individual articular chondrocytes are known to express type X collagen, as distinguished from hypertrophic chondrocytes having temporary cartilage properties (Arthritis Rheum 1992; 35 (7): 806-811). In addition, in diseased joints, arthritis is accompanied by destruction of articular cartilage and may be a factor in clinical pain (J Rheumatotol 2001; 28 (6): 1330-1337). Inhibition of these changes, i.e., reduction or restoration of the number of articular cartilage matrix and articular chondrocytes, and inhibition of arthritis in osteoarthritis are considered to be useful in the development of therapeutic agents.

It is an object of the present invention to provide a novel therapeutic or prophylactic agent for arthritis, including osteoarthritis, or a method for treating said arthritis.

It is another object of the present invention to provide an articular chondrocyte growth promoter or method.

It is another object of the present invention to provide methods for inhibiting arthritis, including osteoarthritis.

It is another object of the present invention to provide a method for screening an arthritis therapeutic agent.

It is another object of the present invention to provide a method for screening an articular chondrocyte growth promoter.

Disclosure of Invention

The inventors of the present invention prepared CNP transgenic mice overexpressing C-type natriuretic peptide (CNP), a guanylate cyclase B (GC-B) activator, to study the effect on articular cartilage, and obtained the following results: in the CNP transgenic mice, the thickness of articular cartilage and the number of articular chondrocytes were significantly increased; in the osteoarthritis model prepared from the CNP transgenic mouse, there was tolerance to joint swelling, in which degeneration of articular cartilage was reduced, synovial cell growth, granulation tissue formation and inflammatory cell infiltration were slightly changed, and proteoglycan content in articular cartilage was not reduced, whereas in the osteoarthritis model prepared from normal mice, there was a significant change in synovial cell growth, granulation tissue formation and inflammatory cell infiltration. From these findings, the inventors of the present invention have found that the GC-B activator has an anti-arthritic effect and an anabolic effect on articular cartilage.

Accordingly, the present invention includes the following inventions.

In a first aspect, the present invention provides a therapeutic or prophylactic agent for arthritis, comprising a guanylate cyclase B (GC-B) activator as an active ingredient.

In one embodiment of the invention, the arthritis is osteoarthritis.

In another embodiment of the invention, the osteoarthritis is weight bearing or non-weight bearing osteoarthritis.

In another embodiment of the invention, the osteoarthritis is degenerative knee joint disease.

In another embodiment of the invention, the osteoarthritis is degenerative hip osteoarthritis.

In another embodiment of the invention, the osteoarthritis is temporomandibular arthritis.

In another embodiment of the invention, the arthritis is caused by rheumatoid arthritis.

In another embodiment of the invention, the arthritis is caused by osteoarthritis.

In another embodiment of the invention, the GC-B activator is C-type natriuretic peptide (CNP) or a derivative thereof.

In another embodiment of the invention, the CNP as described above is selected from CNP-22 and CNP-53 from mammals including humans or birds.

In another embodiment of the present invention, the CNP is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

In another embodiment of the invention, said CNP derivative has the amino acid sequence shown in SEQ id no:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or more amino acids in the amino acid sequence of 2.

In another embodiment of the present invention, the therapeutic or prophylactic agent for arthritis comprises at least one non-steroidal anti-inflammatory drug.

In a second aspect, the present invention provides an articular chondrocyte growth promoter comprising a GC-B activator as an active ingredient.

In one embodiment of the invention, the GC-B activator is CNP or a derivative thereof.

In another embodiment of the present invention, the CNP is CNP-22 and CNP-53 derived from mammals including humans or birds.

In another embodiment of the invention, said CNP derivative has the amino acid sequence shown in SEQ ID no

NO:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or more amino acids in the amino acid sequence of 2.

In another embodiment of the present invention, the articular chondrocyte growth promoter further comprises at least one non-steroidal anti-inflammatory drug.

In a third aspect, the present invention provides a method for inhibiting arthritis, wherein said arthritis is inhibited by activating GC-B.

In one embodiment of the invention, the GC-B is activated by CNP or a derivative thereof.

In another embodiment of the invention, said GC-B is activated by CNP or a derivative thereof in combination with at least one non-steroidal anti-inflammatory drug.

In a fourth aspect, the present invention provides a method for promoting growth of articular chondrocytes, wherein the growth is promoted by activation of GC-B.

In another embodiment of the invention, the CNP as described above is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

In another embodiment of the invention, the derivative as described above has the amino acid sequence shown in SEQ id no:1 or SEQ ID NO:2, and has a CNP activity.

In a fifth aspect, the present invention provides a method for screening an agent for promoting the growth of articular chondrocytes, which comprises screening a candidate agent capable of promoting the growth of articular chondrocytes using GC-B activity as an index.

In one embodiment of the present invention, the method comprises preparing a GC-B-expressing cultured cell or a cell derived from an articular chondrocyte, culturing the cell in the presence of a candidate drug, and screening for a candidate agent capable of promoting the growth of the articular chondrocyte using the GC-B activity of the cell as an index.

In another embodiment of the invention, the GC-B activity is determined by the intracellular amount of cGMP produced.

In another embodiment of the present invention, the method comprises preparing a cultured cell line that has been forced to express GC-B, culturing the cell line in the presence or absence of a candidate agent, measuring the intracellular production of cGMP, and screening for a candidate agent capable of promoting the growth of articular chondrocytes using the difference between the intracellular production of cGMP in the presence and absence of the candidate agent as an index.

In a sixth aspect, the present invention provides a method of screening for a therapeutic agent for osteoarthritis, rheumatoid arthritis or arthritis, comprising screening for a candidate agent for osteoarthritis, rheumatoid arthritis or arthritis using GC-B activity as an index.

In one embodiment of the invention, the method comprises preparing cultured cells expressing GC-B or cells derived from articular chondrocytes, culturing the cells in the presence of a candidate agent, and screening the candidate agent for an agent capable of treating osteoarthritis, rheumatoid arthritis, or other arthritis using the GC-B activity of the cells as an indicator.

In another embodiment of the present invention, the GC-B activity is determined as the amount of intracellular cGMP produced.

In another embodiment of the present invention, the method comprises preparing a cultured cell line that has been forced to express GC-B, culturing the cell line in the presence or absence of a candidate agent, measuring the intracellular production of cGMP, and screening the candidate agent for an agent capable of treating osteoarthritis, rheumatoid arthritis or other arthritis using the difference between the intracellular production of cGMP in the presence and absence of the candidate agent as an index.

In a seventh aspect, the present invention provides a therapeutic or prophylactic agent for osteoarthritis, comprising a GC-B activator as an active ingredient.

In one embodiment of the present invention, the therapeutic or prophylactic agent for osteoarthritis as described above further comprises at least one non-steroidal anti-inflammatory drug.

In an eighth aspect, the present invention provides a therapeutic or prophylactic agent for rheumatoid arthritis, which comprises a GC-B activator as an active ingredient.

In one embodiment of the present invention, the therapeutic or prophylactic agent for rheumatoid arthritis as described above further comprises at least one non-steroidal anti-inflammatory drug.

In a ninth aspect, the present invention provides an activation promoter for a guanylate cyclase B (GC-B) activator, comprising a non-steroidal activator.

In another embodiment of the invention, the CNP is selected from the group consisting of CNP-22 and CNP-53 from mammals including humans or birds.

In another embodiment of the present invention, the non-steroidal activator is a cyclooxygenase inhibitor.

In another embodiment of the invention, the cyclooxygenase inhibitor is selected from the group consisting of indomethacin, ibuprofen, piroxicam, salicylic acid, diclofenac, ketoprofen, naproxen, and piroxicam.

In a tenth aspect, the present invention further provides a method for activating a GC-B activator, wherein an activation promoter as described above is used.

The description of the present application includes the disclosure of the specification and/or drawings of Japanese patent application 2004-.

Drawings

FIG. 1 shows the construction of vectors for the preparation of CNP transgenic mice. FIG. 1A: the cDNA of mouse CNP, which had been incorporated into the pGEM-T Easy vector, was cut out with Pst I and both ends were smoothed. FIG. 1B: pSG1 was treated with EcoR I and the cut ends were smoothed. FIG. 1C: the cDNA of mouse CNP as prepared in fig. 1A was incorporated into pSG1 as obtained in fig. 1B.

FIG. 2 shows a DNA fragment for injection. A fragment (about 2.3kb) containing the CNP gene was excised from pSG1-CNP prepared in FIG. 1C by digestion with Hind III and Xho I, and used as a fragment for injection.

FIG. 3 shows the results of genotyping CNP transgenic mice. In wild-type mice (WT), 3 signals (designated "wild-type CNP gene") were detected, while in transgenic mice (Tgm), 2 signals (designated "transgene") derived from the transgene were detected in addition to the wild-type CNP gene.

Fig. 4 is a graph showing the thickness of articular cartilage in CNP transgenic mice. The thickness of articular cartilage of the femoral knee facet was compared between normal homoabdominal mice (wild type) and CNP transgenic mice (CNP tgm). This figure indicates that CNP transgenic mice have statistically significantly thicker articular cartilage. **: p < 0.01, unpaired Student's t-test.

FIG. 5 shows the increase in the number of articular chondrocytes in CNP-transgenic mice. The number of chondrocytes per field under light microscopy in articular cartilage of femoral knee facet was compared between normal homoabdominal mice (wild type) and CNP transgenic mice (CNP tgm). This figure indicates that CNP transgenic mice have a statistically significant greater number of chondrocytes per field of view. **: p < 0.05, unpaired Student's t-test.

Fig. 6 is a graph showing tolerance to joint swelling in a collagenase-induced OA model in CNP transgenic mice. After 3% collagenase or physiological saline was administered to the right knee joint of the CNP transgenic mice (Tgm) and the wild type mice (WT), the width of the left and right knee joints was measured, and the difference in the width of the knee joint was used as an index of knee joint swelling to evaluate the progress of the disease (fig. 6A) and to find the area under the curve (AUC) (fig. 6B). The CNP transgenic mice tended to have weak swelling in the right knee and had significantly less AUC than in the wild type. **: p < 0.01, N.S.: there was no significance. Unpaired Student's t test.

Fig. 7 shows histological changes in the synovium of the right knee in the collagenase OA model. This figure is a histological image of the synovial membrane of the right knee joint 28 days after administration of 3% collagenase physiological saline into the right knee joint of CNP transgenic mice and wild type mice. When 3% collagenase was administered, wild type mice showed synovial epithelial cell proliferation, granulation tissue formation and inflammatory cell infiltration (fig. 7B). On the other hand, these occurred slightly in CNP transgenic mice (fig. 7C). Fig. 7A is an image of normal synovial tissue.

FIG. 8 shows histological changes in articular cartilage of the right intrafemoral condyle in the collagenase OA model. This figure is a histological image of the right femoral inner condyle (medial femoral condyle) 28 days after administration of 3% collagenase physiological saline to the right knee joint of CNP transgenic mice and wild type mice. The reduction in staining capacity of safranin O for cartilage matrix indicated a reduction in proteoglycan content in wild type mice (fig. 8B), while the staining capacity of safranin O remained unchanged in CNP transgenic mice (fig. 8C). Fig. 8A is an image of normal articular cartilage.

Fig. 9 shows the effect of inhibiting joint swelling in a CNP collagenase OA mouse model receiving infusion administration. This figure shows the swelling of the right knee joint measured 6 days after the administration of 1.5% collagenase, contained in physiological saline, into the right knee joint of C57BL/6J Jcl mice receiving the continuous subcutaneous administration of CNP-22. CNP-22, at 60 and 600 ng/day, significantly inhibited swelling of the right knee joint over the solvent control group (vehicle). Unpaired Student's t test.

FIG. 10 shows the effect of CNP infusion on the inhibition of joint swelling in a surgical OA mouse model. C57BL/6J Jcl mice were continuously administered subcutaneously CNP-22 and subjected to a surgical procedure to induce osteoarthritis by resection of the anterior cruciate ligament in the right knee, resection of the medial collateral ligament in the tibia, and total resection of the medial meniscus. The width of the right and left knees was measured at 4, 8 and 11 days post-surgery and the difference in AUC is shown. CNP-22, at 60 and 600 ng/day, significantly inhibited swelling of the right knee joint over the solvent control group (vehicle). Unpaired Student's t test.

FIG. 11 shows the inhibition of knee joint swelling by CNP-22(6 ng/day, sustained subcutaneous administration), indomethacin (indo., 1mg/kg, oral administration), and combinations thereof in a C57BL/6J Jcl collagenase OA mouse model. Figure 11A shows the change in swelling of the right knee joint in 7 days after administration of 0.15% and 1.5% collagenase physiological saline to the right knee joint in mice. FIG. 11B shows the area under the curve (AUC) of the graph in FIG. 11A. When comparing AUC, CNP-22 significantly inhibited swelling of the right knee joint compared to the solvent control group (vehicle) without indomethacin at the same time. On the other hand, the combination of CNP-22 and indomethacin showed a significantly stronger significant inhibitory effect than CNP22 alone. Unpaired Student's t test. *: p < 0.05 (to vehicle). + -: p < 0.01 (for vehicle).

FIG. 12 shows the effect of CNP-22 on extremity arthritis and weight changes in a mouse model of adjuvant arthritis. FIG. 12A shows the change in the scores for extremity arthritis, which indicates a lower arthritis score for the CNP-22 group. Fig. 12B shows the body weight change, which indicates that the CNP-22 group showed significantly heavier body weight than the solvent control group (vehicle) on days 7 and 10 from antigen sensitization. Unpaired Student's t test. *: p < 0.05 (for vehicle).

FIG. 13 shows the effect of CNP-22 on body weight in a murine model of collagen arthritis. It is indicated that although the solvent control group (vehicle) showed significantly lighter body weight than the normal group from days 21, 24 and 28 of antigen sensitization, the CNP-22 group (CNP 6 μ g/day) showed significantly heavier body weight than the solvent control group. Unpaired Student's t test. # #: p < 0.01 (for normal group), x: p < 0.05 (for vehicle).

Detailed description of the invention

The invention is further described with reference to the accompanying drawings.

The inventors of the present invention analyzed the genotype of the CNP-transgenic mouse (CNP Tgm) prepared in example 2 as described below by the southern blotting method. As a result, as shown in fig. 3, the inventors of the present invention detected 3 signals ("wild-type CNP gene") in wild-type mice, whereas in CNP Tgm, 2 signals ("transgene") derived from the transgene were detected in addition to the wild-type CNP gene. CNP levels were measured in the liver, an organ predicted to highly express the transgene, and in plasma to study CNP expression in CNP Tgm. As a result, it was found that CNP Tgm showed 10-fold and about 24-fold higher CNP levels than the wild type in liver and plasma, respectively, showing statistically significant over-expression of CNP peptide (table 1 of example 4).

In addition, the thickness and chondrocyte number of the articular cartilage were histologically examined to perform histological analysis of the articular cartilage of CNPTgm, and the results showed that the articular cartilage was statistically significantly thick (fig. 4) and the articular chondrocyte number was statistically significantly large in CNP Tgm (fig. 5). These results indicate that GC-B activators such as CNP can increase the thickness of articular cartilage by increasing the number of chondrocytes.

In example 6 described hereinafter, an osteoarthritis animal model was established by injecting collagenase into the knee joint of mice to destabilize the knee ligament and meniscus and induce osteoarthritis (am.J. Pathol.1989; 135: 1001-14). The CNP Tgm and an osteoarthritis animal model from normal mice were used to evaluate the resistance of the CNP Tgm to arthritis and articular cartilage degeneration. In the animal model of CNP Tgm origin, knee joint swelling was significantly milder, articular cartilage degeneration was significantly more inhibited, synovial cell growth, granulation tissue formation and inflammatory cell infiltration in synovium were all very mild, and there was almost no change in proteoglycan content in articular cartilage, when compared with the animal model of normal mouse origin (fig. 6 to 8). These results indicate that the GC-B activator has an inhibitory effect on arthritis and degeneration of articular cartilage in osteoarthritis.

In addition, an osteoarthritis model was established using normal mice transplanted with an osmotic pump to examine the therapeutic effect of CNP infusion on the osteoarthritis model. In the CNP group, animals of this group were found to be resistant to knee joint swelling, they had significantly reduced degeneration of articular cartilage, and showed rather slight changes in synovial cell growth, granulation tissue formation and inflammatory cell infiltration in the synovium (fig. 9). These results indicate that the GC-B activator has a therapeutic effect on osteoarthritis.

In addition, normal mice transplanted with an osmotic pump were subjected to a surgical procedure, the anterior cruciate ligament was excised in the right knee joint, the medial collateral ligament of the tibia was excised, and the medial meniscus was completely excised to induce osteoarthritis, and the therapeutic effect of CNP infusion on an osteoarthritis model was examined. The results show that AUC (area under the curve) at both doses was less significant in the CNP group than the solvent control group (fig. 10). These results indicate that the GC-B activator is also effective in inhibiting arthritis in osteoarthritis induced by physical overload resulting from a surgical procedure.

In addition, when CNP was administered to collagenase OA mouse model, CNP alone significantly inhibited joint swelling, but not NSAID alone, or in combination with non-steroidal anti-inflammatory drugs (NSAIDs), and the combination of CNP and NSAIDs showed even stronger synergistic anti-swelling effect (example 9, fig. 11).

In addition, when the effects of CNP were further examined using a secondary Arthritis and collagen Arthritis model, which are generally used in the laboratory as a Rheumatoid Arthritis (RA) model (Arthritis & Rheumatism, 27: 797-. These results demonstrate the role of CNP for arthritis in rheumatoid arthritis.

From these confirmatory examples, the inventors of the present invention found that, without being limited by any particular theory or experiment, GC-B activators, such as CNP, have an antiarthritic effect and an anabolic effect on articular cartilage.

Accordingly, the present invention provides a therapeutic or prophylactic agent for arthritis, which comprises a GC-B activator as an active ingredient.

Examples of arthritis that may be treated or prevented according to the present invention include, but are not limited to: those involving articular cartilage, in particular arthritis such as that associated with osteoarthritis, synovitis, rheumatoid arthritis (adult) and juvenile rheumatoid arthritis (children)), osteoarthritis, Systemic Lupus Erythematosus (SLE), gout, scleroderma, psoriasis (psoriatic arthritis), fungal infections such as blastomycosis, ankylosing spondylitis, reiter's syndrome, septic arthritis, adult still disease, lyme disease (late stage), tuberculosis (tuberculous arthritis), viral infections (viral arthritis) and arthritis caused by infection with gonorrhea (gonococcal arthritis) and bacteria (non-gonococcal bacterial arthritis).

In one embodiment of the invention, the preferred arthritis is osteoarthritis or arthritis associated with osteoarthritis.

Osteoarthritis is a disease caused by degeneration and destruction of articular cartilage, and examples of applicable osteoarthritis include, for example, (1) osteoarthritis of weight-bearing joints, such as knee joint degenerative disease in knee joints, hip joint disease in hip joints, foot osteoarthritis in feet, and spinal osteoarthritis in spine, and (2) osteoarthritis of non-weight-bearing joints, such as shoulder osteoarthritis in shoulders, elbow osteoarthritis in elbows, hand osteoarthritis in hands (e.g., herberten's tubercle, buchard's tubercle, thumb CM osteoarthritis), and temporomandibular arthritis in jaws.

In one embodiment of the invention, the osteoarthritis is osteoarthritis affecting a weight bearing joint, preferably degenerative knee joint disease or degenerative hip joint disease.

In another embodiment of the invention, the osteoarthritis is osteoarthritis affecting non-weight bearing joints, preferably temporomandibular arthritis.

The therapeutic or prophylactic agent of the present invention can also be used for treating or preventing rheumatoid arthritis. Rheumatoid arthritis is considered to be an autoimmune disease, although it has a different etiology from osteoarthritis, and it involves, as osteoarthritis progresses, degeneration of the articular cartilage surface and destruction of cartilage. Thus, administration of the therapeutic agent of the present invention can inhibit or alleviate arthritis.

The terms "treatment", "treatment method" and "therapeutic agent" as used herein mean the elimination, inhibition or alleviation of the symptoms of a patient suffering from arthritis according to the present invention. Or a method or medicament for this purpose. In addition, the terms "prevention" and "prophylactic agent" mean prevention of arthritis or a drug for this purpose.

As used herein, the term "guanylate cyclase B (GC-B)" has the same meaning as natriuretic peptide receptor B (NPR-B).

As used herein, the term "GC-B activity" has the same meaning as guanylate cyclase activity. In the present invention, guanylate cyclase B (GC-B) activators or GC-B activators are low molecular weight compounds, either peptidic or non-peptidic, preferably CNP peptides or derivatives thereof, which can bind to and activate GC-B, known as the CNP receptor. As used herein, a peptide refers to a substance consisting of amide linkages of multiple (L-, D-, and/or modified) amino acids, including polypeptides and proteins. GC-B activators can be identified as follows: for example, GC-B receptor is expressed in a cultured cell line such as COS-7, a candidate agent is added to the culture, the cell line is cultured for a certain period of time and at a certain temperature (e.g., 37 ℃ for 5 minutes), and the amount of intracellular cGMP production is measured (Science 1991; 252: 120-. With such a test system and using the amount of intracellular cGMP production as an index, GC-B activators can be identified and used in the present invention.

According to one embodiment of the invention, the GC-B activator is a peptide, and preferably CNP or a derivative thereof. Preferred CNPs are selected from the group consisting of CNP-22 and CNP-53 from mammals including humans or birds, and more preferably the amino acid sequence of SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

According to another embodiment of the invention, the CNP derivative as described above has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 by deletion, substitution or addition of one or several amino acids in the amino acid sequence of 2 while maintaining the CNP activity. Alternatively, the CNP derivative comprises a peptide having a sequence identical to SEQ ID NO:1 or SEQ ID NO:2, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more, and retains the activity of CNP.

The term "one or several" as used herein generally denotes any integer from 1 to 10, preferably from 1 to 5, more preferably from 1 to 3. The "percentage of identity" between two amino acid sequences can be determined using techniques well known to those skilled in the art, such as the BLAST protein assay (Altschul, S.F., Gish, W.Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic Local Alignment Search Tool" J.mol.biol.215: 403-.

Examples of CNP usable in the present invention include CNP obtained from mammals including humans (CNP-22: biochem. Biophys. Res. Commun.1990; 168: 863-870, International publication WO 91/16342, CNP-53: biochem. Biophys. Res. Commun.1990; 170: 973-979, Japanese patent publication 4-74198A (1992), Japanese patent publication 4-139199A (1992), Japanese patent publication 4-121190A (1992)), CNP obtained from birds (Japanese patent publication 4-120094A (1992)), CNP obtained from amphibians (Japanese patent publication 4-120095A (1992)), and CNP derivatives disclosed in Japanese patent publication 6-9688A (1994) and International publication WO02/074234, such as CNP-like peptides.

CNP-22 and CNP-53, consisting of 22 and 53 amino acid residues, respectively, are known as naturally occurring CNPs. Since CNPs have high homology in terms of their sequences between birds and mammals including humans, i.e., regardless of the species of animals, CNPs derived from birds and mammals including humans, preferably CNPs derived from mammals including humans, and more preferably CNPs derived from humans, can be used in the present invention. The amino acid sequence of human CNP-22 or CNP-53 has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, which is represented as:

gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly SerMet Ser Gly Leu Gly Cys (human CNP-22; SEQ ID NO: 1); or

Asp Leu Arg Val Asp Thr Lys Ser Arg Ala Ala Trp Ala Arg Leu LeuGln Glu His Pro Asn Ala Arg Lys Tyr Lys Gly Ala Asn Lys Lys Gly LeuSer Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser GlyLeu Gly Cys (human CNP-53; SEQ ID NO: 2),

each of which has an intermolecular disulfide bond, i.e., forms a cyclic peptide structure between 6-Cys and 22-Cys of human CNP-22, or between 37-Cys and 53-Cys of human CNP-53.

Porcine CNP-22 and rat CNP-22 have the same amino acid sequence as human CNP-22, whereas in porcine CNP-53 and rat CNP-53 the amino acid residues at positions 17 and 28 are His and Gly, respectively, whereas in human CNP-53 they are Gln and Ala, respectively, i.e. on CNP-53 between human and pig or rat, two amino acids are different (japanese patent laid-open No. 4-139199a (1992), japanese patent laid-open No. 4-121190a (1992) and japanese patent laid-open No. 4-74198A (1992)). Further, chicken CNP-22 has the same primary structure as human CNP-22 except that the amino acid residue at position 9 is Val (Japanese patent laid-open No. 4-120094A (1992)).

CNPs that can be used in the present invention include CNPs purified from natural sources, recombinant CNPs prepared by known genetic engineering techniques, and CNPs prepared by known chemical syntheses (e.g., solid phase synthesis using a peptide synthesizer), preferably human CNP-22 and human CNP-53 prepared by genetic engineering techniques. The preparation of human CNP by genetic engineering techniques includes, for example, the steps of incorporating the DNA sequence of human CNP-22 or CNP-53 (Japanese patent laid-open No. 4-139199A (1992)) into a vector such as a plasmid or phage, transferring the vector into a prokaryotic or eukaryotic host cell such as an E.coli mother, and expressing the DNA in a suitable medium, preferably allowing the cell to extracellularly secrete the CNP peptide, and collecting and purifying the prepared CNP peptide. Polymerase Chain Reaction (PCR) techniques can also be used to amplify the target DNA.

Various basic techniques are well known to those skilled in the art, such as gene recombination, site-directed mutagenesis, and PCR techniques, described, for example, in J.Sambrook et al, Molecular Cloning, Alaberration Manual, second edition, Cold Spring Harbor laboratory Press (1990); ausubel et al, Current Protocols In Molecular Biology, John Wiley & Sons (1998), and the techniques disclosed therein can be used In the present invention. As the carrier, a commercially available carrier or a carrier disclosed in a publication can also be used.

As seen in human CNP-22 or CNP-53, the CNP derivatives useful in the present invention have CNP activity and have a cyclic peptide structure having a disulfide bond between two cysteine residues. Examples of CNP derivatives include: CNP fragments as described above; a peptide having one amino acid substituted with at least another amino acid in the above CNP or a fragment thereof; a peptide having at least one amino acid deletion in the above-mentioned CNP or a partial peptide thereof; and a peptide having at least one amino acid attached to the above-mentioned CNP or a partial peptide thereof. As used herein, substitution, deletion, or addition of amino acids means that a certain number of amino acids are substituted, deleted, or added by a known method, such as site-directed mutagenesis, provided that the CNP activity is not lost. For example, the CNP-22 or CNP-53 derivative has the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, which has the activity of CNP, by substitution, deletion or addition of one or several amino acids in the amino acid sequence of 2.

In general, amino acid substitutions are preferably conservative amino acid substitutions. Conserved amino acids may be classified according to, for example, polarity (or hydrophobicity) or charge type. Examples of non-polar, uncharged amino acids include glycine, alanine, valine, leucine, isoleucine, proline, and the like; aromatic amino acids include phenylalanine, tyrosine, and tryptophan; polar, uncharged amino acids include serine, threonine, cysteine, methionine, asparagine, glutamine, and the like; negatively charged amino acids include aspartic acid and glutamic acid; and positively charged amino acids including lysine, arginine and histidine.

The CNP activity as referred to in the present invention means: the activity of acting on GC-B to increase guanylate cyclase activity means an activity to eliminate, inhibit or alleviate arthritis including osteoarthritis, and means an activity to promote the growth of articular cartilage. The CNP activity can be measured by measuring cellular guanylate cyclase activity, such as intracellular cGMP production, and/or by administering CNP or a derivative thereof to a mouse or rat model of arthritis, osteoarthritis or rheumatoid arthritis for a certain period of time and measuring the arthritis or articular cartilage degeneration inhibitory effect as described in examples 7-10 below.

Examples of the CNP-22-like peptide include cyclic peptides (in which underlining indicates a difference from human CNP-22) described in Japanese patent laid-open No. 6-9688A (1994) and International publication No. WO02/074234 below.

Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly AlaMet Ser Gly Leu Gly Cys(SEQ ID NO：3)

Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly SerGln Ser Gly Leu Gly Cys(SEQ ID NO：4)

Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly SerAla Ser Gly Leu Gly Cys(SEQ ID NO：5)

Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu GlyCys(SEQ ID NO：6)

Ser Leu Arg Arg Ser Ser Cys Phe Gly Leu Lys Leu Asp Arg Ile GlySer Met Ser Gly Leu Gly Cys(SEQ ID NO：7)

Gly Leu Ser Lys Gly Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly SerMet Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr(SEQ ID NO：8)

Cys Phe Gly Leu Lys Leu Asp Arg Ile Gly Ser Gln Ser Gly Leu GlyCys Asn Ser Phe Arg Tyr(SEQ ID NO：9)

Cys Phe Gly Xaa Xbb Xcc Asp Arg Ile Gly Xdd Xee Ser Xff XggGly Cys

(wherein Xaa ═ Leu, Ile, Val; Xbb ═ Lys, Leu, Met; Xcc ═ Leu, Ile, Ala, Val; Xdd ═ Ser, Ala, Gly, Thr, Asn; Xee ═ Met, Ala, Trp, His, Lys, Ser, Gly; Xff ═ Gly, Lys, Als, Leu; Xgg ═ Leu, Met) (SEQ ID NO: 10).

Examples of CNP-53-like peptides include cyclic peptides comprising amino acid changes similar to CNP-22-like peptides.

The present invention also provides an articular chondrocyte growth promoter comprising a GC-B activator as an active ingredient. The present invention is based on the action of a GC-B activator to increase articular chondrocytes. An example of a GC-B activator is CNP or a derivative thereof as defined above. The CNP is preferably mammalian, including human, or avian CNP-22 or CNP-53, and more preferably SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2. The CNP derivative has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 by deletion, substitution or addition of one or several amino acids in the amino acid sequence of 2 while maintaining the CNP activity. Other GC-B activators can be identified as follows: for example, by expressing a GC-B receptor in a cultured cell line such as COS-7, adding a candidate agent to the medium, culturing the cell line at a certain time and temperature (e.g., 37 ℃ for 5 minutes), and measuring the amount of intracellular cGMP produced (Science 1991, 252: 120-. Thus, using such a test system and using the amount of intracellular cGMP production as an indicator, GC-B activators can be identified and used in the present invention.

The present invention also provides a method of inhibiting arthritis, wherein said arthritis is inhibited by activating GC-B. The present invention also provides a method for promoting growth of articular chondrocytes, comprising promoting the growth by activating GC-B. The present invention is based on the discovery that: arthritis, preferably osteoarthritis, as defined above may be inhibited and growth of articular chondrocytes may be enhanced by using a GC-B activator or by activating GC-B. In one embodiment of the invention, said GC-B is activated by CNP or a derivative thereof as defined above.

The present invention further provides a method for screening an agent for promoting the growth of articular chondrocyte, which comprises screening a candidate agent capable of promoting the growth of articular chondrocyte using GC-B activity as an index. Since it is known that the synthesis of the second messenger, cGMP, from GTP is catalyzed by GC-B through the activity of guanylate cyclase, which can be determined by the amount of intracellular cGMP production.

According to an embodiment of the present invention, the screening method as described above may comprise the steps of: a GC-B-expressing cultured cell or a cell derived from an articular chondrocyte is prepared, the cell is cultured in the presence of a candidate agent, and the candidate agent capable of promoting the growth of the articular chondrocyte is screened using the guanylate cyclase activity of the cell, for example, the intracellular amount of cGMP produced, as an index.

According to a preferred embodiment of the present invention, the screening method comprises: preparing a cultured cell line that has been forced to express GC-B, culturing the cell line in the presence or absence of a test substance, determining the amount of intracellular cGMP production, and screening for a candidate agent capable of promoting the growth of articular chondrocyte using the difference between the amount of intracellular cGMP production in the presence and absence of the candidate agent as an index.

The screening method of the present invention can be used for screening of the articular chondrocyte growth promoter, for example, by expressing GC-B in cultured cells such as COS-7, adding a candidate agent to the culture, culturing the cells for a certain period of time and at a certain temperature (e.g., 37 ℃ C., 5 minutes), and measuring the amount of cGMP produced intramolecularly (Science 1991, 252: 120-.

The present invention further provides a method of screening for a therapeutic agent for osteoarthritis, rheumatoid arthritis or other arthritis, which comprises screening for a candidate agent for an agent capable of treating osteoarthritis, rheumatoid arthritis or other arthritis using GC-B activity as an index. As described above, GC-B activity can be determined as the activity of guanylate cyclase, such as the amount of intracellular cGMP produced.

In one embodiment of the present invention, the screening method as described above may comprise the steps of: a method for screening a drug candidate for a drug capable of treating osteoarthritis, rheumatoid arthritis or other arthritis, which comprises preparing GC-B-expressing cultured cells or cells derived from articular chondrocytes, culturing the cells in the presence of the drug candidate, and screening the drug candidate using the guanylate cyclase activity of the cells, for example, the intracellular amount of cGMP produced, as an index.

According to a preferred embodiment of the present invention, the screening method comprises: preparing a cultured cell line that has been forced to express GC-B, culturing the cell line in the presence or absence of a test substance, determining the amount of intracellular cGMP produced, and screening a candidate agent for a drug capable of treating osteoarthritis, rheumatoid arthritis, or other arthritis using the difference in the amount of intracellular cGMP produced in the presence and absence of the candidate agent as an index.

The screening method of the present invention can be used for screening therapeutic agents for osteoarthritis, rheumatoid arthritis or other arthritis, for example, by expressing GC-B in cultured cells such as COS-7, adding a candidate agent to the culture, culturing the cells for a certain period of time and at a certain temperature (e.g., 37 ℃ C., 5 minutes), and measuring the amount of cGMP produced within the molecule (Science 1991, 252: 120-.

The therapeutic or prophylactic agent for arthritis such as osteoarthritis of the present invention is formulated into a preparation for oral or parenteral administration by combining the GC-B activator as an active ingredient as defined above with a pharmaceutically acceptable carrier, excipient, additive, etc.

Examples of carriers and excipients for use in formulations include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cocoa butter, ethylene glycol, and other commonly used agents.

Examples of solid compositions for oral administration include tablets, pills, capsules, powders, granules and the like. In these solid compositions, at least one active ingredient is mixed with at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminosilicate, and the like. The composition may also contain additives other than inert diluents, for example, lubricants such as magnesium stearate, disintegrants such as calcium fibrous glycolate, and cosolvents such as glutamic acid or aspartic acid, according to conventional methods. Tablets or pills, if desired, may be coated with a glycocalyx, such as sucrose, gelatin or hydroxypropylmethylcellulose phthalate, or with a gastric or enteric film, or with two or more layers. Capsules of absorbable material, such as gelatin, are also included.

Liquid compositions for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs, and may also include conventional inert diluents such as purified water and ethanol. The composition may contain adjuvants which are not inert diluents, such as wetting and suspending agents, sweetening, flavoring, perfuming and preservative agents.

Exemplary kit for parenteral injectionIncluding sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of the aqueous solution and suspension include water for injection and physiological saline for injection. Examples of non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80. These compositions may further comprise adjuvants such as preservatives, humectants, emulsifiers, dispersants, stabilizers (e.g. lactose), and cosolvents (e.g. glutamic acid or aspartic acid). The above substances can be sterilized by a conventional sterilization method such as filtration sterilization using a microfiltration membrane, heat sterilization such as autoclaving, or addition of a sterilizing agent. Injectables can be liquid formulations, or lyophilized formulations that can be reconstituted prior to use. Examples of excipients for lyophilization include sugar alcohols and sugars, such as mannitol and glucose.

The therapeutic or prophylactic agent of the present invention is administered by a pharmaceutically commonly used oral or parenteral administration method. Parenteral administration methods are preferred, such as injection (e.g., subcutaneous, intravenous, intramuscular, and intraperitoneal injection), transdermal administration, transmucosal administration (e.g., nasal and rectal), and pulmonary administration. Oral administration may also be employed.

The dosage of the GC-B activator, preferably CNP or its derivative as defined above, as an active ingredient contained in the composition of the present invention may be determined according to the type of disease to be treated, the severity of the disease, the age of the patient, etc., and the amount thereof may be generally 0.005 μ g/kg to 100mg/kg, preferably 0.02 μ g/kg to 5mg/kg, more preferably 0.02 μ g/kg to 0.25 mg/kg. However, the drugs containing the CNP activators of the present invention are not limited in their dosage.

The therapeutic or prophylactic agent of the present invention can be combined with conventional or novel therapeutic agents such as anti-inflammatory agents, hyaluronic acid and adrenocortical steroids, as well as with surgical procedures such as arthroscopic (arthrosopic) surgery, artificial joint replacement and osteotomy.

Anti-inflammatory drugs, in particular, for example, a combination of at least one non-steroidal anti-inflammatory drug with a GC-B activator (e.g., CNP or a derivative thereof as defined above) may provide synergistic inhibition of arthritis (example 10).

As used herein, "non-steroidal anti-inflammatory drug" refers to an anti-inflammatory drug having no steroidal skeleton, and preferably has an effect of inhibiting cyclooxygenase involved in prostaglandin production. Examples of non-steroidal anti-inflammatory drugs that can be used in the present invention include, but are not limited to, indomethacin (e.g., Indacin)^TM) Ibuprofen (e.g. Brufen)^TM) Piroxicam, salicylic acid, diclofenac (e.g. Voltaren)^TM) Ketoprofen, naproxen and piroxicam.

In addition, the synergistic effect of the combination of a GC-B activator and a non-steroidal anti-inflammatory drug as described above means that the GC-B activity is enhanced compared to the GC-B activator alone, or in other words, the active ingredient of the non-steroidal anti-inflammatory drug as described above acts as an activation promoter in the activation of GC-B with the GC-B activator.

Accordingly, the present invention further provides an activation enhancer for GC-B activators comprising a non-steroidal activator.

The GC-B activator comprises CNP or a derivative thereof as defined above. Examples of CNP are CNP-22 or CNP-53 from mammals including humans or birds, more specifically SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2. Examples of CNP derivatives include those having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 by deletion, substitution or addition of one or several amino acids in the amino acid sequence of 2, and retains the CNP activity.

In the present invention, the non-steroidal activator as described above is preferably a cyclooxygenase inhibitor. Examples of such cyclooxygenase inhibitors include, but are not limited to, indomethacin, ibuprofen, piroxicam, salicylic acid, diclofenac, ketoprofen, naproxen, and piroxicam.

The dosage forms, dosages, and administration methods described above for the therapeutic and prophylactic agents of the present invention can be used for the activation promoters of the present invention.

The invention further provides a process for activating a GC-B activator, wherein an activation promoter as described above is used.

The activation promoters and methods of the present invention as described above may be used to effectively treat a disease, such as arthritis, in a patient, for example, by activating GC-B.

The present invention includes, but is not limited to, the following aspects.

(1) A therapeutic or prophylactic agent for arthritis, which comprises a guanylate cyclase B (GC-B) activator as an active ingredient.

(2) The therapeutic or prophylactic agent according to (1) above, wherein the arthritis is osteoarthritis.

(3) The therapeutic or prophylactic agent according to (2) above, wherein the osteoarthritis is osteoarthritis of a weight-bearing or non-weight-bearing joint.

(4) The therapeutic or prophylactic agent according to (3) above, wherein the osteoarthritis is a degenerative knee disease.

(5) The therapeutic or prophylactic agent according to (3) above, wherein the osteoarthritis is a hip degenerative disease.

(6) The therapeutic or prophylactic agent according to (3) above, wherein the osteoarthritis is temporomandibular arthritis.

(7) The therapeutic or prophylactic agent according to (1) above, wherein the arthritis is caused by rheumatoid arthritis.

(8) The therapeutic or prophylactic agent of the above (1), wherein the arthritis is caused by osteoarthritis.

(9) The therapeutic or prophylactic agent according to any one of (1) to (8) above, wherein the GC-B activator is C-type natriuretic peptide (CNP) or a derivative thereof.

(10) The therapeutic or prophylactic agent according to (9) above, wherein the CNP is selected from the group consisting of CNP-22 and CNP-53 derived from mammals including humans or birds.

(11) The therapeutic or prophylactic agent of (9) above, wherein the CNP is a peptide of SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

(12) The therapeutic or prophylactic agent of the above (9), wherein the derivative has an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and having a CNP activity.

(13) The therapeutic or prophylactic agent according to any one of (1) to (12) above, which further comprises at least one non-steroidal anti-inflammatory drug.

(14) An articular chondrocyte growth promoter comprising a GC-B activator as an active ingredient.

(15) The accelerating agent of the above (14), wherein the GC-B activator is CNP or a derivative thereof.

(16) The accelerating agent of the above (15), wherein the CNP is CNP-22 or CNP-53 derived from a mammal including a human or an avian.

(17) The accelerating agent of the above (15), wherein the CNP is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

(18) The accelerating agent of the above (15), wherein the derivative has an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or several amino acids in the amino acid sequence of 2.

(19) The accelerating agent according to (14) to (18) above, which further comprises at least one non-steroidal anti-inflammatory drug.

(20) A method for inhibiting arthritis, wherein said arthritis is inhibited by activating GC-B.

(21) The method for inhibiting according to (20) above, wherein the GC-B is activated by CNP or a derivative thereof.

(22) The method of inhibiting according to (21) above, wherein the CNP is CNP-22 or CNP-53 derived from a mammal including a human being or an avian.

(23) The method of inhibiting according to (21) above, wherein the CNP is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

(24) The method of inhibiting according to (21) above, wherein the derivative has an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or several amino acids in the amino acid sequence of 2.

(25) The method of inhibiting according to (20) to (24) above, wherein said GC-B is activated by a combination of CNP or a derivative thereof and at least one non-steroidal anti-inflammatory drug.

(26) A method for promoting growth of articular chondrocyte, wherein the growth of articular chondrocyte is promoted by activating GC-B.

(27) The method of the above (26), wherein the GC-B is activated by CNP or a derivative thereof.

(28) The method of the above (27), wherein the CNP is CNP-22 or CNP-53 derived from a mammal including human or bird.

(29) The method of (27) above, wherein the CNP is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

(30) The method of (27) above, wherein the derivative has an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or more amino acids in the amino acid sequence of 2.

(31) The method of any one of (26) to (30) above, wherein the GC-B is activated by a combination of CNP or a derivative thereof and at least one non-steroidal anti-inflammatory drug.

(32) A method for screening an agent for promoting the growth of articular chondrocyte, which comprises screening a candidate agent capable of promoting articular chondrocyte using GC-B activity as an index.

(33) The method of the above (32), which comprises preparing a cultured cell expressing GC-B or a cell derived from an articular chondrocyte, culturing the cell in the presence of a candidate agent, and screening for a candidate agent capable of promoting the growth of an articular chondrocyte using the GC-B activity of the cell as an index.

(34) The method of the above (32) to (33), wherein the GC-B activity is determined by the intracellular production amount of cGMP.

(35) The method of the above (32) to (34), which comprises preparing a cultured cell line that has been forcedly expressed with GC-B, culturing the cell line in the presence or absence of a test substance, determining the intracellular production amount of cGMP, and screening a candidate agent capable of promoting the growth of articular chondrocytes using the difference in the intracellular production amount of cGMP between in the presence and in the absence of the candidate agent as an index.

(36) A method of screening for a therapeutic agent for osteoarthritis, rheumatoid arthritis or other arthritis, comprising screening for a candidate agent for an agent capable of treating osteoarthritis, rheumatoid arthritis or other arthritis using GC-B activity as an index.

(37) The method of (36) above, which comprises preparing cultured cells expressing GC-B or cells derived from articular chondrocytes, culturing the cells in the presence of a candidate agent, and screening the candidate agent for an agent capable of treating osteoarthritis, rheumatoid arthritis or other arthritis using the GC-B activity of the cells as an index.

(38) The method of the above (36) to (37), wherein the GC-B activity is determined by the intracellular production amount of cGMP.

(39) The method of any one of the above (36) to (38), which comprises preparing a cultured cell line that has been forcedly expressed with GC-B, culturing the cell line in the presence or absence of a test substance, determining the intracellular production amount of cGMP, and screening a candidate agent for an agent capable of treating osteoarthritis, rheumatoid arthritis or other arthritis using the difference in the intracellular production amount of cGMP between the presence and absence of the candidate agent as an index.

(40) A therapeutic or prophylactic agent for osteoarthritis, comprising a guanylate cyclase B (GC-B) activator as an active ingredient.

(41) The therapeutic or prophylactic agent for osteoarthritis according to (40) above, further comprising at least one non-steroidal anti-inflammatory drug.

(42) A therapeutic or prophylactic agent for rheumatoid arthritis, which comprises a guanylate cyclase B (GC-B) activator as an active ingredient.

(43) The therapeutic or prophylactic agent for rheumatoid arthritis according to (42) above, further comprising at least one non-steroidal anti-inflammatory drug.

(44) A method of treating arthritis comprising administering a GC-B activator to a patient in need of arthritis treatment.

(45) The method of the above (44), wherein the GC-B activator is CNP or a derivative thereof.

(46) The method of (44) or (45) above, wherein the arthritis is osteoarthritis.

(47) The method of (46) above, wherein the osteoarthritis is osteoarthritis of a weight-bearing or non-weight-bearing joint.

(48) The method of (47) above, wherein the osteoarthritis is degenerative knee joint disease, degenerative hip joint disease or temporomandibular arthritis.

(49) The method of (44) above, wherein the arthritis is caused by rheumatoid arthritis.

(50) The method of (44) above, wherein the arthritis is caused by osteoarthritis.

(51) The method of any one of (45) to (50) above, wherein the CNP is selected from CNP-22 or CNP-53 derived from a mammal including a human or a bird.

(52) The method of any one of (45) to (50) above, wherein the CNP is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

(53) The method of any one of (45) to (50) above, wherein the derivative has an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or several amino acids in the amino acid sequence of 2.

(54) The method of any one of (44) to (53) above, wherein said GC-B activator is comprised in combination with at least one non-steroidal anti-inflammatory drug.

(55) An activation enhancer for a GC-B activator comprising a non-steroidal activator.

(56) The activation accelerator according to the above (55), wherein the GC-B activator is CNP or a derivative thereof.

(57) The activation accelerator according to the above (56), wherein the CNP is selected from CNP-22 or CNP-53 derived from mammals including humans or birds.

(58) The activation accelerator according to the above (56), wherein the CNP is SEQ ID NO:1 CNP-22 or SEQ ID NO: CNP-53 of 2.

(59) The activation accelerator according to the above (56), wherein the derivative has an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and having a CNP activity, by deletion, substitution, or addition of one or several amino acids in the amino acid sequence of 2.

(60) The activation accelerator according to the above (55), wherein the non-steroidal activator is a cyclooxygenase inhibitor.

(61) The activation accelerator according to the above (60), wherein the cyclooxygenase inhibitor is selected from the group consisting of indomethacin, ibuprofen, piroxicam, salicylic acid, diclofenac, ketoprofen, naproxen, and piroxicam.

(62) A method for activating a GC-B activator, wherein the activation accelerator according to any one of the above (55) to (61) is used.

The present invention will be described in more detail by the following examples, which are for illustrative purposes and are not intended to limit the scope of the present invention. Therefore, the present invention is not limited by these examples.

Examples

Example 1: construction of vector for preparing CNP transgenic mice

As shown in FIG. 1A, murine CNP cDNA (526 bp; FEBS Lett.276: 209-213, 1990) was subcloned into pGEM-T easy vector (Promega), which was then cut with Pst I and blunt-ended to prepare mouse CNP cDNA. As shown in FIG. 1C, the vector PSG1 (Promega; FIG. 1B) was cleaved with EcoRI, the cleaved ends were smoothed and ligated (ligation) with the murine CNP cDNA to prepare an SAP-mCNP vector (pSG 1-CNP).

Example 2: preparation of CNP transgenic mice

DNA fragments for injection were prepared as follows. The SAP-mCNP vector (pSG 1-CNP; FIG. 1C) having the CNP gene inserted therein was first treated with Hind III and Xho I to cut out a fragment (about 2.3kb) containing the CNP gene. This fragment was then collected with the Gel Extraction Kit (QIAGEN) and diluted with PBS-to a concentration of 3 ng/. mu.l, thus obtaining a DNA fragment for injection (FIG. 2).

The eggs of the mice in the pronuclear stage into which the DNA fragments were injected were collected as follows. C57BL/6 female mice (clean Japan, Inc.) were first injected intraperitoneally with 5i.u. pregnant horse serum gonadotropin (PMSG) and 48 hours later with 5i.u. human chorionic gonadotropin (hCG) to induce superovulation. This mouse was crossed with a syngeneic male mouse. The next morning of the cross, the presence of emboli was determined in female mice, and then oviducts were perfused to collect pre-nucleated mouse ova.

The injection DNA fragment was injected into the pronucleus mouse ovum with a micromanipulator (test Technology in Gene Targeting (Yodosha, Japan), 190-. Specifically, the DNA fragment was injected into the 660C57BL/6J embryo, and in the following days, 561 embryos at 2-cell stage were transplanted into the oviducts of recipient mice on day 1 of pseudopregnancy, about 10 on each side of the oviducts (about 20 per animal).

Recipient female mice that did not lay during the expected period of birth were subjected to caesarean delivery from a foster mother. A total of 136 litters were obtained, 5 of which were transgenic mice with the introduced CNP gene (hereinafter designated as "Tgm"). Hereinafter, the initially obtained mouse was designated as a Founder (Founder).

All founder mice were male, and subsequent litters (i.e., F1 mice) were obtained from four of the five lines.

Example 3: genotype analysis of CNP transgenic mice

Genotyping was performed by southern blotting according to the method described below.

Tails (about 15mm) were taken from 3-week-old mice and treated with proteinase K (shaking overnight at 100rmp at 55 ℃) to obtain a lysate. The obtained lysate was then subjected to an automated nucleic acid separator (KURABO NA-1000; Kurabo, Japan) to prepare genomic DNA. The genomic DNA (15. mu.g) was treated with Pvu II (200U), then treated with phenol-chloroform to remove the restriction enzyme, and precipitated with ethanol to collect the DNA. The obtained DNA was dissolved in 25. mu.l of TE and subjected to electrophoresis on a 0.7% agarose gel (at 50V constant voltage), and then the gel was treated with a 0.25M HCl solution for 15 minutes to cleave the DNA, washed with water, and blotted overnight on a nylon membrane in a 0.4M NaOH solution. The DNA on the membrane is then immobilized by UV cross-linking. The membrane was treated with a hybridization solution (50% formamide, 0.5x Denhard's, 0.5% SDS, 5x SSPE) and using the CNP cDNA as a template, the membrane that had been prepared with BcaBEST labelling Kit (TaKaRa, Japan) was used³²P-labeled probe was added to the membrane to effect hybridization overnight at 42 ℃. After treatment with a washing solution (2x SSC, 0.1% SDS) at 55 ℃ for 20 minutes, the membrane was exposed to Imaging Plate (Fuji Film) overnight to detect the signal of the transgene with BAS2000(Fuji Film, japan) (fig. 3). In wild type mice (WT), 3 signals (wild type CNP gene) were detected, whereas in transgenic mice (Tgm), in addition to the wild type CNP gene, 2 signals (transgene) derived from the transgene were detected.

Example 4: CNP expression in CNP transgenic mice

The CNP-22EIA measurement kit (PHOENIX PHARMACEUTICAL SINC.) was used for the determination of CNP levels.

Three each of 7-week old male and female CNP transgenic mice, and three each of male and female homoabdominal mice, were euthanized by exsanguination from the posterior vena cava under ether anesthesia.

The liver, an organ expected to show high expression of the transgene, was excised, and the EIA test buffer obtained from the measurement kit described above was added thereto in an amount of 1mL per 0.1g of liver weight, followed by cooling on ice. The liver was homogenized in a Waring blender (Physcotron) and the supernatant was used as a sample for determination of CNP-22 levels after centrifugation (2,000rpm, 5 minutes).

One mg of ethylenediaminetetraacetic acid tetrasodium salt (Junsei Chemical co., ltd., japan) and 2 trypsin inhibitory units of aprotinin (Sigma) were added to the drawn blood and agitated to separate plasma, which was used as a sample for determining the CNP-22 level.

The results are shown in Table 1.

Table 1: CNP expression in CNP transgenic mice

**: p < 0.01 (unpaired Student t test)

#: p < 0.05(Wilcoxon rank sum test)

CNP transgenic mice had CNP-22 levels in liver and plasma that were about 10-fold and about 24-fold higher than wild type mice, respectively, when compared to the mean ± SD values of wild type mice. The difference in each case was statistically significant. From these results, it was confirmed that the CNP peptide was overexpressed in the CNP transgenic mice.

Example 5: histological analysis of articular cartilage of CNP transgenic mice.

To histologically analyze the thickness of the growing cartilage, 5 female CNP transgenic mice 9 weeks old each and female normal homoabdominal mice were euthanized by exsanguination from the posterior vena cava under ether anesthesia. And the femur was fixed with 20% formalin for one week. After decalcification by soaking in a 20% aqueous EDTA-4Na (Junsei Chemical Co., Ltd., Japan) solution (pH 4.4), the femoral patellar surface was subjected to a midline sagittal section and embedded in paraffin by a conventional method to prepare a paraffin block. The 4 μm-thick section was further sectioned with a microtome to prepare a paraffin section, which was stained with hematoxylin and eosin. For the thickness of the growing cartilage, one microscopic field observed with an eyepiece (x 10) was combined with image analysis software (IPAP, Sumika technosphere, japan), and the thickness of each layer of the resting layer, the proliferation layer, and the hypertrophic layer was measured at 5 points in the microscopic field with the same software, and the calculated average value was taken as the thickness of each layer of the individual. The total thickness of the three layers is considered to be the thickness of the individual's growing cartilage. The mean and standard deviation of these items were calculated between CNP transgenic mice and normal homoabdominal mice (using Microsoft Excel2000, Microsoft) and statistical analysis was performed using unpaired Student's t-test (SAS version 6.12; SASInstitute Japan, Japan).

CNP transgenic mice, male and female, all demonstrated statistically significant thicker articular chondrocytes (fig. 4). In addition, statistically significant numbers of articular chondrocytes per microscopic field were shown in both male and female CNP transgenic mice (fig. 5).

These results indicate that GC-B (NPR-B) activating substances such as CNP can increase the thickness of articular cartilage by increasing the number of chondrocytes, and by increasing the number of cells generally known to be caused by excessive growth of chondrocytes in an individual (J Biol Chem 2003; 278 (21): 18824-32).

Example 6: tolerance of CNP transgenic mice to osteoarthritis models

An animal model of osteoarthritis was established by injecting collagenase into the knee joint of mice to destabilize the knee ligaments and meniscus (am.j. pathol.1989; 135: 1001-14). Resistance to arthritis and articular cartilage degeneration was evaluated in this animal model using CNP transgenic mice to determine the prophylactic and therapeutic effects on osteoarthritis. Mu.l of 3% collagenase type II (Sigma) in physiological saline solution were injected twice (initial day of administration and 7 days later) into the right knee joints of CNP transgenic mice and homoabdominal wild type C57BL/6 line mice. The widths of both the left and right knee joints were measured in time with a vernier caliper (Mitutoyo corp., japan) 28 days after the administration, and the difference between the right and left knee joints was calculated to represent the degree of swelling of the knee joint. The area under the time-phase curve (AUC) of the continuous change was calculated by the trapezoidal method and CNP transgenic mice and wild type mice were compared by Student t-test. As a result, AUC in the transgenic mice was significantly smaller than in the wild type, indicating that the CNP transgenic mice were resistant to swelling of the knee joint by collagenase (fig. 6). For histological evaluation of arthritis and articular cartilage degeneration, euthanasia was performed by exsanguination from the posterior vena cava under ether anesthesia at day 28 after collagenase administration, and then the knee was excised, and hematoxylin-eosin stained and safranin O stained samples were prepared and subjected to histological analysis as described in example 5. As a result, wild-type mice showed that in synovium, collagenase induced significant synovial cell growth, granulation tissue formation and inflammatory cell infiltration, and these changes were significantly reduced in CNP transgenic mice (fig. 7). For the degeneration of articular cartilage, wild-type mice showed reduced staining capacity of safranin O and reduced proteoglycan content in articular cartilage, while these changes were slight in CNP transgenic mice, which provided histological evidence of CNP transgenic mice resistant to degenerative changes in articular cartilage caused by collagenase administration (fig. 8). The mean plasma CNP levels determined using the EIA kit (Phoenix Pharmaceutical) were 0.21ng/mL in wild-type mice and 0.50ng/mL in CNP transgenic mice.

These results suggest that CNP has an inhibitory effect on arthritis and degenerative changes in articular cartilage in osteoarthritis.

Example 7: therapeutic Effect of CNP infusion on osteoarthritis models (1)

Osmotic pumps (2004, Durect) containing the following solutions were implanted subcutaneously on the back of 9-week old male C57BL/6J strain mice.

Solvent: distilled water containing 5% dextrose (Junsei Chemical co., ltd., japan), 10% mannose (Nacalai Tesque inc., japan), and 5mmol/L hyaluronic acid (Wako pure Chemical industries, japan).

10. mu.g/mL of a solution of CNP-22(Calbiochem Novabiochem) (60 ng/day).

100. mu.g/mL of a solution of CNP-22(Calbiochem Novabiochem) (600 ng/day).

6 days after the transplantation, 6. mu.L of 1.5% collagenase type II (Sigma) solution was injected into the right knee joint, and the widths of the right and left knee joints were measured with a vernier caliper (Mitutoyo Corp., Japan) at 28 days after the injection in time, and the difference between the right and left knee joints was calculated. This difference represents the degree of knee joint swelling and AUC was compared between the solvent control group and the CNP group by Student's t-test (SAS version 6.12). The results show that the AUC values were significantly lower at each dose in the CNP-22 group compared to the solvent control group. Hematoxylin-eosin stained and safranin O stained samples were prepared and histologically analyzed according to the procedure in example 5.

As a result, the solvent control group showed significant synovial cell growth, granulation tissue formation and inflammatory cell infiltration induced by collagenase in the synovium, and these changes were significantly reduced in the CNP group (fig. 9). These results, which are derived from synovial tissue, show that CNP has a therapeutic effect on osteoarthritis.

Example 8: therapeutic Effect of CNP infusion on osteoarthritis models (2)

An osmotic pump (2004, Durect) containing the following solution was subcutaneously transplanted on the back of 9-week-old male C57BL/6J strain mice (CLEA Japan, Japan).

10mg/mL of a solution of CNP-22(Calbiochem Novabiochem) (60 ng/day).

100mg/mL of a solution of CNP-22(Calbiochem Novabiochem) (600 ng/day).

On the next day after implantation, mice were anesthetized with ether and subjected to a surgical procedure to remove the anterior cruciate ligament in the right knee, remove the medial collateral ligament of the tibia, and remove the medial meniscus in its entirety to induce osteoarthritis. The widths of the right and left knee joints were measured in time with a vernier caliper (Mitutoyo Corp.) 11 days after the administration, and the difference between the right and left knee joints was calculated. This difference indicates the degree of swelling of the knee joint, and the AUC between the solvent control group and the CNP group was compared by Student's t-test (SAS clinical package, Institute Japan). The results showed that the AUC values were significantly lower at each dose in the CNP-22 group compared to the solvent control group (fig. 10).

These results indicate that CNP is also effective in inhibiting arthritis in osteoarthritis caused by excessive physical loading of the knee joint as a result of the surgical procedure.

Example 9: combined action of non-steroidal anti-inflammatory drugs (NSAIDs) and CNP in collagenase OA model

1. mu.g/mL of a solution of CNP-22(Calbiochem Novabiochem) (6 ng/day).

In addition, to examine the effect of the NSAID indomethacin (Sigma) when used alone and in combination with CNP, a suspension of indomethacin in 0.2% carboxymethylcellulose (Nacalai Tesque inc., japan) was administered 1mg/kg 1 time a day for 4 consecutive days in a forced oral manner starting from the date of pump implantation as described above.

The experimental groups were set up as follows.

Solvent control group (infusion solvent, oral administration solvent)

CNP 6 ng/day

Indometacin 1mg/kg

CNP 6 ng/day + Indometacin 1mg/kg

On the day of pump implantation and the next day, 6 μ L of 0.15% collagenase type II (Sigma) and 6 μ L of 1.5% collagenase type II solution were infused into the right knee, respectively, and 7 days after infusion, the width of the right knee and left knee was measured daily with a vernier caliper (Mitutoyo Corp.) to calculate the difference between the right knee and left knee. This difference represents the degree of knee joint swelling and AUC was compared between the solvent control group and the CNP group by Student's t-test (SAS version 6.12).

As a result, swelling of the knee joint was not inhibited when indomethacin was used alone. The CNP group administered 6 ng/day significantly suppressed knee swelling. The group given the combination of CNP and indomethacin showed significantly stronger knee joint swelling inhibition effect than the group given CNP alone (fig. 11). These results show that CNP has a more effective knee joint swelling inhibition effect than NSAIDs when used alone, is a standard anti-arthritic drug, and also has a synergistic effect when used in combination with NSAIDs.

Example 10: effect of CNP on the adjuvant arthritis rat model

Osmotic pumps (2004, Durect) containing the following solutions were implanted subcutaneously on the back of 9-week old male LEW/Crj-line rats (Charles River Laboratories Japan, inc., Japan).

10. mu.g/mL of a solution of CNP-22(Calbiochem Novabiochem) (60 ng/day).

On the next day of transplantation, powder of tuberculosis bacteria (M.TUBERCULOSIS DES.H37 RA DIFCO LABORATORIES) which is no longer infectious was suspended in liquid paraffin (Junsei Chemical Co., Ltd., Japan) at a concentration of 3mg/mL, and 50. mu.L of the suspension was inoculated into the root skin of rat tail. After inoculation, the condition of the extremities was evaluated daily using a scoring system according to the following criteria, and the sum of the extremities scores was calculated to represent the individual's arthritis score.

0 minute: without damage

1 minute: flare/swelling was observed in one or more of the knuckles. Or redness in the back of the paw without swelling.

And 2, dividing: a slight swelling occurred in the back of the forelimb or hindlimb.

And 3, dividing: severe swelling occurred in the back of the forelimbs or hind limbs, but not in all the fingers.

And 4, dividing: severe swelling occurs in the back and fingers of the forelimbs or hind limbs.

The results showed somewhat lower arthritis scores in the CNP group than in the solvent control group (fig. 12A).

Daily based weight changes were also measured. The results showed a significant increase in body weight in the CNP group compared to the solvent control group (fig. 12B).

These results suggest that CNP can also inhibit arthritis and improve overall symptoms in the helper rat model.

Example 11: effect of CNP on collagen arthritis rat model

Osmotic pumps (2004, Durect) containing the following solutions were implanted subcutaneously in the back of 10-week-old female DA/Slc-line rats (Japan Slc, inc., Japan).

1mg/mL of a solution of CNP-22(Calbiochem Novabiochem) (6. mu.g/day).

Immediately after the transplantation, bovine type II collagen was dissolved in 0.1mol/L acetic acid aqueous solution to make 1.5mg/mL, and suspended in an equal volume of incomplete Freund's adjuvant (DIFCOLABORATORIES), and 400. mu.L of the suspension was inoculated into the skin of the back of the rat. Daily based weight changes were also measured. In addition, daily-based body weight change was also measured in the normal group with neither pump inhibition nor vaccination.

As a result, the body weight was significantly reduced in the solvent control group compared to the normal group, whereas the body weight reduction was significantly smaller in the CNP group than in the low-solvent control group (fig. 13). These results indicate that CNP improves overall symptoms in a collagen arthritis rat model.

INDUSTRIAL APPLICABILITY

Since the therapeutic or prophylactic agents of the present invention comprising a GC-B activator as an active ingredient can increase the thickness of articular cartilage and the number of articular chondrocytes, provide tolerance to joint swelling, inhibit degenerative changes in articular cartilage, significantly reduce changes in synovial cell growth, granulation tissue formation and inflammatory cell infiltration, and avoid a reduction in proteoglycan content in articular cartilage, they are useful for the treatment or prevention of arthritis including osteoarthritis, such as degenerative knee disease, degenerative hip disease, elbow osteoarthritis, spinal osteoarthritis and temporomandibular joint disease. Administration of the pharmaceutical composition of the present invention results in the inhibition of reduction or regeneration of articular cartilage matrix and chondrocytes at the affected joint part, and the inhibition of degenerative changes in articular cartilage and swelling in the joint part, which results in the inhibition or reduction of arthritic diseases. In particular, since the therapeutic agents for osteoarthritis of the present invention cause less load and pain to patients compared to conventional orthopedic surgery such as arthroscopic surgery, artificial joint replacement, and osteotomy, they provide excellent therapeutic agents having a qqql satisfying patients.

The new finding that GC-B activators have the efficacy as described above means that it is possible to inhibit arthritis and promote the growth of articular chondrocytes by activating GC-B. In addition, it is also possible to screen for articular chondrocyte growth promoters and arthritis therapeutic agents using GC-B activity (for example, the amount of intracellular cGMP production) as an index.

All publications, patents and patent applications cited herein are incorporated by reference in their entirety.

Free text of sequence listing

SEQ ID NO: description of 1: disulfide bonds are formed between 6-Cys and 22-Cys.

SEQ ID NO:2, description: disulfide bonds are formed between 37-Cys and 53-Cys.

SEQ ID NO: 3 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 6-Cys and 22-Cys.

SEQ ID NO: 4 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 6-Cys and 22-Cys.

SEQ ID NO: 5 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 6-Cys and 22-Cys.

SEQ ID NO: 6 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 1-Cys and 17-Cys.

SEQ ID NO: 7 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 7-Cys and 23-Cys.

SEQ ID NO: 8 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 6-Cys and 22-Cys.

SEQ ID NO: 9 manual sequence description: a CNP-22 derivative wherein a disulfide bond is formed between 1-Cys and 17-Cys.

SEQ ID NO: 10 manual sequence description: CNP-22 derivatives, wherein 4-Xaa ═ Leu, Ile, Val; 5-Xaa ═ Lys, Leu, Met; 6-Xaa ═ Leu, Ile, Ala, Val; 11-Xaa ═ Ser, Ala, Gly, Thr, Asn; 12-Xaa ═ Met, Ala, Trp, His, Lys, Ser, Gly; 14-Xaa ═ Gly, Lys, Ala, Leu; 15-Xaa ═ Leu, Met, and disulfide bonds are formed between 1-Cys and 17-Cys.

Claims

1. Use of a guanylate cyclase B activator for the preparation of a therapeutic or prophylactic agent for arthritis,

wherein the guanylate cyclase B activator is a C-type natriuretic peptide or a derivative thereof,

wherein said C-type natriuretic peptide is selected from the group consisting of CNP-22 of SEQ ID NO. 1 and CNP-53 of SEQ ID NO. 2, and

wherein the derivative is selected from the group consisting of a guanylate cyclase B activating peptide having deletion, substitution or addition of 1 to 5 amino acids in the amino acid sequence of SEQ ID NO. 1 and having an activity to activate guanylate cyclase B and a guanylate cyclase B activating peptide having deletion, substitution or addition of 1 to 5 amino acids in the amino acid sequence of SEQ ID NO. 2 and having an activity to activate guanylate cyclase B.

2. Use according to claim 1, wherein the 1 to 5 amino acids are 1 to 3 amino acids.

3. The use of claim 1, wherein the arthritis is osteoarthritis.

4. The use of claim 3, wherein the osteoarthritis is of a weight-bearing or non-weight-bearing joint.

5. The use of claim 4, wherein the osteoarthritis is knee degenerative disease.

6. The use of claim 4, wherein the osteoarthritis is a degenerative hip disease.

7. The use of claim 4, wherein the osteoarthritis is temporomandibular arthritis.

8. The use of claim 1, wherein the arthritis is caused by rheumatoid arthritis.

9. The use of claim 1, wherein the arthritis is caused by osteoarthritis.

10. The use of claim 1, wherein the therapeutic or prophylactic agent further comprises indomethacin.

11. The application of guanylate cyclase B activator in preparing articular cartilage cell growth promoter,

wherein the derivative is selected from the group consisting of a guanylate cyclase B activating peptide having 1 to 5 amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO. 1 and having an activating guanylate cyclase B activity and a guanylate cyclase B activating peptide having 1 to 5 amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO. 2 and having an activating guanylate cyclase B activity.

12. The use of claim 11, wherein 1 to 5 amino acids are 1 to 3 amino acids.

13. Use according to claim 11, wherein the accelerating agent further comprises indomethacin.

14. Use of a guanylate cyclase B activator for the preparation of a therapeutic or prophylactic agent for rheumatoid arthritis,

15. The use of claim 14, wherein 1 to 5 amino acids are 1 to 3 amino acids.

16. The use of claim 14, wherein the therapeutic or prophylactic agent further comprises indomethacin.