NZ624870B2 - Therapeutic agent for arthrosis - Google Patents

Abstract

therapeutic agent comprising a compound of formula I for treatment of joint diseases, such as arthrosis, which has an activity of preventing the destruction of articular cartilages and has a high therapeutic effect. According to the present disclosure, a therapeutic agent for joint diseases can be provided, which comprises cyclic phosphatidic acid or carbacyclic phosphatidic acid as an active ingredient. provided, which comprises cyclic phosphatidic acid or carbacyclic phosphatidic acid as an active ingredient.

Description

DESCRIPTION Title ofInvention: THERAPEUTICAGENTFORARTHROSIS Technical Field The present invention relates to a therapeutic agent for sis comprising, as an active ingredient, cyclic phosphatidic acid or carbacyclic phosphatidic acid.

BackgroundArt Arthrosis es include, for example, osteoarthritis, articular rheumatism, and rheumatic fever, and the number of patients with osteoarthritis and articular tism is particularly large.

Accordingly, osteoarthritis and articular tism are considered to be major arthrosis subtypes.

Osteoarthritis is classified as congenital or secondary osteoarthritis or primary osteoarthritis caused by articular cartilage deformation resulting fiom aging. The number of patients with primary osteoarthritis is increasing as the elderly population increases. There are significant differences between osteoarthritis and lar rheumatism in terms of causes of diseases and clinical conditions, although these es have the following in common: Joint functions are impaired as a result of articular cartilage destruction at the end. To date, anti—inflammatory analgesic agents, such as n and indomethacin, have been used as therapeutic agents for tic diseases, such as osteoarthritis. r, such anti—inflammatory analgesic agents do not exert any inhibitory effects on articular cartilage ction. In addition, the inhibitory effects of therapeutic agents such as gold preparations, immunosuppressive , and steroid preparations on articular cartilage destruction have not yet been confirmed in clinical settings.

Articular cartilage is composed of chondrocytes and cartilage es. Cartilage matrices have a complicated three—dimensional structure formed by collagens, which are fibrous proteins produced by chondrocytes, and proteoglycans (protein—polysaccharide complexes) bound to hyaluronic acids. Normal joint functions are maintained with the retention of a large quantity of water in the cartilage matrices.

In addition to the eutic agents for osteoarthritis described above, intraarticular injection of hyaluronic acid, which has been proven to have effects of protection and repair of articular cartilage and effects of lubrication in joints, has been employed in clinical settings.

However, such que is invasive and thus is not satisfactory from the viewpoint of patients’ QOL. y of the Invention Object to Be ed by the Invention An object of the present invention is to provide a therapeutic agent for arthrosis which exerts inhibitory effects on articular cartilage destruction and high therapeutic effects on arthrosis, or at least provides a useful alternative to known treatments of arthrosis.

Means for Attaining the Object The present inventors considered that articular cartilage destruction may be inhibited by rating hyaluronic acid production in articular chondrocytes, and that it may function as an effective therapeutic means for osteoarthritis. They have conducted concentrated studies and, as a consequence, ered that cyclic phosphatidic acids and derivatives thereof would accelerate hyaluronic acid production in chondrocytes derived from patients with osteoarthritis at significant levels, and that such effects would be observed in animal models of osteoarthrosis. This has led to the completion of the present ion.

Thus, the present invention provides a therapeutic agent for sis which ses, as an active ingredient, a compound represented by formula (I): [Formula 1] wherein R represents a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkynyl group having 2 to 30 carbon atoms, which may contain a cycloalkane or aromatic ring; X and Y each ndently represent an oxygen atom or a ene group, provide that X and Y do not simultaneously represent a methylene group; and M represents a hydrogen atom or an alkali metal atom. [00 1 0] Preferably, in Formula (I), X and Y ent an oxygen atom.

Preferably, in Formula (I), either X or Y ents an oxygen atom and the other represents a methylene group.

Preferably, the compound represented by Formula (I) is carbacyclic phosphatidic acid of 1-oleoyl—cyclic atidic acid, 1—palmitoleoy1—cyclic phosphatidic acid, or a derivative thereof. [001 1] The present invention fiirther provides a method for treatment of arthrosis comprising administering a compound represented by the aforementioned Formula (I) to a t with arthrosis.

The present invention further provides use of a compound represented by the aforementioned Formula (I) for production of a therapeutic agent for arthrosis.

Effects ofthe ion [00 1 3] The present invention can provide a therapeutic agent for arthrosis which inhibits lar cartilage destruction and has high therapeutic effects on arthrosis.

Brief Description ofthe Drawings [Fig.1] Fig. 1 shows the results of examination of effects of a cyclic phosphatidic acid derivative on expression of the hyaluronic acid synthase gene (HAS1) in chondrocytes derived from a patient with rthritis.

[Fig.2] Fig. 2 shows the results of examination of effects of a cyclic phosphatidic acid derivative on sion of the hyaluronic acid synthase gene (HASZ) in chondrocytes derived from a patient with osteoarthritis.

[Fig.3] Fig. 3 shows the results of examination of effects of a cyclic phosphatidic acid derivative on expression of the hyaluronic acid synthase gene (HAS3) in chondrocytes derived from a patient with osteoarthritis.

] Fig. 4 shows the results of examination of effects of a cyclic phosphatidic acid derivative on expression of the hyaluronidase gene (HYAL1) in chondrocytes derived from a patient with osteoarthritis.

[Fig.5] Fig. 5 shows the results of examination of efiects of a cyclic phosphatidic acid derivative on expression of the onidase gene (HYALZ) in chondrocytes derived fiom a patient with osteoarthritis. - [Fig.6] Fig. 6 shows the results of examination of effects of a cyclic phosphatidic acid derivative on hyaluronic acid tion in chondrocytes derived from a patient with osteoarthritis.

[Fig.7] Fig. 7 shows the results of examination of effects of a cyclic phosphatidic acid derivative (test compound: ScPA) on expression of the onic acid se genes (HASl, HAS2, and HAS3) in ocytes derived from a patient with osteoarthritis.

] Fig. 8 shows the results of examination of efiects of a cyclic phosphatidic acid derivative on expression of the hyaluronidase genes (HYAL1 and HYALZ) in synoviocytes derived from a patient with osteoarthritis.

[Fig.9] Fig. 9 shows changes in pain assessment (weight distribution across both hind limbs).

Mean d: standard error, n = 6; in comparison with the group 1 (the e administered group) *,**; significance levels at p < 0.05 and p < 0.01 (the Student’s t test and the Aspin—Welch t test).

[Fig.10] Fig. 10 shows the results of swelling assessment (articular swelling of both hind limbs).

Mean :t standard error, n = 6; in ison with the group 1 (the vehicle administered group) *,**; significance levels at p < 0.05 and p < 0.01 (the Student’s t test and the Aspin-Welch t test).

[Fig.ll] Fig. 11 shows the histopathological scores of the e of the femur and those of the tibia.

[Fig.12] Fig. 12 shows histopathological images of representative examples. Image A shows the vehicle administered group (Animal No. 101), in which disorganization of chondrocytes (indicated by an arrow) and cluster formation (indicated by an arrow) are observed in the joint cartilage (the medial aspect of the right femur, HE staining, magnification X200). Image B shows the test substance stered group (Animal No. 201), in which cartilage erosion is observed (slight) in the joint cartilage (the medial aspect ofthe right femur, HE staining, magnification X200). Image C shows the vehicle administered group (Animal No. 101), in which lowered proteoglycan stainability (slight) is observed in the joint cartilage (the medial aspect of the right femur, SO staining, magnification X200). Image D shows the test substance stered group (Animal No. 201), in which no change is observed in the joint cartilage (the medial aspect of the right femur, SO staining, magnification X200).

Embodiments for Carrying out the ion Hereafter, the present invention is described in greater detail.

The therapeutic agent for arthrosis ing to the present invention can be used for treatment of arthrosis, such as osteoarthritis, articular rheumatism, and rheumatic fever (and osteoarthritis, in particular). Such agent comprises, as an active ingredient, cyclic phosphatidic acid, carbacyclic phosphatidic acid, or a salt thereof. Any compound can be used as cyclic phosphatidic acid, carbacyclic phosphatidic acid, or a salt thereof Without particular tion, provided that such compound ts the effects of the present invention. Preferable examples include compounds ented by a (I) below: [00 1 6] [Formula 2] CH2.”(3H0R éH—x CHz-Y Ffom wherein R ents a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkynyl group having 2 to 30 carbon atoms, Which may contain a cycloalkane or aromatic ring; X and Y each independently represent an oxygen atom or a methylene group, provided that X and Y do not simultaneously ent a methylene group; and M represents a hydrogen atom or an alkali metal atom. [001 8] In Formula (I), specific examples of a linear or ed alkyl group having 1 to 30 carbon atoms represented by a substituent R include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, a undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, and an eicosyl group.

Specific examples of linear or branched alkenyl groups having 2 to 30 carbon atoms ented by a substituent R include an allyl group, a butenyl group, an octenyl group, a decenyl group, a dodecadienyl group, and a hexadecatrienyl group. More specific examples thereof include an 8-decenyl group, an 8—undecenyl group, an 8—dodecenyl group, an 8-tridecenyl group, an 8-tetradecenyl group, an 8—pentadecenyl group, an 8-hexadecenyl group, an adecenyl group, an 8—octadecenyl group, an 8—icocenyl group, an 8-docosenyl group, a heptadeca—8,11—dienyl group, a heptadeca—8,l 1,14-trienyl group, a nonadeca—4,7,10,l3-tetrenyl group, a nonadeca—4,7,10,13,16-pentenyl group, and a henicosa—3,6,9,12,15,l 8-hexenyl group.

Specific examples of linear or branched alkynyl groups having 2 to 30 carbon atoms represented by a substituent R include an 8-decynyl group, an cynyl group, an cynyl group, an 8—tridecynyl group, an 8—tetradecynyl group, an 8-pentadecynyl group, an 8-hexadecynyl group, an 8-heptadecynyl group, an 8—octadecynyl group, an 8-icocynyl group, an 8—dococynyl group, and a heptadeca—8, ll—diynyl group.

Specific es ofthe cycloalkane ring, which may be contained in the above—described alkyl, alkenyl, or alkynyl group, include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, and a cyclooctane ring. The lkane ring may contain one or more hetero atoms, and es thereof include an oxylane ring, an oxetane ring, a tetrahydrofiiran ring, and an N—methylprolidine ring.

Specific es of the aromatic ring, which may be contained in the above-described alkyl, l, or alkynyl group, include a benzene ring, a naphthalene ring, a pyridine ring, a furan ring, and a thiophene ring.

When the substituent R is an alkyl group substituted with a cycloalkane ring, accordingly, specific examples include a cyclopropyhnethyl group, a cyclohexylethyl group, and an 8,9-methanopentadecyl group.

When the substituent R is an alkyl group substituted with an aromatic ring, specific es include a benzyl group, a phenetyl group, and a p—pentylphenyloctyl group.

Preferably, R represents a linear or branched alkyl group having 9 to 17 carbon atoms, a linear or branched alkenyl group having 9 to 17 carbon atoms, or a linear or ed alkynyl group having 9 to 17 carbon atoms. More preferably, R represents a linear or ed alkyl group having 9, ll, 13, 15, or 17 carbon atoms or a linear or branched alkenyl group having 9, 11, 13, 15, or 17 carbon atoms. Particularly preferably, R represents a linear or branched alkenyl group having 9, 11, 13, 15, or 17 carbon atoms.

X and Y in the compound represented by Formula (I) each independently represent an oxygen atom () or a methylene group (-CH2-), provided that X and Y do not simultaneously represent a methylene group. That is, combinations of X and Y e the following three patterns: (1) X ents an oxygen atom and Y represents an oxygen atom; (2) X represents an oxygen atom and Y represents a methylene group; or (3) X represents a methylene group and Y represents an oxygen atom.

M in the cyclic phosphatidic acid derivative represented by Formula (1) represents a hydrogen atom or an alkali metal atom. Examples of alkali metal atoms include m, sodium and potassium, with sodium being particularly preferable.

Specifically, the compound represented by Formula (I) in the present invention is particularly preferably a cyclic phosphatidic acid or a carbacyclic phosphatidic acid derivative , as an acryl group at position 1, an oleoyl group in which the substituent R ents an alkenyl group having 17 carbon atoms (abbreviated as “C18:1”) or a palmitoleoyl group in which the tuent R represents an alkenyl group having 15 carbon atoms (abbreviated as “C16: 1”).

The compound represented by Formula (I) may comprise an isomer, such as a positional , geometric isomer, er, or optical isomer, in accordance with the type of a substituent thereof. All possible s and mixtures comprising two or more types of such s at a certain ratio are within the scope ofthe present invention.

In addition, the compound represented by Formula (I) may be in the form of an adduct composed of the compound and water or various types of solvents (hydrates or solvates). Such adduct is also within the scope of the present invention. Moreover, any crystal forms of the compound represented by Formula (I) and salts thereof are also within the scope of the present invention.

A compound represented by Formula (I) in which both X and Y represent oxygen atoms can be chemically synthesized in accordance with the method bed in, for example, JP Patent Publication (Kokai) No. HOS-230088 A (1993), H07—149772 A (1995), H07—258278 A (1995), or H09-25235 A .

Also, a compound represented by Formula (I) in which both X and Y ent oxygen atoms can be synthesized in accordance with the method described in JP Patent Publication (Kokai) No. 2001—178489 A by allowing phospholipase D to react with lysophospholipid.

Lysophospholipid used herein is not particularly d, so long as it is capable of reacting with phospholipase D. Many types of lysophospholipids are known, molecular species having different types of fatty acids or having ether or vinyl ether bonds are known, and such lysophospholipids are commercially available. As phospholipase D, those d from higher—order plants such as cabbage or peanuts or those derived from microorganisms such as Streptomyces chromofuscus or Actinomadula Sp, are commercially available as reagents, although a cyclic phosphatidic acid is synthesized by an enzyme derived fiom the madula Sp. No. 362 strain in a very selective manner (JP Patent Publication (Kokai) No. Hll~367032 A (1999)). The reaction between lysophospholipid and phospholipase D may be carried out under any conditions, so long as an enzyme is able to exert its activity. For example, the reaction can be carried out in an acetate buffer containing calcium chloride (pH: about 5 to 6) at room temperature or higher (preferably 37°C) for l to 5 hours. The resulting cyclic phosphatidic acid derivative can be purified in accordance with a conventional technique by means of, for example, extraction, column chromatography, or thin-layer chromatography (TLC).

A compound represented by Formula (I) in which X represents an oxygen atom and Y represents a methylene group can be synthesized in accordance with the method described in literature (Kobayashi, S. et al., Tetrahedron Letters, 34, 050, 1993) or .

An example ofa specific synthetic pathway is shown below.

[Formula 3] Bta‘ 015121 OBn mom-Awhile):‘? 0B“ PPTS HQ R , o /OMc CH2___P \ -> 1 THE OMe toluene 03“ RCO H ii 0%).)"0 Ham/C OH was“? eke“R Meow . , 0a» ’0 MP --—+ 0% «0 mo“ MeO MQOWP a 4 5 “wear 0—C WR O ”C “R N OH W0% ’0 Ma 0% 10:!“ PIC/P Mao/P 7 [003 5] In the above formulae, at the outset, commercially available (R)—benzyl glycidyl ether (1) is activated with the aid of BF3-Et20, n—BuLi is d to react with dimethyl phosphonate, and the resulting lithiated form is subjected to the on to obtain an alcohol (2).

The resulting alcohol is subjected to reaction in toluene with the use of an excessive amount of a pyridinium salt of p-toluenesulfonic acid at 80°C to obtain a cyclized form (3). The resulting ed form is hydrolyzed under a hydrogen atmosphere with the use of20% Pd(OH)2-C to perform debenzylation (4). l—Ethyl—3—(3—dimethylaminopropyl)carbodiimide hloride, as a ser, is allowed to react with a fatty acid to obtain a d form (5). uently, bromotrimethylsilane is used as a nucleophile to exclusively remove a methyl group in a position—selective , thereby obtaining a cyclic phosphonic acid (6). The resultant is introduced into a separatory funnel with the aid of ether, and a small amount of an aqueous solution of 0.02 N sodium hydroxide is added dropwise thereto to separate liquids. The compound of interest is extracted and purified as a sodium salt (7).

A compound represented by Formula (I) in which X represents a methylene group and Y represents an oxygen atom can be synthesized in accordance with the method described in JP Patent Publication (Kokai) No. 2004-010582 A or ational Publication W0 2003/1 04246.

The therapeutic agent for arthrosis according to the present invention is preferably provided in the form of a pharmaceutical composition that comprises one or more pharmaceutically acceptable additives and the nd represented by Formula (I) as an active ingredient.

The therapeutic agent for arthrosis according to the present invention can be administered in various forms, and administration may be carried out orally or parenterally (for example, intravenous, intramuscular, subcutaneous or intracutaneous injection, rectal administration, and permucosal administration may be ed). Examples of dosage forms for pharmaceutical itions suitable for oral stration include a tablet, a granule, a capsule, a powder, a solution, a suspension, and syrup. Examples of dosage forms for pharmaceutical itions suitable for parenteral administration include an injection, an infusion, a suppository, and a percutaneous absorption agent. The dosage form for the agent of the present invention is not limited thereto. Further, the agent can also be made into sustained-release formulations in accordance with methods known in the art.

Types of pharmaceutical additives used for producing the therapeutic agent for arthrosis according to the present invention are not particularly limited, and a person skilled in the art can select adequate additives. Examples of additives that can be used include an excipient, a egration agent or a disintegration auxiliary agent, a binder, a lubricant, a coating agent, a base, a dissolving agent or a solubilizer, a dispersant, a suspension agent, an fier, a bufier, an antioxidant, an antiseptic, an isotonic agent, a pH adjusting agent, a dissolving agent, and a stabilizer.

Each specific ient used for the above purposes is well known to a person skilled in the art.

Examples of pharmaceutical additives that can be used for the production of oral preparations include: an excipient, such as glucose, lactose, D—mannitol, starch, or crystalline cellulose; a disintegration agent or a disintegration ary agent, such as carboxymethyl cellulose, starch, or carboxymethyl cellulose m; a binder, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, or gelatin; a lubricant, such as magnesium stearate or talc; a g agent, such as hydroxypropyl methylcellulose, white sugar, polyethylene glycol, or um oxide; and a base, such as Vaseline, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, or hard fat.

Examples ofthe pharmaceutical additives that can be used for tion of an injection or an infusion preparation include: a dissolving agent or a lizer that can be used for an s ion or a use—time dissolution type injection, such as injection distilled water, physiological saline, ene glycol, or a surfactant; an isotonic agent, such as glucose, sodium chloride, D—mannitol, or glycerin; and a pH adjusting agent, such as an inorganic acid, an organic acid, an inorganic base, or an organic base.

The therapeutic agent for arthrosis according to the present invention can be administered to mammals, including humans.

A dose of the therapeutic agent for arthrosis according to the present invention should be increased or decreased in ance with conditions such as age, sex, body weight, symptoms of a patient, and the route of administration. The dose of the active ingredient per day per adult is generally 1 rig/kg to 1,000 mg/kg, and preferably 10 ug/kg to 100 mg/kg. The agent may be administered in the amounts mentioned above once a day or several separate times (for example, about 2—4 times) a day.

The present invention is described in greater detail with reference to the following examples, although the present invention is not limited to the examples.

Example 1 (1) Method (1-1) e of articular chondrocytes and synoviocytes Hyaline articular cartilage that was sampled fiom a patient with knee osteoarthrosis at the time of artificial knee joint ement was used. Articular cartilage pieces were washed and subjected to enzyme treatment with pronase and collagenase to decompose cartilage matrices.

Thereafter, cells were sampled, cultured, and then cryopreserved. Synoviocytes were also d.

In this example, such articular chondrocytes and synoviocytes obtained from a t with osteoarthritis were used, such cells were subjected to monolayer, high-density culture in order to avoid transformation, and the ed cells were then subjected to the experiment. A medium composed ofDMEM, 10% FBS, and 1% antibiotics/antifimgus was used, the medium was replaced with a serum-free medium upon reaching confluence, and the experiment was then initiated. (1-2) Addition of cyclic phosphatidic acid or carbacyclic phosphatidic acid (CPA) As cyclic atidic acid and carbacyclic phosphatidic acid, Cl6:l—cPA (CPA) and native cPA (NCPA) were used, respectively, and these substances were examined in terms of production of onic acid (HA), expression of hyaluronic acid (HA)—synthetic enzymes (HASl, HASZ, and HAS3), and hyaluronic acid (HA)~degrading s (HYALl and HYALZ) at concentrations of 0 to 50 nM for 0 to 48 hours.

The chemical structure of Cl6zl-cPA (indicated as “cP ” in the figure) is as shown below.

[Formula 4] ICH2”O"C“ C15 H29 CH—CH2 o l )P¢ CHz—O \oNa Native CPA (indicated as “NcPA” in the figure) was prepared in the manner described below (see Examples 1 and 3 ofJP Patent Application No. 2011-126901).

Soybean phospholipid (10 g; in content: 70%) was dissolved in 100 ml of 1 M acetate buffer (pH 6.5) containing 0.3 M calcium chloride, 6,000 units of Streptomyces—derived phospholipase A2 were added, and the e was subjected to reaction at 40°C for 18 hours with stirring. The pH level ofthe reaction solution was ed to 2.5 to inactivate the enzyme, 100 ml of chloroform and 50 ml of methanol were added and thoroughly mixed via stirring, and lipid components were extracted. The chloroform layer was sampled and solidified to dryness under d pressure in a rotary evaporator. Acetone (100 ml) was added to the solid component to precipitate phospholipids, and fiee fatty acids were removed. The precipitate (5 g) was dissolved in 40 ml of chloroform, 10 ml of 1 M e bufi‘er (pH 5.5) was added thereto, 1,500 units of Acfinomadura—derived phospholipase D were further added, and the mixture was subjected to reaction at 40°C for 18 hours with ng. To the reaction solution, 20 ml of 3 M sodium chloride and 20 ml of 0.1 M EDTA solution were added, and the ant was subjected to stirring at 40°C for 1 hour. r, 20 ml of methanol was added thereto, the mixture was thoroughly stirred, and the resultant was fuged at 3,000 rpm for 5 minutes to collect the chloroform layer. The solution was fied to dryness under reduced pressure in a rotary evaporator and 3.8 g of sodium salt of cyclic phosphatidic acid was obtained. The yield was 54.3% since 3.8 g of cyclic phosphatidic acid Na was obtained from soybean phospholipid with lecithin t of70% (i.e., 7 g of lecithin in 10 g of soybean phospholipid). Purity of a sodium salt of cyclic phosphatidic acid was analyzed using a silica gel plate, the sample was spread on the plate with chloroformzmethanolzacetic acid:5% sodium disulfite (100:40:12:5, V/V), and the plate was soaked in a mixture of 5% copper acetate, 8% phosphoric acid, and 2% sulfuric acid for a short period of time. The plate was air dried and heated at 180°C for about 10 minutes, and the formed spots were inspected using a scanner (manufactured by ATTO Corporation). Specifically, a reference product (purity: 97%) was used as a control , and spots in the thin-layer tograph were examined using a densitometer, followed by quantification based on the area ratio. The purity of the sodium salt of cyclic phosphatidic acid in the product obtained in the above step was 54%.

The sodium salt of cyclic phosphatidic acid (500 mg) was dissolved in 5 ml of form containing 10% methanol, applied to a silica gel column, spread with the aid ofthe solvent described above, further spread with the aid of form containing 20% methanol, and fractionated to fiactions of 10 ml each. Fractions containing sodium salt of cyclic atidic acid were collected in accordance with the TLC method described above and solidified to dryness under d pressure in a rotary evaporator. Thus, 320 mg of sodium salt of cyclic atidic acid powder was obtained. The purity of the sodium salt of cyclic phosphatidic acid in the sample was 95%. (1 —3) Measurement ofHA-synthetic enzyme expression and HA-degrading enzyme expression A ocyte culture was replaced with a serum—flee medium 24 hours before the initiation ofthe experiment, and cPA or N—cPA at various trations (0, 5, 10, 25, or 50 M) was added. Also, a periosteal cell culture was replaced with a serum—free medium 24 hours before the initiation of the experiment, and CPA at various trations (0, 10, or 25 M was added. Total RNA was isolated from the cell culture 0, 0.5, l, 2, and 4 hours later, cDNA was synthesized, and HASl, HAS2, HAS3, HYLl, and HYLZ expression levels were quantified by real—time PCR. The expression level was determined relative to the B actin gene (i.e., the control gene) and it was represented relative to the control value without the addition of CPA or N—cPA or the value before addition thereof, which was normalized to 1.

Specifically, the expression ratio was determined by the AACt method comprising comparing the differences in cycle number threshold (Ct value) obtained for the target and the control in a sample with the CT—value obtained for a control sample, as described below. 1) ACt is determined using Ct obtained for a relevant sample: ACt = Ct (target gene) — Ct (control gene) 2) AACt is determined: AACt = ACt (target sample) — ACt (control sample) 3) Target gene expression level in the target sample is normalized: 2(-AACt) 4) Changes in target gene expression levels are ined with reference to the control sample value ized to l. (1—4) Measurement ofhyaluronic acid (HA) production A chondrocyte e was replaced with a serum—free medium 48 hours before the initiation of the experiment, and CPA and N-cPA at various concentrations (0, 10, or 50 uM) was added. A e supernatant was sampled 0, 6, 12, 24, and 48 hours later. By the sandwich ELISA method involving the use of HA—binding ns d from bovine nasal age (QnE Hyaluronic Acid (HA) ELISA Assay kit; Biotech Trading Partners, Inc), HA production was quantified. (2) Results (2—1) Results of expression assay ofHA-synthetic enzymes and HA—degrading enzymes Fig. 1 to Fig. 5 each show the results of expression assay of HA-synthetic enzymes and HA—degrading s in chondrocytes. As shown in Fig. 1 to Fig. 5, C16:l—cPA (cPA) continuously induced HAS2 expression in a concentration—dependent manner. While HASl and HAS3 expression was transiently d 2 hours after the addition, the expression level was lowered 4 hours later. NcPAproduced r results. The expression ofHA—degrading enzymes (HYLl, HYL2, and HYL3) was not influenced. Such results demonstrate that CPA and NcPA induce expression of HA—synthetic enzymes. Also, Fig. 7 shows the results of expression assay of HA—synthetic enzymes in synoviocytes and Fig. 8 shows the results of expression assay of HA—degrading enzyme in synoviocytes. (2-2) Results ofmeasurement ofhyaluronic acid (HA) production Fig. 6 shows the results of measurement of HA production. As shown in Fig. 6, C16:l-cPA (CPA) accelerated the HA synthesis in chondrocytes with the elapse oftime and released HA to the outside of the cells. Forty eight hours later, the amount of HA prodUced in the group to which cPA had been added at 50 nM was approximately 3 times greater than that produced in the the vehicle administered group. NcPA produced similar results. Such results demonstrate that C16:1-cPA (CPA) and NcPA accelerate HA production in lar chondrocytes with osteoarthritis (0A). (3) Conclusions The cyclic phosphatidic acid or yclic phosphatidic acid represented by Formula (I) was found to induce expression of HA—synthetic enzymes and to accelerate production of HA in articular ocytes ofhumans with CA, as with the case al fibroblasts.

Example 2: Evaluation of eflects of ScPA on osteoarthritis in rabbit knee (1) Method (1 -1) Animals used and rearing conditions Twelve 11— to lZ—week—old male s (KbszNZW) were used. Rabbits were reared in separate cages (one rabbit per cage) at 144°C to 249°C under light (12 hours from 7:00 am to 7:00 pm) with continuous ventilation. Rabbits were allowed to eat 150 g of feed (CR—3, CLEA Japan, Inc.) per day and to drink tap water. (1—2) Substances to be administered (9Z)—9-Octadecenoic acid—(2—hydroxy—2—oxide—l,2-oxaphospholan—4-yl)methyl ester sodium salt (Cl 8:1-cPA; hereafter, referred to as “‘ScPA”) was used.

[Formula 5] (IDHg-O-C— C17 H33 CH“CH2\P¢O/ CH2— 0 \O Na Physiological saline was used as a vehicle. (1-3) Preparation of osteoarthritis model Hair in the vicinity of the knee joint of the right hind limb of a rabbit was shaved using electrical clippers under deep anesthesia, and the shaved area was ected with Isodine. The outer coat on the medial aspect ofthe right hind limb was incised with a al knife, the ry n the medial aspect and the articular capsule of the patellofemoral ligament was further incised, and the medial patellofemoral ligament was then ted. Thereafter, the articular capsule was spread wide open to expose the medial meniscus, which was then completely removed.

Following the removal of the meniscus, tissue and epidermis in the vicinity of the articular capsule were sutured. At the time of suturing, the site of operation was washed with physiological saline (titer: 500 mg/20 ml) containing antibiotics (Viccillin, Meiji Seika Pharma Co., Ltd).

On the day of surgical treatment (day 0), rabbits were allowed to freely drink water under fasting conditions. Awakening was confirmed upon observation of spontaneous movement of heads. In order to t the animals from losing body temperature, the animals were kept warm by wrapping their trunks with towels until awakening had been confirmed. Also, body positions were adequately changed in order to t blood from pooling. For the purpose of infection control, an antibiotic (Viccillin: 3 units/kg) was intramuscularly administered once a day up to 5 days after the treatment (day 5).

After the model animals were ed, they were d into two groups each consisting of 6 individuals while averaging body temperature (i.e., the vehicle stered group of Animal Numbers 101 to 106 and the test substance (ScPA) administered group of Animal Numbers 201 to 206). (1 -4) Administration oftest compound and vehicle into joint cavity The test compound and the vehicle were administered in the manner described below.

Route of administration: into the joint cavity Site of administration: right hind limb (treated limb) Timing ofadministration: on days 7, 11, 14, 18, 21, 25, 28, 32, 35, and 39 Dosage: Test compound: 10 rig/rabbit (volume: 0.2 ml) Vehicle : 0.2 ml/rabbit Means ofadministration: with the use of a 1.0—ml syringe (Terumo Corporation) and a 27G injection needle (Terumo Corporation) (1-5) Pain assessment t distribution across both hind limbs) Frequency ofmeasurement: Measurement was carried out seven times in total: i.e., before treatment and l, 2, 3, 4, 5, and 6 weeks after the ent.

Method ofmeasurement: Body weights loaded on the right hind limb and the left hind limb were separately measured using a weight scale, and the weight distribution on the treated limb (the right hind limb) was determined using the following equation.

Weight distribution (%) on treated limb (right hind limb) = [right hind limb (kg) / (right hind limb (kg) + left hind limb (kg))] x 100 (1—6) Swelling ment (articular swelling ofboth hind limbs) Frequency ofmeasurement: Measurement was carried out 6 weeks after the treatment.

Method ofmeasurement: The Widest areas at the joints of the right and lefl hind limbs were measured using digital calipers, and the lar swelling induced by osteoarthritis was determined using the following equation.

Swelling (%) of treated limb (right hind limb) = [(right hind limb (mm) - left hind limb (mm)) / (left hind limb (mm) + left hind limb (mm))] X 100 (l—7) Sampling ofbiomaterials (day 42) and post-sampling treatment Four limbs were dissected from the animals under deep anesthesia, g them to bleed to death. Thereafter, the femur condyle and the tibial condyle were removed fiom the knee joint of the d limb (the right hind limb), followed by fixation in a 10% neutral buffered formalin solution. (1-8) Preparation ofpathological specimens and histopathological tion thereof The femur and the tibia that had been soaked and fixed in a 10% neutral buffered formalin solution were subjected to demineralization with EDTA. After the completion of demineralization, the d sites identified below were embedded in paraffin in accordance with a conventional technique, and the resultant was sliced to a thickness of 4 mm each. The slices were subjected to hematoxylin—eosin (HE) staining and safranin O (proteoglycan) staining and histopathologically examined under an optical cope (BXSlTF; OLYMPUS). The degree of cartilage ration of the pathological specimens was evaluated in accordance with the ia shown in Table 1 below (Kikuchi, T, Yoneda, H. et al., rthritis cartilage, 4; p. 99 and continuing pages, 1996). Specifically, the specimens were evaluated in terms of loss of the following 8 items according to a five—grade evaluation (0 to +4): superficial layer; cartilage erosion; fibrillation and/or fissure; lowered proteoglycan stainability (safranin O stainability); disorganization of chondrocytes; loss of chondrocytes; exposure of subchondral bone; and cluster ion. The sum total score of all items was defined as the overall score. Observation items without specific ia defined in Table 1 were evaluated in accordance with the criteria shown in Table 2 below (Naoki ro et al., l of y, 29: p. 112 and continuing pages, 2010).

[Tabkal] Observation/ Score 1 2 3 4 Loss of superficial layer <sigl_1t Moderate Focally severe ive severe Erosion of cartilage <Detectable Moderate Focally severe Extensive severe <Noticeable Moderate Marked (2 small or ive (3 small, Fibrillation and/or fissures g<1very mall} g1 small) 1 medium) 2 medium or 1 <Paler stain Moderate loss of Marked loss of Total loss of L°SS “mew-yea“ than control safraniophilia safraniophilia safraniophilia Disorgam'zatlon of chondrothe Noticeable Moderate, with Marked loss of No recom’able <Noticeable Moderate decrease Marked decrease in Very extensive Loss of chondrocyte , . .

Decrease in cells in cells cells decrease in cells <Foca1 Moderate Fairly extensive Very extensive Exposure of subchondral bone Exgosure ofbone Exposure ofbone exposure ofbone exgosure ofbone 953mg mmm‘gn ‘9 <3-4 small] or 1-2 5—6 smallI 3—4 7 or more small: 5- 7 or more medium a) Small: 2-4 cells, Medium: 5—8 cells, Large: 9 or more cells [Table 2] Observation Score Criteria 0 No change Lesions limited to the superficial to intermediate layers are observed throughout 1/3 or less of the area of the evaluation site in the width direction Lesions limited to the superficial to intermediate layers are observed throughout 1/3 to 2/3 of the area or lesions Cartilage erosion reaching the deep layer are observed throughout 1/3 or less ofthe area ofthe evaluation site in the width direction Lesions limited to the superficial to intermediate layers are observed throughout 2/3 or more of the area or lesions reaching the deep layer are observed throughout 1/3 to 2/3 ofthe area Lesions limited to the superficial to intermediate layers are observed throughout the entire area or lesions reaching the deep layer are observed throughout 2/3 or more ofthe area t—‘O No change Lesions limited to the superficial to intermediate layers are observed throughout 1/3 or less of the area of the tion site in the width direction N Lesions limited to the superficial to intermediate layers are observed throughout 1/3 to 2/3 of the area or lesions Fibrillation/fissure ng the deep layer are observed throughout 1/3 or less ofthe area ofthe evaluation site in the width ion m Lesions limited to the cial to intermediate layers are observed throughout 2/3 or more of the area or lesions ng the deep layer are observed throughout 1/3 to 2/3 ofthe area 4:. Lesions limited to the superficial to intermediate layers are observed throughout the entire area or s reaching the deep layer are ed hout 2/3 or more ofthe area No change Lesions account for 1/4 or less ofthe area ofthe evaluation site Loss ofsuperficial Lesions account for 1/4 to 2/4 ofthe area ofthe evaluation site layer Lesions account for 2/4 to 3/4 ofthe area ofthe evaluation site Lesions account for 3/4 or more ofthe area ofthe evaluation site No change Lowered Lesions account for 1/4 or less ofthe area ofthe evaluation site proteoglycan Lesions t for 1/4 to 2/4 ofthe area ofthe evaluation site stainability Ioath—‘OI-waIt—IOJBUJNHO Lesions account for 2/4 to 3/4 ofthe area ofthe evaluation site Lesions account for 3/4 or more ofthe area ofthe evaluation site No change Lesions t for 1/4 or less ofthe area ofthe evaluation site Disorganization of s account for 1/4 to 2/4 ofthe area ofthe evaluation site chondrocytes Lesions account for 2/4 to 3/4 ofthe area ofthe evaluation site Lesions account for 3/4 or more ofthe area ofthe evaluation site No change Lesions account for 1/8 or less ofthe area ofthe evaluation site Loss ofchondrocytes Lesions account for 1/8 to 1/3 ofthe area ofthe evaluation site JAUJNt—‘O-PWNb-‘O-bw Lesions account for 1/3 to 2/3 ofthe area ofthe tion site I Lesions account for 2/3 or more ofthe area ofthe evaluation site I No change I s account for 1/8 or less ofthe area ofthe tion site re of Lesions account for 1/8 to 1/3 ofthe area ofthe evaluation site subchondral bone Lesions account for U3 to 2/3 ofthe area ofthe evaluation site Lesions account for 2/3 or more ofthe area ofthe evaluation site No change Lesions account for 1/8 or less ofthe area ofthe evaluation site r formation Lesions account for 1/8 to 1/3 ofthe area ofthe evaluation site Lesions t for 1/3 to 2/3 ofthe area ofthe evaluation site Lesions account for 2/3 or more ofthe area ofthe evaluation site (1—9) Data processing and statistical analysis A group mean (mean) and its standard error (SE) for the weight distribution across both hind limbs and for articular swelling of both hind limbs were separately determined. Thereaiter, the test nce administered group and the e administered group were subjected to the . When there was no variance between the samples, the Student’s t—test was carried out.

When there was variance between the s, the Aspin—Welch t—test was carried out. Concerning the overall scores ofthe evaluation items ofthe histopathological test, the group mean (mean) and its standard error (SE) were determined, and the difierence between the mean values for the two groups was then determined by the Mann-Whitney U test. The two-sided significance levels were set at % and 1%. (2) Results (2-1) Pain assessment (weight distribution across both hind limbs) Table 3 and Fig. 9 show changes in weight distribution across both hind limbs fiom the day on which model animals were prepared (day 0) to the day on which biomaterials were sampled (day 42). In the e administered group, recovery fiom surgical invasion was observed up to day 14, osteoarthritis was induced fter, the weight loaded on the treated limb (the right hind limb) was decreased because of the pain caused by osteoarthritis, and such weight was decreased to as low as 28.0% on the day on which biomaterials were sampled (day 42). In the test substance administered the vehicle group, in contrast, recovery from surgical invasion was observed up to day 14, as with administered group, and, thereafter, the weight loaded on the treated limb (the right hind limb) was maintained at higher levels up to the day on which biomaterials were sampled (day 42), compared with the vehicle stered group. A cant difference was observed as a result of measurement on day 42 (p = 0.0053).

[Table 3] Table 3: Pain assessment (weight distribution across both hind limbs) 'Gmu p, Am No-—_fl——__' M , ”WET-mm” 101 “MW-5m- ' “WEN—M“ mwmmm—m 103 670 740 Venice mmmmm administer 104 wwmmm—m edgroup wswmm “mam—“— Fmiaauinaai mm— 105MM mum-m- —‘IE_——W 106 WWW“ smai —-m_-ummm§- ——so.3"32.1m_39.4“I‘m—sac "_-4.60—3.4—2.87 3.14 5.07 “$- —Pis?rih&mi§mb{si4-19.3-"m3"" .fl-Ifi-H' Ci. 201 510mm _m—————m mm,410W __m__“-§§-“ —w-nflum- “ 540 _m_— WEE“ 7 "Emma-film“ 204 —um--_——mm Wflnm mm380 700 Mackinaw “mm-mu _m-—-——mm 310 750 mum—u Wigwam “ls-“Wm- 0-6486 0-9960“M“ (2-2) Swelling assessment (articular swelling ofboth hind limbs) Table 4 and Fig. 10 show the percentage values for articular swelling ofboth hind limbs on the day on which biomaterials were d (day 42). The width of the joint of an individual animal was measured, and the variance between groups was determined. As a result, articular swelling was inhibited more significantly in the test substance stered group, compared With the vehicle administered group (p = 0.0164).

[Table 4] Table 4: Swelling assessment (articular swelling of both hind limbs) s ”111913! 7 24‘ '55 terms first: mm 23, 98 n...—enemas n.“— n...“ “——,7 “-I- 201 mm 203 —m 206 IaumuMflflhllllflnflEI -0. ‘__ i mil.” “5?" (2-3) Histopathological evaluation Tables 5 and 6 and Fig. 11 each Show a summary ofthe results ofhistopathological tests, and Fig. 12 shows histopathological images esentative examples.

In both the vehicle administered group and the test substance administered group, substantially no damages resulting from the ation of osteoarthritis models were observed in lateral condyles ofthe femur or in those ofthe tibia.

Concerning the medial e of the femur, loss of articular cartilage superficial layer, lowered proteoglycan stainability, disorganization of chondrocytes, loss of chondrocytes, and cluster formation were observed in all 6 s of the vehicle administered group, and age erosion was observed in 5 of the 6 samples thereof. The degree of cluster formation was ularly high in all samples (mean = 2.50 i: 0.43). The histopathological scores of the observation items were 9, 21, 17, 11, 8, and 20 (mean = 14.33 :t 2.33), and slight to severe changes were observed in the articular cartilage of all samples. Concerning the medial condyle of the femur, cluster formation was observed in all 6 samples of the test substance administered group, and loss of cartilage superficial layer, cartilage erosion, and disorganization of chondrocytes were observed in 5 of the 6 samples thereof. The athological scores were 5, 6, 10, 3, 20, and 6 (mean = 8.33 i 2.51), and such s observed in 4 of the 6 samples were lesser than those observed in the vehicle administered group in the same regions. That is, the histopathological scores of the test substance administered group were lower than those ofthe vehicle administered group (p = 0.0649).

Concerning the medial condyle of the tibia, loss of articular cartilage cial layer, cartilage fibrillation and/or fissure, and disorganization of chondrocytes were observed in all 6 samples of the vehicle administered group, and lowered proteoglycan stainability was ed in 5 ofthe 6 samples thereof. The histopathological scores were 8, 27, 14, 5, 3, and 18 (mean = 12.50 i 3 .69), and slight to severe changes in the articular cartilage were observed in 4 ofthe 6 samples. In the test substance stered group, cartilage fibrillation and/or fissure was observed in all 6 samples, and loss of cartilage cial layer and lowered proteoglycan stainability were observed in 5 of the 6 samples. The histopathological scores were 4, 16, 6, 1, 27, and 9 (mean = 10.50 2‘: 3.91).

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Claims (5)

1. [Claim 1] Use a compound represented by formula (I): wherein R represents a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear or branched alkynyl group having 2 to 30 carbon atoms, which may contain a cycloalkane or aromatic ring; X and Y each ndently represent an oxygen atom or a ene group, provide that X and Y do not aneously represent a methylene group; and M represents a hydrogen atom or an alkali metal atom, in the manufacture of a medicament for the treatment of arthrosis.
2. [Claim 2] The use ing to claim 1, wherein, in Formula (I), X and Y represent an oxygen atom.
3. [Claim 3] The use according to claim 1 or 2, wherein, in Formula (I), either X or Y represents an oxygen atom and the other represents a ene group.
4. [Claim 4] The use according to any one of claims 1 to 3, wherein the compound represented by Formula (I) is carbacyclic phosphatidic acid of 1-oleoyl-cyclic phosphatidic acid, 1-palmitoleoyl-cyclic phosphatidic acid, or a derivative thereof.
5. [Claim 5] The use according to claim 1, substantially as herein described with reference to any one of the Examples and/or