JP2007326824A - Synoviocyte-growth inhibitor, pharmaceutical composition and therapy using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a synoviocyte-growth inhibitor which can inhibit growth of a synoviocyte without induction of apoptosis and immunosuppression, and a pharmaceutical composition and a therapy which can treat and prevent diseases involving growth of the synoviocyte. <P>SOLUTION: The synoviocyte-growth inhibitor comprises a compound represented by structural formula (A) or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition comprises the synoviocyte-growth inhibitor and the therapy adopts the synoviocyte-growth inhibitor. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a synovial cell proliferation inhibitor containing as an active ingredient at least one of a compound represented by the structural formula (A) described below and a pharmaceutically acceptable salt thereof, and the synovial cell proliferation inhibitor And a therapeutic method using the synovial cell proliferation inhibitor.

The synovial membrane is originally composed of a single layer of synovial cells, but the synovial cells may proliferate excessively due to some inflammatory mechanism, resulting in synovial hyperplasia.
Synovial cells are activated in the increased synovium, and various inflammatory mediators and tissue destructive proteolytic enzymes are produced from the synovial cells. This series of inflammatory reactions is referred to as proliferative synovitis, and the proliferative synovitis results in serious dysfunction such as cartilage degeneration and bone tissue destruction. Examples of diseases in which such synovial cell proliferation is involved in the disease state include rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout.

  Rheumatoid arthritis is an autoimmune disease, and inflammatory mediators (eg, IL-1, IL-6, TNF-α, etc.) from lymphocytes and the like are excessively present in the vicinity of joints, resulting in tissue inflammation. It is a disease where bone destruction progresses and pathology appears. One of the etiologies is the proliferation of the synovial cells, but it is thought that there are multiple etiologies, so a multifaceted treatment method is required. Development is desired.

Conventionally, as a treatment method for rheumatoid arthritis, symptomatic treatment by the administration of analgesics and anti-inflammatory agents has been carried out. However, symptomatic treatment can delay the onset, but stops the progression of symptoms. Furthermore, it was impossible to cure the disease itself.
On the other hand, in recent years, based on the fact that some of the drugs originally used as anticancer agents are also effective as lymphocyte proliferation inhibitors, by administering them, anti-TNF-α antibodies are further used in combination. It has been reported that the treatment improves the inflammation, sometimes stops the progression of the disease, and increases the improvement rate and cure rate of the disease.
However, the administration of these drugs has a high therapeutic effect, but also has a high immunosuppressive effect due to suppression of lymphocyte proliferation, so that the administered patient is always in an immunosuppressed state and is extremely vulnerable to infection. is there.

  In addition to the anticancer drug and anti-TNF-α antibody, there are a plurality of drugs for treating rheumatoid arthritis by suppressing inflammation. For example, even if it is very effective for a specific patient, it is effective for other patients. There are many drugs that are low, and a drug that is highly versatile and has a complete healing effect is not yet known.

On the other hand, as one of the therapeutic agents for rheumatoid arthritis, a drug (synovial cell growth inhibitor) that suppresses the proliferation of the synovial cells, which is one of its etiology, has been proposed.
Examples of the synovial cell growth inhibitor include diterpene compounds (see Patent Document 1) and FADD genes (see Patent Document 2) that suppress synovial cell proliferation by inducing apoptosis of synovial cells. , Fas ligand (see Patent Document 3), anti-Fas antibody (see Patent Document 4), 3,6-anhydrogalactose and its derivatives (see Patent Document 5), etc. As what suppresses cell growth, an imidazole derivative (see Patent Document 6), a quinoline derivative, a quinolone derivative (see Patent Document 7), and the like have been proposed. In addition to these, as the synovial cell proliferation inhibitor, an association product of hyaluronic acid and zinc (see Patent Document 8), a quinolonecarboxylic acid derivative (see Patent Document 9), and a substance in milk whey protein (Patent Document 10) Reference), bFGF antagonist (see patent document 11), IL-6 antagonist (see patent document 12), cAMP derivative (see patent document 13), vitamin D3 and its derivative (see patent document 14), c-erbB-2 activity Inhibitors (see Patent Document 15), γ-secretase-like protease inhibitors (Patent Document 16), and the like have been proposed.

These synovial cell proliferation inhibitors are not only for rheumatoid arthritis but also for preventing the onset, reducing the symptoms, and increasing the severity of diseases involving the proliferation of synovial cells such as osteoarthritis, psoriatic arthritis, and gout. Expected to be a useful substance for suppression.
However, without inducing apoptosis, that is, a synovial cell proliferation inhibitor that can more safely suppress the proliferation of synoviocytes, and without causing immunosuppression, The present condition is that the pharmaceutical composition and the treatment method which can treat or prevent the disease in which synovial cell proliferation is involved without reducing resistance have not yet been found.

JP 2004-168713 A JP 2000-198737 A Japanese Patent Laid-Open No. 11-060501 JP-A-08-208515 WO99 / 064424 JP 2003-040888 A Japanese Patent Laid-Open No. 2002-371078 JP 2003-089647 A JP 2002-293745 A JP 2001-011094 A JP 2000-229883 A JP-A-08-208514 Japanese Unexamined Patent Publication No. 07-324035 Japanese Unexamined Patent Publication No. 07-145042 WO01 / 076630 WO01 / 073211 publication

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention relates to a synovial cell proliferation inhibitor capable of suppressing the proliferation of synoviocytes without inducing apoptosis and causing immune suppression, and synoviocyte proliferation. It is an object of the present invention to provide a medicinal composition and a treatment method capable of treating or preventing a disease.

In order to solve the above-described problems, the present inventors diligently studied and as a result, obtained the following knowledge. That is, a compound represented by the structural formula (A) described later (N- {5- [2- (cyclohexyloxy) -6-methyl-4-pyrimidinyl] -1,3-thiazol-2-yl} -5- [ (4-methyl-1-piperazinyl) methyl] -2-pyrazinamine (sometimes referred to herein as “compound A”) has a strong inhibitory effect on synovial cell proliferation, while apoptosis It is the knowledge that it does not induce immune action such as lymphocyte activity.
The compound A is an aminothiazole skeleton-containing compound disclosed in, for example, WO2006 / 008874. For example, in the publication, the compound has a selective inhibitory activity against CDK4 / 6. In addition, it has been disclosed that it has an inhibitory effect on cell proliferation of clinically isolated cancer cells.
However, it has not been known at all that the compound A can suppress the proliferation of synovial cells without inducing apoptosis and without suppressing immune action such as lymphocyte activity. This is a new finding of the present inventors.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A synovial cell proliferation inhibitor comprising, as an active ingredient, at least one of a compound represented by the following structural formula (A) and a pharmaceutically acceptable salt thereof.
<2> A medicinal composition comprising the synovial cell proliferation inhibitor according to <1>.
<3> The medicinal composition according to <2>, which is at least one of an anti-rheumatoid arthritis agent, an anti-osteoarthritis agent, an anti-psoriatic arthritis agent, and an anti-gout agent.
<4> The medicinal composition according to any one of <2> to <3>, which is used for the treatment of at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout.
<5> A therapeutic method comprising administering the synovial cell proliferation inhibitor according to <1> to treat at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout. .

  According to the present invention, various conventional problems can be solved, and a synovial cell proliferation inhibitor capable of inhibiting the proliferation of synovial cells without inducing apoptosis and causing immune suppression. In addition, it is possible to provide a medicinal composition and a treatment method capable of treating or preventing a disease involving synovial cell proliferation.

(Synovial cell growth inhibitor)
The synovial cell proliferation inhibitor of the present invention comprises a compound represented by the following structural formula (A) (N- {5- [2- (cyclohexyloxy) -6-methyl-4-pyrimidinyl] -1,3-thiazol- 2-yl} -5-[(4-methyl-1-piperazinyl) methyl] -2-pyrazineamine; may be referred to herein as “Compound A”), and pharmaceutically acceptable salts thereof Is contained as an active ingredient, and further contains other ingredients as necessary.

  The salt is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include inorganic salts and organic salts. More specifically, hydrochlorides, hydrobromides, Examples thereof include sulfate, phosphate, acetate, oxalate, tartrate, citrate, maleate, and fumarate.

-Compound A-
There is no restriction | limiting in particular as a manufacturing method of the compound (compound A) represented by the said structural formula (A), According to the objective, it can select suitably, For example, refer to the method as described in WO2006 / 008874 gazette Can be manufactured.
The content of the compound A in the synovial cell growth inhibitor is not particularly limited and may be appropriately selected depending on the purpose. The synovial cell proliferation inhibitor is the compound A itself. May be.

-Other ingredients-
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include DMSO (dimethyl sulfoxide) and citrate buffer for dissolving and diluting the compound A. . Moreover, there is no restriction | limiting in particular as content of the said other component in the said synovial cell proliferation inhibitor, According to the objective, it can select suitably.

-Synovial cells-
The synoviocytes to be targeted by the synovial cell proliferation inhibitor are not particularly limited as long as they are proliferative cells derived from synovial tissue, and can be appropriately selected according to the purpose. Examples include fibroblasts. The synoviocytes may be in vivo cells or in vitro cultured cells. The animal species derived from the synovial cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, and the like. The synovial cells can also be collected from, for example, patients suffering from arthritis accompanied by synovial cell proliferation, experimental model animals induced arthritis accompanied by synovial cell proliferation, and the like.

The synovial cell proliferation inhibitor can suppress the proliferation of the synovial cells without inducing apoptosis. Therefore, the synovial cell proliferation agent is highly safe and has no side effects. Diseases in which cell proliferation is involved in the pathology can be treated or prevented.
The fact that the synovial cell proliferation inhibitor can inhibit the proliferation of the synovial cells is confirmed using, for example, a known cell proliferation analysis method such as BrdU incorporation measurement or flow cytometry, or a cell cycle analysis method. be able to. The fact that the synovial cell proliferation inhibitor does not induce apoptosis can also be confirmed using a known apoptosis detection method such as SubG1 analysis, TUNEL analysis, or agarose gel electrophoresis.

In addition, the synovial cell proliferation inhibitor can suppress the proliferation of the synovial cells without suppressing immune action such as lymphocyte activity. For this reason, the synovial cell proliferation inhibitor is a patient. Thus, it is possible to treat or prevent a disease in which the proliferation of synovial cells is involved in the disease state without causing a decrease in immunity.
It can be confirmed that the synovial cell proliferation inhibitor does not suppress the immunity using, for example, a known immunoactivity measurement technique such as lymphocyte count measurement.

In addition, since the active ingredient of the synovial cell proliferation inhibitor (compound A) has a lower molecular weight than that of a peptide drug or the like, for example, when administered to a living body, it shifts to a site where target synovial hyperplasia occurs. It is advantageous in that it is easy.
The synovial cell proliferation inhibitor can be suitably used, for example, for the medicinal composition of the present invention described later, the treatment method of the present invention described later, and the like.

(Medicinal composition)
The medicinal composition of the present invention contains at least the synovial cell proliferation inhibitor of the present invention, and further contains other components as necessary.

  There is no restriction | limiting in particular as content of the said synovial cell growth inhibitor in the said medicinal composition, For example, according to the dosage form of the said medicinal composition mentioned later, the grade of the symptom of the patient who is an administration object, etc. Can be selected as appropriate.

-Other ingredients-
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include pharmaceutically acceptable carriers. Moreover, there is no restriction | limiting in particular as said carrier, For example, it can select suitably according to the dosage form etc. of the said medicinal composition.
There is no restriction | limiting in particular as content of the said other component in the said medicinal composition, For example, according to the dosage form etc. of the said medicinal composition mentioned later, it can select suitably.

-Dosage form-
There is no restriction | limiting in particular as a dosage form of the said medicinal composition, According to the desired administration method, it can select suitably, For example, an oral solid agent (a tablet, a coated tablet, a granule, a powder, a capsule etc.), Oral solutions (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for erection, etc.), suppositories, ointments, patches, gels, creams, external powders, sprays And inhalable powders. Among these, as the dosage form, oral solid preparations, oral liquid preparations, and injections are preferable.

Examples of the oral solid preparation include, for example, excipients and further additives such as a binder, a disintegrant, a lubricant, a coloring agent, a flavoring and flavoring agent, etc., to the synovial cell proliferation inhibitor. In addition, it can be produced by a conventional method.
The excipient is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. .
Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include , Purified talc, stearate, borax, polyethylene glycol, and the like. Examples of the colorant include titanium oxide and iron oxide. Te is, for example, white sugar, orange peel, citric acid, and tartaric acid.

The oral solution can be produced by a conventional method, for example, by adding additives such as a taste-masking / flavoring agent, a buffer, and a stabilizer to the synovial cell growth inhibitor.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like, examples of the buffering agent include sodium citrate, and examples of the stabilizer include tragacanth. Arabic gum, gelatin and the like.

As the injection, for example, a pH regulator, a buffer, a stabilizer, an isotonic agent, a local anesthetic, and the like are added to the synovial cell growth inhibitor, and are used subcutaneously and intramuscularly by a conventional method. Injectables for intravenous use can be produced.
Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

  Examples of the suppository include, for example, the above-mentioned synovial cell proliferation inhibitor, known carriers for suppository preparations such as polyethylene glycol, lanolin, cocoa butter, and fatty acid triglyceride, and if necessary, Tween (registered trademark). After adding the surfactant, etc., it can be produced by a conventional method.

The ointment can be produced, for example, by blending a known base, stabilizer, wetting agent, preservative and the like with the synovial cell proliferation inhibitor and mixing them by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

  As the patch, for example, a cream, gel or paste as the ointment can be applied to a known support by a conventional method. Examples of the support include woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane and other films made of cotton, suf, and chemical fibers, and foam sheets.

-Application-
The medicinal composition is suitable, for example, as a therapeutic or prophylactic agent for diseases in which the proliferation of the synovial cells is involved in the pathology, and examples of the diseases include rheumatoid arthritis, osteoarthritis, psoriatic arthritis, A gout etc. are mentioned suitably. Moreover, the said medicinal composition can be used conveniently for the treatment method of this invention etc. which are mentioned later, for example.

(Method of treatment)
The treatment method of the present invention includes administering at least one of the synovial cell proliferation inhibitor and the medicinal composition (the synovial cell proliferation inhibitor and / or the medicinal composition), and further if necessary. Including other steps as appropriate, and treating at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout.

-Administration-
There is no restriction | limiting in particular as an administration object of the said synovial cell proliferation inhibitor and / or the said medicinal composition, According to the objective, it can select suitably, For example, a human, a mouse | mouth, a rat, a cow, a pig, a monkey etc. Is mentioned.
Moreover, there is no restriction | limiting in particular as an administration method of the said synovial cell growth inhibitor and / or the said medicinal composition, For example, it can select suitably according to the dosage form etc. of the said medicinal composition.
In addition, the dosage of the synovial cell proliferation inhibitor and / or the medicinal composition can be appropriately selected according to the age, weight, sex, symptom, etc. of the patient to be administered. For example, when the medicinal composition is an oral preparation (oral solid preparation, oral liquid preparation), the amount of the compound A that is an active ingredient per day for an adult is preferably 10 to 20 g. Moreover, when the said medicinal composition is an injection, 1-2 g is preferable as the quantity of the said compound A which is an active ingredient per day for an adult.
Moreover, there is no restriction | limiting in particular as administration frequency of the said synovial cell growth inhibitor and / or the said medicinal composition, According to the objective, it can select suitably, For example, the dosage per day is 1 It may be administered once a day or divided into multiple times (for example, about 2 to 4 times).

  Moreover, there is no restriction | limiting in particular also as an administration time of the said synovial cell proliferation inhibitor and / or the said medicinal composition, According to the objective, it can select suitably, For example, even if it administers before onset of the said disease. It may be administered after onset. The synovial cell proliferation inhibitor and / or the medicinal composition can exert a synovial cell proliferation inhibitory effect in both administration before onset and administration after onset.

-Other processes-
The other steps are not particularly limited and may be appropriately selected depending on the purpose. For example, other conventionally known anti-rheumatoid arthritis agents, anti-osteoarthritis agents, anti-psoriatic arthritis agents, The process etc. which administer a gout agent etc. are mentioned. That is, although the synovial cell proliferation inhibitor and / or the medicinal composition alone has an excellent therapeutic or preventive effect, it can be used in combination with other conventionally known drugs as described above.

(effect)
The synovial cell proliferation inhibitor and medicinal composition of the present invention can inhibit synovial cell proliferation without inducing apoptosis, and synovial cell proliferation is in a pathological state without inhibiting immune activity. Anti-rheumatoid arthritis, anti-osteoarthritis, anti-psoriatic arthritis that is safe and has few side effects and does not cause a decrease in patient immunity because it can treat or prevent the disease involved It is useful as an agent, an anti-gout agent and the like. In addition, according to the treatment method of the present invention, the synovial cell proliferation can be safely performed without decreasing the immunity of the patient by administering the synovial cell proliferation inhibitor and / or the medicinal composition. It is possible to treat or prevent diseases related to the pathological condition.
Since the synovial cell proliferation inhibitor and medicinal composition of the present invention can directly inhibit synovial cell proliferation without suppressing an immune reaction, as described above, for example, It is believed to be effective in treating arthritis that occurs independently of activation. In addition, most of the currently provided anti-rheumatic drugs are intended for inhibition of immune reaction, whereas the synovial cell proliferation inhibitor and medicinal composition of the present invention can be used without inhibition of immune reaction. Since the treatment or prevention from different aspects of inhibiting synovial cell proliferation is possible, the synovial cell proliferation inhibitor and / or medicinal composition of the present invention and a conventional antirheumatic drug are used in combination. A synergistic therapeutic or preventive effect is also expected.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

<Preparation of Compound A>
A compound represented by the following structural formula (A) (N- {5- [2- (cyclohexyloxy) -6-methyl-4-pyrimidinyl] -1,3-thiazol-2-yl} -5-[(4- methyl-1-piperazinyl) methyl] -2-pyrazinamine; in this specification, “compound A” may be referred to as “Compound A” obtained from Ariyu Pharmaceutical Co., Ltd. Acid buffer (pH 4)). This was used in the following Examples as the synovial cell proliferation inhibitor or medicinal composition of the present invention. The concentration of Compound A was as described in each Example, and was diluted with DMSO as necessary to prepare a desired concentration for use.

Example 1
<Inhibitory effect on synovial fibroblast proliferation>
In Example 1, the effect of Compound A on human and mouse synovial fibroblast proliferation and cell cycle progression was evaluated.

-Preparation of synovial fibroblasts-
Human rheumatoid synovial fibroblasts (RSF) have been found in patients with rheumatoid arthritis (RA) who underwent total joint replacement or synovectomy at Tokyo Medical and Dental University Hospital or National Hospital Organization Shimotsu Hospital. It was collected from the synovial tissue and prepared. The patients' rheumatoid arthritis (RA) was diagnosed according to American College of Rheumatology criteria (see Arnett et al .; Arthritis Rheum. 1988; 31: 315-324). In addition, consent from the patient was obtained before surgery. All the experimental means in this example have been approved by the ethics committee of RIKEN and Tokyo Medical and Dental University.
In addition, mouse synovial fibroblast (MSF) was collected from the knee joint synovial tissue of CIA mice (see below, Example 2) immunized with type II collagen and induced arthritis. (See Abad et al .; J Immunol 2001; 167 (6): 3182-3189).
The RSF and MSF were cultured in DMEM medium (Sigma) supplemented with L-glutamine, penicillin, streptomycin and 10% fetal calf serum (Sigma), respectively.

-Proliferation analysis-
RSF and MSF proliferation analysis was performed by BrdU incorporation. The RSF and MSF were respectively seeded in a 96-well plate at 2 to 5,000 cells / well, cultured overnight, and then stimulated with 10 μg / L IL-1β and 10 μg / L TNFα for 24 hours. did. Human and mouse IL-1β and TNFα were purchased from Wako. Each cytokine concentration is a value determined as an optimal dose in a preliminary experiment.
The compound A (0.01 to 10 μM) or 10 mmol / L citrate buffer (pH 4) containing 5% glucose as a control was added to the wells. After 24 hours, bromodeoxyuridine (BrdU) was added and the culture was continued. Twenty hours later, the incorporated BrdU was quantified using a BrdU Cell Proliferation ELISA kit (Exalpha Biologics). The results are shown in FIG.
In FIG. 1, the BrdU incorporation in the compound A addition group at each concentration is shown as a percentage when the BrdU incorporation in the compound A non-addition group (control) is 100%. Average values of 3 wells are shown with error bars indicating standard deviation (SD). Representative results of three experiments performed independently were shown.

-Cell cycle analysis-
The cell cycle analysis of the RSF and MSF was performed as follows. The RSF and MSF were stimulated with cytokine for 24 hours in the same manner as described above in the presence of the compound A (2.5 μmol / L) or in the presence of 10 mmol / L citrate buffer (pH 4) containing 5% glucose as a control. After washing with phosphate buffered saline (PBS), it was resuspended in 0.15% Triton X / PBS. After staining with propidium iodide (50 μg / ml), the DNA content was examined using a FACS caliber flow cytometer (Becton Dickinson). The results are shown in FIG.
FIG. 2 shows a histogram of DNA content and a percentage group indicating the proportion of cells in each cell cycle stage. Representative results of three experiments performed independently were shown.

As a result of the proliferation analysis, Compound A inhibited the proliferation of RSF and MSF in a dose-dependent manner (FIG. 1). Also, no phenotypic cell death due to the addition of Compound A was observed at all concentrations tested.
Further, as a result of the cell cycle analysis, the compound A increased the cell group in the G0 / G1 phase without increasing the sub-G1 group serving as an index of apoptosis in any type of cells. That is, cell cycle arrest in G1 phase was induced (FIG. 2).
From these results, it was found that the compound A has an action capable of strongly suppressing the proliferation of synovial fibroblasts without inducing apoptosis.

(Example 2)
<Inhibitory action against collagen-induced arthritis>
-Production of collagen-induced arthritis (CIA) mice-
Collagen-induced arthritis (CIA) is a disease in which synovitis with proliferation of synovial cells occurs as a result of autoimmunity to type II collagen, and the joint is destroyed like rheumatoid arthritis. Yes, it is a good model for proliferative synovitis. The collagen-induced arthritis (CIA) mouse was prepared by the following method, and the inhibitory action of arthritis by the compound A was evaluated.
200 μg of bovine type II collagen (CII) (collagen technology workshop) emulsified in 7-week-old male DBA / 1 mice (Nippon Charles River Co., Ltd.) with complete Freund adjuvant (CFA) (Difco) Were immunized to the base of the tail. After the first immunization, immunization was performed again 21 days later to prepare CIA mice.

-Administration of Compound A-
The CIA mice were orally administered 200 mg / kg of Compound A every 12 hours (twice a day) for 7 days. The oral administration was started from the day when CIA mouse arthritis was confirmed (24 days after the first immunization).
Moreover, since administration of Compound A was able to be reduced to 30 mg / kg intraperitoneal administration once a day without losing the therapeutic effect, this treatment was assumed to be an actual arthritis treatment and after the onset of arthritis Of the CIA mice. Once a day, 30 mg / kg of Compound A was intraperitoneally administered for 5 consecutive days, separated by 1 day, and again for 5 consecutive days.
In addition, as a control, CIA mice administered with 10 mmol / L citrate buffer (pH 4) containing 5% glucose in place of Compound A were also prepared.

-Arthritis evaluation-
The degree of arthritis development in each limb of the CIA mouse was evaluated at 4 points or less per limb, that is, 16 points or less per individual, according to the following arthritis score evaluation criteria.
[Arthritis score evaluation criteria]
0: No swelling is observed.
Swelling is observed at 1: 1 joint.
2: Swelling is observed in 2 or more joints, but not severe.
3: Severe swelling is observed in 2 or more joints.
4: Severe swelling is observed in 2 or more joints (including numerical swelling).
The arthritis score was analyzed statistically by Student t-test.
In addition, the degree of joint swelling of the CIA mice administered orally was quantified by measuring the thickness of the hind limb and the width of the ankle using a micrometer (Ozaki Seisakusho). The results are shown in FIGS.

  3A to 3C show the arthritis score and the changes over time of the limb thickness and the ankle width with the oral administration of the compound A, respectively. FIG. 3D shows the change over time in the arthritis score due to intraperitoneal administration of Compound A after the onset of arthritis. Average values of 8 mice were shown together with error bars indicating standard deviation (SD) (**: p <0.05, **: p <0.01, ***: p <0.001).

  Oral administration of Compound A reduced the arthritis score and limb and ankle swelling in CIA mice (FIGS. 3A-C). Moreover, since administration of Compound A could be reduced to 30 mg / kg intraperitoneal administration once a day without losing the therapeutic effect, this treatment was started after the onset of arthritis. The arthritis score was reduced (Figure 3D). In addition, the present inventors also confirmed that administration of the compound A does not cause any abnormality in the physical findings, behaviors, etc. of the mice, except for the recovery of arthritis. From these results, it was found that Compound A can suppress arthritis without adversely affecting normal biological functions, and can also exert a therapeutic effect against ongoing arthritis.

(Example 3)
<Action on immune response>
The effect of Compound A on the immune response of the living body was evaluated by examining the presence or absence of an effect on the proliferation of type II collagen-reactive T cells.

Mice were immunized with type II collagen in the same manner as in Example 2. Simultaneously with the initiation of immunization, Compound A 30 mg / kg was administered intraperitoneally for 5 consecutive days, separated by 1 day, and again administered for 5 consecutive days. As a control, a mouse to which Compound A was not administered (type II collagen immunization only) was also prepared. On the 12th day from the start of the immunization, the lymph nodes were removed, the cell group was seeded in a 96-well plate at 400,000 cells / well, and each amount (0, 4, 20, 100 μg / ml) of type II collagen was used for 72 hours. After culture, BrdU was added and further cultured. After 17 hours, the incorporated BrdU was quantified by ELISA to examine the proliferation of type II collagen-reactive T cells. The results are shown in FIG.
FIG. 4 shows the BrdU incorporation in each group as a percentage when the amount of BrdU incorporation was 100% when no type II collagen was added during culture (0 μg / ml).

As a result, the group administered with compound A together with type II collagen (FIG. 4, compound A) depends on the amount of type II collagen added at the time of culture, as in the group administered with type II collagen alone (FIG. 4, control). It was shown that type II collagen-reactive T cells were increased (FIG. 4).
From this, it was found that Compound A does not suppress T cell proliferation due to immune reaction, that is, does not cause immunosuppression.

  The synovial cell proliferation inhibitor and medicinal composition of the present invention can inhibit synovial cell proliferation without inducing apoptosis, and synovial cell proliferation is in a pathological state without inhibiting immune activity. Anti-rheumatoid arthritis, anti-osteoarthritis, anti-psoriatic arthritis that is safe and has few side effects and does not cause a decrease in patient immunity because it can treat or prevent the disease involved It is useful as an agent, anti-gout agent and the like. In addition, according to the treatment method of the present invention, the synovial cell proliferation can be safely performed without decreasing the immunity of the patient by administering the synovial cell proliferation inhibitor and / or the medicinal composition. It is possible to treat or prevent diseases related to the pathological condition.

FIG. 1 is a graph showing the effect of compound A on the proliferation of synovial fibroblasts. FIG. 2 is a histogram showing the effect of Compound A on the cell cycle progression of synovial fibroblasts. FIG. 3A is a graph showing changes in arthritis score due to oral administration of Compound A. FIG. 3B is a graph showing changes in limb thickness due to oral administration of Compound A. FIG. 3C is a graph showing changes in the width of the ankle by oral administration of Compound A. FIG. 3D is a graph showing changes in arthritis score due to intraperitoneal administration of Compound A. FIG. 4 is a graph showing the effect of Compound A on type II collagen (CII) reactive T cell proliferation.

Claims (5)

A synovial cell proliferation inhibitor comprising, as an active ingredient, at least one of a compound represented by the following structural formula (A) and a pharmaceutically acceptable salt thereof.
  A medicinal composition comprising the synovial cell proliferation inhibitor of claim 1.   The medicinal composition according to claim 2, which is at least one of an anti-rheumatoid arthritis agent, an anti-osteoarthritis agent, an anti-psoriatic arthritis agent, and an anti-gout agent.   The medicinal composition according to any one of claims 2 to 3, which is used for the treatment of at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout. A synovial cell growth inhibitor according to claim 1 and a medicinal composition according to any one of claims 2 to 4 are administered, and rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout A treatment method characterized by treating at least one of them.