WO2007010946A1 - Synovial cell proliferation inhibitor - Google Patents

Abstract

Disclosed is a synovial cell proliferation inhibitor which does not induce apoptosis or immunosuppression and can inhibit the proliferation of a synovial cell. Also disclosed is a pharmaceutical composition or therapeutic method which can treat a disease involving the synovial cell proliferation without reducing the resistance against a infection. A synovial cell proliferation inhibitor comprising at least one of flavopiridol (IUPAC name: alvocidib), which is a compound of the structural formula (1), and a pharmaceutically acceptable salt thereof as an active ingredient; a pharmaceutical composition comprising at least the synovial cell proliferation inhibitor; and a therapeutic method comprising at least administering the synovial cell proliferation inhibitor.

Description

 Specification

 Synovial cell growth inhibitor

 Technical field

 [0001] The present invention relates to a synovial cell proliferation inhibitor comprising as an active ingredient at least one of flavopiridol (flavopiridol or IUPAC name: alvocidib) and a pharmaceutically acceptable salt thereof.

The present invention also relates to a pharmaceutical composition containing the synovial cell proliferation inhibitor and a treatment method using the synovial cell proliferation inhibitor.

 Background art

 [0002] The synovial membrane is originally composed of a single layer of synovial cells. However, the synovial cells may proliferate excessively due to some inflammatory mechanism, resulting in synovial hyperplasia.

 The 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 as a result of the proliferative synovitis, there is a serious dysfunction such as cartilage degeneration or bone tissue destruction. As diseases in which such synovial cell proliferation is involved in the pathology, for example, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gout and the like are known.

 [0003] Rheumatoid arthritis is an autoimmune disease, and inflammatory mediators (eg, IL-1, IL-6, TNF-a, etc.) of lymphocyte isotropic force are excessively present in the vicinity of the joint. Is a disease in which inflammation and bone destruction of the tissue progress and a pathological condition appears. As one of the etiological factors, proliferation of the synovial cells is cited. However, since it is thought that there are a plurality of etiological factors, a multifaceted treatment method is required. Drug development is desired.

 [0004] Conventionally, as a method for treating rheumatoid arthritis, symptomatic treatment has been carried out by administering an analgesic and an anti-inflammatory agent. In symptomatic treatment, although the onset can be delayed, It was impossible to stop the progression and even cure the disease itself.

In contrast, based on the fact that some of the drugs originally used as anticancer agents in recent years are also effective as lymphocyte proliferation inhibitors, administration of these drugs further increases anti-TNF- It has been reported that treatment with a combination of spleen antibodies subsides inflammation and, in some cases, stops disease progression and increases the rate of improvement and cure 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 immunosuppressed, extremely vulnerable to infection, etc. There will be problems!

 [0005] 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, There are many drugs that are less effective for the elderly, and drugs that are highly versatile and have a complete healing effect are not yet known.

 On the other hand, as one of the therapeutic agents for rheumatoid arthritis, a drug (synovial cell proliferation inhibitor) that suppresses the proliferation of the synovial cells, which is one of its etiology, has been proposed.

 Examples of the synovial cell proliferation inhibitor include diterpene compounds (see Patent Document 1), FADD gene (Patent Document 1) that suppress synovial cell proliferation by inducing apoptosis of synovial cells. 2), Fas ligand (see Patent Document 3), anti-Fas antibody (see Patent Document 4), and 3,6-anhydrogalactose and its derivatives (see Patent Document 5), etc. As an inhibitor of proliferation of synovial cells by inhibiting adhesion, imidazole derivatives (see Patent Document 6), quinoline derivatives, quinolone derivatives (see Patent Document 7), and the like have been proposed. In addition to these, the synovial cell proliferation inhibitor includes 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). BFGF antagonist (see patent document 11), IL 6 antagonist (see patent document 12), cAMP derivative (see patent document 13), vitamin D3 and its derivatives (see patent document 14), c-erbB-2 activity inhibition Agents (see Patent Document 15), y-secretase-like protease inhibitors (Patent Document 16) and the like have been proposed.

 [0007] These synovial cell proliferation inhibitors are used not only for rheumatoid arthritis, but also for prevention of the onset of diseases involving the proliferation of synovial cells such as osteoarthritis, psoriatic arthritis, and gout. It is expected to be a useful substance for controlling aging.

In other words, it suppresses lymphocyte proliferation and activity, that is, it causes immune suppression. A synovial cell growth inhibitor that can suppress the proliferation of synovial cells without rubbing, and a medicinal composition and a treatment method for treating a disease involving synovial cell proliferation without reducing resistance to infection are still available. It has been discovered and the current situation is.

 Patent Document 1: Japanese Patent Application Laid-Open No. 2004-168713

 Patent Document 2: JP 2000-198737 A

 Patent Document 3: Japanese Patent Laid-Open No. 11-060501

 Patent Document 4: Japanese Patent Laid-Open No. 08-208515

 Patent Document 5: WO99Z064424

 Patent Document 6: Japanese Unexamined Patent Publication No. 2003-040888

 Patent Document 7: Japanese Unexamined Patent Publication No. 2002-371078

 Patent Document 8: Japanese Patent Laid-Open No. 2003-089647

 Patent Document 9: Japanese Patent Laid-Open No. 2002-293745

 Patent Document 10: Japanese Patent Laid-Open No. 2001-011094

 Patent Document 11: Japanese Unexamined Patent Publication No. 2000-229883

 Patent Document 12: Japanese Patent Laid-Open No. 08-208514

 Patent Document 13: Japanese Patent Application Laid-Open No. 07-324035

 Patent Document 14: Japanese Patent Application Laid-Open No. 07-145062

 Patent Document 15: WO0lZ076630

 Patent Document 16: WO0lZ072321

 Disclosure of the invention

 [0009] An object of the present invention is to meet the above-mentioned demand, solve the conventional problems, and achieve the following objects. That is, the present invention provides a synovial cell proliferation inhibitor capable of suppressing the proliferation of synoviocytes without inducing apoptosis and causing immune suppression, and also has resistance to infection. It is an object of the present invention to provide a medicinal composition and a method for treating a disease involving synovial cell proliferation without reducing it.

 [0010] As a result of intensive studies by the present inventors in order to solve the above problems, the following knowledge was obtained.

That is, flavopiridol (flavopiridol or IUPAC name: alvocidib) force It has a strong inhibitory activity on synovial cell proliferation without inducing apoptosis, but on the activity of lymphocytes Is the knowledge that it has no effect.

 Flavopyridol is a flavonoid that induces apoptosis against cancer cells, Dysoxylum binectariferum from the mahogany family, and a novel flavonoid synthesized with hints of components extracted from crusts. Its physiological activity is cyclin. Dependent kinase inhibitory activity, smooth muscle growth inhibitory activity, etc. are known!

 However, it has not been known at all to suppress synovial cell proliferation without inducing apoptosis without inhibiting the proliferation of flavopiridol power lymphocytes. This is a new finding.

 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> It is characterized by containing at least one of flavopiridol (flavopiridol or IUPAC name: alvocidib), which is a compound represented by the following structural formula (1), and a pharmaceutically acceptable salt thereof as an active ingredient. It is a synovial cell proliferation inhibitor.

[Chemical 1]

)

)

 <2> A medicinal composition comprising at least the synovial cell proliferation inhibitor according to <1>.

<3> The medicinal composition according to <2> comprising a pharmaceutically acceptable carrier. <4> The medicinal composition according to any one of <2> to <3>, wherein at least one of anti-rheumatoid arthritis, anti-osteoarthritis, anti-psoriatic arthritis, and anti-gout is at least It is. <5> 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.

 <0013> <6> At least the synovial cell proliferation inhibitor according to <1> is administered to treat at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout, It is a treatment method.

 <7> The treatment according to <6>, wherein the pharmaceutical composition according to <2> to <5> is administered to treat at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout Is the method.

[0014] According to the present invention, the conventional problems can be solved, and the proliferation of synovial cells can be suppressed without inducing apoptosis and without causing immune suppression. It is possible to provide an inhibitor, and a medicinal composition and a treatment method for treating a disease involving synovial cell proliferation without reducing resistance to infection. Brief Description of Drawings

 [0015] FIG. 1 is a graph showing the effect of flavopiridol on the proliferation of synovial fibroblasts.

 FIG. 2 is a histogram showing the effect of flavopiridol on cell cycle progression of synovial fibroblasts.

 FIG. 3 is a graph showing changes in arthritis scores in CIA mice by intraperitoneal administration of flavopiridol.

 FIG. 4 is a graph showing changes in arthritis scores in CIA mice by intraperitoneal administration of flavopiridol.

 FIG. 5 is a tissue staining diagram and an X-ray photograph showing the effect of flavopiridol force on arthritis in mice. A, D shows the hind limbs of CIA mice treated with DMSO only,

B, E is lmg / kg flavopiridol containing DMSO

C and F represent hind limbs of CIA mice intraperitoneally administered with DMSO containing 2.5 mgZkg flavopiridol.

[Fig. 6] Fig. 6 shows the joints of RAG gene-deficient mice by intraperitoneal administration of KZBxN mouse serum. It is a graph which shows a flame point change.

 FIG. 7 is a histogram showing the effect of flavopiridol on antibody response in CIA mice.

 FIG. 8 is a histogram showing the effect of flavopiridol force on type 1 collagen-reactive T cell proliferation.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0016] (Endotoxin activity inhibitor)

 The synovial cell growth inhibitor of the present invention contains at least one of flavopiridol, which is a compound represented by the following structural formula (1), and a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical 2]

 [0017] The salt is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include inorganic salts and organic salts. Specific examples include hydrochlorides and hydrobromides. Sulfate, phosphate, acetate, oxalate, tartrate, kenate, maleate, and fumarate are preferred.

 [0018] The production method of flavopiridol is not particularly limited, and a known method force can be appropriately selected according to the purpose. As flavopiridol, a commercially available product may be used.

[0019] The synovial cell proliferation inhibitor of the present invention can suppress the proliferation of synovial cells without inducing apoptosis. As a result, the synovial cell proliferating agent can treat or prevent a disease in which the proliferation of synovial cells is involved in its pathological condition without the side effect of high safety. This ability to inhibit the proliferation of synovial cells that does not induce apoptosis can be confirmed, for example, using a known cell proliferation analysis method such as measurement of BrdU incorporation or flow cytometry, and a cell cycle analysis method. Further, the fact that the synovial cell proliferation inhibitor of the present invention does not induce apoptosis can be confirmed by using known apoptosis detection methods such as SubGl analysis, TUNEL analysis, and agarose gel electrophoresis.

[0020] The synovial cell proliferation inhibitor has an action of suppressing proliferative synovitis by suppressing the proliferation of synoviocytes without inhibiting the proliferation and activity of lymphocytes. Treat or prevent cartilage degeneration and bone tissue destruction without inhibition.

 This ability to suppress synovial cell proliferation without immunosuppression is achieved by, for example, transferring sera from mice that spontaneously develop arthritis (eg, KZBx N mice) to lymphocyte-deficient mice (eg, RAG gene-deficient mice). To evaluate the severity of inflammation between the group of mice administered with the synovial cell proliferation inhibitor of the present invention and the group of mice not administered with the model mouse. Can do.

 In this evaluation, for example, since dose-dependent suppression of inflammation is observed in the group administered with the synovial cell growth inhibitor of the present invention, the mechanism of action of the synovial cell proliferation inhibitor of the present invention is lymph. It can be seen that this is not due to suppression of sphere proliferation.

[0021] The synovial cell proliferation inhibitor of the present invention has low cytotoxicity and high safety. In addition, since the molecular weight is small, for example, when administered to a living body, it is advantageous to immediately shift to a target site where synovial hyperplasia occurs.

 Moreover, the synovial cell proliferation inhibitor of the present invention is suitable as an active ingredient of the medicinal composition of the present invention described below, and is also suitably used for the treatment method of the present invention described below.

[0022] (medicinal composition)

 The medicinal composition of the present invention contains at least the synovial cell proliferation inhibitor of the present invention and, if necessary, other components appropriately selected from known medium strengths.

Examples of the other components include a pharmaceutically acceptable carrier, and the carrier can be appropriately selected according to the dosage form of the medicinal composition without particular limitation. [0023] The dosage form of the medicinal composition can be appropriately selected depending on the administration method without any particular restrictions. For example, oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.) , Oral solutions (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for dissolution, etc.), suppositories, ointments, patches, gels, creams, powders for external use, Examples include sprays and inhaled powders.

 [0024] Examples of the oral solid preparation include excipients to the synovial cell proliferation inhibitor, and additives such as binders, disintegrants, lubricants, coloring agents, and flavoring agents as necessary. And can be produced by a conventional method.

 Examples of the excipient include lactose, sucrose, sodium chloride salt, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.

 The additive is not particularly limited and can be appropriately selected according to the purpose. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose. , Hydroxypropyl cellulose, hydroxypropinorestarch, methinoresenorelose, ethinoresenorelose, shellac, calcium phosphate, polybulurpyrrolidone and the like. Examples of the disintegrant include dry starch and alginic acid. Sodium, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like, and examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like. Before As the colorant, titanium oxide, iron oxide and the like, examples of the flavoring 'flavoring agent include sucrose, orange peel, Kuen acid, and tartaric acid.

 [0025] The oral liquid preparation can be produced by a conventional method, for example, by adding additives such as a flavoring agent, a buffering agent, and a stabilizer to the synovial cell proliferation inhibitor.

 The additive is not particularly limited and can be appropriately selected according to the purpose. Examples of the flavoring agent include sucrose, orange peel, citrate, tartaric acid, and the like. Examples of the stabilizer include sodium quenate and the like, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.

[0026] As the injection, for example, a pH regulator, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the synovial cell proliferation inhibitor, and subcutaneous, muscle, etc. are added by a conventional method. Intra and vein An internal injection 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, and thiolactic acid. Examples of the isotonic agent include sodium chloride salt and glucose. Examples of the local anesthetic include pro-in hydrochloride hydrochloride and lidocaine hydrochloride.

[0027] As the suppository, the synovial cell proliferation inhibitor, a known suppository formulation carrier, for example, polyethylene glycol, lanolin, cocoa butter, fatty acid triglyceride and the like, and TWEEN (TWEEN) as necessary. : A registered trademark) and the like, and then can be produced by a conventional method.

 [0028] The ointment can be produced by mixing 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, honey beeswax, otatildodecyl alcohol, paraffin and the like. Examples of the preservative include methyl noxybenzoate, ethyl paraoxybenzoate, and propyl noraoxybenzoate.

 [0029] The patch can be produced, for example, by applying a cream, gel, paste or the like as the ointment to a known support by a conventional method. Examples of the support include cotton, suf, woven fabric made of chemical fiber, non-woven fabric, soft vinyl chloride, polyethylene, polyurethane and other films, and foam sheets.

 [0030] The content of the synovial cell proliferation inhibitor in the pharmaceutical composition can be appropriately selected depending on the symptom of the patient to whom the dosage form is administered, but is usually per dosage unit. In the case of the oral preparation, about 1-1, OOOmg is preferred. In the case of the injection, about 0.1-50Omg is preferred, and in the case of the suppository, about 5-1, OOOmg is preferred.

 In addition, the dosage of the pharmaceutical composition is a force that can be appropriately selected according to the patient's symptoms, weight, age, sex, etc. Usually, about 0.1 to 5, OOOmg per day for an adult is preferable, ~: L, more preferred than OOOmg power! / ヽ.

As the administration frequency of the pharmaceutical composition, the daily dose is preferably administered once or divided into 2 to 4 times. [0031] Further, the medicinal composition of the present invention is suitable as a therapeutic or prophylactic agent for diseases in which the proliferation of synovial cells is involved in its pathology, such as anti-rheumatoid arthritis agents, anti-degenerative arthropathy agents, Suitable as anti-psoriatic arthritis agent and anti-gout agent.

 [0032] (Treatment method)

 The therapeutic method of the present invention comprises administering at least the synovial cell proliferation inhibitor and rheumatoid arthritis.

, Osteoarthritis, psoriatic arthritis, and gout, and it is preferable to administer the pharmaceutical composition.

[0033] In the treatment method, the method for administering the synovial cell proliferation inhibitor or the pharmaceutical composition can be appropriately selected according to the dosage form and the like, which are not particularly limited.

[0034] In addition, as the treatment method, other known anti-rheumatoid arthritis agents, anti-osteoarthritis agents, anti-psoriatic properties in the treatment methods for rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout. It can be used in combination with an arthritis agent and an anti-gout agent.

 Example

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

[Example 1]

 <Inhibition of synovial fibroblast proliferation>

 The effect of the synovial cell proliferation inhibitor of the present invention on the proliferation and cell cycle progression of the synovial fibroblasts was evaluated by the following method.

[0037] Rheumatoid synovial fibroblasts (RSF) in human rheumatoid synovial fibroblasts (RSF) have undergone total joint replacement or synovectomy at Tokyo Medical and Dental University Hospital or National Hospital Organization Shimotsu Hospital. RA) The patient's synovial tissue strength was collected and prepared. Rheumatoid arthritis (RA) is a standard of the American College of Rheumatology (Arnett et al .; Arthritis Rheum. 1988;

31: 315-324). We also obtained patient consent before surgery. In addition

All experimental means in this example have been approved by RIKEN and the ethics committee of Tokyo Medical and Dental University.

In addition, mouse synovial fibroblasts (MSFs) from the knee joints of CIA mice immunized with type II collagen and induced arthritis (see Example 2 below) Synovial tissue force was also collected and prepared (see Abad et al .; J Immunol 2001; 167 (6): 3182-31 89).

 RSF and MSF were cultured in DMEM medium (Sigma) supplemented with L-glutamine, penicillin, streptomycin and 10% urine fetal serum (Sigma), respectively.

[0038] Proliferation analysis of the RSF and MSF was performed by BrdU incorporation. After RSF and MSF were seeded in 96-well plates at 2 to 5,000 cells Z-well, and cultured in sputum, 10 g / L IL—lj8 and 10 g / L TNF a were added. Used for 24 hours to stimulate the site force. Human and mouse IL1β and TNFa were purchased from Wako. Further, each of the site force in concentrations is a value determined as an optimum dose in a preliminary experiment.

 Flavopyridol (0.01-10 M) or 5% glucose-containing 1 Ommol / L citrate buffer (pH 4) well was added as a control. After 24 hours, bromodexuridine (BrdU) was added and the culture was continued. After 20 hours, the incorporated BrdU was quantified using a Brd U Cell Proliferation ELISA kit (Exalpha Biologicals). The results are shown in Figure 1.

 Figure 1 shows the percentage of BrdU uptake in flavopiridol-added cocoon at each concentration when the amount of BrdU incorporation in flavopiridol-free cocoon (control) is 100%. . Average values of 3 wells are shown with error bars indicating standard deviation (SD). Representative results from three independent experiments are shown.

 [0039] The cell cycle analysis of the RSF and MSF was performed as follows. The RSF and MSF were treated in the same manner as described above in the presence of the flavopiridol (2.5 mol / L) or in the presence of 10 mmol / L citrate buffer (pH 4) containing 5 o / o glucose as a control. After stimulation with cytodynamic force for 24 hours, the cells were washed with phosphate buffered saline (PBS) and resuspended in 0.15% TritonXZPBS. The DNA content was stained using a FACS caliber flow cytometer (Becton Dickinson) after staining with propidium iodide (50 μg / ml). The result is shown in figure 2.

FIG. 2 shows a histogram of DNA content and a percentage group indicating the proportion of cells in each cell cycle stage. Representative results from three independent experiments are shown. [0040] As a result of the proliferation analysis, flavopiridol inhibited the proliferation of RSF and MSF in a dose-dependent manner (Fig. 1). At all concentrations tested, phenotypic cell death due to the addition of the flavopiridol was not observed.

 In addition, as a result of the cell cycle analysis, flavopiridol increased the number of cells in the GOZG1 phase without increasing the number of sub-G1 groups, which is an index of apoptosis, in any type of cells, that is, Led to cell cycle arrest in the G1 phase (Figure 2).

 From these results, it was found that the synovial cell proliferation inhibitor of the present invention has an action capable of strongly suppressing the proliferation of synovial fibroblasts without inducing apoptosis.

 [0041] (Example 2)

 <Inhibition of collagen-induced arthritis in mice>

 Collagen-induced arthritis in mice is a disease in which synovitis with the proliferation of synovial cells occurs as a result of autoimmunity to type II collagen, and the joint is destroyed like rheumatoid arthritis. A good model. The arthritis model mouse was prepared by the following method, and the suppression of arthritis by the synovial cell proliferation inhibitor of the present invention was evaluated.

 [0042] Urushi type II collagen (CII) (collagen) emulsified in complete Freund adjuvant (CFA) (Difco) in 7-week-old male DBAZ1 mice (Nippon Charles' Ribaichi Co., Ltd.) Technical workshop) 200 g was immunized to the base of the tail. After the first immunization, 21 days later, immunization (booster immunization) was performed again to produce CIA mice.

 [0043] The arthritis model mouse was intraperitoneally administered with flavopiridol dissolved in 0.01% DMSO for 10 consecutive days from 4 days after booster immunization (25 days after the first immunization). The administered group was divided into two groups, and the dose of flavopiridol was set to 1 mg / kg or 2.5 mg Zkg, respectively. In addition, as a comparison target group, DMSO alone was administered intraperitoneally in an equal amount.

 [0044] The degree of arthritis onset in the arthritis model mice was evaluated by an arthritic index. -Evaluation was performed according to the following criteria, with 4 points or less on the limb and 16 points or less per individual. The results are shown in Figure 3. Figure 3 shows the mean values of 7 mice with error bars indicating standard deviation (SD) (*: p <0.05, * *: p <0.01, * * *: p <0 . 001

) o

[Arthritis score evaluation criteria] 0: No swelling is observed.

 1: Swelling is observed at one 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's t test. In addition, the degree of swelling of the joints of the CIA mice administered with the abdominal cavity was quantified by measuring the thickness of the hind limbs and the width of the ankles using a micrometer (Ozaki Seisakusho).

[0045] (Example 3)

 The arthritis model mice prepared in the same manner as in Example 2 were treated twice a week for 5 weeks from 3 days after booster immunization (24 days after the first immunization) (28, 34, 38, 42, 4 from the first immunization). 5, 48, 52, 55, 59, and 62 days later) Flavopiridol dissolved in 0.01% DMSO was administered intraperitoneally. There are 3 administration groups, with flavopiridol doses of lmg / kg and 2.5 mg / kg, and 2.5 mg / kg for which administration was discontinued 34 days after the first immunization. did. In addition, as a comparison target group, DMSO alone was administered intraperitoneally in an equal amount. The results of evaluating the degree of arthritis development in the same manner as above are shown in FIG.

 In addition, Fig. 4 also shows the average values of 7 mice with error bars indicating standard deviation (SD) (*: p <0.05, water: p <0.01, water: p <0. 001).

 [0046] From the results of FIGS. 3 and 4, the degree of arthritis was improved in mice administered with flavopiridol compared to mice administered only DMSO without flavopiridol. It was dose-dependent and persisted for 5 weeks.

 In addition, in the mice in which the administration of flavopiridol in Example 3 was discontinued (FIG. 4, open squares), after the administration was discontinued and the force was about 8 days later, joint inflammation similar to that in the comparative group was observed. Came to show. This result suggests that the immune response against ushi type II collagen (CII) was suppressed during flavopiridol administration!

 [Example 4]

Arthritis was induced in the same manner as in Example 2, and the hind limbs of a model mouse to which flavopiridol was intraperitoneally administered at the same dose as in Example 2 was fixed with 10% formalin buffer, and 10% E Decalcified with DTA solution and embedded in norafine. The paraffin embedding was adjusted to a 4 m section with a microtome, placed on a slide glass, and deparaffinized. Next, after staining with hematoxylin solution and eosin solution (HE staining), a tissue specimen was prepared by covering with a cover glass using an encapsulant and observed with an optical microscope. Further, a tissue X-ray photograph of the formalin-fixed hind limb was obtained with a soft X-ray imaging apparatus: CMB-2 (Softex), and observed with an optical microscope. The results are shown in FIG.

 [0048] Fig. 5A-C light microscope image (HE staining), Fig. 5D- F light microscope image (X-rays), cyst formation, severe bone with joint stiffness seen in DMSO-only mice It was found that destruction (FIGS. 5A, D) was suppressed in a dose-dependent manner with flavopiridol (FIGS. 5B, C, E, F).

 [0049] (Example 5)

 <Inhibition of arthritis in lymphocyte-deficient mice>

 Mice lacking the RAG gene have no immune response due to lymphocytes due to lack of lymphocytes.However, when mice with model mice (KZBx N) that spontaneously develop arthritis very similar to rheumatoid arthritis (KZBx N serum) are administered, It is known that the proliferation of membrane cells is promoted and arthritis is caused. Using this model mouse, the inhibition of arthritis by the synovial cell proliferation inhibitor of the present invention was evaluated.

 [0050] An arthritis model was prepared by intraperitoneally administering 300 L of serum of KZBxN mice to RAG gene-deficient mice (9-10 weeks old, RAG2ZB6 KO, C57BLZ6 genetic background). One day after the transfer of serum, flavopiridol dissolved in DMSO was administered intraperitoneally as a synoviocyte growth inhibitor for 10 consecutive days. The administration group was divided into two groups, and the dose of flavopiridol was 1 mg / kg or 2.5 mgZkg, respectively. As a comparison group, DMSO alone was given an equivalent amount of abdominal cavity.

 The degree of arthritis onset in the arthritis model mice was evaluated in the same manner as in Example 2. The result is shown in FIG.

[0051] From the results shown in FIG. 6, the degree of arthritis was improved in the mice administered with the flavopiridol compared to the mice administered only with DMSO not containing the flavopiridol. The improvement was dependent on the dose. From this, it was also found that the suppression of proliferative arthritis was not due to the suppression of immune reactions such as the inhibition of lymphocyte activity or the suppression of proliferation of the synovial cell proliferation inhibitor of the present invention. .

 [0052] (Example 6)

 <Antibody response in collagen-induced arthritis in mice>

 As in Example 2, flavopiridol was administered to model mice in which arthritis was induced in the same manner as in Example 2, and the administration start date (day 0, immediately before administration), day 11 after administration, and after administration 39 On the day, antibody responses of mouse subclasses (IgG1, IgG2a, IgG2b) were measured by the following method.

 [0053] Ushi type II collagen diluted with PBS was placed in a 96-well plate and allowed to stand at 4 ° C. The 96-well plate was washed with 0.05% Tween20-containing PBS, and then washed with 2% Blocking was carried out with PBS containing serum serum albumin to prepare 96-well plates coated with ushi type II collagen.

 [0054] The antibody response is measured by placing a diluted serum solution collected from the mouse in a 96-well plate coated with the Ushi type II collagen and reacting at room temperature for 2 hours, and then the 96-well plate. Next, peroxidase-conjugated anti-mouse IgGl, IgG2a, or IgG2b antibody (manufactured by Zymed) is added and reacted, and these are developed with TMBZ (3, 3, 5, 5, 1 Tetramethylbenzidine) substrate. The measurement was performed by measuring the absorbance at a wavelength of 450 nm.

 [0055] From the results of Fig. 7, it was found that the synovial cell proliferation inhibitor of the present invention did not suppress the type II collagen-reactive antibody response.

 [Example 7]

 <Proliferation of type II collagen-reactive T cells>

In the same manner as in Example 2, mice were immunized with type II collagen. Simultaneously with the initiation of immunization, flavopiridol 5 mg Zkg was administered intraperitoneally for 5 consecutive days, separated by 1 day, and again administered for 5 consecutive days. As a control, mice not administered with flavopiridol were also prepared (type I collagen immunization only). On the 12th day after the start of immunization, the spleen was removed, and the cells were seeded on 96 well plates with 200,000 cell Z well and each amount (0, 4, 20, 100 μ & ml) of type II collagen for 72 hours. After culture, BrdU was added and further cultured. 17 hours Subsequently, 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. 8 shows the percentage of BrdU incorporation in each group when the amount of BrdU incorporation was 100% when no type II collagen was added during culture (0 gZml).

[0057] As a result, in the group administered with flavopiridol together with type II collagen (Fig. 8, flavopiridol), similar to the group administered with type II collagen alone (Fig. 8, control), type II collagen reactivity T It was shown that the cells increased (Fig. 8).

 This indicates that flavopiridol does not suppress type II collagen-reactive T cell proliferation due to immune responses. Therefore, including the results of Examples 3, 5, and 6, the synovial cell proliferation inhibitor of the present invention does not inhibit lymphocyte activation or proliferation, that is, without causing immune suppression. It was revealed that proliferative arthritis was strongly suppressed. Industrial applicability

[0058] The synovial cell proliferation inhibitor comprising flavopiridol as an active ingredient and the medicinal composition comprising the synovial cell proliferation inhibitor as an active ingredient of the present invention can increase synovial cell proliferation when administered. Anti-rheumatoid, anti-osteoarthritis, anti-psoriatic, anti-rheumatoid arthritis, anti-rheumatoid arthritis, anti-rheumatoid Suitable as arthritis agent and anti-gout agent. In addition, by administering the synovial cell proliferation inhibitor and the medicinal composition, it is possible to prevent or treat diseases in which synovial cell proliferation is involved in pathological conditions without suppressing immunity.

Claims

The scope of the claims  A slipping agent characterized by containing at least one of flavopiridol (flavopiridol or IUPA C name: alvocidib), which is a compound represented by the following structural formula (1), and a pharmaceutically acceptable salt thereof as an active ingredient. Membrane cell growth inhibitor.  [Chemical 3]

 [2] A medicinal composition comprising at least the synovial cell growth inhibitor of claim 1.  [3] 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. [4] The medicinal composition according to claim 2, which is used for the treatment of at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout. [5] A therapeutic method comprising administering at least the synovial cell proliferation inhibitor according to claim 1 and treating at least one of rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and gout.