JP2016216452A - Therapeutic agent or preventive agent for arthrosis or arthritis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel arthrosis therapeutic agent or a preventive agent having therapeutic effect or preventive effect to arthrosis or arthritis.SOLUTION: An therapeutic agent or preventive agent for arthrosis or arthritis contains ascorbic acid derivatives expressed by the following general formula (1) or pharmacologically acceptable salts thereof, or a solvate thereof as an active ingredient [In Formula (1), R denotes an acyl group derived from saturated or unsaturated aliphatic carboxylic acid]. It is preferable that the aliphatic carboxylic acid is lauric acid, myristic acid, palmitic acid, stearic acid, 2-hexyldecanic acid or isostearic acid.SELECTED DRAWING: None

Description

  The present invention relates to a therapeutic or preventive agent for arthropathy or arthritis.

Arthritis or arthritis is a disease related to joints, and an increase in the number of patients with the aging of society is a social problem. Among them, osteoarthritis is a joint disease mainly composed of cartilage degeneration. Aging and mechanical load are known as the cause of osteoarthritis, and cartilage is worn away, resulting in great friction. Injured cartilage tissue also causes inflammation in the synovium in the joint, accumulating joint fluid, and joint pain and impairment of joint function. If this state is left unattended, it progresses chronically, leading to a decline in the quality of life.
Non-steroidal drugs (NSAIDs) having anti-inflammatory and analgesic effects have been used for the treatment of osteoarthritis. In addition to these, treatments for draining joint fluid and injecting protective agents for joint cartilage such as corticosteroids and sodium chondoitin sulfate have been performed. However, the current situation is that a sufficient effect has not been obtained. Although some meta-analysis has been reported so far about the effect of intraarticular injection of hyaluronic acid for knee osteoarthritis, the effect is not clear (Non-patent Documents 1 to 6).

CT Wang, J. Lin, CJ Chang, YT Lin, SM Hou, Therapeutic effects of hyaluronic acid on osteoarthritis of the knee.A meta-analysis of randomized controlled trials.J Bone Joint Surg Am 86-A, 538-545 (2004 ). N. Bellamy et al., Viscosupplementation for the treatment of osteoarthritis of the knee.Cochrane Database Syst Rev, CD005321 (2006). G. H. Lo, M. LaValley, T. McAlindon, D. T. Felson, Intra-articular hyaluronic acid in treatment of knee osteoarthritis: a meta-analysis.JAMA 290, 3115-3121 (2003). A. Modawal, M. Ferrer, H. K. Choi, J. A. Castle, Hyaluronic acid injections relieve knee pain.J Fam Pract 54, 758-767 (2005). J. Arrich et al., Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee: systematic review and meta-analysis.CMAJ 172, 1039-1043 (2005). J. M. Medina, A. Thomas, C. R. Denegar, Knee osteoarthritis: should your patient opt for hyaluronic acid injection? J Fam Pract 55, 669-675 (2006).

  An object of the present invention is to provide a novel therapeutic or preventive agent for arthritis or arthritis.

The inventors of the present invention administered a higher fatty acid ester salt of ascorbic acid-2-phosphate to a model animal of osteoarthritis, which reduced the active oxygen in the chondrocytes of the joint, The present inventors have found that the disease state is improved and have completed the present invention.
That is, the present invention provides the following means.
[1] An arthropathy or arthritis characterized by containing an ascorbic acid derivative represented by the following general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient: A therapeutic or prophylactic agent.
[In the formula (1), R represents an acyl group derived from a saturated or unsaturated aliphatic carboxylic acid. ]
[2] The therapeutic or preventive agent for arthropathy or arthritis according to [1], wherein the aliphatic carboxylic acid is lauric acid, myristic acid, palmitic acid, stearic acid, 2-hexyldecanoic acid or isostearic acid.
[3] The therapeutic or preventive agent for arthritis or arthritis according to [1], wherein the aliphatic carboxylic acid is palmitic acid.
[4] The therapeutic or preventive agent for arthropathy or arthritis according to [1], wherein the aliphatic carboxylic acid is 2-hexyldecanoic acid.
[5] A pharmacologically acceptable salt of the ascorbic acid derivative represented by the general formula (1) or a solvate thereof as an active ingredient, wherein the salt is a Na salt, a K salt, a Mg salt or The therapeutic or preventive agent for arthritis or arthritis according to any one of [1] to [4], which is a Zn salt.
[6] The therapeutic or preventive agent for arthritis or arthritis according to any one of [1] to [5], wherein the arthropathy or arthritis is arthritis and osteoarthritis.
[7] The therapeutic or preventive agent for arthritis or arthritis according to any one of [1] to [6], which is an injection or an external preparation.

  According to the therapeutic agent or preventive agent for arthritis or arthritis of the present invention, arthropathy or arthritis can be treated or prevented.

The results of comparison of cartilage degeneration in the knee joint between mice (Col2Al-Cre; Sod2 ^{fl / fl} , Sod2 cKO) with 12 months old chondrocyte-specific deletion in the Examples and control mice were shown. FIG. It is a figure which shows the result of having compared the state of the cartilage degeneration in a knee joint with a chondrocyte specific Sod2 deficient mouse | mouth when a mechanical stress was induced | guided | derived surgically, and a control mouse | mouth. It is a figure which shows the inhibitory effect of the active oxygen level by administration of the salt of an ascorbic acid derivative in the cultured cell of the cartilage derived from a Sod2 cKO mouse. It is a figure which shows the inhibitory effect of the active oxygen level by administration of the salt of an ascorbic acid derivative in the chondrocyte of the knee joint of a Sod2 cKO mouse | mouth. It is a figure which shows the inhibitory effect of a cartilage degeneration by administration of the salt of an ascorbic acid derivative in the knee joint of a Sod2 cKO mouse | mouth. It is a figure which shows the inhibitory effect of the reactive oxygen level in the chondrocyte of a wild type mouse | mouth knee joint by administration of the salt of an ascorbic acid derivative. It is a figure which shows the effect regarding the cell number increase of a chondrocyte cell line by administration of the salt of an ascorbic acid derivative. It is a figure which shows the effect regarding the increase in intracellular ascorbic acid amount by administration of the salt of an ascorbic acid derivative to a human fibroblast cell line. It is a figure which shows the effect regarding the increase in intracellular ascorbic acid amount by administration of the salt of an ascorbic acid derivative to a human fibroblast cell line.

  The therapeutic or prophylactic agent for arthritis or arthritis of the present invention is an ascorbic acid derivative represented by the following general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient. contains.

[In the formula (1), R represents an acyl group derived from a saturated or unsaturated aliphatic carboxylic acid. ]

<Ascorbic acid derivative>
First, the ascorbic acid derivative according to the present invention will be described.
The ascorbic acid derivative according to the present invention is a compound represented by the general formula (1). The ascorbic acid derivative according to the present invention is a compound in which phosphoric acid is ester-bonded to the 2-position hydroxyl group of ascorbic acid and an aliphatic carboxylic acid is ester-bonded to the 6-position hydroxyl group.
In the general formula (1), R is an acyl group derived from a saturated or unsaturated aliphatic carboxylic acid, and more specifically, OH bonded from the aliphatic carboxylic acid to the carbonyl carbon of the aliphatic carboxylic acid. Is excluded. Derived from a saturated or unsaturated aliphatic carboxylic acid or derived from a saturated or unsaturated aliphatic carboxylic acid means that the aliphatic carboxylic acid forms an ester bond with the hydroxyl group at the 6-position of ascorbic acid. It means that the structural change necessary for The aliphatic carboxylic acid may be linear or branched, and is preferably an aliphatic monocarboxylic acid, more preferably a saturated aliphatic carboxylic acid, and more preferably a saturated aliphatic monocarboxylic acid. preferable.

The aliphatic carboxylic acid preferably has 5 to 30 carbon atoms, more preferably 10 to 20 carbon atoms, and still more preferably 14 to 18 carbon atoms.
Preferable examples of the aliphatic carboxylic acid having 5 to 30 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, 2-hexyldecanoic acid, and isostearic acid. Of these, palmitic acid and 2-hexyldecanoic acid are more preferable.

  The ascorbic acid derivative according to the present invention has improved stability due to the ester linkage of phosphoric acid to the hydroxyl group at the 2-position, and improved cell permeability due to the ester linkage of aliphatic carboxylic acid to the hydroxyl group at the 6-position. It is considered that an excellent therapeutic or preventive effect for arthropathy or arthritis is exhibited.

An example of the ion of the ascorbic acid derivative according to the present invention includes an anion represented by the following formula (2).
Examples of the pharmacologically acceptable salt of the ascorbic acid derivative represented by the general formula (1) according to the present invention include a salt of an anion and a counter ion represented by the following formula (2).

As said counter ion, a metal ion is preferable and an alkali metal ion is more preferable.
The salt of the ascorbic acid derivative according to the present invention is not particularly limited as long as it is a pharmacologically acceptable salt, and specifically, Na salt, K salt, Ca salt, Mg salt, Zn salt and the like are preferable. Among these, Na salt is more preferable.

  The solvate of the ascorbic acid derivative or pharmacologically acceptable salt thereof according to the present invention is not particularly limited as long as it is a pharmacologically acceptable solvate, and is a hydrate or an organic solvate. Etc.

  The ascorbic acid derivative represented by the general formula (1) can be produced by phosphorylating the 2-position hydroxyl group of ascorbic acid and then esterifying the 6-position hydroxyl group with an aliphatic carboxylic acid. As the palmitate body, commercially available Apleshier (registered trademark) manufactured by Showa Denko KK can be used as a raw material.

  The ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof is one of the therapeutic or preventive agents for arthropathy or arthritis of the present invention. It may be contained independently and may be contained in combination of 2 or more types.

  As long as the therapeutic agent or preventive agent for arthritis or arthritis of the present invention is a therapeutic agent for arthritis or arthritis, its purpose of administration, administration subject, administration route, administration method, dosage form, and amount of active ingredient Further, the dosage, the administration interval, etc. are not particularly limited.

  The administration route of the therapeutic agent or prophylactic agent of the present invention is not particularly limited as long as the above purpose is achieved. Examples of the administration method include injection (intra-articular cavity, intravenous, intramuscular, subcutaneous, intradermal, abdominal cavity). , Intrathoracic, intraspinal, etc.), infusion, oral, transdermal, inhalation, enema, transrectal, nasal, instillation, nasal drop, ear drop, application to mucous membrane, etc. The administration method can be appropriately selected depending on the site where treatment or the like is desired. Among the above administration route methods, administration to a specific site by injection or application administration is preferable, administration to a specific site by injection is more preferable, and direct administration into the joint cavity by injection is particularly preferable.

Examples of the dosage form of the therapeutic agent or prophylactic agent of the present invention include liquids (solutions, suspensions, emulsions, solid agents for dissolution when used), dispersion preparations, semi-solid preparations, granular preparations, molded preparations, and leaching. Specific examples include tablets, coated tablets, dragees, pills, troches, hard capsules, microcapsules, liposizing agents, implants, powders, powders, granules, fine granules , Injection, liquid, elixir, emulsion, syrup, water, emulsion, suspension, liniment, lotion, aerosol, spray, inhalant, spray, ointment, plaster, patch , Pasta, cataplasm, tape, gel, cream, oil, suppository, tincture, skin solution, eye drops, nasal drop, ear drops, coating agent, infusion solution, lyophilized formulation, injection Powders for functional agents, functional foods and the like.
Among these dosage forms, from the viewpoint that an excellent effect on arthritis or arthritis is easily exerted, the dosage form of the therapeutic agent or prophylactic agent of the present invention is for external use such as an injection, a coating agent, a patch and the like. It is preferable that the dosage form of the therapeutic agent or prophylactic agent of the present invention is an injection from the viewpoint that an excellent effect on arthritis or arthritis is easily exhibited. The injection may be a liquid or a gel.

  The injection is preferably an injection used for injection into the joint cavity. As shown in the examples described later, the ascorbic acid derivative represented by the general formula (1) has a cell growth promoting effect on chondrocytes. The compound injected into the joint cavity is effectively taken into fibroblasts, chondrocytes, etc., and is excellent in effect against arthropathy or arthritis.

The liquid agent is, for example, an ascorbic acid derivative represented by the above general formula (1) or a pharmacologically acceptable salt thereof as an active ingredient in a suitable aqueous solvent or a solvent commonly used in the pharmaceutical field, Can be produced by dissolving the solvate. Examples of such a solvent include distilled water, water for injection, buffer solution, physiological saline, water containing a water-miscible organic solvent, and the like.
The gel can be produced, for example, by adding a gelling agent to the liquid to form a gel.
When provided as an injection, the form may be any of a solution, a frozen product, a dried product, a lyophilized product, and the like. This can be filled and sealed in an appropriate container such as an ampoule, vial, injection syringe, etc., distributed or stored as it is, and provided as an injection for administration.

  A known method can be used for formulating the therapeutic agent or prophylactic agent of the present invention. In preparation of the preparation, other drugs may be used as long as they do not adversely affect the action of the ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof. Active ingredients (for example, anti-inflammatory agents, analgesics, vitamins, antibacterial agents, growth factors, adhesion factors, etc.), stabilizers, emulsifiers, osmotic pressure adjusting agents, pH adjusting agents, buffering agents, isotonic agents, Components commonly used in the pharmaceutical field such as preservatives, soothing agents, coloring agents, excipients, binders, lubricants, disintegrating agents and the like can be used.

  The ascorbic acid derivative represented by the general formula (1) in the preparation or a pharmacologically acceptable salt thereof, or the blending amount of the solvate, the dose per administration, the administration interval, etc. , Subject of administration, administration route, administration method, dosage form, specific symptoms of patient, age, sex, body weight, etc., and should not be specifically limited.

  In the case of direct administration into the joint cavity by injection, the concentration of the ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof or a solvate thereof in the injection is 0. 0.0001 to 30% (w / v) is preferable, 0.001 to 10% (w / v) is more preferable, and 0.01 to 3% (w / v) is more preferable. When the ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof or a solvate thereof is contained in an injection at such a concentration, arthritis or arthritis This is preferable because the efficacy effect required for a therapeutic agent or a preventive agent is more effectively exhibited.

In the case of direct administration into the joint cavity by injection, the ascorbic acid derivative represented by the above general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof is administered once per adult. 0.000005 to 1.5 g is preferably administered, 0.00005 to 0.5 g is more preferably administered, and 0.0005 to 0.2 g is more preferably administered.
In addition, the treatment agent of the present invention may be administered once or multiple times. When administered multiple times, it may be administered every day, or may be administered at intervals of about 1 day to 3 months. Moreover, it may be administered once a day, or may be divided into 2 to 3 times a day.

In the present invention, the therapeutic agent for arthritis or arthritis is used in a meaning including those that have a curative effect, an improvement effect, a reduction effect, or a maintenance effect (aggravation prevention effect) for arthropathy or arthritis. In the present invention, the prophylactic agent for arthritis or arthritis is used in a sense including those that preventively exert the effect. The effect on arthropathy or arthritis can be evaluated based on medical or pathological indicators.
For example, an ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or an administration population of an animal administered with a solvate thereof, and the general formula (1) A cartilage degeneration score (pathological index) of articular cartilage in a non-administered population of an animal to which the ascorbic acid derivative represented or the pharmacologically acceptable salt thereof, or a solvate thereof is not administered calculate. Here, the cartilage degeneration score indicates that the higher the score, the more the cartilage is denatured. When the cartilage degeneration score of the administration group is significantly lower than the cartilage degeneration score of the non-administration group, the ascorbic acid derivative or a pharmacologically acceptable salt thereof, or a solvation thereof It can be judged that a thing has the effect of the treatment effect, improvement effect, reduction effect, or maintenance effect (deterioration prevention effect) with respect to arthropathy or arthritis.
As for the preventive effect, the symptoms of arthritis or arthritis that may occur after administration of the ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof. Judging about

  In addition, for example, an ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or an administration cell group of cells administered with a solvate thereof, and the general formula (1) The number of proliferated cells is counted with a non-administered cell group of cells not administered with the ascorbic acid derivative represented by the formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof. When the cell proliferation of the administration cell group is significantly higher than the cell proliferation of the non-administration cell group, the ascorbic acid derivative or a pharmacologically acceptable salt thereof, or a solvate thereof is It can be determined that the therapeutic effect, the improvement effect, the reduction effect, or the maintenance effect (deterioration prevention effect) for arthropathy or arthritis is obtained. The cells are preferably chondrocytes or fibroblasts.

  Examples of joint sites related to arthropathy or arthritis include knee joints, shoulder joints, elbow joints, hip joints, finger joints, toe joints, temporomandibular joints, spinal joints, and intervertebral discs. Not limited.

Examples of the arthropathy include osteoarthritis such as knee osteoarthritis, hip osteoarthritis, osteoarthritis, osteoarthritis, and neurogenic arthropathy such as Charcot joint, Examples include traumatic arthropathy (meniscus injury, anterior cruciate ligament injury, lateral collateral ligament injury, medial collateral ligament injury, patella tendon injury) and temporomandibular arthrosis due to trauma or surgery.
Examples of arthritis include rheumatoid arthritis, tuberculous arthritis, periarthritis (fifty shoulders, calcified tendonitis, Achilles tendonitis, bursitis, subpatellar panniculitis), and other gouty arthritis Can be mentioned.
The arthropathy or arthritis according to the present invention is preferably a disease accompanied by damage or degeneration of articular cartilage. Among the above-mentioned arthropathy or arthritis, osteoarthritis is more preferable, and knee osteoarthritis is more preferable.

In one embodiment, the present invention provides an arthrosis comprising a step of administering an ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof to a mammal. Alternatively, a method for treating or preventing arthritis is provided.
In one embodiment, the present invention comprises a step of administering an ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof to a non-human mammal. A method for treating or preventing arthritis or arthritis is provided.
In one embodiment, the present invention includes a step of injecting an ascorbic acid derivative represented by the general formula (1) or a pharmaceutically acceptable salt thereof, or a solvate thereof into a joint cavity of a mammal. A method for treating or preventing arthritis or arthritis is provided.
In one embodiment, the present invention provides an injection of an ascorbic acid derivative represented by the general formula (1) or a pharmaceutically acceptable salt thereof, or a solvate thereof into a joint cavity of a non-human mammal. And a method for treating or preventing arthritis or arthritis.

  In one embodiment, the present invention provides an ascorbic acid derivative represented by the above general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof for the treatment or prevention of arthropathy or arthritis. Offer things.

  In one embodiment, the present invention provides an ascorbic acid derivative represented by the general formula (1) or a pharmacologically acceptable salt thereof for producing a therapeutic or preventive agent for arthritis or arthritis, or Use of these solvates is provided.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited at all by these Examples.

<Experimental example 1> Effect of Sod2 deficiency in chondrocytes on cartilage degeneration Sod2 (superoxide dismutase 2) is known as an enzyme that degrades active oxygen in mitochondria. The inventors created a mouse (Col2Al-Cre; Sod2fl ^{/ fl} , Sod2cKO) in which the Sod2 gene was deficient in a chondrocyte-specific manner using the cre-loxP system. To confirm whether the Sod2 deficiency promotes cartilage degeneration due to aging, a 12-month-old Sod2 cKO mouse and a 12-month-old Sod2 ^{fl / fl} mouse (control mouse) as a control were used. Histological analysis was performed and the results were compared. At 12 months of age, there was no significant difference in body weight and motor function between Sod2 cKO mice and control mice.
The results are shown in FIG. The histological analysis was digitized based on the OARSI score (Glasson et al. “The OARSI histopathology initiative e recommendations for histological assessments of osteoarthritis in the mouse.” Osteoarthritis Cartilage 18, 17-23 (2010)). The OARSI score is shown in Table 1. The Sod2 cKO mouse joint showed a significant loss of the safranin O-stained portion showing the chondrocyte portion compared to the knee joint of the control mouse. Among the joints, the analysis results for the femoral condyle (MFC) and the medial tibial plateau (MTP) are shown in FIG. 1 [In FIG. 1, each scale bar in the micrograph represents 1 mm. In FIG. 1, the values in the graph indicate mean values ± standard deviation (n = 4), and ** P <0.01 and *** P <0.001 (vs. control, t-test)]. The MFC and MTP of Sod2 cKO mice showed a significant decrease in safranin O staining cartilage tissue components compared to control mice.
From these results, it was shown that, in mice lacking the Sod2 gene specifically in cartilage, cartilage degeneration of the knee joint is spontaneously promoted with aging.

<Experimental example 2> Effect on the cartilage degeneration induced by Sod2-deficient chondrocytes in the presence of mechanical load The inventors have examined whether cartilage degeneration is promoted by applying mechanical load to articular cartilage without Sod2 Based on previous reports (SS Glasson, TJ Blanchet, EA Morris, The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129 / SvEv mouse. Osteoarthritis Cartilage 15, 1061-1069 (2007)) Surgery of the media meniscus (DMM) that destabilizes the medial meniscus was performed on the knees of 8 week old control mice and Sod2 cKO mice. DMM is an operation that destabilizes the medial meniscus by cutting off the medial meniscotibial ligament (MMTL). The histological analysis was digitized based on the aforementioned OARSI score. The analysis results for the femoral condyle (MFC) and the medial tibial plateau (MTP) are shown in FIG. 2 [In FIG. 2, each scale bar in the upper micrograph represents 200 μm, and each scale bar in the middle and lower micrographs. Represents 20 μm. The middle and lower photos are enlarged images of the upper photo. In FIG. 2, the values in the graph indicate the mean value ± standard deviation (n = 5), and indicate * P <0.05 and *** P <0.001 (vs. control, t-test)]. In control mice in which the medial meniscus was destabilized, a decrease in safranin O staining was observed up to the tidemark level at 8 weeks postoperatively, but no structural failure was observed. On the other hand, in the mouse articular cartilage without Sod2 that destabilized the inner meniscus, a remarkable defect of the cartilage layer was recognized up to the tide mark level.
From these results, it was clarified that in mice deficient in the Sod2 gene specifically in cartilage, cartilage degeneration induced by mechanical load is accelerated.

<Experimental Example 3> Administration of APPS to Sod2 cKO Chondrocytes Vitamin C is known as an antioxidant having an antioxidant action. The effect of ascorbyl phosphate 3Na (APPS), which is a salt of a vitamin C derivative, to weaken the active oxygen level in chondrocytes was examined. Chondrocytes were isolated and cultured from the Sod2 cKO mice. APPS was administered to Sod2 cKO chondrocytes and cultured in the presence of APPS at a final concentration of 10 μM for 24 hours. As a control, chondrocytes were isolated and cultured from Sod2 ^{fl / fl} mice in the same manner, and cultured for 24 hours in the presence of APPS at a final concentration of 10 μM. The chondrocytes after 6 days of culture were stained with dihydroethidium (DHE), and the amount of active oxygen in the chondrocytes of each mouse was measured using flow cytometry. The results are shown in FIG. 3. [In FIG. 3, the values in the graph indicate mean ± standard deviation (n = 4), and ** P <0.01 (vs. control, t-test)]. It was found that administration of APPS can suppress mitochondrial reactive oxygen levels in Sod2 cKO chondrocytes.

<Prescription Example 1> Preparation of therapeutic or preventive agent for arthritis or arthritis Each component was mixed according to the following formulation to produce an injection containing a salt of an ascorbic acid derivative according to the present invention.
・ Injection 1
Phosphate buffered saline (PBS) 100 mL
Ascorbyl palmitate 3Na (APPS) (Apressie (registered trademark) Showa Denko) 1g

Administration of APPS to knee joint of mouse with Sod2 cKO <Experimental example 4>
It was verified whether APPS can suppress the mitochondrial active oxygen also in vivo. DMM surgery was performed on the knee joint of an 8-week-old Sod2 cKO mouse. At 2 weeks after the DMM operation, 20 μL each of the injection of the above-mentioned Formulation Example 1 or PBS was injected into the joint space on the operation side (DMM side). Twenty-four hours after joint injection, articular cartilage was isolated from the tibial plateau of each mouse knee joint. Mitochondrial reactive oxygen in chondrocytes was quantified by flow cytometry after DHE staining. The results are shown in FIG. 4 [in FIG. 4, the values in the graph are mean values ± standard deviation (n = 5), * P <0.05, ** P <0.01 (vs. control, t-test). ]]. Administration of 1% (w / v) APPS to the knee joint cavity resulted in Sod2
It became clear that the mitochondrial active oxygen level of articular cartilage of cKO mice was suppressed.

<Experimental example 5>
In order to evaluate the therapeutic effect of APPS on knee cartilage, the following experiment was conducted. An 8-week-old Sod2 cKO mouse was subjected to DMM surgery, and 20 μL of the injection of Formulation Example 1 was injected into the joint space on the DMM side once a week from 1 to 8 weeks after the DMM surgery. As a control, the same experiment was conducted except that PBS was injected in place of the injection of Formulation Example 1 into the joint space on the DMM side of Sod2 cKO mice. The results of histological analysis are shown in FIG. Moreover, the result of having digitized the histological analysis result based on the OARSI score is shown in the graph of FIG. 5 [in FIG. 5, the value of the graph shows the average value ± standard deviation (n = 5-7), * P < 0.05 (vs. control, t-test)]. It became clear that administration of 1% (w / v) APPS to the knee joint cavity suppressed the degeneration of articular cartilage in Sod2 cKO mice.

<Experimental Example 6> APPS Administration to Knee Joints of Wild Type Mice Wild type mice were subjected to DMM surgery, and after 2 weeks of DMM surgery, the injection of Formulation Example 1 was placed in the joint space on the DMM side. 20 μL was injected. After 24 hours from the injection, the chondrocytes were collected from the DMM side joint of the wild type mouse. As a control, cartilage was collected from the knee joint of a wild type mouse that had not undergone DMM surgery. In addition, as a control, a similar experiment was performed except that PBS was injected into the joint space on the DMM side of the wild-type mouse instead of the injection of Formulation Example 1, and cartilage was collected from the knee joint of the mouse. .
The amount of mitochondrial active oxygen in the cells of the tibial plateau cartilage of each mouse was measured by flow cytometry after DHE staining. The results are shown in FIG. 6 [in FIG. 6, the values in the graph indicate mean ± standard deviation (n = 3), * P <0.05 (vs. control, t-test)]. It was revealed that administration of 1% (w / v) APPS to the knee joint cavity suppressed the mitochondrial reactive oxygen level in articular chondrocytes of wild-type mice.

<Medium preparation examples 1 to 3>
Each component was mixed by the prescription shown below, and the culture medium containing the salt of the ascorbic acid derivative based on this invention was manufactured.
-Medium 1
DMEM / F12 + ITS (insulin-transferrin-selenite) (manufactured by Gibco by life technologies, DMEM / F12: 1750070 ITS: 4140045)
Ascorbyl palmitate 3Na (APPS) (Apressie (registered trademark) manufactured by Showa Denko KK) Final concentration 10 μmol / L
Medium 2
DMEM / F12 + ITS
Ascorbyl palmitate 3Na final concentration 30 μmol / L
-Medium 3
DMEM / F12 + ITS
Ascorbyl palmitate 3Na final concentration 40 μmol / L

<Comparison Medium Preparation Examples 1-3>
Each component was mixed by the prescription shown below, and the culture medium for comparison was manufactured.
Comparative medium 1
DMEM / F12 + ITS (insulin-transferrin-selenite) (manufactured by Gibco by life technologies, DMEM / F12: 1750070 ITS: 4140045)
L-ascorbic acid (manufactured by SIGMA-ALDRICH, L-Ascorbic acid # SLBG5136V) final concentration 10 μmol / L
Comparative medium 2
DMEM / F12 + ITS
L-ascorbic acid final concentration 30 μmol / L
Comparative medium 3
DMEM / F12 + ITS
L-ascorbic acid final concentration 40 μmol / L

<Experimental example 7> Administration of APPS to chondrocyte cell line The cells of ATDC5 chondrocyte cell line were placed in wells containing the above mediums 1 to 3 or comparative mediums 1 to 2 at 10 ⁵ cells / ml per well. Sowing. The cell number was measured 24 hours after seeding.
The results are shown in FIG. 7. [In FIG. 7, the values in the graph indicate mean value ± standard deviation (n = 3) and * P <0.05 (t test)]. Compared with the medium supplemented with ascorbic acid (AA), the number of cells significantly increased in the chondrocytes cultured in the medium supplemented with APPS.

<Experimental Example 8> Administration of APPS to fibroblasts Human fibroblasts (TIG118) were cultured for 1 hour in the above-mentioned DMEM / F12 + ITS medium, medium 1 or comparative medium 1, and ascorbic acid (AA) in the cells The amount was measured.
The results are shown in FIG. 8. [In FIG. 8, the values in the graph indicate mean ± standard deviation (n = 4), * P <0.05 (t test)]. In FIG. 8, “Ctrl.” Represents the above-mentioned DMEM / F12 + ITS medium, “APPS” represents the above-mentioned medium 1 to which APPS was added, and “AA” represents the above-mentioned medium to which AA was added. This represents Comparative Medium 1. In addition, the molecular weight of AA is 176.12, and the molecular weight of APPS is 560. Compared to the AA-added medium, the amount of intracellular AA was significantly (approximately 1.8 times) higher in human fibroblasts cultured in the APPS-added medium.

<Experimental Example 9> Administration of Ascorbic Acid Transporter Inhibitor and APPS to Fibroblasts Human fibroblasts (TIG118) were treated with PMA [AA transporter inhibitor (final concentration: 10 mM Phorbol 12) using the above DMEM / F12 + ITS medium. -Myristate 13-acetate)] and / or Glc [glucose (final concentration 10 mM Glucose)] for 1 hour. Thereafter, AA or APPS was added to a final concentration of 10 mM, followed by further culturing for 1 hour, and the amount of intracellular AA was measured. The final contents of AA, APPS, PMA, and Glc in each medium after addition of AA or APPS are nine combinations shown in FIG.
The results are shown in FIG. 9 [in FIG. 9, the values in the graph indicate mean ± standard deviation (n = 4), * P <0.05, and ** P <0.01 (t test). ]. The increase in the amount of intracellular AA due to the addition of AA was significantly suppressed by the inhibitor pretreatment. On the other hand, the increase in intracellular AA amount due to the addition of APPS was not suppressed by inhibitor pretreatment. From this, it can be concluded that APPS can increase the amount of intracellular AA independently of the AA transporter.

  From the results shown in the above Experimental Examples 7 to 9, it was shown that APPS has an advantage in increasing the amount of intracellular AA over AA. The method of directly injecting, applying or sticking the therapeutic agent or prophylactic agent of the present invention to an affected area as an external preparation such as an injection, a coating agent, a patch, etc. is very useful as a method for increasing the intracellular AA amount in a local tissue. It became clear that.

Claims (7)

A therapeutic agent for arthritis or arthritis, comprising ascorbic acid derivative represented by the following general formula (1) or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient: Preventive agent.
[In the formula (1), R represents an acyl group derived from a saturated or unsaturated aliphatic carboxylic acid. ]   The therapeutic or preventive agent for arthritis or arthritis according to claim 1, wherein the aliphatic carboxylic acid is lauric acid, myristic acid, palmitic acid, stearic acid, 2-hexyldecanoic acid or isostearic acid.   The therapeutic or preventive agent for arthropathy or arthritis according to claim 1, wherein the aliphatic carboxylic acid is palmitic acid.   The therapeutic or preventive agent for arthritis or arthritis according to claim 1, wherein the aliphatic carboxylic acid is 2-hexyldecanoic acid.   A pharmacologically acceptable salt of the ascorbic acid derivative represented by the general formula (1) or a solvate thereof is contained as an active ingredient, and the salt is a Na salt, a K salt, a Mg salt, or a Zn salt. The therapeutic agent or preventive agent of the arthropathy or arthritis as described in any one of Claims 1-4.   The therapeutic or preventive agent for arthropathy or arthritis according to any one of claims 1 to 5, wherein the arthropathy or arthritis is arthropathy and is osteoarthritis.   It is an injection or an external preparation, The therapeutic agent or preventive agent of the arthropathy or arthritis as described in any one of Claims 1-6.