JP5756498B2 - Osteoarthritis prevention and treatment - Google Patents

Description

  The present invention relates to a novel preventive and therapeutic agent for osteoarthritis.

  Osteoarthritis is an arthropathy that occurs when the cartilage or meniscus, which acts as a cushion for joints, is worn and deformed little by little over a long period of time. Typical risks include aging, obesity, intense exercise, and poor posture. Is a factor. Treatment includes exercise therapy, surgical therapy, and drug therapy. As pharmacotherapy, intraarticular injection of hyaluronic acid and its modified form (Patent Documents 1 to 3), chondroitin sulfate production promoter (Patent Document 4), administration of anti-inflammatory analgesic and the like have been reported.

Japanese Patent Publication No. 5-74571 International Publication No. 00/53194 JP 2007-291133 A JP 2009-274955 A

However, conventional osteoarthritis preventive and therapeutic drugs are symptomatic treatments or temporarily supplemented with cartilage components, and do not regenerate cartilage components over a long period of time.
Accordingly, an object of the present invention is to provide a novel preventive and therapeutic agent for osteoarthritis that can regenerate the cartilage component of the joint over a long period of time.

Accordingly, the present inventors have made various studies paying attention to actions such as promotion of production of cartilage components and inhibition of ossification or degradation of cartilage tissue. As a result, 3′-phosphoadenosine-5 ′, which is an in vivo sulfate group donor, has been studied. -Phosphosulfate or a salt thereof significantly improves the suppression of joint extension angle in a meniscal osteoarthritis model, and quite unexpectedly promotes the production of type II collagen, which is one of the cartilage components, In addition, the inventors have found that the production of a signal transduction factor of Notch pathway involved in ossification and degradation of cartilage tissue has been found, and the present invention has been completed.

That is, the present invention provides a preventive and / or therapeutic drug for osteoarthritis comprising 3′-phosphoadenosine-5′-phosphosulfate or a salt thereof as an active ingredient.
The present invention also provides a type II collagen production promoter containing 3′-phosphoadenosine-5′-phosphosulfate or a salt thereof as an active ingredient.
Furthermore, the present invention provides a Notch signaling factor production inhibitor comprising 3′-phosphoadenosine-5′-phosphosulfate or a salt thereof as an active ingredient.

  According to the present invention, administration of 3′-phosphoadenosine-5′-phosphosulfate (hereinafter referred to as PAPS) or a salt thereof promotes the production of aggrecan which is a kind of cartilage component of joints, chondroitin sulfate proteoglycan. Moreover, as a result of promoting the production of type II collagen and suppressing the production of Notch signaling factor in cartilage tissue, the symptoms of osteoarthritis can be remarkably improved. Furthermore, osteoarthritis prophylaxis and / or treatment drugs that simultaneously have the two actions of promoting the production of cartilage components such as aggrecan and type II collagen and inhibiting the ossification and degradation of cartilage tissue have been conventionally achieved. It was something that was not known.

The effect of PAPS with respect to the joint extension angle in an osteoarthritis model rat is shown. The effect of PAPS on the expression of aggrecan is shown. The effect of PAPS on the expression of type II collagen is shown. Figure 2 shows the effect of PAPS on the expression of Notch signaling factor ( Hes 1). Figure 6 shows the effect of PAPS on the expression of Notch signaling factor ( Jag 1).

  The active ingredient of the osteoarthritis preventive and / or therapeutic agent of the present invention is 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or a salt thereof. PAPS is an in vivo sulfate group donor and can be produced by the methods described in International Publication No. 2006/080313, Japanese Patent Application Laid-Open No. 2002-78498, and the like. Moreover, the stable salt is disclosed by international publication 2007/058278. The PAPS is known to be useful as a hair restoring agent (International Publication No. 2012/057336), a moisturizing agent (Japanese Patent Laid-Open No. 2012-201665), etc., but no effect on cartilage components has been reported. . Examples of the salt of PAPS include metal salts such as sodium salts, ammonium salts, various amine compound salts, amino acid salts, imidazole salts, and the like, and in particular, trimethylamine, triethylamine, triethanolamine and the like described in International Publication No. 2007/058278. The amine compound salt is preferred.

  As shown in Examples below, PAPS improved the suppression of joint extension angle in a dose-dependent manner in the evaluation using a meniscal resection osteoarthritis model. Therefore, PAPS has an effect of improving various symptoms of osteoarthritis, such as joint pain, joint movement and the like.

  In cultured articular cartilage cells, PAPS had an effect of promoting the production of aggrecan, a chondroitin sulfate proteoglycan, and an effect of enhancing the expression of type II collagen gene. In addition to the cartilage component proteoglycan, PAPS also promotes the production of protein type II collagen.

PAPS suppressed the expression of Notch pathway signaling factors that promoted the deterioration of osteoarthritis pathology in chondrocytes. Notch signal is a signal pathway that promotes the ossification of cartilage tissue and the expression of cartilage tissue degrading enzymes such as aggrecanase (PNAS Vol.110, No.5 p.1875-1880). Specific examples of Notch signaling factors Examples include Notch ligands (Delta-like 1, 3, 4, Jagged-1 and 2), Notch receptors (Notch 1 to 4), Notch target genes (Hes, Myc, p21 family) and the like. It is known that Jagged-1 ( Jag 1) examined in this test is a gene related to activation of Notch signal, and Hes1 is related to ossification of cartilage tissue and induction of cartilage tissue degrading enzyme. Cartilage ossification and cartilage tissue degradation are both important factors causing osteoarthritis. On the other hand, it can be seen that administration of PAPS can suppress the expression of signal transduction factors related to these, and consequently has an effect of suppressing damage to the cartilage tissue.

  That is, PAPS or a salt thereof (1) has an effect of promoting the production of structural components of cartilage tissue itself (proteoglycan such as aggrecan and type II collagen), and (2) suppresses ossification of cartilage and cartilage tissue degradation. It was found that the mechanism of effect (Notch signal related) is highly likely to improve the symptoms of osteoarthritis.

  The osteoarthritis preventive and / or therapeutic agent, type II collagen production promoter, and Notch signaling factor production inhibitor of the present invention can be used as a pharmaceutical product for mammals including humans.

  The administration method of the medicament of the present invention is preferably by injection. The dosage is preferably about 0.001 to 0.5 μmol / kg per day as PAPS. The number of administration is preferably about 1 to 4 times a week.

In the present invention, when PAPS or a salt thereof is administered by injection, an aqueous injection, an aqueous suspension injection, a fat emulsion, a liposome injection and the like are preferable.
In aqueous injections and aqueous suspension injections, PAPS or a salt thereof is mixed with purified water, and if necessary, a water-soluble or water-swellable polymer, a pH adjusting agent, a surfactant, an osmotic pressure adjusting agent, Preservatives, preservatives and the like are added, mixed, dissolved or suspended while heating as necessary, sterilized, filled and sealed in an injection container, and made into an aqueous injection or aqueous suspension injection. Aqueous injections can be administered intravenously, subcutaneously, intramuscularly, intradermally, intraarticularly, and the like. Aqueous suspension injections can be administered subcutaneously, intramuscularly, intradermally, intraarticularly, and the like.
As water-soluble or water-swellable polymers, gelatin, cellulose derivatives, acrylic acid derivatives, povidone, macrogol, polyamino acid derivatives, and polysaccharides are preferable. Purified gelatin is used for gelatins, and methylcellulose and hydroxypropylmethylcellulose are used for cellulose derivatives. 2910, hydroxypropylmethylcellulose 2208, hydroxypropylmethylcellulose 2906, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, carmellose sodium, acrylic acid derivatives, aminoacryl methacrylate copolymers, methacrylic acid copolymers, polyamino acid derivatives, Polylysine and polyglutamic acid are preferred. As the polysaccharide, hyaluronic acid, dextran, and dextrin are particularly preferable. The amount of the water-soluble or water-swellable polymer added varies depending on the amount of PAPS or its salt, and the nature, molecular weight, and application site of the water-soluble or water-swellable polymer, but is generally 0.01% to the total amount of the preparation. It can be used in the range of 10% by mass.
An acid or alkali that is harmless to the human body is used as the pH adjuster, and a nonionic surfactant, an anionic surfactant, or an amphoteric surfactant is used as the surfactant. Examples of the osmotic pressure regulator include sodium chloride and glucose, examples of the preservative include parabens, and examples of the preservative include ascorbic acid and sulfites. Although there are no particular limitations on the amount of these used, it is used in a range where the action can be exhibited. If necessary, a local anesthetic such as procaine hydrochloride, a soothing agent such as benzyl alcohol, a chelating agent, a buffering agent, or a water-soluble organic solvent may be added.
For example, a fat emulsion is prepared by adding an emulsifier and PAPS or a salt thereof to an appropriate oil and fat, adding purified water, and if necessary, a water-soluble or water-swellable polymer, a pH adjusting agent, a surfactant, an osmotic pressure adjusting agent. It can be prepared by adding preservatives, preservatives, etc., emulsifying with a suitable emulsifying device, sterilizing and filling and sealing the injection container. Fat emulsions can be administered intravenously, subcutaneously, intramuscularly, intradermally, intraarticularly, and the like.
The fats and oils used are preferably vegetable oils or synthetic fats and oils such as soybean oil, corn oil, cocoon oil, sesame oil, cottonseed oil, safflower oil, medium chain fatty acid triglyceride, and isopropyl myristate. As the emulsifier, a nonionic surfactant, an anionic surfactant, an amphoteric surfactant, and a fatty acid can be used. Water-soluble or water-swellable polymers, pH adjusters, surfactants, osmotic pressure adjusters, preservatives, preservatives and the like can be used.
Liposome injection is prepared by, for example, dissolving lipids such as phosphatidylcholine, phosphatidylethanolamine, sphingolipid, and cholesterol in a suitable organic solvent, placing them in a flask, and removing the organic solvent with a rotary evaporator. Form a film. An aqueous solution in which esculetin, a derivative thereof or a pharmacologically acceptable salt thereof is dissolved is added thereto, and if necessary, a water-soluble or water-swellable polymer, a pH adjuster, a surfactant, an osmotic pressure adjuster, an antiseptic It can be prepared by adding an agent, a preservative, etc., sterilizing an emulsion obtained by vigorous shaking and stirring, and filling and sealing it in an injection container. The liposome preparation can be administered intravenously, subcutaneously, intramuscularly, intradermally, intraarticularly, or the like.

  EXAMPLES Next, an Example is given and this invention is demonstrated in detail.

(Example 1) Osteoarthritis ameliorating action in osteoarthritis model rats The animals were raised by free feeding (solid feed CE-2, Nippon Claire), automatic water supply, and 12-hour light-dark cycle. SD rats (produced by CLEA Japan) (male, 6-7 weeks old) acclimated until 14 weeks of age under free feeding (solid feed CE-2, Japan CLEA), automatic water supply, and 12-hour light-dark cycle environment did. After pentobarbital was dissolved in physiological saline and anesthetized by intraperitoneal injection into rats at a volume of 100 mg / kg, the body hair around the right hind knee joint was shaved, sterilized with 70% ethanol, and the medial meniscus Was excised. Antibiotics (0.5 mg / ml sulfomethoxazole, 0.1 mg / ml trimethoprim) were mixed with drinking water and ingested every other day for one week after excision of the inner half-moon. From 11 days after excision of the medial meniscus, the following four kinds of test substances prepared by dissolving in physiological saline were administered once by 50 μl, twice a week for 4 weeks by intra-articular injection. The first group (n = 11) received physiological saline as a control. The second group (n = 12) was administered with 0.1 mM PAPS, 4Na. The third group (n = 13) received 1 mM PAPS, 4Na. Group 4 (n = 10) received 0.5 mg / ml hyaluronic acid as a positive control. After the administration period, an excessive amount of pentobarbital anesthesia was performed and the mouse was euthanized. Then, the right hind limb was excised, the muscle was removed without damaging the joint follicle, and the joint extension angle of the knee was measured. The measurement results of knee joint extension angles are shown in FIG.

  While the average extension angle of the left hind limb knee joint without meniscus resection was 152 °, the extension angle of the right hind knee joint of the first group was 139 °, which was caused by osteoarthritis. It was confirmed that the operating range of was limited. The operating ranges of the second group (0.1 mM PAPS group) and the third group (1 mM PAPS group) are 141 ° and 143 °, respectively, and are larger depending on the capacity of PAPS than the control. The extension angle of group 3 is statistically significantly larger than the equivalent value of group 1 (p value <0.05 by Dunnett's multiple comparison test), and PAPS has an effect of improving osteoarthritis. It was. Furthermore, the extension angle of the 1 mM PAPS group was larger than the same value of 142 ° of the hyaluronic acid group as the positive control group, and an osteoarthritis improving action superior to hyaluronic acid was observed.

(Example 2) Promotion of chondrocyte production of chondrocytes Japanese white (male, 3 kg) was euthanized by an excessive amount of pentobarbital anesthesia, and femoral epiphysis, tibia epiphysis and patella were collected and iced. After washing with cold 1% ampicillin and 1% streptomycin-containing Dulbecco's modified MEM medium (D-MEM), articular cartilage was isolated by a microsurgery technique under a stereomicroscope. The articular cartilage was minced and then suspended in D-MEM containing 0.05% hyaluronidase (Sigma) and treated at 37 ° C. for 10 minutes. Next, after removing the enzyme solution by suction, the cartilage pieces were suspended in 0.2% trypsin (Gibco) and treated at 37 ° C. for 30 minutes. Further, after removing the enzyme solution by suction, the cartilage pieces were suspended in 0.2% collagenase type II (Worthington Biochemical) and treated at 37 ° C. for 3 hours. The enzyme-treated solution was filtered with a stainless steel filter (150 μm) and then washed 3 times with PBS to prepare rabbit hyaline cartilage-derived chondrocytes. Chondrocytes were suspended in D-MEM containing 1% ampicillin, 1% streptomycin, 2.5 μg / ml fungizone (Invitrogen), 10% fetal bovine serum (FBS), and a cell density of 4.0 × 10 ⁴ cells / cm ² . So that it was inoculated on a collagen coat plate and cultured in a carbon dioxide incubator (carbon dioxide concentration 5%) adjusted to 37 ° C. After culturing until confluent, the cells were washed twice with a buffered saline solution, replaced with serum-free D-MEM, and various concentrations of PAPS and physiological saline as a control were added. Four hours after the addition of the test substance, the medium was removed, and total RNA was extracted from the cells using RNeasy Mini kit (QIAGEN). Using this total RNA as a template, prepare a reverse transcription reaction solution using ReverTra Ace (R) qPCR RT Kit (Toyobo), reverse transcription reaction solution and GoTaq (R) qPCR Master Mix (Promega), real-time PCR device Thermal MRNA expression levels of aggrecan and type II collagen, which are cartilage substrates, and Gapdh as an internal standard were measured by real-time PCR using Cycler Dice Real Time System (Takara Bio). After correcting with the value of the expression level of Gapdh in the same sample, the expression levels of aggrecan and type II collagen were compared with the control group. The results are shown in FIGS.

  As shown in FIGS. 2 and 3, the expression levels of aggrecan and type II collagen mRNA were remarkably increased in a concentration-dependent manner by the addition of PAPS. From the above results, it was confirmed that PAPS has a very excellent cartilage matrix production promoting action.

(Example 3) Inhibition of Notch signaling factor expression Chondrocyte culture, RNA extraction, and real-time PCR were performed in the same manner as in Example 2 to measure Jagged-1 and Hes1 and Gapdh mRNA expression levels as internal standards. did. After correction with the value of the expression level of Gapdh in the same sample, the expression level was compared with the control group. The results are shown in FIGS.
As shown in FIGS. 4 and 5, mRNA expression levels of H es 1 and Jagged-1 (J ag 1) was reduced in a concentration-dependent manner and significantly by PAPS added. From the above results, it was confirmed that PAPS has a very excellent Notch signaling factor expression inhibitory action.

Claims (3)

A cartilage matrix production promoter comprising 3′-phosphoadenosine-5′-phosphosulfate or a salt thereof as an active ingredient. The cartilage matrix production promoter according to claim 1, wherein the cartilage matrix is type II collagen and aggrecan .   A Notch signaling factor production inhibitor comprising 3′-phosphoadenosine-5′-phosphosulfate or a salt thereof as an active ingredient.