JP2007161688A - Cartilage formation promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a chondrogenesis promoter useful for promoting bone extension, treating and ameliorating arthritis, etc. <P>SOLUTION: The chondrogenesis promoter comprises a II type collagen biosynthetic raw material component and a proteoglycan biosynthetic raw material component and is administered just before sleeping-four hours before sleeping. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cartilage production promoter useful for treatment and / or improvement of cartilage and bone diseases such as bone elongation disorder, arthropathy, arthritis, cartilage trauma, and fracture.

Bone elongation occurs through cell proliferation of growth plate cartilage, extracellular matrix synthesis, and calcification. In addition, joint cartilage matrix synthesis is required for joint health or regeneration. The main components of the extracellular matrix (ECM) of these cartilages are type II collagen and aggrecan (proteoglycan), which are produced in large quantities through the expression and post-translational modification of type II collagen and aggrecan core protein genes by chondrocytes. Synthesized and secreted. From this point of view, for the purpose of improving symptoms such as arthritis, health foods and drugs containing ingredients such as chondroitin sulfate and mannosamine are used as materials for the extracellular matrix of cartilage.
Nature, 195: 82-83, 1962 Nature, 202: 906-907, 1964

However, the effectiveness of conventional arthritis therapeutic agents for promoting cartilage production is low, and the development of more effective drugs or foods is desired.
Accordingly, an object of the present invention is to provide a cartilage production promoter useful for the treatment and / or improvement of cartilage and bone diseases such as bone elongation disorder, arthropathy, arthritis, cartilage trauma, and fracture.

Therefore, the present inventor has paid attention to circadian rhythm, that is, an intrinsic rhythm of about 24 hours that is almost equal to the rotation period of the earth of most living things. Circadian fluctuations have been observed in many biological functions such as blood pressure, body temperature, and hormones, and elucidation at the gene level is progressing. In cartilage, it is suggested that there is a circadian rhythm in collagen synthesis, proteoglycan synthesis and cell division (Non-Patent Documents 1 and 2). Assessment is difficult, and further analysis at the genetic level has not been made, and the experimental conditions / animals differ from study to study (thesis), so the phase of circadian rhythms of collagen synthesis, proteoglycan synthesis and cell division Could not be compared.
Therefore, the present inventor comprehensively and simultaneously analyzed circadian rhythm genes including all genes involved in collagen synthesis, proteoglycan synthesis and cell division in a cartilage growth plate using rats reared under the same conditions. As a result, the cartilage growth plate has a unique peripheral clock system different from other tissues, and type II collagen and its related genes, proteoglycan and its related genes have circadian rhythms, and they are genes in the same time zone. It was found that expression reached a peak. Then, further investigation revealed that these components were used so that the components used as biosynthetic raw materials for type II collagen and proteoglycan were at a sufficiently high blood concentration at the time when the expression of type II collagen and proteoglycan related genes peaked. It was found that administration of the material significantly promotes cartilage production, and the present invention was completed.

  That is, the present invention provides a cartilage production promoter for administration from immediately before going to bed to 4 hours before, containing a type II collagen biosynthetic raw material component and a proteoglycan biosynthetic raw material component.

  If the cartilage formation promoter of the present invention is administered immediately before bedtime to 4 hours before bedtime, both the type II collagen biosynthesis raw material and the proteoglycan production raw material have sufficiently high blood concentrations after two hours after bedtime. Therefore, the circadian rhythm of cartilage efficiently promotes the biosynthesis of type II collagen and proteoglycan at the same time, thereby significantly promoting the production of cartilage. Therefore, according to the cartilage production promoter of the present invention, symptoms of cartilage and / or bone diseases such as bone elongation disorder, arthropathy, arthritis, cartilage trauma, and fracture can be improved.

  The cartilage production promoter of the present invention contains a type II collagen biosynthesis raw material component and a proteoglycan biosynthesis raw material component. The main components of the extracellular matrix constituting cartilage are type II collagen and proteoglycan, and the production of cartilage is fully promoted only when the production of both is promoted. As shown in the examples below, the circadian rhythm of the expression of type II collagen, proline hydroxylase, lysyl oxidase (necessary for collagen cross-linking) and proteoglycan gene in the cartilage growth plate is synchronized (shows the same phase). Because. Furthermore, the present invention is a gene involved in calcification that the rhythm of type II collagen and aggrecan synthesis in the growth plate is consistent with the rhythm of bone elongation that grows at night in humans, which has been reported so far. Since the phosphate transporter Pit1 and the ankylosis gene also have a slightly delayed circadian rhythm, we found that calcification after cartilage formation can be supported by combining calcium and phosphate simultaneously.

  Type II collagen biosynthetic raw material components include amino acids selected from glycine, proline, alanine, glutamic acid, arginine, aspartic acid and essential amino acids, vitamin C or derivatives thereof, iron or iron compounds, α-ketoglutaric acid, collagen and gelatin The component chosen is mentioned.

Of these, the amino acid is particularly important because it is the main constituent amino acid of type II collagen. Here, essential amino acids include valine, isoleucine, leucine, threonine, lysine, methionine, phenylalanine, and tryptophan. Vitamin C is a reducing agent necessary for the hydroxylation of proline. Derivatives of vitamin C, C ₁ -C ₂₄ fatty acid esters of vitamin C, phosphorus acid esters of vitamin C, and metal salts such as vitamin C. As the iron compound, divalent iron compounds such as ferrous fumarate, ferrous sulfate, and ferrous sodium citrate are preferable. Iron and α-ketoglutaric acid are compounds necessary for hydroxylation of proline and lysine. Gelatin is a modified substance of collagen.

  Of these type II collagen biosynthetic raw material components, it is more preferable to add proline and vitamin C or a derivative thereof, more preferably proline, vitamin C or a derivative thereof, dartamic acid and arginine, particularly proline, Vitamin C or a derivative thereof, glutamic acid, arginine, aspartic acid, glycine and an iron compound are preferably blended.

  The total amount of the type II collagen biosynthesis raw material component is preferably 1 to 80% by mass, particularly 1 to 50% by mass in the cartilage formation promoter of the present invention.

  Examples of the proteoglycan biosynthesis raw material component include components selected from chondroitin sulfate, chondroitin, N-acetylglucosamine, N-acetylgalactosamine, glucose, glucosamine, galactose, glucuronic acid, mannose, mannoseamine, xylose and sulfate compounds. Examples of the sulfuric acid compound include metal sulfates such as magnesium sulfate.

  Of these proteoglycan biosynthetic raw materials, it is preferable to mix glucosamine, more preferably glucosamine, glucuronic acid and mannosamine, particularly glucosamine, glucuronic acid, mannosamine, xylose, chondroitin sulfate and glucose. preferable.

  The proteoglycan biosynthetic raw material component is preferably contained in a total amount in the cartilage formation promoter of the present invention in an amount of 1 to 80% by mass, particularly 1 to 50% by mass.

From the research of the present inventor, the collagen synthesis and proteoglycan synthesis of the bone growth plate are synchronized with the circadian rhythm of bone growth, so that calcium compounds, phosphate compounds, vitamin D or derivatives thereof necessary for bone growth, etc. These vitamins may be added at the same time. Here, as calcium compounds, raw materials such as calcium carbonate, calcium hydrogen phosphate, calcium lactate, calcium gluconate, shellfish calcium derived from oyster shells, shellfish, shellfish, algae, mammalian bones and egg shells, etc. Are listed. Examples of the phosphoric acid compound include phosphoric acid and phosphate. The vitamin D derivative, seocalcitol, calcipotriol, calcitriol, tacalcitol, maxacalcitol, paricalcitol, file Leka Cie Torr include active vitamin D ₃ and the like. Vitamin K is mentioned as another vitamin. Among these components, the calcium compound is preferably contained in the present cartilage production promoter in an amount of 1 to 50% by mass, particularly 1 to 20% by mass. The phosphoric acid compound is preferably contained in an amount of 0.1 to 20% by mass, particularly 0.1 to 10% by mass. Vitamin D or a derivative thereof is preferably contained in an amount of 0.00001 to 1% by mass, more preferably 0.00001 to 0.1% by mass.

  The cartilage production promoter of the present invention can contain components involved in the promotion of cartilage production, such as hyaluronic acid, other polysaccharides, and proteins. In addition, other minerals such as magnesium, copper, iodine, manganese, selenium, zinc, chromium, molybdenum, other vitamins, citric acid, sugar, maltose, inositol, sorbitol, polyphenols, caffeine, flavoring, fruit juice, etc. can do.

  As shown in the examples described later, in the cartilage growth plate, the circadian rhythms of the biosynthetic genes of type II collagen and proteoglycan are synchronized, and in the rat, it reaches a peak 2 hours after becoming bright. Since rats are nocturnal, this peak in humans corresponds to 2 hours after bedtime. Therefore, the present invention cartilage production promoter is the blood of the type II collagen biosynthetic raw material component and the aggrecan biosynthetic raw material component 2 hours after bedtime when the circadian rhythm relating to the expression of type II collagen and proteoglycan gene in the cartilage growth plate peaks. It is administered so that the concentration is sufficiently high. In order for the blood concentration of these components to be sufficiently high after 2 hours of bedtime, it is desirable to administer the cartilage promoter of the present invention immediately before bedtime to 4 hours before bedtime. This dosing schedule does not necessarily coincide with the conventional usual dosing schedule, which is within 30 minutes after a meal.

  The agent for promoting cartilage formation of the present invention may be an agent for oral administration, an agent for injection, a suppository, or an external agent, but an agent for oral administration is preferred. Examples of the agent for oral administration include liquid preparations such as liquids, syrups, elixirs, suspensions and emulsions in addition to solid preparations such as tablets, powders, granules, pills and capsules. it can.

  In the administration form of the cartilage production promoter of the present invention, both of the active ingredients may be contained in one preparation, and the two active ingredients only need to have a high blood concentration at the same time. Therefore, each active ingredient may be contained in a separate preparation. For example, a combination of a tablet containing a type II collagen biosynthesis raw material component and a tablet containing an aggrecan biosynthesis raw material component may be used.

  The cartilage production promoter of the present invention may be used as a pharmaceutical, and may be used as a health food, functional food, or quasi drug.

  The cartilage production promoter of the present invention can be made into the above-mentioned various preparations by blending a pharmaceutically acceptable carrier with the active ingredient. As the pharmaceutically acceptable carrier to be used, for example, a binder, a disintegrant, a dissolution accelerator, a lubricant, a filler, an excipient and the like can be selected and used as necessary.

  The present invention cartilage production promoter is administered at a dose of 0.1 to 150 g, further 0.3 to 50 g per adult day as the total amount of type II collagen biosynthetic raw material component and proteoglycan biosynthetic raw material component. Is preferred.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these at all.

Example 1
Six-week-old male SD rats are reared for 2 weeks under light / dark conditions (LD, light: 12 hours, dark: 12 hours), then 3 rats every 4 hours for 48 hours continuously under LD or constant darkness (DD) conditions. Was sacrificed, and a costal cartilage growth plate was collected and total RNA was extracted. Next, all collected points (24 points) of growth plate RNA samples were subjected to DNA microarray (GeneChip Rat Expression Array 230A / B: Affymetrix) and analyzed to comprehensively identify genes showing circadian rhythm. The presence or absence of circadian rhythm was determined based on the coincidence rate of cosine curve at the mRNA level of each gene under LD and DD conditions and the coincidence rate of rhythm in LD and DD.

  As a result, 264 genes with circadian rhythm were identified, of which 109 were known, 74 were identified from homology with mice and humans, and 81 were unknown genes. Among these genes, genes related to transcription factors, growth factors, cell cycle, extracellular matrix, and intracellular transmission showed circadian rhythms, in addition to clock genes Bmal1, Per2, and Dec2. Moreover, when these rhythm genes were classified according to peak time, ZT14, that is, most genes showed a peak in the time zone when 2 hours passed after darkening. This time indicates the time at which the nocturnal rat started to act. On the other hand, Prolyl-4-hydroxylase 1 (P4ha1), Lysyl oxidase (Lox), and MT-MMP1, which are cartilage matrix-related genes identified by DNA microarray, showed expression peaks in ZT2 (at the beginning of bedtime) (Figure). 1). ZT2 means resting (night in humans, day in rats), consistent with the observation that bone grows at rest.

  Among these genes, several types of clock genes forming the core feedback loop of the molecular clock system also showed a circadian rhythm in cartilage (FIG. 2). However, other than the clock gene, the circadian rhythm gene of cartilage hardly overlapped with the circadian rhythm gene of the suprachiasmatic nucleus or liver. In other words, although the molecular clock system is common to each tissue, it has been shown that the output genes from the clock are tissue-specific, and the growth plate has its own circadian rhythm expression. did.

(Example 2)
Among the 264 circadian rhythm genes identified in Example 1, which are known genes and have real-time PCR (Q-PCR) primer and probe information, Taq Man Low Density Custom Arrays or The rhythm was reconfirmed by quantitative real-time PCR using Taq Man Gene expression assays (Applied Biosystems). At the same time, DNA microarrays were not determined to have circadian rhythms, but circadian rhythms of genes that show circadian variation in LD or DD and play an important role in cartilage growth and differentiation are also quantitative real-time PCR It was examined in.

  As a result, the results of the DNA microarray were confirmed by real-time PCR, and the results of the DNA microarray obtained this time were shown to be highly reliable (FIGS. 2 and 3).

Furthermore, it was found by real-time PCR that Aggrecan and Collagen type II, which are cartilage-specific genes, also have a circadian rhythm with a peak of ZT2 (FIG. 4 and Table 1). Further, Prolyl-4-hydroxylase 1 (P4ha1), Lysyl oxidase (Lox), and MT-MMP1 described in Example 1 were also confirmed to have a circadian rhythm with a peak at ZT2 in the real-time PCR, as a result of DNA microarray. Therefore, these results reveal that the circadian rhythm of cartilage matrix synthesis is controlled at the transcription / post-transcriptional stage, and the mRNA peaks of these cartilage matrix synthesis-related genes are concentrated in ZT2. Turned out to be.
On the other hand, Aggrecan, Collagen type II Timp3 (Fig. 5) and Timp4 also showed circadian rhythms, but these showed peaks in ZT14, indicating that the cartilage matrix synthesis and degradation system peaks. It was found that the phase was shifted by about 12 hours.

It is a figure which shows the rhythm expression of a cartilage matrix related gene (Prolyl-4-hydroxylase 1, Lysyl oxidase, and MT-MMP1) by a DNA microarray. It is a figure which shows the rhythm expression of the clock gene (Per2, DEC2, DBP, Cry1, Per3, DEC2, Rev-erbβ, Bmal1) in the growth plate. It is a figure which shows the rhythm expression of other genes (Gilz, PSD-Zip10, TEF, Thrd, lgfbp5, Tgfα, Slc39a1, Gadd45α) excluding the clock gene. It is a figure which shows the rhythm expression in real-time PCR of a cartilage matrix related gene (Collagen typeII, Aggrecan, Prolyl-4-hydroxylase 1, Lysyl oxdase, MT-MMP1). It is a figure which shows the rhythm expression of Timp3.

Claims (6)

  An agent for promoting cartilage formation, which contains a type II collagen biosynthetic raw material component and a proteoglycan biosynthetic raw material component, for administration immediately before going to bed to 4 hours before sleep.   Type II collagen biosynthetic raw material components are amino acids selected from glycine, proline, alanine, glutamic acid, arginine, aspartic acid and essential amino acids, vitamin C or derivatives thereof, iron or iron compounds, α-ketoglutaric acid, collagen, and gelatin The cartilage production promoter according to claim 1, which is a selected component.   The proteoglycan biosynthetic raw material component is a component selected from chondroitin sulfate, chondroitin, N-acetylglucosamine, N-acetylgalactosamine, glucose, glucosamine, galactose, glucuronic acid, mannose, mannosamine, xylose and sulfate compounds. The cartilage formation promoter as described.   The cartilage formation promoter according to any one of claims 1 to 3, further comprising a component selected from a phosphate compound, a calcium compound, vitamin D and a derivative thereof.   It is an agent for oral administration, The cartilage formation promoter of any one of Claims 1-4. The cartilage production promoter according to any one of claims 1 to 5, which is for improving symptoms of cartilage and / or bone disease selected from bone elongation disorder, arthropathy, arthritis, cartilage trauma and fracture.

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