JP4575105B2 - Osteoblast activity inhibitor - Google Patents

Description

The present invention relates to an osteoblast activity inhibitor having a function of suppressing calcium mineralization and calculus formation in human bones and organs.

  Traditionally, the mechanism of human bone formation involves osteoblasts that make bones and osteoclasts that break bones. The cause of osteoporosis is thought to be due to the imbalance of these cells. Yes. There are various theories as a mechanism of bone formation, but the matrix vesicle theory is generally accepted as a promising one.

  Matrix vesicles are secreted by osteoblasts and abundant in the matrix at the site of endochondral ossification, and many matrix vesicles generated from hard tissue-forming cells migrated into the matrix during the initial calcification process Later, the accumulation of amorphous calcium phosphate was observed in the inside, and gradually, these were converted to hydroxyapatite crystals and increased / expanded to promote calcification. Since strong alkaline phosphatase activity exists in this substrate vesicle, the concentration of calcium or phosphate locally increases, and when a certain dissolved concentration is exceeded, inorganic salts are precipitated, and calcification occurs. It is considered.

On the other hand, as disclosed in Patent Documents 1 and 2, an agent for preventing or ameliorating symptoms caused by leukotriene B ₄ containing omega-9 unsaturated fatty acid as an active ingredient, or for preventing or preventing diseases caused by abnormalities in cartilage tissue Therapeutic drugs have been proposed.
Japanese Patent Laid-Open No. 7-41421 WO97 / 5863 Publication

  However, it has been experimentally known that omega-9 unsaturated fatty acids have an anti-inflammatory effect, but in Patent Document 2 as well, omega-9 unsaturated fatty acids act on cartilage by any mechanism. I haven't made it clear. In addition, since the mechanism of bone formation and stone formation in the human body, which is completely different from the aforementioned abnormalities such as inflammation of the cartilage tissue, has not been fully elucidated, prophylactic and therapeutic drugs for these diseases are also available. There is a tendency to become symptomatic treatment, and the emergence of a fundamental therapeutic agent has been desired.

The present invention has been made in view of the above-described conventional problems, and is based on the mechanism of calcium calcification in the human body, and suppresses osteoblast activity that suppresses calcification of cartilage and calcium calcification in the body. The purpose is to provide an agent.

  In order to solve the above-mentioned problems, the present inventors have studied various unsaturated fatty acids, have an excellent calcification-inhibiting action, and are extremely useful for the prevention or prevention of calcification in the human body. A 9-series unsaturated fatty acid was found and the present invention was completed.

The present invention is an osteoblast activity inhibitor comprising omega-9 unsaturated fatty acid, particularly 5,8,11-cis-eicosatrienoic acid (mead acid) as an active ingredient. Calcium mineralization refers to kidney stones, gallbladder stones, pancreatic duct stones, and cartilage mineralization.

The osteoblast activity inhibitor of the present invention is easy to ingest and has no side effects, effectively inhibits unnecessary calcification in the body, and can be used for the prevention or treatment of various diseases resulting therefrom.

  Hereinafter, embodiments of the present invention will be described in detail. 5,8,11-cis-eicosatrienoic acid (hereinafter referred to as mead acid), which is an active ingredient of the present invention, is an omega-9 unsaturated fatty acid. The double bond closest to the methyl end is between the 9th and 10th carbons, counting from the methyl group, and has 2 or more double bonds, preferably 18 to 22 carbon atoms. It is what you have. For example, 6,9-octadecadienoic acid, 8,11-eicosadienoic acid, 5,8,11-eicosatrienoic acid and the like can be mentioned, and these can be used alone or in combination. Since all naturally occurring omega-9 unsaturated fatty acids are cis type, it is preferable to use mead acid which is a cis type omega-9 unsaturated fatty acid in the present invention.

  The omega-9 unsaturated fatty acids of the present invention can be used in the form of free fatty acids, but pharmaceutically acceptable salts such as sodium salts, potassium salts, lithium salts, or other alkali metal salts, zinc salts, It may be used in the form of other metal salts such as calcium salts and magnesium salts, and various forms such as mono-, di-, triglycerides, esters of lower alcohols, phospholipids, glycolipids, amides, especially ethyl esters. And triglycerides are preferred.

  The source of the omega-9 unsaturated fatty acid used in the present invention may be anything. It may be produced by microorganisms capable of producing omega-9 unsaturated fatty acids, animal tissues that are deficient in essential fatty acids, or cultured animal cells that are deficient in essential fatty acids, chemically or enzymatically. It may be a synthesized product or a natural product such as one extracted, separated and purified from animal cartilage.

Here, the microorganism capable of producing an omega-9 unsaturated fatty acid specifically has Δ5 desaturase activity and Δ6 desaturase activity and a decrease in Δ12 desaturase activity or Deficient microorganisms such as Mortierella alpina SAM1861 (FERM
BP-3590) can be used.

  Extraction / separation / purification of omega-9 unsaturated fatty acids or their esters free from these microorganisms was first obtained from cells by organic solvent extraction or supercritical carbon dioxide extraction with, for example, n-hexane. Oils and fats are subjected to hydrolysis and esterification operations to obtain a free fatty acid mixture or a fatty acid ester mixture. Thereafter, free fatty acid or fatty acid such as 5,8,11-cis-eicosatrienoic acid, which is the target omega-9 unsaturated fatty acid, by urea fractionation method, liquid-liquid partition chromatography, column chromatography, etc. Esters can be obtained with a purity of 80% or more.

  Moreover, the omega-9 unsaturated fatty acid which is an active ingredient of the present invention is not necessarily limited to a high-purity purified product, and fats and oils containing omega-9 unsaturated fatty acids (the omega-9 unsaturated fatty acids are contained in the fats and oils). Use of fatty acid triglycerides, diglycerides, monoglycerides, phospholipids, glycolipids, free omega-9 unsaturated fatty acids), and free fatty acid mixtures or fatty acid ester mixtures containing omega-9 unsaturated fatty acids it can. As described above, the fats and oils containing omega-9 unsaturated fatty acids can destroy the cells from cultured microorganisms capable of producing omega-9 unsaturated fatty acids. Moreover, a free fatty acid mixture or a fatty acid ester mixture containing an omega-9 unsaturated fatty acid can be obtained by subjecting the oil and fat to hydrolysis and esterification operations.

  When the omega-9 unsaturated fatty acid of the present invention is used as a pharmaceutical, the dosage form may be any dosage form such as oral administration or parenteral administration, for example, injection, infusion, powder, granule Tablets, capsules, enteric solvents, troches, liquids for internal use, suspensions, emulsions, syrups, liquids for external use, poultices, nasal drops, ear drops, eye drops, inhalants, ointments, lotions, Suppositories and the like can be mentioned, and these can be used alone or in combination depending on the symptoms.

  These various preparations are prepared by using known adjuvants that can be usually used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, and corrigents according to the purpose according to conventional methods. Can be The dose varies depending on the purpose of administration and the situation of the administration subject (gender, age, weight, etc.), but is usually 1 to 1000 mg per day, preferably 1 to 500 mg when administered orally to an adult. More preferably in the range of 1 to 200 mg, and in the case of parenteral administration, 0.1 to 100 mg per day, preferably 0.1 to 50 mg, more preferably 0.1 to 20 mg is suitably adjusted and administered. be able to.

  The omega-9 unsaturated fatty acid, which is an active ingredient of the present invention, is known to be biosynthesized in the living body when essential fatty acids are deficient, and 2 g / day / Kg for 7-week-old IRC male mice. Even in an experiment in which the drug was continuously administered (oral administration) for 2 weeks, there was a result that no abnormal symptom was observed, and it is clear that it is excellent in terms of safety.

  When the fatty acid of the present invention is used in the form of food or drink, it may be in the form of the above-mentioned preparation, but the required amount of omega-9 unsaturated fatty acid is used as a raw material for food and drink, particularly the omega 9 unsaturated fatty acid of the present invention. In addition to the food / beverage raw material which does not contain substantially, it can process and manufacture by a general manufacturing method. The blending amount varies depending on the dosage form and the morphological properties of the food, but generally 0.001 to 50% by weight is preferable with respect to the total amount of food, but is not particularly limited.

  In particular, ingestion as health foods and functional foods, for example, proteins (such as milk proteins with high amino acid balance, soy protein, egg albumin and the like are widely used as protein sources, but these proteins are decomposed. A mixture of amino acids alone, sugars, fats, trace elements, vitamins, emulsifiers, fragrances, etc., and the natural flow of the present invention. Examples of processing forms include foods, semi-digested nutritional foods and component nutritional foods, and drinks. Moreover, under the management of a dietitian based on a doctor's meal note, the fatty acid of the present invention can be added to any food during cooking in a hospital meal and given to the patient in the form of a functional food adjusted on the spot.

  The form of food and drink includes solid or liquid foods or luxury products such as bread, noodles, rice, confectionery (biscuits, cakes, candy, chocolate, Japanese confectionery), agricultural products such as tofu and processed products thereof, and sake. , Medicinal liquor, mirin, vinegar, soy sauce, miso, dressing, and other fermented foods, yoghurt, ham, bacon, sausage, mayonnaise and other livestock foods, kamaboko, fried tempura, marinated foods such as hampen, fruit juice drinks, soft drinks And beverages such as sports drinks, alcoholic drinks, and tea.

  The experimental result regarding the calcification of the omega-9 unsaturated fatty acid of this invention is shown below.

  In this experiment, goldfish scales were used. In scales, osteoclasts and osteoblasts are present at the same time, and fish regulates the concentration of calcium in the blood by taking calcium in and out of scales, not from vertebrae. This scale calcium is also present in the same hydroapatite form as human calcium. Accordingly, the scale is the same as that obtained by slicing a human bone, and the scale can be used as a model of the human bone. Therefore, in this example, the following experiment was performed using an easily available goldfish. The mead acid used in this experiment has a purity of 98% or higher from Cayman Chemical, MI, USA.

A goldfish female (weight around 30g) is anesthetized with MS222 (Aldrich) and the scale is peeled off. The scale is washed twice with Eagles minimal medium (Dainippon Pharmaceutical) containing 1% antibiotics. 1 ml each of the medium is put into a 24-well plate, and a plurality of the scales (usually 8 pieces) are placed, and 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ are placed in each hole. M (mol) of Mead acid is added respectively. An additive-free control is also provided to compare the action on bone cells. At this time, two groups for osteoclasts and osteoblasts are provided.

Therefore, a total of 12 wells of the additive-free control, 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ M medic acid (2 holes each) are prepared.

In order to compare the effects of fatty acid addition, oleic acid was also added in the same manner as in the non-added control, 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ M oleic acid addition group (total 12 holes ). Furthermore, in order to provide culture time of 6 hours and 18 hours, two 24-well plates are prepared. That is, one sheet is used for 6-hour culture and one is used for 18-hour culture. In addition to oleic acid, the same experiment was conducted with palmitic acid.

  Then, after culturing at 15 ° C. for each, the medium is removed, and 0.05 M cacodylate buffer (pH 7.4) containing 10% formalin is added and fixed. The scale is stored at 4 ° C. in 0.05 M cacodylate buffer until measurement of enzyme activity.

(1) Effect of osteoclasts: Activity measurement of Tartrate-resistant acid phosphatase (TRAP) (Tartrate-resistant acid phosphatase) The scales subjected to the above-mentioned fixing treatment are taken out, and the scale weight is measured. After the measurement, place the scales in a 96-well microplate, add 200 μl of 100 mM acetate buffer containing 20 mM tartaric acid and 10 mM paranitrophenol phosphate (substrate) to each well, and react at 20-25 ° C. for 1 hour. The reaction is then stopped with 2N sodium hydroxide (50 μl). Thereafter, 150 μl is transferred to another microplate, and the amount of nitrophenol (abbreviated as pNP) generated by TRAP is measured with a spectrophotometer (405 nm). Osteoclast activity is expressed as the amount of paranitrophenol produced by degrading paranitrophenol phosphate per 1 mg of scale.

  The result is shown in FIG. Thus, TRAP, which shows osteoclast activity, was not significantly different between the groups of mead acid and oleic acid, and it was confirmed that these fatty acids had no effect on osteoclasts.

  Similarly, FIG. 2 shows a comparison with mead acid when palmitic acid is used instead of oleic acid. Also in this case, no significant difference was observed between mead acid and palmitic acid, and it was confirmed that there was no effect on osteoclasts.

(2) Effects of osteoblasts: Alkaline phosphatase (ALP) activity measurement The scales subjected to the above-mentioned fixing treatment are taken out and the scale weight is measured. After the measurement, place the scales in a 96-well microplate, and add 100 mM Tris-HCl buffer (pH 9.5) containing 10 mM paranitrophenol phosphate (substrate), 1 mM magnesium chloride, and 0.1 mM zinc chloride to each hole. Add 200 μl, react at 20-25 ° C. for 1 hour, and stop with 2N sodium hydroxide (50 μl). Thereafter, 150 μl was transferred to another microplate, and the amount of nitrophenol (abbreviated as pNP) generated by ALP was measured with a spectrophotometer (405 nm) to determine the activity.

As a result, ALP, which shows osteoblast activity after 6 hours and 18 hours, was significantly reduced with mead acid compared to oleic acid at concentrations of 10 ⁻⁶ , 10 ⁻⁵ , and 10 ⁻⁴ M, respectively. It was confirmed that the activity of osteoblasts was suppressed.

Similarly, FIG. 4 shows a comparison with mead acid when palmitic acid is used instead of oleic acid. Also in this case, it was confirmed that at the concentrations of 10 ⁻⁵ and 10 ⁻⁴ M, the activity of osteoblasts was suppressed by significantly lowering with mead acid as compared with palmitic acid.

  In this experiment, it was confirmed that alkaline phosphatase activity was suppressed by administration of mead acid. From this, it was found that administration of mead acid to humans leads to suppression of calcification. It can be applied as a remedy for diseases involving calcification, and specifically used as a preventive or therapeutic agent for various diseases caused by calcification in kidney stones, gallbladder stones, pancreatic duct stones, and cartilage. Can do.

The results of measuring the activity of TRAP, which shows the activity of osteoclasts, in the culture solution to which mead acid and oleic acid were added at various concentrations are shown. Paranitrophenol per hour of scales cultured for 6 hours and 18 hours It is a graph which shows a production amount. The results of measuring the activity of TRAP, which shows the activity of osteoclasts, in the culture medium to which mead acid and palmitic acid were added at various concentrations are shown, and paranitrophenol per hour of scales cultured for 6 hours and 18 hours. It is a graph which shows a production amount. The results of measuring the activity of ALP showing the activity of the osteoblasts of scales in the culture medium to which mead acid and oleic acid were added at various concentrations are shown. Paranitrophenol per hour of scales cultured for 6 hours and 18 hours It is a graph which shows a production amount. The results of measuring the activity of ALP, which shows osteoblast activity, in the culture solution to which mead acid and palmitic acid were added at various concentrations are shown. Paranitrophenol per hour of scales cultured for 6 hours and 18 hours It is a graph which shows a production amount.

Claims (2)

An osteoblast activity inhibitor comprising 5,8,11-cis-eicosatrienoic acid as an active ingredient. The osteoblast activity inhibitor according to claim 1, wherein the 5,8,11-cis-eicosatrienoic acid suppresses calcium mineralization by suppressing the activity of osteoblasts in cartilage.

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