JP5338016B2 - Biologically active substance-containing octacalcium crystal, method for producing the same, and pharmaceutical composition containing the same - Google Patents

Description

  The present invention relates to an octacalcium phosphate crystal containing a biologically active substance, a pharmaceutical composition containing the same, and a method for producing the same. According to the present invention, an osteoclast dissolves an octacalcium phosphate crystal containing a biologically active substance in an affected part of a bone disease accompanied by an increased function of osteoclasts. It is possible to provide a sustained-release pharmaceutical composition that is released and corresponds to the enhanced function of osteoclasts.

  As a treatment method for a chronic disease or a disease occurring locally in a living body, a treatment method using a sustained-release pharmaceutical preparation in which a biologically active substance such as a drug is gradually released in the living body is known. Since the sustained-release pharmaceutical preparation gradually releases the biologically active substance in vivo, the effect can be sustained by a small number of administrations. In addition, since the biologically active substance can be released locally in the living body, it is possible to increase the concentration of the biologically active substance in the affected area and enhance the therapeutic effect of the biologically active substance. As such sustained-release pharmaceutical preparations, those in which a biologically active substance is supported on a carrier, administered into a living body, and gradually released are known. For example, as a sustained-release pharmaceutical preparation for chronic diseases, a pharmaceutical preparation that is sustainedly released in vivo by carrying a protein on apatite nanoparticles and injecting subcutaneously has been reported (Patent Document 1, Non-patent Document 1). Patent Document 1).

  Examples of local bone diseases include metastatic bone tumors and osteoporosis, which are intractable bone diseases, and rheumatoid arthritis, which is a bone immune disease. In such diseases, osteoblasts that form bone And the osteoclasts that resorb bone are disrupted and normal bone metabolic function is inhibited. In particular, the development of a treatment method that can enhance osteoclast function at the diseased site, suppress osteoclast function, and promote bone regeneration is desired. For such a bone disease, for example, a method has been reported in which a drug is supported on a hydroxyapatite porous body baked at high temperature to act on the affected area (Patent Document 2, Non-Patent Document 2, Non-Patent Document). Reference 3 and Non-Patent Document 4) However, the hydroxyapatite porous material has affinity for bone, but since it is fired at a high temperature, the amount capable of carrying the drug is small, and the affected part Could not release a sufficient amount of drug.

  In general, the sustained-release pharmaceutical preparation uses apatite nanoparticles, hydroxyapatite porous material, calcium carbonate compound or polylactic acid / glycolic acid (PLGA) as a carrier, and on the surface of these carriers, Since biologically active substances such as drugs are adsorbed by hydrophobic bonds, the drugs and the like are gradually released in the living body. However, since the drug is supported by adsorption, there is a problem such as an initial burst release in which the drug is excessively released at the initial stage of administration of the pharmaceutical preparation, and the amount of the released drug must be reduced over time. I didn't get it.

Japanese Patent Laid-Open No. 2005-8545 Japanese Patent Laid-Open No. 2003033044 “Journal of Controlled Release” (Netherlands), 2006, 110, p. 260-265 “Materials Science and Engineering C” (Netherlands), 2005, Vol. 25, p. 207-213 “Journal of Biomedical Materials Research Part B: Applied Biomaterials Research (B): Applied Biomaterials” (USA), 2004, 70B, p. 240-249 “Osteoaritris and Cartilage” (Netherlands), 2005, Vol. 13, p. 405-417

The inventors of the present invention have provided sustained release capable of acting locally in affected areas in metastatic bone tumors and osteoporosis, which are intractable bone diseases in which normal bone metabolic function is inhibited, or rheumatoid arthritis, which is a bone immune disease. As a result of intensive investigations on the active pharmaceutical composition, the apatite-like layered compound octacalcium phosphate [Ca ₈ H ₂ (PO ₄ ) ₆ · 5H ₂ O; hereinafter sometimes referred to as OCP] is used as a drug, etc. It has been found that the release amount of drugs and the like can be controlled in response to the severity of bone disease symptoms by using as a carrier for containing the biologically active substance. Specifically, by replacing the surface of the calcium octaphosphate crystal and the HPO ₄ ^2- group of the hydrated layer with a biologically active substance capable of treating bone disease, a sufficient amount of biological It was possible to contain active substances. In addition, by adjusting the pH and temperature at the time of production, it was possible to contain calcium octaphosphate crystals without impairing the activity of biologically active substances such as drugs. When calcium octaphosphate crystals containing this biologically active substance are administered to the affected area of bone disease, osteoclasts with enhanced function recognize and dissolve calcium octaphosphate crystals. It was found that the biologically active substance is efficiently released in the affected area. Therefore, the more the function of the osteoclast that causes the disease is increased, the more biologically active substance is released and the concentration of the drug or the like at that site can be increased. And found that the treatment can be performed efficiently.
The present invention is based on these findings.

Accordingly, the present invention relates to an octacalcium phosphate crystal containing a biologically active substance in a hydrated layer.
In a preferred embodiment of the octacalcium phosphate crystal according to the present invention, the biologically active substance is a biologically active substance having an anionic group in its structure, preferably a bone disease treatment compound, More preferred is bisphosphonate.
In another preferred embodiment of the octacalcium phosphate crystal according to the present invention, a part of the calcium ion of the octacalcium phosphate crystal is substituted with a metal cation, and in particular, the metal cation is magnesium. A metal cation selected from the group consisting of strontium, barium, platinum, manganese, copper, zinc and iron.

The present invention also relates to a pharmaceutical composition in which a physiologically active substance is released according to disease symptoms.
In a preferred embodiment of the pharmaceutical composition according to the present invention, the symptom of the disease is a symptom due to hyperfunction of cells in a living body, and in particular, the cells in the living body are osteoclasts.
In another preferred embodiment of the pharmaceutical composition according to the present invention, the octacalcium phosphate crystal is included as an active ingredient, and in particular, the disease is a bone disease.
The present invention also provides a phosphorous containing a biologically active substance, which is mixed and stirred at a temperature of 25 to 80 ° C. while maintaining a solution containing calcium ions and a solution containing phosphate ions at pH 4.5 to 8.5. A method for producing octacalcium acid, wherein the solution containing calcium ions and / or the solution containing phosphate ions contains a biologically active substance, The present invention also relates to a method for producing calcium-based crystals.
In a preferred embodiment of the method for producing octacalcium phosphate crystals according to the present invention, the solution containing calcium ions and / or the solution containing phosphate ions contains a metal cation.
Furthermore, the present invention is a solution containing a biologically active substance held in _{pH4.5~8.5, CaHPO 4 · 2H 2 O} , CaHPO 4, αCa 3 (PO 4) 2, or βCa ₃ (PO ₄ It also relates to a method for producing octacalcium phosphate containing a biologically active substance, wherein ₂ is added and stirred at 25-80 ° C.
In a preferred embodiment of the method for producing octacalcium phosphate crystals according to the present invention, the solution containing the biologically active substance contains a metal cation.
In another preferred embodiment of the method for producing octacalcium phosphate crystals according to the present invention, the biologically active substance is a biologically active substance having an anionic group in its structure, preferably for treating bone diseases. Compounds, more preferably bisphosphonates.

  According to the method of the present invention, the biologically active substance can be contained in the hydrated layer or surface of the octacalcium phosphate crystal without reducing the effect of the biologically active substance. The weight ratio of the contained biologically active substance is a maximum of 70% by weight of the octacalcium phosphate crystal, and is a content capable of long-term sustained release. Moreover, the octacalcium phosphate crystal containing the biologically active substance according to the present invention can release the biologically active substance selectively under acidic conditions, slowly under neutral conditions. Therefore, in a normal state, almost no biologically active substance is released under normal conditions. However, osteoclasts, which are the cause of refractory bone disease and rheumatoid arthritis, are in acidic conditions in which octacalcium phosphate crystals are dissolved. In response to the action of osteoclasts, biologically active substances can be released efficiently and bone diseases can be treated. Furthermore, octacalcium phosphate, which is a carrier, has excellent biocompatibility and exhibits osteoconductivity, and is used as a material for repairing the affected bone absorbed by osteoclasts.

Octacalcium phosphate is a hydrate and forms layered crystals including a hydrated layer. The octacalcium phosphate crystal in the present invention is characterized in that part or all of the HPO ₄ ^2- group of the hydrated layer is substituted with a biologically active substance. Furthermore, a biologically active substance is also supported on the crystal surface by binding with calcium.

  The biologically active substance contained in the octacalcium phosphate crystal according to the present invention is not particularly limited as long as it is a substance that can exhibit a physiologically active function in vivo. Substances showing medicinal properties against diseases in can be used. Specific examples include drugs such as proteins, peptides, low molecular compounds, and nucleic acids such as DNA and RNA. When the octacalcium phosphate crystal of the present invention is used as an active ingredient in a sustained-release pharmaceutical composition, it is possible to release a biologically active substance under acidic conditions, and in a phosphate buffer. Biologically active substances can also be released when converted to apatite under neutral conditions. Therefore, it can be used as a sustained-release pharmaceutical composition for various diseases by containing a biologically active substance having a medicinal effect for a specific disease. The lower limit of the content of these biologically active substances in the octacalcium phosphate crystals is preferably 3% by weight or more, and more preferably 8% by weight or more. The amount of 3% by weight or more is preferable in that the biologically active substance can be efficiently and locally released. Further, the upper limit of the content of the biologically active substance is preferably 70% by weight or less, more preferably 50% by weight or less, and most preferably 15% by weight.

Since the biologically active substance is structurally inserted into the hydrated layer of the octacalcium phosphate crystal, it preferably has an anionic group in its structure. This is because by having an anionic group, it interacts with calcium ions and the biologically active substance is easily inserted into the hydrated layer. As anionic group is not particularly limited, for example, -HPO ₄ ^2-group, _-PO ^{3 2-group,} -COO ^- group, -OH ^- group, -Cl ^- group, _-SO ^{3 2-} Examples of the group include -PO ₃ ^2- group.

  The octacalcium phosphate crystal according to the present invention can be used as an active ingredient in a sustained-release pharmaceutical composition for bone diseases. Examples of the bone disease include osteoporosis, which is an intractable bone disease, metastatic bone tumor, and rheumatoid arthritis, which is a bone immune disease. These bone diseases are characterized by increased bone resorption by osteoclasts. In addition to these diseases, pathological bone resorption by osteoclasts includes arthritis and periodontal disease. (Periodontitis), inflammatory bowel disease, diabetes and autoimmune diseases such as lupus erythematosus, metastatic breast cancer and lung cancer.

  Examples of the biologically active substance having a medicinal effect on the bone disease include osteoclast inhibitors such as estrogen, calcitonin, activated vitamin D3, vitamin K, ipriflavone, misramycin, pricamycin, gallium nitrate, and bisphosphonate. Can be mentioned. In addition to the osteoclast inhibitors, biologically active substances having a medicinal effect on rheumatoid arthritis include cytokine inhibitors (for example, anti-TNF), interferons, and anti-rheumatic drugs (for example, hydroxychloroquine, azathioprine). , Methotrexate, cyclophosphoamide, leflunomide, sulfasalazine, robenzaritni sodium, bucillamine, acralit salazosulfapyridine, and prednisolone farnesylate). Moreover, raloxifene which has an antiestrogenic action can be mentioned as a biologically active substance which shows the medicinal effect with respect to osteoporosis. Furthermore, an anticancer agent can be used as a biologically active substance having a medicinal effect on metastatic bone tumor, metastatic breast cancer and lung cancer.

  Examples of the bisphosphonate that is an inhibitor of osteoclasts include etidronate, alendronate, risedronate, incadronate, zoledronate, ibandronate, clodronate, pamidronate, tiludronate, olpadronate, neridronate, minodronate, 1-hydroxy-3 Mention may be made of-(1-pyrrolidinyl) -propylidene bisphosphonate, cimadronate, risedronate, pyridronate, pamidronate, their pharmaceutically acceptable salts and mixtures thereof.

  Furthermore, the biologically active substance that can be contained in the octacalcium phosphate crystal according to the present invention includes a drug that can enhance the action of osteoblasts, or the coupling of osteoclasts and osteoblasts. It is also possible to use a drug that can bring the normal state to the normal state.

The calcium ion contained in the octacalcium phosphate crystal according to the present invention can be substituted with a metal cation. The metal cation is not particularly limited, and examples thereof include magnesium, strontium, barium, platinum, manganese, copper, zinc, iron, and a mixture thereof. By substituting calcium ions with other metal cations, it is possible to control the interlayer distance of the hydrated layer of octacalcium phosphate crystals, and hydrate the biologically active substances of different sizes. Can be inserted into. In particular, by substitution with strontium ions and barium ions, the interlayer distance of the hydrated layer is increased, and a biologically active substance having a relatively large molecular weight can be introduced into the hydrated layer. The content of metal cations contained in octacalcium phosphate-based crystals, especially but not limited to, [Ca 8 _{H 2} of 2 molecules _{(PO 4) 6 · 5H 2} O ] unit cell to calcium atoms per Is preferably 1 to 4, more preferably 1 to 3. If the substitution exceeds 4, there is a possibility of exhibiting biotoxicity. If the biocompatibility is deteriorated, recognition by osteoclasts tends to be weakened, which is not preferable.

  The size of the octacalcium phosphate crystal of the present invention is not particularly limited, but is preferably a size at least capable of recognizing osteoclasts in order to treat bone diseases. Moreover, it is preferable that it is a magnitude | size which can administer by injection or implantation to the local in-vivo. The octacalcium phosphate crystal of the present invention can take a form such as a cube, a rectangular parallelepiped, or particles, but the particle size is preferably 1 μm to 1 mm, more preferably 1 μm to 100 μm.

  The octacalcium phosphate crystal containing the biologically active substance in the hydrated layer according to the present invention can be produced by a coprecipitation method or a hydrolysis method. It is preferable not to perform rapid pH changes, rapid ion concentration changes, preparation at high production temperatures, vigorous stirring, etc. so that biologically active substances are not inactivated during production. For example, it is preferable to maintain the pH in the range of 4.5 to 8.5 and not to change it abruptly. Also, the reaction temperature is preferably kept constant without changing abruptly, preferably at a constant temperature in the temperature range of 25-80 ° C, more preferably 30-60 ° C, most preferably 35-50 ° C. For example, when a stirrer or the like is used, it is preferable to perform rotation at 3000 rpm or less. Furthermore, the pH used for the reaction is preferably kept constant without changing rapidly, preferably 4.5 to 8.5, more preferably 5.0 to 7.5, and most preferably 5.0 to 6. Perform at a constant pH in the range of 0.0. By producing the octacalcium phosphate crystal of the present invention under such mild conditions, a sufficient amount of biologically active substance can be added to the water of the octacalcium phosphate crystal without causing a decrease in medicinal effect. It is possible to introduce into the sum layer.

  For example, when producing an octacalcium phosphate crystal containing a biologically active substance in the hydrated layer by a coprecipitation method, the pH and ionic strength can be controlled as follows. While maintaining the pH at 4.5 to 8.5, a solution containing phosphate ions and a solution containing calcium ions are slowly mixed to produce octacalcium phosphate crystals. For mixing, a solution containing calcium ions is dropped into a solution containing phosphate ions, or a solution containing phosphate ions is dropped into a solution containing calcium ions slowly at 25 to 80 ° C. A biologically active substance such as a drug can be contained in a solution containing phosphate ions and / or a solution containing calcium ions, but is preferably contained in a solution containing phosphate ions. As phosphate ion, orthophosphoric acid or ammonium phosphate is preferable, and as calcium ion, calcium hydroxide, calcium chloride, calcium nitrate, calcium acetate or calcium carbonate is preferable. Furthermore, when a metal cation is added, it is preferably contained in a solution containing phosphate ions and / or a solution containing calcium ions in the same manner as the biologically active substance and dissolved uniformly.

When producing octacalcium phosphate crystals containing biologically active substances in the hydrated layer by hydrolysis, the biological activity is adjusted to a solution adjusted to pH 4.5 to 8.5 with acetate or the like. The substance is dissolved, and the powder of brushite [CaHPO ₄ .2H ₂ O], monetite [CaHPO ₄ ] αCa ₃ (PO ₄ ) ₂ , or βCa ₃ (PO ₄ ) ₂ is dispersed in this solution at 25 to 80 ° C. Then, it is slowly stirred and dissolved to form octacalcium phosphate crystals. Furthermore, when adding a metal cation, it is preferable to add it to the solution together with the biologically active substance and dissolve it uniformly.

  Also in the coprecipitation method and the hydrolysis method, by adding a metal cation to the solution, the calcium ion of the octacalcium phosphate crystal is replaced with the metal cation, and the interlayer distance of the hydrated layer can be controlled. It has become possible. In particular, by substitution with strontium ions and barium ions, the interlayer distance of the hydrated layer is increased, and a biologically active substance having a relatively large molecular weight can be introduced into the hydrated layer.

  Furthermore, in order to adjust the particles of the octacalcium phosphate crystal according to the present invention to a size that can be recognized by osteoclasts, it is also possible to use a spray drying method or granulation drying. The prepared suspension of drug-containing octacalcium phosphate crystals is adjusted to a concentration of 0.1 to 15% by weight, the spray pressure is 0.005 to 0.6 MPa, and the inlet temperature and outlet temperature are 100 to 180 ° C., respectively. And spray drying by adjusting in the range of 40-90 ° C. It is also possible to perform granulation drying. By these methods, the size of the particles can be freely adjusted to a size of 1 μm to 3 mm.

  The octacalcium phosphate crystal containing the biologically active substance can efficiently release the biologically active substance under acidic conditions (for example, pH 6.0 or lower). The rate of release increases with increasing acidity. For example, by immersion in a solution for 1 hour, 70% of the content is released at pH 3.0 and 40% of the content is released at pH 4.5. The closer it is, the slower the release rate.

  In addition, in a pseudo-biological environment similar to the in-vivo environment (neutral, 37 ° C., in the presence of phosphoric acid), the octacalcium phosphate crystal containing a biologically active substance takes more than one month, It gradually changes to apatite similar to biological tissue. At this time, the contained biologically active substance is slowly released into the solution with the change. Furthermore, no clear rejection reaction or marked inflammatory reaction was observed for the octacalcium phosphate crystal containing the biologically active substance administered in vivo, and the calcium phosphate crystal of the present invention is biocompatible. Indicates.

  The pharmaceutical composition comprising an octacalcium phosphate crystal containing a biologically active substance in the hydration layer of the present invention as an active ingredient is a carrier, excipient, or other It can be adjusted using additives.

  The pharmaceutical composition of the present invention is a parenteral agent, for example, an injection, subcutaneous injection, an organ, a joint, etc., a solid preparation such as an embedding agent, a granule or a powder, a liquid agent or an ointment such as a suspension. As such, it can be administered. Injections include sterile water-soluble or non-aqueous solutions, suspensions, or emulsions. The water-soluble solution or suspension includes, for example, distilled water for injection or physiological saline as a diluent. Examples of the diluent for the water-insoluble solution or suspension include alcohols (for example, ethanol), glycols (for example, propylene glycol or polyethylene glycol), or polysorbetol 80 (trade name). . The composition may further contain a wetting agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a dissolving or solubilizing aid, or a preservative.

  When the pharmaceutical composition of the present invention is used for the treatment of bone disease, the pharmaceutical composition can be directly administered by injection or the like to the affected area of osteoporosis, the affected area of rheumatoid arthritis, or the site of bone tumor. Accordingly, the size of the octacalcium phosphate crystal may be in a range that can ensure the degree of dispersion and needle penetration when used as a suspension injection, for example. For example, the average particle size of the crystals is preferably 0.1 to 300 μm, more preferably 1 to 150 μm, and most preferably 2 to 100 μm.

  The dosage of the pharmaceutical composition of the present invention varies depending on the type and content of the biologically active substance, the dosage form, the duration of active substance release, the animal to be administered, etc., but includes an effective amount of the biologically active substance. As long as the dose is within the range. For example, as a dose per administration, 0.01 mg to 500 mg, preferably 1 mg to 250 mg, more preferably 5 mg to 50 mg as bisphosphonate per adult (body weight 50 kg) should be administered once a week to 3 months. Can do.

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Osteoclasts adhere to old bones and form resorption pits between the bones and osteoclasts, lowering the pH of those parts and dissolving and resorbing the bones. Under normal conditions, osteoblasts coupled to osteoclasts form new bone in the resorbed area, but in bone diseases such as osteoporosis, bone tumors, rheumatoid arthritis, etc. It is considered that pathological bone resorption is taking place because the coupling between cells and osteoblasts is broken, and the bone resorption effect of osteoclasts is enhanced.
On the other hand, the octacalcium phosphate crystal of the present invention contains a biologically active substance such as bisphosphonate in the hydrated layer in the crystal. However, since the biologically active substance exists in the crystal, Even when administered into the body, in a normal in vivo environment, the biologically active substance is not released rapidly, and problems such as initial burst release are not expected to occur. However, since octacalcium phosphate crystals contain phosphate and calcium, they are recognized by osteoclasts in the same way as bone, and this is especially true in bone disease affected areas where osteoclast function is enhanced. The octacalcium phosphate crystals of the invention are lysed by osteoclasts and the biologically active substance is released. When the biologically active substance is a compound that suppresses the function of osteoclasts, the compound is released in the vicinity of the osteoclast that the compound acts on and suppresses the function of osteoclasts. The effect can be expected.
In addition, biologically active substances contained in the octacalcium phosphate crystals include not only biologically active substances that suppress the function of osteoclasts, but also drugs or osteoclasts that enhance the function of osteoblasts. It is also possible to introduce drugs that restore the normal and osteoblastic coupling, and such substances are also selectively released in the affected area where osteoclast function is enhanced. You can expect an effective therapeutic effect.
Conventionally, there has been no symptom-responsive or pathological-compatible pharmaceutical composition that releases a physiologically active substance such as a drug according to the symptoms and pathologies of such diseases. Symptoms and conditions of the disease may be directly caused by foreign pathogens, etc., but often occur when the body's defense reaction such as the immune response or inflammatory reaction of the living body or the normal living body reaction becomes abnormal Sometimes. It is cells in the living body that are responsible for the symptoms and pathologies of such diseases, and the symptoms and pathologies change as their functions are enhanced or suppressed. The pharmaceutical composition of the present invention is capable of releasing a physiologically active substance such as a drug according to a disease symptom or pathology, and can be expected to have a selective therapeutic effect.

  Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but these do not limit the scope of the present invention.

Example 1
In Example 1, an octacalcium phosphate-based crystal containing bisphosphonate was generated by a hydrolysis method.
Bisphosphonate (15 mg) was dissolved in water, and sodium acetate was added to prepare 100 mL of a pH 5.0 solution. As the solution was stirred slowly, the addition of brushite [CaHPO ₄ · 2H ₂ O] 160 mg. The solution was stirred at 60 ° C. for one day to obtain octacalcium phosphate crystals containing bisphosphonate. Theoretically, the formula below can produce from 100 mg of brushite to 100 mg of octacalcium phosphate crystals, but in practice the stoichiometric response is around 85%, from 160 mg of brushite to 100 mg of Octacalcium phosphate crystals were produced.
[Formula 1]
CaHPO ₄ · 2H ₂ O → Ca ₈ H ₂ (PO ₄ ) ₆ · 5H ₂ O
The obtained bisphosphonate-containing octacalcium phosphate crystal was subjected to powder X-ray diffraction analysis (XRD), thermal analysis (TG-DTA), and scanning electron microscope (SEM) analysis.

<< X-ray diffraction analysis >>
X-ray diffractometer [manufactured by Rigaku Denki Co., Ltd.] Using RINT2200, CuKα rays generated at 40 kV / 40 mA are used, the divergence slit angle is 1 degree, the divergence longitudinal limit slit is 10 mm, the scattering slit is 1.25 mm, the light receiving slit is The measurement was performed under the conditions of 3 mm, a scanning speed of 2 degrees / minute, and a sampling width of 0.02 degrees. The obtained product is a crystal based on octacalcium phosphate [Ca ₈ H ₂ (PO ₄ ) ₆ · 5H ₂ O], compared with the octacalcium phosphate crystal obtained without adding bisphosphonate. Then, the low-angle diffraction line (Cuba: 4.7 degrees) is further moved to the low-angle side, and the inter-layer distance of the hydrated layer is increased by inserting bisphosphonate into the hydrated layer. It was confirmed.

《Thermal analysis》
Using a DTG-50 manufactured by Shimadzu Corporation, about 10 mg of the obtained product was heated from 30 ° C. to 600 ° C. at a rate of temperature increase of 10 ° C./min under an air flow of 70 mL / min. A weight reduction curve was determined as a reference (100%). As a result, a peak characteristic of organic combustion was observed around 400 ° C., and the weight loss was about 12%. From this, it was found that the obtained product contained about 12% of bisphosphonate.

《Analysis of morphology by scanning electron microscope》
The morphology of the bisphosphonate-containing octacalcium phosphate crystal was observed at an acceleration voltage of 10 kV using a field emission scanning electron microscope FE-SEM [HITACHI S-5000]. The obtained product was a plate-like crystal and had a size of about 100 × 10 × 10 μm.

Example 2
In Example 2 , the production of octacalcium phosphate crystals containing bisphosphonate was performed by a coprecipitation method.
40 mL of Na ₂ HPO ₄ and 40 mmol of NaH ₂ PO ₄ were mixed to prepare 250 mL of pH 5.5 phosphate buffer. Bisphosphonate was added to this buffer solution and dissolved with slow stirring. It was added slowly 20mmol acetate mosquitoes Resid um 250 mL, at 37 ° C., and reacted for 6 hours to obtain a octacalcium phosphate-based crystal containing bisphosphonate.
Powder X-ray diffraction analysis (XRD), thermal analysis (TG-DTA) and scanning electron microscope (SEM) analysis of the obtained bisphosphonate-containing octacalcium phosphate crystal were performed using the same method as in Example 1. .
<< X-ray diffraction analysis >>
As in Example 1, when compared with the octacalcium phosphate crystal not containing bisphosphonate, the low-angle diffraction line (Cuba: 4.7 degrees) is shifted to the low-angle side, and bisphosphonate is in the hydrated layer. It was confirmed that the interlaminar distance of the hydrated layer was increased by the insertion. 《Thermal analysis》
A peak characteristic of organic combustion was observed around 400 ° C., but the weight loss was about 8%. From this, it was found that the obtained product contained about 8% of bisphosphonate.
《Analysis of morphology by scanning electron microscope》
Similar to Example 1, the obtained product was a plate-like crystal and had a size of about 100 × 10 × 10 μm.

Example 3
In Example 3, the release of bisphosphonates under acidic conditions of the obtained bisphosphonate-containing octacalcium phosphate crystals was examined.
10 mg of the bisphosphonate-containing octacalcium phosphate crystal obtained in Example 1 was added to 20 mL of an aqueous solution adjusted to pH 3.0 and pH 4.5 with hydrochloric acid, and kept at 37 ° C. The aqueous solution at pH 3.0 released 70% bisphosphonate after 1 hour of immersion, and the aqueous solution at pH 4.5 released 40% bisphosphonate after 1 hour of immersion.

Example 4
In Example 4, the release of bisphosphonates under neutral conditions was examined under the obtained bisphosphonate-containing octacalcium phosphate crystal.
5 mg of the bisphosphonate-containing octacalcium phosphate crystal obtained in Example 1 was added to 5 mL of a phosphate buffer and maintained at 37 ° C. After 1 hour of immersion, almost no bisphosphonate release was detected. When left in this state for one month, 100% of the bisphosphonate was released. Moreover, when the product after one month was analyzed by the same powder X-ray diffraction analysis (XRD) as in Example 1, it was converted to a living tissue-like apatite containing a carbonate group.

  The present invention relates to a bone disease such as metastatic bone tumor and osteoporosis, or bone immune disease by using an octacalcium phosphate crystal containing a biologically active substance in a pharmaceutical composition containing it as an active ingredient. It can be used as a therapeutic agent that can effectively treat rheumatoid arthritis and the like. In addition, by the method for producing an octacalcium phosphate crystal containing a biologically active substance of the present invention, an octacalcium phosphate crystal that can contain a sufficient amount of the biologically active substance as a sustained-release preparation Can be manufactured.

Claims (9)

  An octacalcium phosphate crystal containing bisphosphonate in the hydrated layer.   The octacalcium phosphate crystal according to claim 1, wherein a part of calcium ions of the octacalcium phosphate crystal is substituted with a metal cation.   The octacalcium phosphate crystal according to claim 2, wherein the metal cation is a metal cation selected from the group consisting of magnesium, strontium, barium, platinum, manganese, copper, zinc and iron. Including a pharmaceutical composition as an active ingredient an octacalcium phosphate-based crystal according to any one of claims 1 to 3. The pharmaceutical composition according to claim 4, which is used for bone diseases.   A method for producing octacalcium phosphate containing bisphosphonate, wherein a solution containing calcium ions and a solution containing phosphate ions are mixed and stirred at 25 to 80 ° C. while maintaining the pH at 4.5 to 8.5, A method for producing an octacalcium phosphate-based crystal containing bisphosphonate, wherein the solution containing calcium ions and / or the solution containing phosphate ions contains bisphosphonate. The method for producing an octacalcium phosphate crystal containing bisphosphonate according to claim 6 , wherein the solution containing calcium ions and / or the solution containing phosphate ions contains a metal cation. CaHPO ₄ .2H ₂ O, CaHPO ₄ , αCa ₃ (PO ₄ ) ₂ , or βCa ₃ (PO ₄ ) ₂ is added to a solution containing bisphosphonate maintained at pH 4.5 to 8.5, and 25 to 80 ° C. A method for producing octacalcium phosphate containing bisphosphonate, which is stirred in The method for producing octacalcium phosphate containing bisphosphonate according to claim 8 , wherein the solution containing the bisphosphonate contains a metal cation.