WO2021161987A1 - Bisphosphonate-containing carbon particle composite and method for producing same - Google Patents

Bisphosphonate-containing carbon particle composite and method for producing same Download PDF

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WO2021161987A1
WO2021161987A1 PCT/JP2021/004744 JP2021004744W WO2021161987A1 WO 2021161987 A1 WO2021161987 A1 WO 2021161987A1 JP 2021004744 W JP2021004744 W JP 2021004744W WO 2021161987 A1 WO2021161987 A1 WO 2021161987A1
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cap
oxcnh
carbon
acid
particles
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PCT/JP2021/004744
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French (fr)
Japanese (ja)
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湯田坂　雅子
真紀 中村
直人 齋藤
青木　薫
勝也 上田
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国立研究開発法人産業技術総合研究所
国立大学法人信州大学
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Priority to JP2022500417A priority Critical patent/JPWO2021161987A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

Definitions

  • the present invention relates to a bisphosphonate-containing carbon particle complex and a method for producing the same.
  • BPs Bisphosphonates
  • oral administration of BP has a low absorption rate from the intestinal tract (about several percent), and side effects such as esophageal ulcer and esophagitis become a problem.
  • BP blood pressure
  • side effects such as non-traumatic subtrochanteric femoral and atypical fractures of the proximal femoral shaft due to suppression of bone metabolism, and BP-related osteonecrosis.
  • BP-related osteonecrosis For this reason, there are doubts about the safety and effectiveness of BP during long-term use overseas.
  • a drug delivery system for cancer treatment DDS
  • bone repair scaffold is being studied.
  • DDS and Scaffold are currently produced using biodegradable materials, the environment of metastatic bone cancer is poorly ordered, and the drug is slowly released to control bone repair according to the degree of cancer progression. It is difficult to achieve the purpose of doing so.
  • Patent Document 1 describes a method for producing a nanostructure such as a carbon nanotube having a BP structure.
  • An object of the present invention is to provide a BP-containing carbon particle complex in which the action of BP is improved and side effects are suppressed, and a method for producing the same.
  • the present inventors have determined that among non-biodegradable materials, carbon particles having high biocompatibility and capable of carrying various kinds of therapeutic agents and target substances are suitable. Therefore, the present inventors have focused on imparting bone affinity to carbon particles in vivo, and investigated modifying the surface of carbon particles with calcium phosphate (CaP). As a result, it was determined that by combining BP and CaP with carbon particles, a complex having high bone affinity and capable of killing osteoclasts can be obtained.
  • CaP calcium phosphate
  • the present invention has been completed based on these findings, and according to the present invention, a carbon particle composite containing BP, CaP and carbon particles and a method for producing the same are provided.
  • the BP-containing carbon particle complex in the present invention can improve the action of BP and suppress side effects. Therefore, the BP-containing carbon particle complex in the present invention is effective for osteogenesis-promoting treatment of osteoclast-targeted metastatic bone cancer, osteoporosis, osteogenesis imperfecta, osteoarthritis and the like.
  • Photograph of dispersion of BV-CaP-OxCNH and CaP-OxCNH Photograph of dispersion of BV-CaP-OxCNH and CaP-OxCNH.
  • TEM transmission electron microscope
  • STEM scanning transmission electron microscope
  • A Results of toxicity evaluation of mouse macrophage-like cells (RAW264.7 cells) of BV-CaP-OxCNH.
  • B Results of BV RAW264.7 cytotoxicity assessment.
  • A Results of RAW264.7 cytotoxicity evaluation of BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH.
  • B Results of RAW264.7 cytotoxicity evaluation of BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH.
  • An optical micrograph of osteoclasts obtained by differentiating from RAW264.7 cells (a) and RAW264.7 cells, stained with tartrate-resistant acid phosphatase (TRAP). Fluorescence micrograph showing uptake of BV-CaP-OxCNH by osteoclasts. Results of survival rate evaluation of osteoclasts supplemented with OxCNH, CaP-OxCNH, and BV-CaP-OxCNH. SEM images of BV-CaP-OxCNH, ZO-CaP-OxCNH, and PM-CaP-OxCNH.
  • Tibial CT images of osteoporosis model rats 0, 8 and 12 weeks after sample implantation Total bone mineral density of osteoporosis model rats 0, 4, 8 and 12 weeks after sample implantation.
  • the carbon particle complex in the present invention contains BP, CaP and carbon particles.
  • the carbon particles are, but are not limited to, CNH, carbon nanotubes, nanographene, nanodiamond, or carbon fiber, or a combination thereof.
  • the carbon particles have chemical and physical stability, and it is easy to introduce a functional group such as a carboxyl group (-COOH) suitable for chemical modification on the surface of the carbon particles, so that the carbon particles are suitable for imparting multiple functions. ..
  • CNH is preferable as carbon particles used for a carbon particle complex because it has advantages such as size uniformity, dispersion stability in an aqueous solution, high-purity mass production, and no purification required.
  • the CNH has a spherical shape having a diameter of about 100 nm, in which thousands of carbon nanotubes having a diameter of 2 nm to 5 nm are radially gathered.
  • CNH is robust and does not decompose easily. Further, since CNH has a structure with abundant irregularities on the surface, it does not strongly agglomerate and is isolated and dispersed in an aqueous solution.
  • OxCNH which is obtained by oxidizing CNH with hydrogen peroxide, has holes in the tube wall due to the cleavage of carbon-carbon bonds, and also facilitates the entry and exit of molecules into the internal space. More preferable as carbon particles to be used.
  • OxCNH is hydrophilic because a functional group such as a carboxyl group is introduced into the pore edge or the like, and is more preferable as carbon particles used in a carbon particle complex.
  • CaP may be a compound containing at least phosphate ions and calcium ions. Further, CaP may be amorphous. Calcium ions in the CaP supersaturated solution are attracted to the carboxyl group of the carbon particles by electrostatic interaction, and CaP is precipitated from there to form a complex of CaP and carbon particles. Alternatively, the precipitated calcium ions on the surface of the CaP are attracted to the carboxyl group of the carbon particles by electrostatic interaction to form a complex of the CaP and the carbon particles.
  • the BP contained in the carbon particle composite is etidronic acid, ibandronic acid, zoledronic acid, alendronic acid, minodronic acid, risedronic acid, pamidronic acid, incadronic acid, or salts thereof, or a combination thereof.
  • the BP may contain at least a phosphonic acid group, inhibit the activity of osteoclasts, and prevent bone resorption.
  • the phosphonic acid group of BP attracts calcium ions in the CaP supersaturated solution and calcium ions on the surface of the precipitated CaP by electrostatic interaction, so that CaP and BP are compounded and a complex with carbon particles is further formed.
  • the BP is contained in the internal space of the carbon particles or is supported on the carbon particles by being physically adsorbed on the surface of the carbon particles.
  • the BP-containing carbon particle complex in the present invention can kill osteoclasts with a small amount of BP content.
  • the carbon particle complex in the present invention can be produced by preparing a CaP supersaturated solution containing BP and carbon particles and allowing them to stand to co-precipitate the three.
  • the solution used as a raw material for the CaP supersaturated solution is not particularly limited.
  • a pH adjuster or the like may be appropriately contained in addition to the solution containing calcium ions (calcium-containing solution) and the solution containing phosphate ions (phosphate-containing solution).
  • As the raw material of the CaP supersaturated solution for example, various injection preparations and the like may be used in combination.
  • the carbon particle composite of the present invention can be used as it is as a preparation.
  • a CaP supersaturated solution containing BP and carbon particles can be prepared by mixing BP, carbon particles, a calcium-containing liquid and a phosphoric acid-containing liquid. At the time of mixing, a pH adjuster or the like may be added if necessary.
  • the temperature and time for allowing the CaP supersaturated solution containing BP and carbon particles to stand are not particularly limited. It can be appropriately adjusted in consideration of the size and dispersibility of the carbon particle composite to be produced.
  • the carbon particle complex in the present invention contains BP, it can be applied to a preparation targeting osteoclasts.
  • the formulation in the present invention may appropriately contain additives and the like in addition to the BP-containing carbon particle complex.
  • the pharmaceutical product of the present invention may be administered to a patient by intravenous injection, for example, or may be locally administered to a specific site (affected area).
  • the BP-containing carbon particle complex in the present invention can function as a DDS capable of controlling the drug distribution in the body spatially, quantitatively, and temporally.
  • the BP-containing carbon particle complex in the present invention has increased bone affinity by containing CaP, and can kill osteoclasts with a small amount of BP.
  • the locally administered BP-containing carbon particle complex can stay in the affected area for a long time and continue to release BP.
  • the BP-containing carbon particle complex is localized in lysosomes in osteoclasts, CaP is dissolved in the acidic environment, and BP can be efficiently released.
  • the carbon particles generate heat when irradiated with light, photothermia treatment is possible depending on the affected area.
  • CaP is a component of bone matrix
  • bone repair by osteoblasts can be promoted after bone resorption by osteoclasts is suppressed.
  • the carbon particles themselves fulfill the function of Scaffold having excellent bone formation, it is effective for bone formation promoting treatment. Therefore, the BP-containing carbon particle complex in the present invention can be efficiently treated for promoting bone formation due to the synergistic effect of DDS and Scaffold.
  • OxCNH powder is dispersed in ultrapure water to 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL, and sterilized by autoclave. An OxCNH dispersion was obtained.
  • BV solution a Bombiva intravenous injection 1 mg syringe (Chugai Pharmaceutical Co., Ltd.) containing sodium ibandronate hydrate as an active ingredient was used.
  • a calcium-containing solution, a phosphoric acid-containing solution, and a pH adjuster were prepared as raw materials for the CaP supersaturated solution.
  • the calcium-containing solution was prepared by mixing Ringer's solution “Otsuka” (Otsuka Pharmaceutical Co., Ltd.) (49.361 mL) and Ca chloride correction solution 1 mEq / mL (Otsuka Pharmaceutical Co., Ltd.) (0.639 mL).
  • the phosphoric acid-containing solution was prepared by mixing Clinisalz (registered trademark) infusion solution (Kyowa CritiCare Co., Ltd.) (9.469 mL) and dipotassium phosphate Injection 20 mEq kit "Terumo" (Terumo Corporation) (0.531 mL). ..
  • the pH adjuster was prepared by mixing Meylon (registered trademark) intravenous injection 7% (Otsuka Pharmaceutical Co., Ltd.) (5 mL) and water for injection (Fuso Pharmaceutical Indus Co., Ltd.) (20 mL). Sterilized OxCNH aqueous dispersion (0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL dispersion, 0.250 mL each), BV solution (0.
  • reaction solution 10 mL
  • the reaction solution was allowed to stand in an incubator at 25 ° C. for 30 minutes to coprecipitate BV, CaP, and OxCNH.
  • the obtained sample BV-CaP-OxCNH was collected by centrifugation (6000 rpm, 5 minutes), and washed by repeating the process of dispersing the sample in water for injection and centrifuging.
  • CaP-OxCNH As a control containing no BV, a sample CaP-OxCNH was obtained by performing the same operation as the preparation of BV-CaP-OxCNH except that the reaction solution was prepared using water for injection instead of the BV solution.
  • BV-CaP and CaP As a control containing no CNH, the same operation as the preparation of BV-CaP-OxCNH or the preparation of CaP-OxCNH was performed except that the reaction solution was prepared using water for injection instead of the sterile OxCNH aqueous dispersion, and the BV and A complex of CaP (BV-CaP) and CaP particles (CaP) were obtained.
  • BV-CaP and BV-CaP-OxCNH are shown in FIG.
  • SEM images of CaP and CaP-OxCNH are shown in FIG.
  • BV-CaP, BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, and BV-CaP-OxCNH-4 all have a primary particle size of about 50 nm.
  • CaP-OxCNH / CaP 1.38 to 1.44. It is considered that BV-CaP-OxCNH and BV-CaP supported BV containing phosphorus, so that the phosphorus content was relatively large with respect to calcium and the Ca / P molar ratio was small.
  • the Ca / P molar ratio of CaP in BV-CaP-OxCNH is equivalent to that of CaP-OxCNH
  • the BV content was estimated and found to be 20-30% (0. It was calculated to be 16-0.24 ⁇ mol).
  • the amount ratio of BV to CaP was estimated to be 4-7: 100.
  • BV-CaP-OxCNH BV, CaP and OxCNH form a ternary complex.
  • BV-CaP-OxCNH and BV-CaP all contained less calcium and phosphorus than CaP-OxCNH and CaP. It is considered that this is because in BV-CaP-OxCNH and BV-CaP, the pH of the reaction solution was lowered by the addition of the acidic BV solution to the reaction solution, and the formation of CaP was hindered. It is also possible that the compounding with BV affected the rate of CaP formation. On the other hand, the concentration of OxCNH in the reaction solution did not significantly affect the calcium and phosphorus contents.
  • the amounts of CaP and BV contained in BV-CaP-OxCNH do not differ greatly regardless of the concentration of OxCNH in the reaction solution.
  • the fact that the changes in the amount of CaP and the amount of BV are small even if the amount of OxCNH changes suggests that CaP and BV are bound without being affected by OxCNH. That is, it was presumed that BV was mainly supported on CaP and that BV-CaP had a structure in which a complex was formed with OxCNH.
  • FIG. 5 shows a TEM image of BV-CaP-OxCNH-2 and a STEM-EDX image showing the element distribution of carbon (C), calcium (Ca), oxygen (O), and phosphorus (P).
  • C carbon
  • Ca calcium
  • O oxygen
  • P phosphorus
  • Phagocytic mouse macrophage-like cells (RAW264.7) were seeded on 96-well plates and cultured at 37 ° C. After culturing for 24 hours, the medium is removed, BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, and Medium containing BV-CaP-OxCNH-6 was added.
  • the concentration of each sample in the medium is 0.25 ⁇ g / mL for BV-CaP-OxCNH-1, 0.5 ⁇ g / mL for BV-CaP-OxCNH-2, and 1 for BV-CaP-OxCNH-3. .25 ⁇ g / mL, BV-CaP-OxCNH-4 was 2.5 ⁇ g / mL, BV-CaP-OxCNH-5 was 5.0 ⁇ g / mL, and BV-CaP-OxCNH-6 was 12.5 ⁇ g / mL.
  • the BV concentration in the medium is about 0.5 ⁇ g / mL to 0.75 ⁇ g / mL
  • the Ca concentration derived from CaP is 0.089 mM to 0.11 mM
  • the P concentration is 0.068 mM to 0. It was estimated to be about 081 mM.
  • a normal medium containing no BV-CaP-OxCNH was used as a control. After culturing for 24 hours, the number of surviving cells was determined by measuring the absorbance at 450 nm using the Cell Counting Kit-8 reagent (Dojin Chemical Laboratory). The cell viability was calculated with the absorbance of the control as 100%.
  • 6A shows the results of cytotoxicity evaluation of BV-CaP-OxCNH.
  • BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3 are almost the same, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, BV-CaP-OxCNH- In No. 6, cytotoxicity became more prominent as the OxCNH concentration in the sample increased. The cytotoxicity of BV was evaluated in the same manner as above.
  • FIG. 6B shows the results of RAW264.7 cytotoxicity evaluation of BV. As shown in FIG. 6 (b), BV showed cytotoxicity in a concentration-dependent manner. Since the BV concentration contained in BV-CaP-OxCNH is estimated to be about 0.5 ⁇ g / mL to 0.75 ⁇ g / mL in the medium, the amount of BV-CaP-OxCNH is smaller than that of BV alone.
  • Cytotoxicity was evaluated for BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH by the same method as described above. The concentration of each sample in the medium was diluted so that the OxCNH concentration was 0.17 ⁇ g / mL, 0.5 ⁇ g / mL, and 1.7 ⁇ g / mL. Similarly, cytotoxicity was evaluated for BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH.
  • FIG. 7A shows the results of cytotoxicity evaluation of BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH.
  • FIG. 7 (a) CaP-OxCNH-2 and OxCNH were almost equivalent, and BV-CaP-OxCNH-2 showed stronger cytotoxicity.
  • FIG. 7B shows the results of cytotoxicity evaluation of BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. As shown in FIG.
  • FIG. 8 shows an optical micrograph of RAW264.7 cells cultured for 24 hours in a medium supplemented with BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. As shown in the lower part of FIG.
  • OxCNH was presumed to be taken up by intracellular lysosomes. As shown in the middle part of FIG. 8, it was presumed that CaP-OxCNH formed a large mass in the medium and was difficult to be taken up into cells. As shown in the upper part of FIG. 8, it was confirmed that BV-CaP-OxCNH was taken up into the cells, but most of them were apoptotic dead cells and necrosis dead cells. It was suggested that BV-CaP-OxCNH causes apoptotic death and necrosis death of cells by being taken up into cells and releasing BV, and exhibits cytotoxicity efficiently.
  • RAW264.7 cells were differentiated into osteoclasts.
  • RAW264.7 cells were seeded on a 96-well plate, cultured for 1 day, replaced with a medium containing RANKL (100 ng / mL), and cultured for 5 days to induce differentiation into osteoclasts.
  • tartrate-resistant acid phosphatase which is a marker of osteoclasts, was stained and observed with an optical microscope.
  • FIG. 9 shows an optical micrograph of TRAP-stained osteoclasts (b) obtained by differentiating from RAW264.7 cells (a) and RAW264.7 cells.
  • the RAW264.7 cells before differentiation were not stained by TRAP staining, but were stained by the osteoclasts after differentiation. From this result, it was confirmed that the RAW264.7 cells were normally differentiated into osteoclasts.
  • FIG. 10 shows fluorescence micrographs showing the uptake of BV-CaP-OxCNH by osteoclasts after 24 hours (upper part of FIG. 10) and 48 hours later (lower part of FIG. 10).
  • BV-CaP-OxCNH had OxCNH (arrow) incorporated into the lysosomes of osteoclasts. Mononuclear and multinucleated osteoclasts were observed.
  • FIG. 11 shows the viability of osteoclasts supplemented with OxCNH, CaP-OxCNH, and BV-CaP-OxCNH.
  • BV-CaP-OxCNH-1 OxCNH concentration 2.5 ⁇ g / mL
  • BV-CaP-OxCNH-2 OxCNH concentration 5.0 ⁇ g / mL
  • BV-CaP-OxCNH-3 OxCNH concentration 12.5 ⁇ g
  • the cell viability decreased as the OxCNH concentration in the sample increased.
  • OxCNH, CaP-OxCNH-1, CaP-OxCNH-2, and CaP-OxCNH-3 no decrease in cell viability was observed depending on the OxCNH concentration.
  • BP-CaP-OxCNHs were prepared by changing the type of BP and the composition of the CaP supersaturated solution.
  • BP in addition to the BV used in Example 1, zoledronic acid (Zometa, ZO) and pamidronic acid (PM) were used.
  • concentrations of CaP supersaturated solutions were prepared for each of BV, ZO, and PM to prepare a total of 12 types of BP-CaP-OxCNH.
  • the concentration of the OxCNH aqueous dispersion was adjusted to 2 mg / mL, and the mixture was sterilized by an autoclave to obtain a sterilized OxCNH aqueous dispersion.
  • the BV solution the Bombiva intravenous injection 1 mg syringe used in Example 1 was used.
  • the ZO solution Zoledronic acid hydrate containing zoledronic acid hydrate as an active ingredient was used as an intravenous drip infusion of Zometa 4 mg / 5 mL (Novartis Pharma Co., Ltd.).
  • the calcium-containing solution and the phosphoric acid-containing solution which are the raw materials of the CaP hypersaturated solution, were the same as those used in Example 1, the calcium-containing solution was Ringer's solution "Otsuka” and the Ca chloride correction solution 1 mEq / mL, and the phosphoric acid-containing solution was It was prepared by mixing Clinisarz® infusion solution with dipotassium phosphate Injection 20mEq kit "Termo". By changing the mixing amount of each reagent, four kinds of calcium-containing liquids and phosphoric acid-containing liquids in which the concentrations of Ca and P contained were changed were prepared.
  • Example 2 based on the same calcium-containing solution and phosphoric acid-containing solution used in Example 1 and the concentrations of Ca or P contained in those solutions, the concentrations of Ca and P are 78%, respectively. , 67% and 56% of calcium-containing solution and phosphoric acid-containing solution were prepared. Moreover, the same pH adjuster as in Example 1 was used.
  • These supersaturated solutions were designated as CaP supersaturated solutions a, b, c and d.
  • the reaction solution was allowed to stand in an incubator at 25 ° C. for 30 minutes to coprecipitate BP, CaP, and OxCNH.
  • BP-CaP-OxCNH was collected by centrifugation (6000 rpm, 5 minutes), and washed by repeating the process of dispersing the sample in water for injection and centrifuging.
  • Samples prepared using the CaP supersaturated solutions a, b, c, and d were designated as BP-CaPa-OxCNH, BP-CaPb-OxCNH, BP-CaPc-OxCNH, and BP-CaPd-OxCNH, respectively.
  • BP corresponds to any of BV, ZO, and PM.
  • BP-CaP-OxCNH The structure of BP-CaP-OxCNH was confirmed by SEM. Similar to Example 1, the sample was dropped on a silicon substrate, dried, and gold was vapor-deposited for observation. An SEM image of BP-CaP-OxCNH is shown in FIG. In any of the BP-CaP-OxCNH particles, particles having a primary particle diameter of 50 to 100 nm and particles having a primary particle diameter of 100 to 150 nm were observed. Further, the particles having a primary particle diameter of 100 to 150 nm had an uneven structure considered to be caused by CNH.
  • FIG. 15A shows the results of cytotoxicity evaluation of ZO-CaP-OxCNH. Compared with BV-CaP-OxCNH, ZO-CaP-OxCNH showed strong cytotoxicity. ZO alone shown in FIG.
  • FIG. 16A shows the results of cytotoxicity evaluation of PM-CaP-OxCNH.
  • PM-CaP-OxCNH showed stronger cytotoxicity as compared with BV-CaP-OxCNH.
  • PM-CaP-OxCNH showed almost no toxicity under the condition of 1.7 ⁇ g / mL as the OxCNH concentration in the medium.
  • a hole was made in the tibia of an osteoporosis model rat, and a sample such as BV-CaP-OxCNH was embedded therein, and the effect of the sample on bone formation was evaluated.
  • osteoporosis model rat Bilateral ovaries of Wistar rats (female, 10 weeks old) were removed and the wound was sutured. Osteoporosis model rats were prepared by breeding normally for 8 weeks after the operation. For comparison, sham-surgery rats without removing the ovaries were also prepared. Changes in body weight were recorded, and it was confirmed that the osteoporosis model rats gained weight (a prominent phenomenon in the osteoporosis model rats) as compared with the sham-surgery rats.
  • bone mineral density was measured using X-ray CT for experimental animals, and the cancellous bone density of the osteoporosis model rat was reduced by about 20% compared to the sham-operated rat, that is, the condition of osteoporosis. It was confirmed.
  • BV-CaP-OxCNH-2, CaP-OxCNH-2, BV-CaP, and CaP were prepared in the same manner as in Example 1 to obtain a sample.
  • the OxCNH dispersion which is one of the raw materials for sample preparation, was prepared by the same method as in Example 2 (without freeze-drying).
  • the resulting sample was suspended in a small amount of water for injection.
  • the OxCNH concentration in the suspension is estimated to be 0.83 mg / mL, and the BV concentration is estimated to be about 2.1 mg / mL.
  • OxCNH (0.83 mg / mL), BV (1.0 mg / mL as ibandronic acid), and physiological saline (negative control) were also examined for comparison.
  • Boneal reaming and sample implantation Three osteoporosis model rats were prepared for each sample, and each of the left and right tibias was reamed with an 18-gauge syringe needle (operation of making a hole in the bone, bone hole of about 1 mm). Each sample was prepared in an amount of 1 mL so that the total BV dose per rat body weight was 154 ⁇ g / kg and the total OxCNH dose was 61.7 ⁇ g / kg (body weight per rat was about 200 g). Each sample was placed in the bone holes of the left and right tibias of each osteoporosis model rat in an amount of 50 ⁇ L, and the wound was sutured.
  • Osteoporosis model rats were normally bred for 12 weeks after implantation while performing CT imaging and bone mineral density evaluation, which will be described later, over time.
  • a group in which the BV solution was subcutaneously administered was also prepared.
  • Subcutaneous administration of the BV solution was performed once a day for a total of 12 weeks every 4 weeks at different sites in the rat each time so as not to be a local administration (1 week continuous administration 0, 4, I went 3 times after 8 weeks).
  • the BV solution was prepared so that the total BV dose per rat body weight of the BV solution (1.0 mg / mL) was 154 ⁇ g / kg in 12 weeks.
  • the animals were normally bred while performing CT imaging and bone mineral density evaluation, which will be described later, over time.
  • FIG. 17A shows a laparotomy photograph (upper side) and a photograph of the removed uterus (lower side) of a sham-surgery rat (left side) and an osteoporosis model rat (right side).
  • clear uterine atrophy (a prominent phenomenon in the osteoporosis model rat) was observed as compared with the pseudo-surgery rat.
  • the weight of the uterus removed from the pseudo-surgery rat and the osteoporosis model rat was measured.
  • 17B shows the relative value of the uterine weight removed from the osteoporosis model rat (right side) when the uterine weight removed from the pseudo-surgery rat (left side) is 1.
  • the relative value of the uterine weight removed from the osteoporosis model rat was about 0.3 when the uterine weight removed from the sham-operated rat was 1.
  • a clear decrease in uterine weight was observed as compared with the sham-surgery rats.
  • FIG. 18 shows tibial CT images 0, 8 and 12 weeks after sample implantation in each osteoporosis model rat.
  • CT Computed tomography
  • FIG. 19 shows the total bone density of each osteoporosis model rat tibia
  • FIG. 20 shows the cortical bone density of each osteoporosis model rat tibia
  • FIG. 21 shows the cancellous bone density of each osteoporosis model rat tibia.
  • FIG. 19 in rats subcutaneously administered with BV and rats locally administered with BV-CaP-OxCNH-2, BV-CaP, BV, saline, CaP, OxCNH, or CaP-OxCNH-2 was locally administered.
  • FIG. 22 shows the time course of the relative value when the cancellous bone mineral density of each osteoporosis model rat tibia immediately after sample implantation (0 week later) is set to 1 using the data shown in FIG. 21. As shown in FIG. 22
  • BV-CaP-OxCNH-2 showed the same bone mineral density improving effect as subcutaneous administration of BV administered multiple times every month by one local administration (implantation in the affected area). From this, it is inferred that since BV-CaP-OxCNH-2 is a single dose, the load on the patient is small and the bone formation promoting effect is efficiently exhibited.

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Abstract

Provided are: a BP-containing carbon particle composite having improved BP action and suppressed side effects; and a method for producing the same.　Provided is a carbon particle composite containing a calcium phosphate, a bisphosphonate, and carbon particles. The carbon particles may be carbon nanohorns, carbon nanotubes, nanographenes, nanodiamonds, and carbon fibers, or a combination thereof. The carbon particles may also be carbon oxide nanohorns.

Description

ビスホスホネート含有カーボン粒子複合体及びその製造方法Bisphosphonate-containing carbon particle complex and its production method
　本発明は、ビスホスホネート含有カーボン粒子複合体及びその製造方法に関する。 The present invention relates to a bisphosphonate-containing carbon particle complex and a method for producing the same.
　多くの癌は骨転移の頻度が高く、癌が骨に転移すると破骨細胞による溶骨が進行し、痛みと機能障害が生じる。また、溶骨による高カルシウム血症や、骨からのがん細胞の再放出により、生命予後が短縮する。破骨細胞にアポトーシスを誘発させる治療として、ビスホスホネート（Ｂｉｓｐｈｏｓｐｈｏｎａｔｅ：ＢＰ）剤が処方されている。しかしながら、ＢＰの経口投与では腸管からの吸収率が低く（数％程度）、食道潰瘍や食道炎などの副作用が問題となる。また、長期間にわたるＢＰの投与により、骨代謝抑制に起因する非外傷性の大腿骨転子下及び近位大腿骨骨幹部の非定型骨折、ＢＰ関連骨壊死などの副作用が問題となる。このため海外では、長期使用の際のＢＰの安全性と有効性に疑問がもたれている。しかしながら、転移性骨癌に対して効果的な他の治療薬はない。そこで、癌治療用薬物送達システム（Ｄｒｕｇ　Ｄｅｌｉｖｅｒｙ　Ｓｙｓｔｅｍ：ＤＤＳ）や骨修復Ｓｃａｆｆｏｌｄによる転移性骨癌の治療が検討されている。このようなＤＤＳやＳｃａｆｆｏｌｄは、現在、生体内分解性材料を使って作製されているものの、転移性骨癌の環境は秩序性が乏しく、薬剤を徐放し骨修復を癌進行度に応じて制御するという目的を達成するのは困難である。 Most cancers have a high frequency of bone metastasis, and when the cancer metastasizes to bone, osteoclasts progress to osteoclasts, causing pain and dysfunction. In addition, hypercalcemia due to osteolysis and re-release of cancer cells from bone shorten the prognosis of life. Bisphosphonates (BPs) have been prescribed as treatments that induce apoptosis in osteoclasts. However, oral administration of BP has a low absorption rate from the intestinal tract (about several percent), and side effects such as esophageal ulcer and esophagitis become a problem. In addition, long-term administration of BP causes side effects such as non-traumatic subtrochanteric femoral and atypical fractures of the proximal femoral shaft due to suppression of bone metabolism, and BP-related osteonecrosis. For this reason, there are doubts about the safety and effectiveness of BP during long-term use overseas. However, there are no other effective treatments for metastatic bone cancer. Therefore, treatment of metastatic bone cancer by a drug delivery system for cancer treatment (Drug Delivery System: DDS) and bone repair scaffold is being studied. Although such DDS and Scaffold are currently produced using biodegradable materials, the environment of metastatic bone cancer is poorly ordered, and the drug is slowly released to control bone repair according to the degree of cancer progression. It is difficult to achieve the purpose of doing so.
　一方で、生体分解されにくく、多種多様な化学修飾が可能であるカーボン粒子を用いたＤＤＳの開発が進められている。特許文献１には、ＢＰ構造を有するカーボンナノチューブなどのナノ構造体の製造方法が記載されている。 On the other hand, the development of DDS using carbon particles, which are not easily decomposed by biotechnology and can be chemically modified in a wide variety of ways, is underway. Patent Document 1 describes a method for producing a nanostructure such as a carbon nanotube having a BP structure.
国際公開第２０１３／００５４１５４号International Publication No. 2013/0054154
　本発明は、ＢＰの作用を向上し、副作用を抑制したＢＰ含有カーボン粒子複合体及びその製造方法を提供することを課題とする。 An object of the present invention is to provide a BP-containing carbon particle complex in which the action of BP is improved and side effects are suppressed, and a method for producing the same.
　上記課題を解決するために本発明者らは、非生体分解性材料のなかでも生体親和性の高く、多種類の治療薬や標的物質を担持可能であるカーボン粒子が適していると判断した。そこで本発明者らは、カーボン粒子に生体内で骨親和性を付与することに着目し、カーボン粒子の表面をリン酸カルシウム（ＣａＰ）で修飾することを検討した。この結果、カーボン粒子にＢＰとＣａＰを複合化させることで、骨親和性が高く、破骨細胞を細胞死させることのできる複合体が得られると判断した。 In order to solve the above problems, the present inventors have determined that among non-biodegradable materials, carbon particles having high biocompatibility and capable of carrying various kinds of therapeutic agents and target substances are suitable. Therefore, the present inventors have focused on imparting bone affinity to carbon particles in vivo, and investigated modifying the surface of carbon particles with calcium phosphate (CaP). As a result, it was determined that by combining BP and CaP with carbon particles, a complex having high bone affinity and capable of killing osteoclasts can be obtained.
　本発明はこれらの知見に基づいて完成に至ったものであり、本発明によれば、ＢＰとＣａＰとカーボン粒子を含有するカーボン粒子複合体及びその製造方法が提供される。 The present invention has been completed based on these findings, and according to the present invention, a carbon particle composite containing BP, CaP and carbon particles and a method for producing the same are provided.
　本発明におけるＢＰ含有カーボン粒子複合体は、ＢＰの作用を向上させ、副作用を抑制することができる。このため、本発明におけるＢＰ含有カーボン粒子複合体は、破骨細胞をターゲットとした転移性骨癌、骨粗鬆症、骨形成不全症、変形性骨炎などの骨形成促進治療に有効である。 The BP-containing carbon particle complex in the present invention can improve the action of BP and suppress side effects. Therefore, the BP-containing carbon particle complex in the present invention is effective for osteogenesis-promoting treatment of osteoclast-targeted metastatic bone cancer, osteoporosis, osteogenesis imperfecta, osteoarthritis and the like.
ＢＶ－ＣａＰ－ＯｘＣＮＨ及びＣａＰ－ＯｘＣＮＨの分散液の写真。Photograph of dispersion of BV-CaP-OxCNH and CaP-OxCNH. ＢＶ－ＣａＰ及びＢＶ－ＣａＰ－ＯｘＣＮＨの走査電子顕微鏡（ＳＥＭ）画像。Scanning electron microscope (SEM) images of BV-CaP and BV-CaP-OxCNH. ＣａＰ及びＣａＰ－ＯｘＣＮＨのＳＥＭ画像。SEM images of CaP and CaP-OxCNH. ＢＶ－ＣａＰ－ＯｘＣＮＨ、ＣａＰ－ＯｘＣＮＨ、ＢＶ－ＣａＰ、ＣａＰのエネルギー分散型Ｘ線分光（ＥＤＸ）スペクトル。Energy dispersive X-ray spectroscopy (EDX) spectra of BV-CaP-OxCNH, CaP-OxCNH, BV-CaP, CaP. ＢＶ－ＣａＰ－ＯｘＣＮＨ―２の透過電子顕微鏡（ＴＥＭ）画像及び炭素（Ｃ）、カルシウム（Ｃａ）、酸素（Ｏ）、リン（Ｐ）の元素分布を示す走査型透過電子顕微鏡（ＳＴＥＭ）－ＥＤＸ画像。BV-CaP-OxCNH-2 transmission electron microscope (TEM) image and scanning transmission electron microscope (STEM) -EDX showing element distribution of carbon (C), calcium (Ca), oxygen (O), phosphorus (P) image. （ａ）ＢＶ－ＣａＰ－ＯｘＣＮＨのマウスマクロファージ様細胞（ＲＡＷ２６４．７細胞）毒性評価の結果。（ｂ）ＢＶのＲＡＷ２６４．７細胞毒性評価の結果。(A) Results of toxicity evaluation of mouse macrophage-like cells (RAW264.7 cells) of BV-CaP-OxCNH. (B) Results of BV RAW264.7 cytotoxicity assessment. （ａ）ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－２、ＯｘＣＮＨのＲＡＷ２６４．７細胞毒性評価の結果。（ｂ）ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－５、ＯｘＣＮＨのＲＡＷ２６４．７細胞毒性評価の結果。(A) Results of RAW264.7 cytotoxicity evaluation of BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH. (B) Results of RAW264.7 cytotoxicity evaluation of BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－５、ＯｘＣＮＨを添加した培地で２４時間培養したＲＡＷ２６４．７細胞の光学顕微鏡写真。Optical micrographs of RAW264.7 cells cultured for 24 hours in a medium supplemented with BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. ＲＡＷ２６４．７細胞(ａ)とＲＡＷ２６４．７細胞から分化させて得た破骨細胞(ｂ)を酒石酸抵抗性酸性ホスファターゼ（ＴＲＡＰ）染色した光学顕微鏡写真。An optical micrograph of osteoclasts (b) obtained by differentiating from RAW264.7 cells (a) and RAW264.7 cells, stained with tartrate-resistant acid phosphatase (TRAP). 破骨細胞によるＢＶ－ＣａＰ－ＯｘＣＮＨの取り込みを示す蛍光顕微鏡写真。Fluorescence micrograph showing uptake of BV-CaP-OxCNH by osteoclasts. ＯｘＣＮＨ、ＣａＰ－ＯｘＣＮＨ、ＢＶ－ＣａＰ－ＯｘＣＮＨを添加した破骨細胞の生存率評価の結果。Results of survival rate evaluation of osteoclasts supplemented with OxCNH, CaP-OxCNH, and BV-CaP-OxCNH. ＢＶ－ＣａＰ－ＯｘＣＮＨ、ＺＯ－ＣａＰ－ＯｘＣＮＨ、及びＰＭ－ＣａＰ－ＯｘＣＮＨのＳＥＭ画像。SEM images of BV-CaP-OxCNH, ZO-CaP-OxCNH, and PM-CaP-OxCNH. ＢＶ－ＣａＰ－ＯｘＣＮＨ、ＺＯ－ＣａＰ－ＯｘＣＮＨ、及びＰＭ－ＣａＰ－ＯｘＣＮＨのＥＤＸスペクトル。EDX spectra of BV-CaP-OxCNH, ZO-CaP-OxCNH, and PM-CaP-OxCNH. ＢＶ－ＣａＰ－ＯｘＣＮＨのＲＡＷ２６４．７細胞毒性評価の結果。Results of RAW264.7 cytotoxicity assessment of BV-CaP-OxCNH. （ａ）ＺＯ－ＣａＰ－ＯｘＣＮＨのＲＡＷ２６４．７細胞毒性評価の結果。（ｂ）ＺＯのＲＡＷ２６４．７細胞毒性評価の結果。(A) Results of RAW264.7 cytotoxicity evaluation of ZO-CaP-OxCNH. (B) Results of ZO's RAW264.7 cytotoxicity assessment. （ａ）ＰＭ－ＣａＰ－ＯｘＣＮＨのＲＡＷ２６４．７細胞毒性評価の結果。（ｂ）ＰＭのＲＡＷ２６４．７細胞毒性評価の結果。(A) Results of RAW264.7 cytotoxicity evaluation of PM-CaP-OxCNH. (B) Results of PM RAW264.7 cytotoxicity evaluation. 骨粗鬆症モデルラット及び偽手術ラットの子宮の写真（ａ）と子宮重量の相対値（ｂ）。Photographs (a) of uteri of osteoporosis model rats and sham-surgery rats and relative values of uterine weight (b). 試料埋入から０、８、１２週間後の骨粗鬆症モデルラットの脛骨ＣＴ像。Tibial CT images of osteoporosis model rats 0, 8 and 12 weeks after sample implantation. 試料埋入から０、４、８、１２週間後の骨粗鬆症モデルラットの全骨密度。Total bone mineral density of osteoporosis model rats 0, 4, 8 and 12 weeks after sample implantation. 試料埋入から０、４、８、１２週間後の骨粗鬆症モデルラットの皮質骨密度。Cortical bone mineral density of osteoporosis model rats 0, 4, 8 and 12 weeks after sample implantation. 試料埋入から０、４、８、１２週間後の骨粗鬆症モデルラットの海綿骨密度。Cancellous bone mineral density of osteoporosis model rats 0, 4, 8 and 12 weeks after sample implantation. 試料埋入から０、４、８、１２週間後の骨粗鬆症モデルラットの海綿骨密度比。The cancellous bone mineral density ratio of osteoporosis model rats 0, 4, 8 and 12 weeks after sample implantation.
　以下、本発明におけるＢＰ含有カーボン粒子複合体及びその製造方法について説明するが、本発明は以下に示す実施の形態及び実施例の記載内容に限定して解釈されるものではない。 Hereinafter, the BP-containing carbon particle complex and the method for producing the BP-containing carbon particle complex in the present invention will be described, but the present invention is not construed as being limited to the description of the embodiments and examples shown below.
［カーボン粒子複合体］
　本発明におけるカーボン粒子複合体は、ＢＰとＣａＰとカーボン粒子を含有する。
　ここでカーボン粒子とは、ＣＮＨ、カーボンナノチューブ、ナノグラフェン、ナノダイアモンド、若しくはカーボンファイバー、又はこれらの組み合わせであるが、これに限定されない。カーボン粒子は、化学的・物理的安定性を有し、カーボン粒子表面に化学修飾に適したカルボキシル基（－ＣＯＯＨ）などの官能基を導入させるのが容易であるため多重機能付与に適している。
　中でも、ＣＮＨは、サイズの均一性、水溶液中での分散安定性、高純度大量作製可能、精製不要などの利点を有することから、カーボン粒子複合体に用いるカーボン粒子として好ましい。ＣＮＨは、直径２ｎｍ～５ｎｍのカーボンナノチューブ状のものが数千個放射状に集合した直径１００ｎｍ程度の球状の形態を有する。ＣＮＨは堅固であり容易には分解しない。また、ＣＮＨは、表面が凹凸に富んだ構造を有するため強く凝集することはなく、水溶液中において孤立分散する。過酸化水素を用いてＣＮＨを酸化して得られるＯｘＣＮＨでは、炭素－炭素結合の開裂によってチューブ壁に孔が開き、さらに内部空間への分子の出入りが容易になることから、カーボン粒子複合体に用いるカーボン粒子としてより好ましい。ＯｘＣＮＨは、孔縁などにカルボキシル基などの官能基が導入されることから親水性であり、カーボン粒子複合体に用いるカーボン粒子としてより好ましい。 [Carbon particle complex]
The carbon particle complex in the present invention contains BP, CaP and carbon particles.
Here, the carbon particles are, but are not limited to, CNH, carbon nanotubes, nanographene, nanodiamond, or carbon fiber, or a combination thereof. The carbon particles have chemical and physical stability, and it is easy to introduce a functional group such as a carboxyl group (-COOH) suitable for chemical modification on the surface of the carbon particles, so that the carbon particles are suitable for imparting multiple functions. ..
Among them, CNH is preferable as carbon particles used for a carbon particle complex because it has advantages such as size uniformity, dispersion stability in an aqueous solution, high-purity mass production, and no purification required. The CNH has a spherical shape having a diameter of about 100 nm, in which thousands of carbon nanotubes having a diameter of 2 nm to 5 nm are radially gathered. CNH is robust and does not decompose easily. Further, since CNH has a structure with abundant irregularities on the surface, it does not strongly agglomerate and is isolated and dispersed in an aqueous solution. OxCNH, which is obtained by oxidizing CNH with hydrogen peroxide, has holes in the tube wall due to the cleavage of carbon-carbon bonds, and also facilitates the entry and exit of molecules into the internal space. More preferable as carbon particles to be used. OxCNH is hydrophilic because a functional group such as a carboxyl group is introduced into the pore edge or the like, and is more preferable as carbon particles used in a carbon particle complex.
　カーボン粒子複合体が含有するＣａＰの組成及び構造は限定されない。ＣａＰは、少なくともリン酸イオンとカルシウムイオンを含む化合物であればよい。また、ＣａＰは、アモルファス状であってもよい。ＣａＰ過飽和溶液中のカルシウムイオンがカーボン粒子のカルボキシル基と静電相互作用で引き合い、そこを起点にＣａＰが析出することで、ＣａＰとカーボン粒子の複合体が形成される。あるいは、析出したＣａＰ表面のカルシウムイオンがカーボン粒子のカルボキシル基と静電相互作用で引き合うことで、ＣａＰとカーボン粒子の複合体が形成される。 The composition and structure of CaP contained in the carbon particle composite are not limited. CaP may be a compound containing at least phosphate ions and calcium ions. Further, CaP may be amorphous. Calcium ions in the CaP supersaturated solution are attracted to the carboxyl group of the carbon particles by electrostatic interaction, and CaP is precipitated from there to form a complex of CaP and carbon particles. Alternatively, the precipitated calcium ions on the surface of the CaP are attracted to the carboxyl group of the carbon particles by electrostatic interaction to form a complex of the CaP and the carbon particles.
　カーボン粒子複合体が含有するＢＰは、エチドロン酸、イバンドロン酸、ゾレドロン酸、アレンドロン酸、ミノドロン酸、リセドロン酸、パミドロン酸、インカドロン酸、若しくはこれらの塩、又はこれらの組み合わせであるが、これに限定されない。ＢＰは、少なくともホスホン酸基を含み、破骨細胞の活動を阻害し、骨の吸収を防ぐものであればよい。ＢＰのホスホン酸基が、ＣａＰ過飽和溶液中のカルシウムイオンや析出したＣａＰ表面のカルシウムイオンなどと静電相互作用で引き合うことで、ＣａＰとＢＰが複合化し、さらにカーボン粒子との複合体が形成される。
　さらにＢＰは、カーボン粒子の内部空間に内包される、又は、カーボン粒子表面に物理吸着することでカーボン粒子に担持される。 The BP contained in the carbon particle composite is etidronic acid, ibandronic acid, zoledronic acid, alendronic acid, minodronic acid, risedronic acid, pamidronic acid, incadronic acid, or salts thereof, or a combination thereof. Not limited. The BP may contain at least a phosphonic acid group, inhibit the activity of osteoclasts, and prevent bone resorption. The phosphonic acid group of BP attracts calcium ions in the CaP supersaturated solution and calcium ions on the surface of the precipitated CaP by electrostatic interaction, so that CaP and BP are compounded and a complex with carbon particles is further formed. NS.
Further, the BP is contained in the internal space of the carbon particles or is supported on the carbon particles by being physically adsorbed on the surface of the carbon particles.
　本発明におけるＢＰ含有カーボン粒子複合体は、少量のＢＰ含有量で破骨細胞を細胞死させることができる。 The BP-containing carbon particle complex in the present invention can kill osteoclasts with a small amount of BP content.
［カーボン粒子複合体の製造方法］
　本発明におけるカーボン粒子複合体は、ＢＰと、カーボン粒子と、を含むＣａＰ過飽和溶液を調製し、静置することによって３者を共沈させることにより製造することができる。
　ＣａＰ過飽和溶液の原料として用いられる溶液は特に限定されない。ＣａＰ過飽和溶液の原料としては、カルシウムイオンを含む溶液（カルシウム含有液）およびリン酸イオンを含む溶液（リン酸含有液）以外に、適宜、ｐＨ調整剤などを含んでもよい。ＣａＰ過飽和溶液の原料としては、例えば、各種注射用製剤などを組み合わせて用いてもよい。ＣａＰ過飽和溶液の原料として各種注射用製剤などを組み合わせてＣａＰ過飽和溶液を調製する場合、本発明におけるカーボン粒子複合体をそのまま製剤として用いることもできる。
　ＢＰと、カーボン粒子と、を含むＣａＰ過飽和溶液は、ＢＰ、カーボン粒子、カルシウム含有液およびリン酸含有液を混合することで調製することができる。混合時に、必要に応じてｐＨ調整剤などを加えてもよい。
　ＢＰと、カーボン粒子と、を含むＣａＰ過飽和溶液を静置する際の温度及び時間は特に限定されない。製造するカーボン粒子複合体の大きさ及び分散性を考慮して適宜調整することができる。 [Manufacturing method of carbon particle complex]
The carbon particle complex in the present invention can be produced by preparing a CaP supersaturated solution containing BP and carbon particles and allowing them to stand to co-precipitate the three.
The solution used as a raw material for the CaP supersaturated solution is not particularly limited. As the raw material of the CaP supersaturated solution, a pH adjuster or the like may be appropriately contained in addition to the solution containing calcium ions (calcium-containing solution) and the solution containing phosphate ions (phosphate-containing solution). As the raw material of the CaP supersaturated solution, for example, various injection preparations and the like may be used in combination. When a CaP supersaturated solution is prepared by combining various injection preparations as a raw material of the CaP supersaturated solution, the carbon particle composite of the present invention can be used as it is as a preparation.
A CaP supersaturated solution containing BP and carbon particles can be prepared by mixing BP, carbon particles, a calcium-containing liquid and a phosphoric acid-containing liquid. At the time of mixing, a pH adjuster or the like may be added if necessary.
The temperature and time for allowing the CaP supersaturated solution containing BP and carbon particles to stand are not particularly limited. It can be appropriately adjusted in consideration of the size and dispersibility of the carbon particle composite to be produced.
［製剤］
　本発明におけるカーボン粒子複合体はＢＰを含有することから、破骨細胞をターゲットとした製剤に適用することができる。本発明における製剤は、ＢＰ含有カーボン粒子複合体以外に、適宜、添加剤などを含むことができる。本発明における製剤は、例えば、静脈内注射により患者に投与してもよいし、特定の部位（患部）に局所投与してもよい。 [pharmaceutical formulation]
Since the carbon particle complex in the present invention contains BP, it can be applied to a preparation targeting osteoclasts. The formulation in the present invention may appropriately contain additives and the like in addition to the BP-containing carbon particle complex. The pharmaceutical product of the present invention may be administered to a patient by intravenous injection, for example, or may be locally administered to a specific site (affected area).
　本発明におけるＢＰ含有カーボン粒子複合体は、体内の薬物分布を空間的・量的・時間的に制御することができるＤＤＳとして機能し得る。本発明におけるＢＰ含有カーボン粒子複合体は、ＣａＰを含有することで骨親和性が高まり、少量のＢＰで破骨細胞を細胞死させることができる。さらに、体内で拡散しにくいカーボン粒子を用いたことで、局所投与したＢＰ含有カーボン粒子複合体は長時間患部にとどまり、ＢＰを放出し続けることが可能となる。また、ＢＰ含有カーボン粒子複合体は破骨細胞内のリソソームに局在することから、その酸性環境下でＣａＰが溶解し、効率よくＢＰを放出することができる。さらに、カーボン粒子は光照射により発熱するため、患部によっては光温熱治療も可能となる。
　一方、ＣａＰは骨基質の成分であるため、破骨細胞による骨吸収が抑制された後に、骨芽細胞による骨修復を促進することができる。また、カーボン粒子そのものが骨形成の優れたＳｃａｆｆｏｌｄの機能を果たすため、骨形成促進治療に有効である。
　したがって、本発明におけるＢＰ含有カーボン粒子複合体は、ＤＤＳおよびＳｃａｆｆｏｌｄとしての相乗効果により、効率よく骨形成促進治療することができる。 The BP-containing carbon particle complex in the present invention can function as a DDS capable of controlling the drug distribution in the body spatially, quantitatively, and temporally. The BP-containing carbon particle complex in the present invention has increased bone affinity by containing CaP, and can kill osteoclasts with a small amount of BP. Furthermore, by using carbon particles that are difficult to diffuse in the body, the locally administered BP-containing carbon particle complex can stay in the affected area for a long time and continue to release BP. In addition, since the BP-containing carbon particle complex is localized in lysosomes in osteoclasts, CaP is dissolved in the acidic environment, and BP can be efficiently released. Furthermore, since the carbon particles generate heat when irradiated with light, photothermia treatment is possible depending on the affected area.
On the other hand, since CaP is a component of bone matrix, bone repair by osteoblasts can be promoted after bone resorption by osteoclasts is suppressed. In addition, since the carbon particles themselves fulfill the function of Scaffold having excellent bone formation, it is effective for bone formation promoting treatment.
Therefore, the BP-containing carbon particle complex in the present invention can be efficiently treated for promoting bone formation due to the synergistic effect of DDS and Scaffold.
　以下に、本発明におけるカーボン粒子複合体及びその製造方法についての実施例を示して、より詳細に説明する。本実施例においては、カーボン粒子の一例としてＯｘＣＮＨ、ＢＰの一例としてイバンドロン酸（ボンビバ、ＢＶ）を用いて、ＯｘＣＮＨにＣａＰとＢＶを複合化させたＢＶ－ＣａＰ－ＯｘＣＮＨを作製し、骨形成促進治療への適用を検討した。 Hereinafter, examples of the carbon particle complex and the method for producing the same in the present invention will be shown and described in more detail. In this example, using OxCNH as an example of carbon particles and ibandronic acid (Bombiva, BV) as an example of BP, BV-CaP-OxCNH in which CaP and BV are compounded with OxCNH is produced to promote bone formation. The application to treatment was examined.
［ＯｘＣＮＨの作製］
　アルゴンガス雰囲気（大気圧）下、炭酸ガスレーザー照射によりグラファイトを分解・気化することにより、ＣＮＨを作製した（文献　J. Phys. Chem. C 2008, 112, 1330-1334）。５０ｍＬバイアルビンに作製したＣＮＨ（２０ｍｇ）と３０％過酸化水素水（２０ｍＬ）を加え、超音波処理を５分間行い、ＣＮＨを過酸化水素水に分散させた。このＣＮＨ過酸化水素水分散液にキセノンランプ光を照射しつつ、７０℃で２時間加熱した。ＣＮＨ過酸化水素水分散液を室温で冷却した後、濾過水洗を５回程度繰り返し、過酸化水素を除去した。得られたＯｘＣＮＨ水分散液を凍結乾燥してＯｘＣＮＨ粉末を得た。 [Preparation of OxCNH]
CNH was prepared by decomposing and vaporizing graphite by carbon dioxide laser irradiation in an argon gas atmosphere (atmospheric pressure) (Reference J. Phys. Chem. C 2008, 112, 1330-1334). The prepared CNH (20 mg) and 30% hydrogen peroxide solution (20 mL) were added to a 50 mL vial, and sonication was performed for 5 minutes to disperse the CNH in the hydrogen peroxide solution. The CNH hydrogen peroxide solution dispersion was heated at 70 ° C. for 2 hours while irradiating with xenon lamp light. After cooling the CNH hydrogen peroxide solution dispersion at room temperature, filtration water washing was repeated about 5 times to remove hydrogen peroxide. The obtained OxCNH aqueous dispersion was freeze-dried to obtain OxCNH powder.
［ＢＶ－ＣａＰ－ＯｘＣＮＨの作製］
　ＯｘＣＮＨ粉末を０．１ｍｇ／ｍＬ、０．２ｍｇ／ｍＬ、０．５ｍｇ／ｍＬ、１ｍｇ／ｍＬ、２ｍｇ／ｍＬ、及び５ｍｇ／ｍＬになるように超純水に分散し、オートクレーブにより滅菌して滅菌ＯｘＣＮＨ分散液を得た。
　ＢＶ溶液として、イバンドロン酸ナトリウム水和物を有効成分として含有するボンビバ静注１ｍｇシリンジ（中外製薬株式会社）を使用した。
　ＣａＰ過飽和溶液の原料として、カルシウム含有液、リン酸含有液、ｐＨ調整剤を調製した。カルシウム含有液は、リンゲル液「オーツカ」（大塚製薬株式会社）（４９．３６１ｍＬ）と塩化Ｃａ補正液１ｍＥｑ／ｍＬ（大塚製薬株式会社）（０．６３９ｍＬ）を混合して調製した。リン酸含有液は、クリニザルツ（登録商標）輸液（共和クリティケア株式会社）（９．４６９ｍＬ）とリン酸２カリウム注２０ｍＥｑキット「テルモ」（テルモ株式会社）（０．５３１ｍＬ）を混合して調製した。ｐＨ調整剤はメイロン（登録商標）静注７％（大塚製薬株式会社）（５ｍＬ）と注射用水（扶桑薬品工業株式会社）（２０ｍＬ）を混合して調製した。
　滅菌ＯｘＣＮＨ水分散液（０．１ｍｇ／ｍＬ、０．２ｍｇ／ｍＬ、０．５ｍｇ／ｍＬ、１ｍｇ／ｍＬ、２ｍｇ／ｍＬ、５ｍｇ／ｍＬの分散液を各０．２５０ｍＬ）、ＢＶ溶液（０．２５０ｍＬ）、カルシウム含有液（７．６７４ｍＬ）、リン酸含有液（０．９１７ｍＬ）、及びｐＨ調整剤（０．９０９ｍＬ）を混合し、反応液（１０ｍＬ）を調製した。反応液は、２５℃のインキュベーターで３０分間静置して、ＢＶ、ＣａＰ、ＯｘＣＮＨの三者を共沈させた。得られた試料ＢＶ－ＣａＰ－ＯｘＣＮＨは、遠心操作（６０００ｒｐｍ、５分）により回収し、注射用水に試料を分散して遠心することを繰り返すことによって洗浄した。０．１ｍｇ／ｍＬ、０．２ｍｇ／ｍＬ、０．５ｍｇ／ｍＬ、１ｍｇ／ｍＬ、２ｍｇ／ｍＬ、及び５ｍｇ／ｍＬの滅菌ＯｘＣＮＨ水分散液を用いて作製した試料のそれぞれを、ＢＶ－ＣａＰ－ＯｘＣＮＨ－１、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３、ＢＶ－ＣａＰ－ＯｘＣＮＨ－４、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、及びＢＶ－ＣａＰ－ＯｘＣＮＨ－６とした。 [Preparation of BV-CaP-OxCNH]
OxCNH powder is dispersed in ultrapure water to 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL, and sterilized by autoclave. An OxCNH dispersion was obtained.
As the BV solution, a Bombiva intravenous injection 1 mg syringe (Chugai Pharmaceutical Co., Ltd.) containing sodium ibandronate hydrate as an active ingredient was used.
A calcium-containing solution, a phosphoric acid-containing solution, and a pH adjuster were prepared as raw materials for the CaP supersaturated solution. The calcium-containing solution was prepared by mixing Ringer's solution "Otsuka" (Otsuka Pharmaceutical Co., Ltd.) (49.361 mL) and Ca chloride correction solution 1 mEq / mL (Otsuka Pharmaceutical Co., Ltd.) (0.639 mL). The phosphoric acid-containing solution was prepared by mixing Clinisalz (registered trademark) infusion solution (Kyowa CritiCare Co., Ltd.) (9.469 mL) and dipotassium phosphate Injection 20 mEq kit "Terumo" (Terumo Corporation) (0.531 mL). .. The pH adjuster was prepared by mixing Meylon (registered trademark) intravenous injection 7% (Otsuka Pharmaceutical Co., Ltd.) (5 mL) and water for injection (Fuso Pharmaceutical Indus Co., Ltd.) (20 mL).
Sterilized OxCNH aqueous dispersion (0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL dispersion, 0.250 mL each), BV solution (0. 250 mL), a calcium-containing solution (7.674 mL), a phosphoric acid-containing solution (0.917 mL), and a pH adjuster (0.909 mL) were mixed to prepare a reaction solution (10 mL). The reaction solution was allowed to stand in an incubator at 25 ° C. for 30 minutes to coprecipitate BV, CaP, and OxCNH. The obtained sample BV-CaP-OxCNH was collected by centrifugation (6000 rpm, 5 minutes), and washed by repeating the process of dispersing the sample in water for injection and centrifuging. BV-CaP- It was designated as OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, and BV-CaP-OxCNH-6.
［ＣａＰ－ＯｘＣＮＨの作製］
　ＢＶを含まないコントロールとして、ＢＶ溶液の代わりに注射用水を用いて反応液を調製したこと以外ＢＶ－ＣａＰ－ＯｘＣＮＨの作製と同様の操作を行い、試料ＣａＰ－ＯｘＣＮＨを得た。０．１ｍｇ／ｍＬ、０．２ｍｇ／ｍＬ、０．５ｍｇ／ｍＬ、１ｍｇ／ｍＬ、２ｍｇ／ｍＬ、及び５ｍｇ／ｍＬの滅菌ＯｘＣＮＨ水分散液を用いて作製した試料のそれぞれを、ＣａＰ－ＯｘＣＮＨ－１、ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－３、ＣａＰ－ＯｘＣＮＨ－４、ＣａＰ－ＯｘＣＮＨ－５、及びＣａＰ－ＯｘＣＮＨ－６とした。 [Preparation of CaP-OxCNH]
As a control containing no BV, a sample CaP-OxCNH was obtained by performing the same operation as the preparation of BV-CaP-OxCNH except that the reaction solution was prepared using water for injection instead of the BV solution. CaP-OxCNH- 1. CaP-OxCNH-2, CaP-OxCNH-3, CaP-OxCNH-4, CaP-OxCNH-5, and CaP-OxCNH-6.
［ＢＶ－ＣａＰ及びＣａＰの作製］
　ＣＮＨを含まないコントロールとして、滅菌ＯｘＣＮＨ水分散液の代わりに注射用水を用いて反応液を調製したこと以外ＢＶ－ＣａＰ－ＯｘＣＮＨの作製又はＣａＰ－ＯｘＣＮＨの作製と同様の操作を行って、ＢＶとＣａＰの複合体（ＢＶ－ＣａＰ）及びＣａＰ粒子（ＣａＰ）を得た。 [Preparation of BV-CaP and CaP]
As a control containing no CNH, the same operation as the preparation of BV-CaP-OxCNH or the preparation of CaP-OxCNH was performed except that the reaction solution was prepared using water for injection instead of the sterile OxCNH aqueous dispersion, and the BV and A complex of CaP (BV-CaP) and CaP particles (CaP) were obtained.
［分散性の観察］
　ＢＶ－ＣａＰ－ＯｘＣＮＨ－１、ＣａＰ－ＯｘＣＮＨ－１、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３、ＣａＰ－ＯｘＣＮＨ－３を注射用水に分散させた後の写真を図１に示す。ＣａＰ－ＯｘＣＮＨでは、粒子の沈降が早く、固液分離が観察された。一方で、ＢＶ－ＣａＰ－ＯｘＣＮＨでは粒子の沈降が観察されず、良好な粒子分散性が示唆された。 [Observation of dispersibility]
After dispersing BV-CaP-OxCNH-1, CaP-OxCNH-1, BV-CaP-OxCNH-2, CaP-OxCNH-2, BV-CaP-OxCNH-3, and CaP-OxCNH-3 in water for injection. The photograph is shown in FIG. In CaP-OxCNH, the particles settled quickly and solid-liquid separation was observed. On the other hand, in BV-CaP-OxCNH, no precipitation of particles was observed, suggesting good particle dispersibility.
［走査型電子顕微鏡による観察］
　作製した試料の構造を走査電子顕微鏡（ＳＥＭ）により確認した。ＳＥＭ観察においては、試料をシリコン基板上に滴下し、乾燥させ、金を蒸着して観察した。ＢＶ－ＣａＰ及びＢＶ－ＣａＰ－ＯｘＣＮＨのＳＥＭ画像を図２に示す。ＣａＰ及びＣａＰ－ＯｘＣＮＨのＳＥＭ像を図３に示す。
　図２に示すように、ＢＶ－ＣａＰ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－１、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３、ＢＶ－ＣａＰ－ＯｘＣＮＨ－４ではいずれも一次粒子径５０ｎｍ程度のナノ粒子が観察されたのに対し、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＢＶ－ＣａＰ－ＯｘＣＮＨ－６ではそれに加えて一次粒子径１００ｎｍ～１５０ｎｍの粒子が観察された。さらにＢＶ－ＣａＰ－ＯｘＣＮＨ－６ではひび割れた形状の粒子が目立った。図３に示すように、ＣａＰ及びＣａＰ－ＯｘＣＮＨはいずれも一次粒子径１００ｎｍ～１５０ｎｍ程度のナノ粒子であった。さらにＣａＰ－ＯｘＣＮＨ－４、ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－６では、反応液中のＯｘＣＮＨ濃度の上昇に伴い、ひび割れた形状の粒子が増加した。 [Observation with scanning electron microscope]
The structure of the prepared sample was confirmed by a scanning electron microscope (SEM). In the SEM observation, the sample was dropped on a silicon substrate, dried, and gold was vapor-deposited for observation. The SEM images of BV-CaP and BV-CaP-OxCNH are shown in FIG. The SEM images of CaP and CaP-OxCNH are shown in FIG.
As shown in FIG. 2, BV-CaP, BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, and BV-CaP-OxCNH-4 all have a primary particle size of about 50 nm. In contrast to the nanoparticles of BV-CaP-OxCNH-5 and BV-CaP-OxCNH-6, particles having a primary particle size of 100 nm to 150 nm were also observed. Furthermore, in BV-CaP-OxCNH-6, cracked particles were conspicuous. As shown in FIG. 3, both CaP and CaP-OxCNH were nanoparticles having a primary particle diameter of about 100 nm to 150 nm. Further, in CaP-OxCNH-4, CaP-OxCNH-5, and CaP-OxCNH-6, the number of cracked particles increased as the OxCNH concentration in the reaction solution increased.
［エネルギー分散型Ｘ線分光法による測定］
　ＳＥＭ観察に用いた試料について、エネルギー分散型Ｘ線分光法（ＥＤＸ）を用いて元素分析を行った。元素分析の結果を図４に示す。図４に示すように、いずれの粒子からも、炭素、酸素、リン、カルシウムのピークが検出されたことから、ＣａＰとＯｘＣＮＨが複合体を形成したことが示唆された。また、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＢＶ－ＣａＰ－ＯｘＣＮＨ－６、ＣａＰ－ＯｘＣＮＨ－５、及びＣａＰ－ＯｘＣＮＨ－６ではカルシウム、リンのピークに対して炭素のピークの顕著な増大が確認された。これは、反応液中のＯｘＣＮＨ濃度の上昇に伴い、生成した粒子中のＣａＰに対するＯｘＣＮＨの割合が大きくなったためと考えられた。 [Measurement by energy dispersive X-ray spectroscopy]
Elemental analysis was performed on the sample used for SEM observation using energy dispersive X-ray spectroscopy (EDX). The result of elemental analysis is shown in FIG. As shown in FIG. 4, carbon, oxygen, phosphorus, and calcium peaks were detected in all the particles, suggesting that CaP and OxCNH formed a complex. In addition, in BV-CaP-OxCNH-5, BV-CaP-OxCNH-6, CaP-OxCNH-5, and CaP-OxCNH-6, a remarkable increase in carbon peaks was confirmed with respect to calcium and phosphorus peaks. .. It is considered that this is because the ratio of OxCNH to CaP in the produced particles increased as the OxCNH concentration in the reaction solution increased.
［高周波誘導結合プラズマ発光分光法による測定］
　得られた試料について、高周波誘導結合プラズマ発光分光法（ＩＣＰ）を用いて元素分析を行った。試料を酸で溶解させた溶液を調製し、測定を行った。１０ｍＬの反応液から得られた試料中のカルシウム含有量、リン含有量、及びリン含有量に対するカルシウム含有量のモル比（Ｃａ／Ｐ）を表１に示す。
Figure JPOXMLDOC01-appb-T000001
　表１の結果より、ＢＶ－ＣａＰ－ＯｘＣＮＨおよびＢＶ－ＣａＰではいずれもＣａＰ－ＯｘＣＮＨおよびＣａＰよりＣａ／Ｐモル比が小さかった（ＢＶ－ＣａＰ－ＯｘＣＮＨ／ＢＶ－ＣａＰ：１．２９～１．３６、ＣａＰ－ＯｘＣＮＨ／ＣａＰ：１．３８～１．４４）。ＢＶ－ＣａＰ－ＯｘＣＮＨおよびＢＶ－ＣａＰではリンを含むＢＶが担持されたために、カルシウムに対してリンの含有量が相対的に大きくなり、Ｃａ／Ｐモル比が小さくなったと考えられる。ここで、ＢＶ－ＣａＰ－ＯｘＣＮＨ中のＣａＰのＣａ／Ｐモル比がＣａＰ－ＯｘＣＮＨと同等と仮定し、ＢＶの含有量を推定したところ、反応液に混合した量の２０－３０％（０．１６－０．２４μｍｏｌ）であると算出された。また、ＢＶとＣａＰの量比は４－７：１００と推定された。以上より、ＢＶ－ＣａＰ－ＯｘＣＮＨでは、ＢＶ、ＣａＰ及びＯｘＣＮＨが三元複合体を形成していることが確認された。
　また、ＢＶ－ＣａＰ－ＯｘＣＮＨおよびＢＶ－ＣａＰではいずれもＣａＰ－ＯｘＣＮＨおよびＣａＰに比べて、カルシウム、リン含有量が少なかった。これは、ＢＶ－ＣａＰ－ＯｘＣＮＨおよびＢＶ－ＣａＰでは、酸性のＢＶ溶液が反応液中に加わることで反応液のｐＨが低下し、ＣａＰの形成が妨げられたためと考えられる。また、ＢＶとの複合化がＣａＰ形成速度に影響を及ぼした可能性もある。一方で、反応液中のＯｘＣＮＨの濃度は、カルシウム、リン含有量に大きな影響を与えなかった。従って、ＢＶ－ＣａＰ－ＯｘＣＮＨに含まれるＣａＰ及びＢＶ量は、反応液中のＯｘＣＮＨの濃度に関わらず大きな差はないと考えられる。ＯｘＣＮＨの量が変化してもＣａＰ量、ＢＶ量の変化が少ないということは、ＯｘＣＮＨに影響されずにＣａＰとＢＶが結合していることを示唆している。すなわち、ＢＶは主にＣａＰに担持され、ＢＶ－ＣａＰがＯｘＣＮＨと複合体を形成した構造を取っていると推察された。 [Measurement by high frequency inductively coupled plasma emission spectroscopy]
The obtained sample was subjected to elemental analysis using radio frequency inductively coupled plasma atomic emission spectroscopy (ICP). A solution in which the sample was dissolved in acid was prepared and measured. Table 1 shows the calcium content, the phosphorus content, and the molar ratio (Ca / P) of the calcium content to the phosphorus content in the sample obtained from the 10 mL reaction solution.
Figure JPOXMLDOC01-appb-T000001
From the results in Table 1, the Ca / P molar ratio of BV-CaP-OxCNH and BV-CaP was smaller than that of CaP-OxCNH and CaP (BV-CaP-OxCNH / BV-CaP: 1.29 to 1.36). , CaP-OxCNH / CaP: 1.38 to 1.44). It is considered that BV-CaP-OxCNH and BV-CaP supported BV containing phosphorus, so that the phosphorus content was relatively large with respect to calcium and the Ca / P molar ratio was small. Here, assuming that the Ca / P molar ratio of CaP in BV-CaP-OxCNH is equivalent to that of CaP-OxCNH, the BV content was estimated and found to be 20-30% (0. It was calculated to be 16-0.24 μmol). The amount ratio of BV to CaP was estimated to be 4-7: 100. From the above, it was confirmed that in BV-CaP-OxCNH, BV, CaP and OxCNH form a ternary complex.
In addition, BV-CaP-OxCNH and BV-CaP all contained less calcium and phosphorus than CaP-OxCNH and CaP. It is considered that this is because in BV-CaP-OxCNH and BV-CaP, the pH of the reaction solution was lowered by the addition of the acidic BV solution to the reaction solution, and the formation of CaP was hindered. It is also possible that the compounding with BV affected the rate of CaP formation. On the other hand, the concentration of OxCNH in the reaction solution did not significantly affect the calcium and phosphorus contents. Therefore, it is considered that the amounts of CaP and BV contained in BV-CaP-OxCNH do not differ greatly regardless of the concentration of OxCNH in the reaction solution. The fact that the changes in the amount of CaP and the amount of BV are small even if the amount of OxCNH changes suggests that CaP and BV are bound without being affected by OxCNH. That is, it was presumed that BV was mainly supported on CaP and that BV-CaP had a structure in which a complex was formed with OxCNH.
［透過電子顕微鏡及び走査型透過電子顕微鏡による観察］
　作製した試料の構造を透過電子顕微鏡（ＴＥＭ）及び走査型透過電子顕微鏡（ＳＴＥＭ）－ＥＤＸを用いて観察した。試料はグリッド上に滴下し、乾燥させて観察に用いた。図５に、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２のＴＥＭ画像及び炭素（Ｃ）、カルシウム（Ｃａ）、酸素（Ｏ）、リン（Ｐ）の元素分布を示すＳＴＥＭ－ＥＤＸ画像を示す。図５に示すように、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２のＴＥＭ像では、チューブが放射状に集合した球状のＯｘＣＮＨの周囲にＢＶ－ＣａＰと思われる物質が付着している様子が観察された。元素分布では、ＯｘＣＮＨに由来する炭素の分布の周囲に、ＢＶ－ＣａＰに由来するカルシウム、酸素、リンが分布していた。これらの結果から、ＯｘＣＮＨの周囲にＢＶ－ＣａＰが付着して複合体を形成していることが確認できた。 [Observation with transmission electron microscope and scanning transmission electron microscope]
The structure of the prepared sample was observed using a transmission electron microscope (TEM) and a scanning transmission electron microscope (STEM) -EDX. The sample was dropped onto the grid, dried and used for observation. FIG. 5 shows a TEM image of BV-CaP-OxCNH-2 and a STEM-EDX image showing the element distribution of carbon (C), calcium (Ca), oxygen (O), and phosphorus (P). As shown in FIG. 5, in the TEM image of BV-CaP-OxCNH-2, it was observed that a substance thought to be BV-CaP was attached around the spherical OxCNH in which the tubes were radially assembled. In the element distribution, calcium, oxygen, and phosphorus derived from BV-CaP were distributed around the distribution of carbon derived from OxCNH. From these results, it was confirmed that BV-CaP adhered around OxCNH to form a complex.
［細胞毒性評価］
　作製した試料の細胞毒性を評価した。貪食性のマウスマクロファージ様細胞（ＲＡＷ２６４．７）を９６ウェルプレートに播種し、３７℃で培養した。２４時間培養後、培地を取り除き、ＢＶ－ＣａＰ－ＯｘＣＮＨ－１、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３、ＢＶ－ＣａＰ－ＯｘＣＮＨ－４、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、及びＢＶ－ＣａＰ－ＯｘＣＮＨ－６を含む培地を加えた。培地中のそれぞれの試料濃度は、ＯｘＣＮＨ濃度として、ＢＶ－ＣａＰ－ＯｘＣＮＨ－１は０．２５μｇ／ｍＬ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２は０．５μｇ／ｍＬ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３は１．２５μｇ／ｍＬ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－４は２．５μｇ／ｍＬ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５は５．０μｇ／ｍＬ、及びＢＶ－ＣａＰ－ＯｘＣＮＨ－６は１２．５μｇ／ｍＬであった（ＩＣＰの結果より、培地中のＢＶ濃度は０．５μｇ／ｍＬ～０．７５μｇ／ｍＬ程度であり、ＣａＰ由来のＣａ濃度は０．０８９ｍＭ～０．１１ｍＭ、Ｐ濃度は０．０６８ｍＭ～０．０８１ｍＭ程度であると推定された）。なお、コントロールでは、ＢＶ－ＣａＰ－ＯｘＣＮＨを含まない通常の培地を用いた。２４時間培養後、Ｃｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ－８試薬（同仁化学研究所）により生存細胞数を４５０ｎｍの吸光度測定から求めた。コントロールの吸光度を１００％として、細胞生存率を算出した。
　図６（ａ）は、ＢＶ－ＣａＰ－ＯｘＣＮＨの細胞毒性評価の結果を示す。ＢＶ－ＣａＰ－ＯｘＣＮＨ－１、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３はほぼ同等で、ＢＶ－ＣａＰ－ＯｘＣＮＨ－４、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＢＶ－ＣａＰ－ＯｘＣＮＨ－６は試料中のＯｘＣＮＨ濃度が高くなるにつれて細胞毒性が顕著に現れた。
　ＢＶの細胞毒性を上記と同様の方法で評価した。培地中に添加したＢＶ濃度は、５μｇ／ｍＬ、１０μｇ／ｍＬ、２５μｇ／ｍＬ、５０μｇ／ｍＬ、１００μｇ／ｍＬとした（コントロールは０μｇ／ｍＬ）。図６（ｂ）に、ＢＶのＲＡＷ２６４．７細胞毒性評価の結果を示す。図６（ｂ）に示すように、ＢＶは濃度依存的に細胞毒性を示した。ＢＶ－ＣａＰ－ＯｘＣＮＨ中に含まれるＢＶ濃度は培地中の濃度として０．５μｇ／ｍＬ～０．７５μｇ／ｍＬ程度と推定されることから、ＢＶ単独と比較して、ＢＶ－ＣａＰ－ＯｘＣＮＨでは少量のＢＶしか含まれていないにも関わらず極めて効率的に細胞毒性を示したと言える。
　上記と同様の方法により、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－２、及びＯｘＣＮＨについて細胞毒性を評価した。培地中のそれぞれの試料濃度は、ＯｘＣＮＨ濃度として０．１７μｇ／ｍＬ、０．５μｇ／ｍＬ、１．７μｇ／ｍＬとなるように希釈した。同様に、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－５、及びＯｘＣＮＨについても細胞毒性を評価した。培地中のそれぞれの試料濃度は、ＯｘＣＮＨ濃度として１．７μｇ／ｍＬ、５．０μｇ／ｍＬ、１６．７μｇ／ｍＬとなるように希釈した。
　図７（ａ）は、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－２、及びＯｘＣＮＨの細胞毒性評価の結果を示す。図７（ａ）に示すように、ＣａＰ－ＯｘＣＮＨ－２及びＯｘＣＮＨはほぼ同等で、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２はより強い細胞毒性を示した。図７（ｂ）は、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－５、及びＯｘＣＮＨの細胞毒性評価の結果を示す。図７（ｂ）に示すように、ＯｘＣＮＨ、ＣａＰ－ＯｘＣＮＨ－５、ＢＶ－ＣａＰ－ＯｘＣＮＨ－５の順により強い細胞毒性を示した。これらの結果から、ＢＶ－ＣａＰ－ＯｘＣＮＨでは、ＣａＰ－ＯｘＣＮＨにＢＶが担持されていることで、より強い細胞毒性を示していると言える。
　ＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－５及びＯｘＣＮＨを添加して培養した細胞を、光学顕微鏡を用いて観察した。図８はＢＶ－ＣａＰ－ＯｘＣＮＨ－５、ＣａＰ－ＯｘＣＮＨ－５、ＯｘＣＮＨを添加した培地で２４時間培養したＲＡＷ２６４．７細胞の光学顕微鏡写真を示す。図８下段に示すように、ＯｘＣＮＨは細胞内のリソソームに取り込まれていると推定された。図８中段に示すように、ＣａＰ－ＯｘＣＮＨは、培地内で大きな塊を形成し、細胞内に取り込まれにくくなっていると推定された。図８上段に示すように、ＢＶ－ＣａＰ－ＯｘＣＮＨは、細胞内に取り込まれていることが確認できたが、その多くはアポトーシス死細胞及びネクローシス死細胞であった。ＢＶ－ＣａＰ－ＯｘＣＮＨは、細胞内に取り込まれてＢＶを放出することで、細胞のアポトーシス死及びネクローシス死を引き起こし、効率的に細胞毒性を示すことが示唆された。 [Cytotoxicity assessment]
The cytotoxicity of the prepared sample was evaluated. Phagocytic mouse macrophage-like cells (RAW264.7) were seeded on 96-well plates and cultured at 37 ° C. After culturing for 24 hours, the medium is removed, BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, and Medium containing BV-CaP-OxCNH-6 was added. The concentration of each sample in the medium is 0.25 μg / mL for BV-CaP-OxCNH-1, 0.5 μg / mL for BV-CaP-OxCNH-2, and 1 for BV-CaP-OxCNH-3. .25 μg / mL, BV-CaP-OxCNH-4 was 2.5 μg / mL, BV-CaP-OxCNH-5 was 5.0 μg / mL, and BV-CaP-OxCNH-6 was 12.5 μg / mL. (From the results of ICP, the BV concentration in the medium is about 0.5 μg / mL to 0.75 μg / mL, the Ca concentration derived from CaP is 0.089 mM to 0.11 mM, and the P concentration is 0.068 mM to 0. It was estimated to be about 081 mM). As a control, a normal medium containing no BV-CaP-OxCNH was used. After culturing for 24 hours, the number of surviving cells was determined by measuring the absorbance at 450 nm using the Cell Counting Kit-8 reagent (Dojin Chemical Laboratory). The cell viability was calculated with the absorbance of the control as 100%.
FIG. 6A shows the results of cytotoxicity evaluation of BV-CaP-OxCNH. BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3 are almost the same, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, BV-CaP-OxCNH- In No. 6, cytotoxicity became more prominent as the OxCNH concentration in the sample increased.
The cytotoxicity of BV was evaluated in the same manner as above. The BV concentration added to the medium was 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL (control was 0 μg / mL). FIG. 6B shows the results of RAW264.7 cytotoxicity evaluation of BV. As shown in FIG. 6 (b), BV showed cytotoxicity in a concentration-dependent manner. Since the BV concentration contained in BV-CaP-OxCNH is estimated to be about 0.5 μg / mL to 0.75 μg / mL in the medium, the amount of BV-CaP-OxCNH is smaller than that of BV alone. It can be said that it showed cytotoxicity extremely efficiently even though it contained only BV.
Cytotoxicity was evaluated for BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH by the same method as described above. The concentration of each sample in the medium was diluted so that the OxCNH concentration was 0.17 μg / mL, 0.5 μg / mL, and 1.7 μg / mL. Similarly, cytotoxicity was evaluated for BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. The respective sample concentrations in the medium were diluted to be 1.7 μg / mL, 5.0 μg / mL, and 16.7 μg / mL as OxCNH concentrations.
FIG. 7A shows the results of cytotoxicity evaluation of BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH. As shown in FIG. 7 (a), CaP-OxCNH-2 and OxCNH were almost equivalent, and BV-CaP-OxCNH-2 showed stronger cytotoxicity. FIG. 7B shows the results of cytotoxicity evaluation of BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. As shown in FIG. 7 (b), OxCNH, CaP-OxCNH-5, and BV-CaP-OxCNH-5 showed stronger cytotoxicity in this order. From these results, it can be said that BV-CaP-OxCNH exhibits stronger cytotoxicity because BV is supported on CaP-OxCNH.
Cells cultured with the addition of BV-CaP-OxCNH-5, CaP-OxCNH-5 and OxCNH were observed using an optical microscope. FIG. 8 shows an optical micrograph of RAW264.7 cells cultured for 24 hours in a medium supplemented with BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. As shown in the lower part of FIG. 8, OxCNH was presumed to be taken up by intracellular lysosomes. As shown in the middle part of FIG. 8, it was presumed that CaP-OxCNH formed a large mass in the medium and was difficult to be taken up into cells. As shown in the upper part of FIG. 8, it was confirmed that BV-CaP-OxCNH was taken up into the cells, but most of them were apoptotic dead cells and necrosis dead cells. It was suggested that BV-CaP-OxCNH causes apoptotic death and necrosis death of cells by being taken up into cells and releasing BV, and exhibits cytotoxicity efficiently.
［ＲＡＷ２６４．７細胞から破骨細胞への分化］
　破骨細胞を用いて細胞毒性を評価するため、ＲＡＷ２６４．７細胞から破骨細胞に分化させた。ＲＡＷ２６４．７細胞を９６　ｗｅｌｌ　ｐｌａｔｅに播種し、１日培養後、ＲＡＮＫＬ（１００ｎｇ／ｍＬ）を加えた培地に入れ替え、５日間培養することによって破骨細胞に分化誘導した。分化を確認するため、破骨細胞のマーカーである酒石酸抵抗性酸性ホスファターゼ（Ｔａｒｔｒａｔｅｒｅｓｉｓｔａｎｔ　ａｃｉｄ　ｐｈｏｓｐｈａｔａｓｅ、ＴＲＡＰ）を染色し、光学顕微鏡で観察した。図９に、ＲＡＷ２６４．７細胞(ａ)とＲＡＷ２６４．７細胞から分化させて得た破骨細胞(ｂ)をＴＲＡＰ染色した光学顕微鏡写真を示す。図９に示すように、分化前のＲＡＷ２６４．７細胞はＴＲＡＰ染色により染まらず、分化後の破骨細胞では染色されていた。この結果から、ＲＡＷ２６４．７細胞から正常に破骨細胞に分化したことが確認できた。 [Differentiation of RAW264.7 cells into osteoclasts]
In order to evaluate cytotoxicity using osteoclasts, RAW264.7 cells were differentiated into osteoclasts. RAW264.7 cells were seeded on a 96-well plate, cultured for 1 day, replaced with a medium containing RANKL (100 ng / mL), and cultured for 5 days to induce differentiation into osteoclasts. In order to confirm the differentiation, tartrate-resistant acid phosphatase (TRAP), which is a marker of osteoclasts, was stained and observed with an optical microscope. FIG. 9 shows an optical micrograph of TRAP-stained osteoclasts (b) obtained by differentiating from RAW264.7 cells (a) and RAW264.7 cells. As shown in FIG. 9, the RAW264.7 cells before differentiation were not stained by TRAP staining, but were stained by the osteoclasts after differentiation. From this result, it was confirmed that the RAW264.7 cells were normally differentiated into osteoclasts.
［破骨細胞を用いた細胞毒性評価］
　得られた破骨細胞培養液の培地を取り除き、ＢＶ－ＣａＰ－ＯｘＣＮＨ－１、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３を含む培地を加えた。培地中のそれぞれの試料濃度は、ＯｘＣＮＨ濃度としてＢＶ－ＣａＰ－ＯｘＣＮＨ－１は２．５μｇ／ｍＬ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２は５．０μｇ／ｍＬ、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３は１２．５μｇ／ｍＬであった。なお、コントロールでは、ＢＶ－ＣａＰ－ＯｘＣＮＨを含まない通常の培地を用いた。２４時間及び４８時間培養後、ＢＶ－ＣａＰ－ＯｘＣＮＨの取り込みを確認するため、破骨細胞の核をＨｏｅｃｈｓｔ　３３３４２で、リソソームをＬｙｓｏｓｏｍｅ　ｐａｉｎｔｅｒ　ｏｒａｎｇｅで染色し、蛍光顕微鏡で観察した。図１０に、２４時間後（図１０上段）及び４８時間後（図１０下段）の破骨細胞によるＢＶ－ＣａＰ－ＯｘＣＮＨの取り込みを示す蛍光顕微鏡写真を示す。図１０に示すように、ＢＶ－ＣａＰ－ＯｘＣＮＨは破骨細胞のリソソームに内にＯｘＣＮＨ（矢印）が取り込まれていた。なお、破骨細胞には単核のものと多核のものが観察された。
　これらの破骨細胞の生存率への影響をａｌａｍａｒＢｌｕｅ法により調べた。図１１に、ＯｘＣＮＨ、ＣａＰ－ＯｘＣＮＨ、ＢＶ－ＣａＰ－ＯｘＣＮＨを添加した破骨細胞の生存率を示す。ＢＶ－ＣａＰ－ＯｘＣＮＨ－１（ＯｘＣＮＨ濃度として２．５μｇ／ｍＬ）、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２（ＯｘＣＮＨ濃度として５．０μｇ／ｍＬ）、ＢＶ－ＣａＰ－ＯｘＣＮＨ－３（ＯｘＣＮＨ濃度として１２．５μｇ／ｍＬ）では、試料中のＯｘＣＮＨ濃度が高くなるにつれて細胞生存率が減少した。一方で、ＯｘＣＮＨ、ＣａＰ－ＯｘＣＮＨ－１、ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－３では、ＯｘＣＮＨ濃度に依存的な細胞生存率の低下は見られなかった。 [Cytotoxicity evaluation using osteoclasts]
The medium of the obtained osteoclast culture solution was removed, and a medium containing BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, and BV-CaP-OxCNH-3 was added. The concentration of each sample in the medium was 2.5 μg / mL for BV-CaP-OxCNH-1, 5.0 μg / mL for BV-CaP-OxCNH-2, and 12. for BV-CaP-OxCNH-3. It was 5 μg / mL. As a control, a normal medium containing no BV-CaP-OxCNH was used. After culturing for 24 hours and 48 hours, osteoclast nuclei were stained with Hoechst 33342 and lysosomes were stained with Lysosome painter orange to confirm the uptake of BV-CaP-OxCNH and observed under a fluorescence microscope. FIG. 10 shows fluorescence micrographs showing the uptake of BV-CaP-OxCNH by osteoclasts after 24 hours (upper part of FIG. 10) and 48 hours later (lower part of FIG. 10). As shown in FIG. 10, BV-CaP-OxCNH had OxCNH (arrow) incorporated into the lysosomes of osteoclasts. Mononuclear and multinucleated osteoclasts were observed.
The effect of these osteoclasts on the viability was investigated by the alamarBlue method. FIG. 11 shows the viability of osteoclasts supplemented with OxCNH, CaP-OxCNH, and BV-CaP-OxCNH. BV-CaP-OxCNH-1 (OxCNH concentration 2.5 μg / mL), BV-CaP-OxCNH-2 (OxCNH concentration 5.0 μg / mL), BV-CaP-OxCNH-3 (OxCNH concentration 12.5 μg) At / mL), the cell viability decreased as the OxCNH concentration in the sample increased. On the other hand, in OxCNH, CaP-OxCNH-1, CaP-OxCNH-2, and CaP-OxCNH-3, no decrease in cell viability was observed depending on the OxCNH concentration.
　本実施例では、ＢＰの種類ならびにＣａＰ過飽和溶液の組成を変化させて、種々のＢＰ－ＣａＰ－ＯｘＣＮＨを作製した。ＢＰとしては、実施例１で用いたＢＶのほか、ゾレドロン酸（ゾメタ、ＺＯ）ならびにパミドロン酸（ＰＭ）を用いた。ＢＶ、ＺＯ、ＰＭそれぞれに対して４つの濃度のＣａＰ過飽和溶液を調製し、計１２種類のＢＰ－ＣａＰ－ＯｘＣＮＨを作製した。 In this example, various BP-CaP-OxCNHs were prepared by changing the type of BP and the composition of the CaP supersaturated solution. As the BP, in addition to the BV used in Example 1, zoledronic acid (Zometa, ZO) and pamidronic acid (PM) were used. Four concentrations of CaP supersaturated solutions were prepared for each of BV, ZO, and PM to prepare a total of 12 types of BP-CaP-OxCNH.
［ＢＰ－ＣａＰ－ＯｘＣＮＨの作製］
　実施例１と同様の方法で、ＣＮＨを過酸化水素により酸化し、水洗して、ＯｘＣＮＨ水分散液を得た。得られたＯｘＣＮＨ水分散液は、凍結乾燥せずにそのまま使用した。ＯｘＣＮＨ水分散液の濃度は、紫外可視分光光度計で７００ｎｍの吸光度を測定し、事前に濃度の判明しているＣＮＨ水分散液を用いて作成した検量線（ＣＮＨ濃度－吸光度プロット）を使って算出した。ＯｘＣＮＨ水分散液は、２ｍｇ／ｍＬになるように濃度調製し、オートクレーブにより滅菌して滅菌ＯｘＣＮＨ水分散液を得た。
　ＢＶ溶液として、実施例１で用いたボンビバ静注１ｍｇシリンジを使用した。ＺＯ溶液として、ゾレドロン酸水和物を有効成分として含有するゾメタ点滴静注４ｍｇ／５ｍＬ（ノバルティスファーマ株式会社）を使用した。ＰＭ溶液として、パミドロン酸二ナトリウム水和物を有効成分として含有するパミドロン酸二Ｎａ点滴静注用１５ｍｇ「サワイ」（沢井製薬株式会社）を使用した。これらのＢＶ、ＺＯ、ＰＭ溶液は、注射用水を用いて２．９４ｍＭとなるように調製した。
　ＣａＰ過飽和溶液の原料となるカルシウム含有液とリン酸含有液は、実施例１で用いたのと同様、カルシウム含有液はリンゲル液「オーツカ」と塩化Ｃａ補正液１ｍＥｑ／ｍＬを、リン酸含有液はクリニザルツ（登録商標）輸液とリン酸２カリウム注２０ｍＥｑキット「テルモ」を混合して調製した。それぞれの試薬の混合量を変化させることで、含有するＣａおよびＰの濃度を変化させた４種類のカルシウム含有液およびリン酸含有液を調製した。具体的には、実施例１で用いたのと同じカルシウム含有液とリン酸含有液、および、それらの溶液中に含まれるＣａまたはＰの濃度を元として、Ｃａ、Ｐの濃度がそれぞれ７８％、６７％、５６％となるようなカルシウム含有液とリン酸含有液を調製した。また、ｐＨ調整剤は実施例１と同じものを用いた。
　滅菌ＯｘＣＮＨ水分散液（２ｍｇ／ｍＬ、０．２５０ｍＬ）、ＢＶ、ＺＯ、又はＰＭ溶液（２．９４ｍＭ、０．２５０ｍＬ）、カルシウム含有液（７．６７４ｍＬ）、リン酸含有液（０．９１７ｍＬ）、及びｐＨ調整剤（０．９０９ｍＬ）を混合し、ＣａＰ過飽和溶液（反応液、１０ｍＬ）を調製した。それぞれのＣａＰ過飽和溶液中のＣａ、Ｐの濃度は、実施例１で用いたのと同じカルシウム含有液とリン酸含有液を使用した場合が、Ｃａ：６．６０ｍＭ、Ｐ：３．３０ｍＭ、また、７８％、６７％、５６％となるように調製したカルシウム含有液とリン酸含有液を使用した場合が、Ｃａ：５．１４ｍＭ、Ｐ：２．５６ｍＭ（７８％）、Ｃａ：４．４０ｍＭ、Ｐ：２．２０ｍＭ（６７％）、Ｃａ：３．６８ｍＭ、Ｐ：１．８３ｍＭ（５６％）であった。これらの過飽和溶液をＣａＰ過飽和溶液ａ、ｂ、ｃ、ｄとした。
　反応液は、調製後すぐ、２５℃のインキュベーターで３０分間静置し、ＢＰ、ＣａＰ、ＯｘＣＮＨの三者を共沈させた。得られた試料ＢＰ－ＣａＰ－ＯｘＣＮＨは、遠心操作（６０００ｒｐｍ、５分）により回収し、注射用水に試料を分散して遠心することを繰り返すことによって洗浄した。ＣａＰ過飽和溶液ａ、ｂ、ｃ、ｄを用いて作製した試料を、それぞれ、ＢＰ－ＣａＰａ－ＯｘＣＮＨ、ＢＰ－ＣａＰｂ－ＯｘＣＮＨ、ＢＰ－ＣａＰｃ－ＯｘＣＮＨ、ＢＰ－ＣａＰｄ－ＯｘＣＮＨとした。ここで、ＢＰはＢＶ、ＺＯ、ＰＭのいずれかに該当する。 [Preparation of BP-CaP-OxCNH]
CNH was oxidized with hydrogen peroxide and washed with water in the same manner as in Example 1 to obtain an OxCNH aqueous dispersion. The obtained OxCNH aqueous dispersion was used as it was without freeze-drying. For the concentration of the OxCNH aqueous dispersion, measure the absorbance at 700 nm with an ultraviolet-visible spectrophotometer and use a calibration curve (CNH concentration-absorbance plot) prepared using a CNH aqueous dispersion whose concentration is known in advance. Calculated. The concentration of the OxCNH aqueous dispersion was adjusted to 2 mg / mL, and the mixture was sterilized by an autoclave to obtain a sterilized OxCNH aqueous dispersion.
As the BV solution, the Bombiva intravenous injection 1 mg syringe used in Example 1 was used. As the ZO solution, Zoledronic acid hydrate containing zoledronic acid hydrate as an active ingredient was used as an intravenous drip infusion of Zometa 4 mg / 5 mL (Novartis Pharma Co., Ltd.). As the PM solution, 15 mg "Sawai" (Sawai Pharmaceutical Co., Ltd.) for intravenous drip infusion of disodium pamidronate containing disodium pamidronate hydrate as an active ingredient was used. These BV, ZO, and PM solutions were prepared to a concentration of 2.94 mM using water for injection.
The calcium-containing solution and the phosphoric acid-containing solution, which are the raw materials of the CaP hypersaturated solution, were the same as those used in Example 1, the calcium-containing solution was Ringer's solution "Otsuka" and the Ca chloride correction solution 1 mEq / mL, and the phosphoric acid-containing solution was It was prepared by mixing Clinisarz® infusion solution with dipotassium phosphate Injection 20mEq kit "Termo". By changing the mixing amount of each reagent, four kinds of calcium-containing liquids and phosphoric acid-containing liquids in which the concentrations of Ca and P contained were changed were prepared. Specifically, based on the same calcium-containing solution and phosphoric acid-containing solution used in Example 1 and the concentrations of Ca or P contained in those solutions, the concentrations of Ca and P are 78%, respectively. , 67% and 56% of calcium-containing solution and phosphoric acid-containing solution were prepared. Moreover, the same pH adjuster as in Example 1 was used.
Sterilized OxCNH aqueous dispersion (2 mg / mL, 0.250 mL), BV, ZO, or PM solution (2.94 mM, 0.250 mL), calcium-containing solution (7.674 mL), phosphoric acid-containing solution (0.917 mL) , And a pH adjuster (0.909 mL) were mixed to prepare a CaP supersaturated solution (reaction solution, 10 mL). The concentrations of Ca and P in each CaP hypersaturated solution were Ca: 6.60 mM, P: 3.30 mM, and when the same calcium-containing solution and phosphoric acid-containing solution used in Example 1 were used. Ca: 5.14 mM, P: 2.56 mM (78%), Ca: 4.40 mM when the calcium-containing solution and the phosphoric acid-containing solution prepared to be 78%, 67%, and 56% were used. , P: 2.20 mM (67%), Ca: 3.68 mM, P: 1.83 mM (56%). These supersaturated solutions were designated as CaP supersaturated solutions a, b, c and d.
Immediately after preparation, the reaction solution was allowed to stand in an incubator at 25 ° C. for 30 minutes to coprecipitate BP, CaP, and OxCNH. The obtained sample BP-CaP-OxCNH was collected by centrifugation (6000 rpm, 5 minutes), and washed by repeating the process of dispersing the sample in water for injection and centrifuging. Samples prepared using the CaP supersaturated solutions a, b, c, and d were designated as BP-CaPa-OxCNH, BP-CaPb-OxCNH, BP-CaPc-OxCNH, and BP-CaPd-OxCNH, respectively. Here, BP corresponds to any of BV, ZO, and PM.
［ＣａＰ－ＯｘＣＮＨの作製］
　ＢＰを含まないコントロールとして、ＢＶ、ＺＯ、又はＰＭ溶液の代わりに注射用水を用いて同様の操作を行い、試料ＣａＰ－ＯｘＣＮＨを得た。ＣａＰ過飽和溶液ａ、ｂ、ｃ、ｄを用いて作製した試料を、それぞれ、ＣａＰａ－ＯｘＣＮＨ、ＣａＰｂ－ＯｘＣＮＨ、ＣａＰｃ－ＯｘＣＮＨ、ＣａＰｄ－ＯｘＣＮＨとした。 [Preparation of CaP-OxCNH]
As a control containing no BP, the same operation was carried out using water for injection instead of the BV, ZO, or PM solution to obtain a sample CaP-OxCNH. Samples prepared using the CaP supersaturated solutions a, b, c, and d were designated as CaPa-OxCNH, CaPb-OxCNH, CaPc-OxCNH, and CaPd-OxCNH, respectively.
［ＳＥＭによる観察］
　ＢＰ－ＣａＰ－ＯｘＣＮＨの構造をＳＥＭにより確認した。実施例１と同様、試料はシリコン基板上に滴下し、乾燥させ、金を蒸着して観察した。ＢＰ－ＣａＰ－ＯｘＣＮＨのＳＥＭ画像を図１２に示す。
　いずれのＢＰ－ＣａＰ－ＯｘＣＮＨでも、一次粒子径５０～１００ｎｍの粒子と、一次粒子径１００～１５０ｎｍの粒子とが観察された。さらに一次粒子径１００～１５０ｎｍの粒子は、ＣＮＨに起因すると考えられる凹凸構造を有していた。 [Observation by SEM]
The structure of BP-CaP-OxCNH was confirmed by SEM. Similar to Example 1, the sample was dropped on a silicon substrate, dried, and gold was vapor-deposited for observation. An SEM image of BP-CaP-OxCNH is shown in FIG.
In any of the BP-CaP-OxCNH particles, particles having a primary particle diameter of 50 to 100 nm and particles having a primary particle diameter of 100 to 150 nm were observed. Further, the particles having a primary particle diameter of 100 to 150 nm had an uneven structure considered to be caused by CNH.
［ＥＤＸによる測定］
　ＢＰ－ＣａＰ－ＯｘＣＮＨをシリコン基板上に滴下し、乾燥させ、ＥＤＸを用いて元素分析を行った。元素分析の結果を図１３に示す。いずれの粒子からも、炭素、酸素、リン、カルシウムのピークが検出されたことから、ＣａＰとＯｘＣＮＨが複合体を形成したことが示唆された。また、ＣａＰ過飽和溶液中のＣａ、Ｐ濃度が低下するにつれて（ＣａＰ過飽和溶液ａからｄ）、炭素のピークに対してカルシウム、リンのピークが顕著に減少した。これは、ＣａＰ過飽和溶液中のＣａ、Ｐ濃度の低下に伴い、生成した粒子中のＯｘＣＮＨに対するＣａＰの割合が小さくなったためと考えられた。 [Measurement by EDX]
BP-CaP-OxCNH was dropped onto a silicon substrate, dried, and elemental analysis was performed using EDX. The result of elemental analysis is shown in FIG. The peaks of carbon, oxygen, phosphorus, and calcium were detected in all the particles, suggesting that CaP and OxCNH formed a complex. Further, as the Ca and P concentrations in the CaP supersaturated solution decreased (CaP supersaturated solutions a to d), the peaks of calcium and phosphorus decreased remarkably with respect to the peak of carbon. It is considered that this is because the ratio of CaP to OxCNH in the produced particles decreased as the concentrations of Ca and P in the CaP supersaturated solution decreased.
［ＩＣＰによる測定］
　ＢＰ－ＣａＰ－ＯｘＣＮＨおよびＣａＰ－ＯｘＣＮＨについて、ＩＣＰを用いて元素分析を行った。試料を酸で溶解させた溶液を調製し、測定を行った。１０ｍＬの反応液から得られた試料中のカルシウム含有量、リン含有量、及びリン含有量に対するカルシウム含有量のモル比（Ｃａ／Ｐ）を表２に示す
Figure JPOXMLDOC01-appb-T000002
　表２の結果より、ＢＰ－ＣａＰ－ＯｘＣＮＨでは、ＢＶ、ＺＯ、ＰＭのいずれを使用した場合も、ＣａＰ－ＯｘＣＮＨよりＣａ／Ｐモル比が小さかった。ＢＰ－ＣａＰ－ＯｘＣＮＨでは、リンを含むＢＰが担持されたために、カルシウムに対してリンの含有量が相対的に大きくなり、Ｃａ／Ｐモル比が小さくなったと考えられる。
　次に、ＢＰ－ＣａＰ－ＯｘＣＮＨ中のＢＰの含有量を推定する。ここで、同じＣａ、Ｐ濃度を有するＣａＰ過飽和溶液を用いて作製したＢＰ－ＣａＰ－ＯｘＣＮＨ中のＣａＰに由来するＣａとＰに対するＣａ／Ｐモル比は、ＣａＰ－ＯｘＣＮＨ中のＣａ／Ｐモル比に等しいと仮定する。この過程をもとにＢＰ－ＣａＰ－ＯｘＣＮＨ中のＢＰの含有量を推定したところ、どのＣａＰ過飽和溶液を用いた場合でも、反応液に混合した量（０．７３５μｍｏｌ）の３０－５０％程度（０．２２－０．３７μｍｏｌ）であると算出された。以上より、ＢＰ－ＣａＰ－ＯｘＣＮＨでは、ＢＰ、ＣａＰ及びＯｘＣＮＨが三元複合体を形成していることが確認された。
　また、ＣａＰ過飽和溶液中のＣａ、Ｐ濃度が低下するにつれて（ａからｄ）、カルシウム、リンの含有量が大幅に減少した。この結果はＥＤＸの結果（図１３）とも相関しており、ＣａＰ過飽和溶液中のＣａ、Ｐ濃度の低下に伴い、ＣａＰの生成量が少なくなり、粒子中のＣａＰ含有量が低下したためと考えられた。 [Measurement by ICP]
Elemental analysis of BP-CaP-OxCNH and CaP-OxCNH was performed using ICP. A solution in which the sample was dissolved in acid was prepared and measured. Table 2 shows the calcium content, the phosphorus content, and the molar ratio (Ca / P) of the calcium content to the phosphorus content in the sample obtained from the 10 mL reaction solution.
Figure JPOXMLDOC01-appb-T000002
From the results in Table 2, the Ca / P molar ratio of BP-CaP-OxCNH was smaller than that of CaP-OxCNH when any of BV, ZO, and PM was used. In BP-CaP-OxCNH, since BP containing phosphorus was supported, it is considered that the phosphorus content was relatively large with respect to calcium and the Ca / P molar ratio was small.
Next, the content of BP in BP-CaP-OxCNH is estimated. Here, the Ca / P molar ratio to Ca and P derived from CaP in BP-CaP-OxCNH prepared using a CaP supersaturated solution having the same Ca and P concentrations is the Ca / P molar ratio in CaP-OxCNH. Is assumed to be equal to. When the content of BP in BP-CaP-OxCNH was estimated based on this process, no matter which CaP supersaturated solution was used, about 30-50% of the amount (0.735 μmol) mixed with the reaction solution (0.735 μmol). It was calculated to be 0.22-0.37 μmol). From the above, it was confirmed that in BP-CaP-OxCNH, BP, CaP and OxCNH form a ternary complex.
Further, as the Ca and P concentrations in the CaP supersaturated solution decreased (a to d), the calcium and phosphorus contents decreased significantly. This result also correlates with the result of EDX (FIG. 13), and it is considered that as the Ca and P concentrations in the CaP supersaturated solution decrease, the amount of CaP produced decreases and the CaP content in the particles decreases. rice field.
［細胞毒性評価］
　実施例１と同様の方法により、ＢＰ－ＣａＰ－ＯｘＣＮＨのＲＡＷ２６４．７に対する細胞毒性を評価した。培地中のそれぞれの試料濃度は、ＯｘＣＮＨ濃度として１．７μｇ／ｍＬ、５．０μｇ／ｍＬ、１６．７μｇ／ｍＬとなるように希釈した。また、ＺＯ単独とＰＭ単独の細胞毒性も評価した。
　図１４にＢＶ－ＣａＰ－ＯｘＣＮＨの細胞毒性評価の結果を示す。ＢＶ－ＣａＰ－ＯｘＣＮＨは、粒子中のＣａＰ含有量に関わらず、同様の細胞毒性を示した。培地中のＯｘＣＮＨ濃度として１６．７μｇ／ｍＬの条件では、培地中のＢＶ濃度は２．３－３．９μｇ／ｍＬ程度と推定されることから、図６（ｂ）に示したＢＶ単独と比較して、ＢＶ－ＣａＰ－ＯｘＣＮＨでは少量のＢＶしか含まれていないにも関わらず、効率的に細胞毒性を示したと言える。
　図１５（ａ）にＺＯ－ＣａＰ－ＯｘＣＮＨの細胞毒性評価の結果を示す。ＢＶ－ＣａＰ－ＯｘＣＮＨと比較して、ＺＯ－ＣａＰ－ＯｘＣＮＨは強い細胞毒性を示した。図１５（ｂ）に示したＺＯ単独では、比較的低濃度（２．５μｇ／ｍＬ）でも細胞毒性を示しており、同濃度ではほとんど細胞毒性を示さないＢＶ単独とは異なる挙動を示している。この、ＺＯ単独とＢＶ単独の細胞毒性の違いが、複合体の細胞毒性にも影響を及ぼしていると考えられる。なお、図１５（ａ）において、培地中のＯｘＣＮＨ濃度として１６．７μｇ／ｍＬの条件では、培地中のＺＯ濃度は２．０－３．４μｇ／ｍＬ程度と推定されることから、図１５（ｂ）に示したＺＯ単独と比較して、ＺＯ－ＣａＰ－ＯｘＣＮＨでは少量のＺＯしか含まれていないにも関わらず、効率的に細胞毒性を示したと言える。粒子中のＣａＰ含有量による細胞毒性の違いは、多少は見られたものの、その違いは顕著ではなかった。
　図１６（ａ）にＰＭ－ＣａＰ－ＯｘＣＮＨの細胞毒性評価の結果を示す。培地中のＯｘＣＮＨ濃度として５．０μｇ／ｍＬ、１６．７μｇ／ｍＬの条件では、ＢＶ－ＣａＰ－ＯｘＣＮＨと比較して、ＰＭ－ＣａＰ－ＯｘＣＮＨは強い細胞毒性を示した。一方で、培地中のＯｘＣＮＨ濃度として１．７μｇ／ｍＬの条件では、ＰＭ－ＣａＰ－ＯｘＣＮＨはほとんど毒性を示さなかった。図１６（ｂ）に示したＰＭ単独では、１０μｇ／ｍＬと２５μｇ／ｍＬの間でＰＭの細胞毒性に大きな違いがあり、培地中のＯｘＣＮＨ濃度として１．７μｇ／ｍＬと５．０μｇ／ｍＬの間でＰＭ－ＣａＰ－ＯｘＣＮＨの細胞毒性に大きな違いが見られる現象と傾向が一致している。なお、図１６（ａ）において、培地中のＯｘＣＮＨ濃度として１６．７μｇ／ｍＬの条件では、培地中ＰＭ濃度は２．０－３．４μｇ／ｍＬ程度と推定されることから、図１６（ｂ）に示したＰＭ単独と比較して、ＰＭ－ＣａＰ－ＯｘＣＮＨでは少量のＰＭしか含まれていないにも関わらず、効率的に細胞毒性を示したと言える。培地中のＯｘＣＮＨ濃度として５μｇ／ｍＬの条件では、粒子中のＣａＰ含有量が高いほど強い細胞毒性が見られた。  [Cytotoxicity assessment]
The cytotoxicity of BP-CaP-OxCNH to RAW264.7 was evaluated by the same method as in Example 1. The respective sample concentrations in the medium were diluted to be 1.7 μg / mL, 5.0 μg / mL, and 16.7 μg / mL as OxCNH concentrations. The cytotoxicity of ZO alone and PM alone was also evaluated.
FIG. 14 shows the results of cytotoxicity evaluation of BV-CaP-OxCNH. BV-CaP-OxCNH showed similar cytotoxicity regardless of the CaP content in the particles. Under the condition that the OxCNH concentration in the medium is 16.7 μg / mL, the BV concentration in the medium is estimated to be about 2.3-3.9 μg / mL, which is compared with the BV alone shown in FIG. 6 (b). Therefore, it can be said that BV-CaP-OxCNH efficiently showed cytotoxicity even though it contained only a small amount of BV.
FIG. 15A shows the results of cytotoxicity evaluation of ZO-CaP-OxCNH. Compared with BV-CaP-OxCNH, ZO-CaP-OxCNH showed strong cytotoxicity. ZO alone shown in FIG. 15B shows cytotoxicity even at a relatively low concentration (2.5 μg / mL), and behaves differently from BV alone, which shows almost no cytotoxicity at the same concentration. .. It is considered that this difference in cytotoxicity between ZO alone and BV alone also affects the cytotoxicity of the complex. In FIG. 15A, under the condition that the OxCNH concentration in the medium is 16.7 μg / mL, the ZO concentration in the medium is estimated to be about 2.0-3.4 μg / mL. It can be said that ZO-CaP-OxCNH efficiently showed cytotoxicity even though it contained only a small amount of ZO as compared with ZO alone shown in b). Although there was some difference in cytotoxicity depending on the CaP content in the particles, the difference was not remarkable.
FIG. 16A shows the results of cytotoxicity evaluation of PM-CaP-OxCNH. Under the conditions of OxCNH concentration in the medium of 5.0 μg / mL and 16.7 μg / mL, PM-CaP-OxCNH showed stronger cytotoxicity as compared with BV-CaP-OxCNH. On the other hand, PM-CaP-OxCNH showed almost no toxicity under the condition of 1.7 μg / mL as the OxCNH concentration in the medium. With PM alone shown in FIG. 16 (b), there is a large difference in the cytotoxicity of PM between 10 μg / mL and 25 μg / mL, and the OxCNH concentrations in the medium are 1.7 μg / mL and 5.0 μg / mL. The tendency is consistent with the phenomenon in which there is a large difference in the cytotoxicity of PM-CaP-OxCNH. In FIG. 16A, under the condition that the OxCNH concentration in the medium is 16.7 μg / mL, the PM concentration in the medium is estimated to be about 2.0-3.4 μg / mL. ), It can be said that PM-CaP-OxCNH efficiently showed cytotoxicity even though it contained only a small amount of PM. Under the condition that the OxCNH concentration in the medium was 5 μg / mL, the higher the CaP content in the particles, the stronger the cytotoxicity was observed.
　本実施例では、骨粗鬆症モデルラットの脛骨に穴をあけ、そこにＢＶ－ＣａＰ－ＯｘＣＮＨ等の試料を埋入し、試料の骨形成に対する影響を評価した。 In this example, a hole was made in the tibia of an osteoporosis model rat, and a sample such as BV-CaP-OxCNH was embedded therein, and the effect of the sample on bone formation was evaluated.
［骨粗鬆症モデルラットの作製］
　Ｗｉｓｔａｒラット（メス、１０週齢）の両側卵巣を摘出し、傷口を縫合した。術後８週間、通常飼育し、骨粗鬆症モデルラットを作製した。なお、比較対象として、卵巣を摘出しない偽手術ラットも作製した。体重の変化を記録し、骨粗鬆症モデルラットでは偽手術ラットと比較して、体重が増加（骨粗鬆症モデルラットにおける著名な現象）していることを確認した。また、実験動物用Ｘ線ＣＴを用いて骨密度計測を行い、骨粗鬆症モデルラットでは偽手術ラットと比較して海綿骨密度が約２０％低下していること、すなわち骨粗鬆症の病態となっていることを確認した。 [Preparation of osteoporosis model rat]
Bilateral ovaries of Wistar rats (female, 10 weeks old) were removed and the wound was sutured. Osteoporosis model rats were prepared by breeding normally for 8 weeks after the operation. For comparison, sham-surgery rats without removing the ovaries were also prepared. Changes in body weight were recorded, and it was confirmed that the osteoporosis model rats gained weight (a prominent phenomenon in the osteoporosis model rats) as compared with the sham-surgery rats. In addition, bone mineral density was measured using X-ray CT for experimental animals, and the cancellous bone density of the osteoporosis model rat was reduced by about 20% compared to the sham-operated rat, that is, the condition of osteoporosis. It was confirmed.
［埋入用試料の作製］
　ＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ、ＣａＰを実施例１と同様の方法で作製し、試料を得た。ただし、試料作製における原料の一つとなるＯｘＣＮＨ分散液については、実施例２と同様の方法（凍結乾燥なし）で作製した。得られた試料は少量の注射用水に懸濁させた。懸濁液中のＯｘＣＮＨ濃度は０．８３ｍｇ／ｍＬ、ＢＶ濃度は２．１ｍｇ／ｍＬ程度と推定される。これらの試料以外に、ＯｘＣＮＨ（０．８３ｍｇ／ｍＬ）、ＢＶ（イバンドロン酸として１．０ｍｇ／ｍＬ）、生理食塩水（ネガティブコントロール）も比較対象として検討した。 [Preparation of sample for embedding]
BV-CaP-OxCNH-2, CaP-OxCNH-2, BV-CaP, and CaP were prepared in the same manner as in Example 1 to obtain a sample. However, the OxCNH dispersion, which is one of the raw materials for sample preparation, was prepared by the same method as in Example 2 (without freeze-drying). The resulting sample was suspended in a small amount of water for injection. The OxCNH concentration in the suspension is estimated to be 0.83 mg / mL, and the BV concentration is estimated to be about 2.1 mg / mL. In addition to these samples, OxCNH (0.83 mg / mL), BV (1.0 mg / mL as ibandronic acid), and physiological saline (negative control) were also examined for comparison.
［脛骨のリーミングと試料の埋入］
　各試料に対して３匹の骨粗鬆症モデルラットを準備し、それぞれの左右脛骨を１８ゲージシリンジ針でリーミング（骨に穴を開ける操作、骨孔１ｍｍ程度）した。各試料は、ラット１匹の体重あたりのＢＶ総投与量が１５４μｇ／ｋｇ、ＯｘＣＮＨ総投与量が６１．７μｇ／ｋｇ（ラット１匹あたりの体重は約２００ｇ）となるようにそれぞれ１ｍＬ調製した。各試料は、それぞれの骨粗鬆症モデルラット左右脛骨の骨孔に５０μＬずつ埋入し、傷口を縫合した。骨粗鬆症モデルラットは、試料を埋入後１２週間、後述するＣＴ撮影および骨密度評価を経時的に行いながら通常飼育した。
　また、ポジティブコントロールとして、ＢＶ溶液を皮下投与した群も作製した。ＢＶ溶液の皮下投与は、局所投与とならないように毎回、ラットの異なる箇所に、１日１回を１週間連続して４週間毎に計１２週間行った（１週間連続投与を０、４、８週後の３回行った）。ＢＶ溶液は、ＢＶ溶液（１．０ｍｇ／ｍＬ）をラット１匹の体重あたりのＢＶ総投与量が１２週間で計１５４μｇ／ｋｇになるように調製した。皮下投与開始から１２週間、後述するＣＴ撮影および骨密度評価を経時的に行いながら通常飼育した。 [Tibial reaming and sample implantation]
Three osteoporosis model rats were prepared for each sample, and each of the left and right tibias was reamed with an 18-gauge syringe needle (operation of making a hole in the bone, bone hole of about 1 mm). Each sample was prepared in an amount of 1 mL so that the total BV dose per rat body weight was 154 μg / kg and the total OxCNH dose was 61.7 μg / kg (body weight per rat was about 200 g). Each sample was placed in the bone holes of the left and right tibias of each osteoporosis model rat in an amount of 50 μL, and the wound was sutured. Osteoporosis model rats were normally bred for 12 weeks after implantation while performing CT imaging and bone mineral density evaluation, which will be described later, over time.
In addition, as a positive control, a group in which the BV solution was subcutaneously administered was also prepared. Subcutaneous administration of the BV solution was performed once a day for a total of 12 weeks every 4 weeks at different sites in the rat each time so as not to be a local administration (1 week continuous administration 0, 4, I went 3 times after 8 weeks). The BV solution was prepared so that the total BV dose per rat body weight of the BV solution (1.0 mg / mL) was 154 μg / kg in 12 weeks. For 12 weeks from the start of subcutaneous administration, the animals were normally bred while performing CT imaging and bone mineral density evaluation, which will be described later, over time.
［子宮の観察と重量測定］
　１２週間経過後、偽手術ラットおよび骨粗鬆症モデルラットは安楽死させ、それぞれの子宮を観察・摘出した。図１７（ａ）に偽手術ラット（左側）および骨粗鬆症モデルラット（右側）の、開腹写真（上側）と摘出した子宮の写真（下側）を示す。骨粗鬆症モデルラットでは、偽手術ラットと比較して明らかな子宮萎縮（骨粗鬆症モデルラットにおける著名な現象）が観察された。次に、偽手術ラットおよび骨粗鬆症モデルラットから摘出した子宮の重量を測定した。図１７（ｂ）に偽手術ラット（左側）から摘出した子宮重量を１としたときの骨粗鬆症モデルラット（右側）から摘出した子宮重量の相対値を示す。偽手術ラットから摘出した子宮重量を１としたときの骨粗鬆症モデルラットから摘出した子宮重量の相対値は約０．３であった。骨粗鬆症モデルラットでは、偽手術ラットと比較して明らかな子宮重量の低下（骨粗鬆症モデルラットにおける著名な現象）が観察された。 [Uterus observation and weight measurement]
After 12 weeks, sham-operated rats and osteoporosis model rats were euthanized, and their uteri were observed and removed. FIG. 17A shows a laparotomy photograph (upper side) and a photograph of the removed uterus (lower side) of a sham-surgery rat (left side) and an osteoporosis model rat (right side). In the osteoporosis model rat, clear uterine atrophy (a prominent phenomenon in the osteoporosis model rat) was observed as compared with the pseudo-surgery rat. Next, the weight of the uterus removed from the pseudo-surgery rat and the osteoporosis model rat was measured. FIG. 17B shows the relative value of the uterine weight removed from the osteoporosis model rat (right side) when the uterine weight removed from the pseudo-surgery rat (left side) is 1. The relative value of the uterine weight removed from the osteoporosis model rat was about 0.3 when the uterine weight removed from the sham-operated rat was 1. In the osteoporosis model rats, a clear decrease in uterine weight (a prominent phenomenon in the osteoporosis model rats) was observed as compared with the sham-surgery rats.
［ＣＴ撮影］
　試料埋入から０、４、８、１２週間後に各骨粗鬆症モデルラットの脛骨のＣＴ（Ｃｏｍｐｕｔｅｄ　Ｔｏｍｏｇｒａｐｈｙ）撮影を行った。図１８に各骨粗鬆症モデルラットの試料埋入から０、８、１２週間後の脛骨ＣＴ像を示す。図１８に示すように、ＢＶを皮下投与（ポジティブコントロール）したラットまたはＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ、ＢＶを局所投与（脛骨に埋入）したラットでは、生理食塩水、ＣａＰ、ＯｘＣＮＨ、またはＣａＰ－ＯｘＣＮＨ－２を局所投与したラットと比較して皮質骨の内側に骨基質の良好な再生が認められた。 [CT photography]
Computed tomography (CT) imaging of the tibia of each osteoporosis model rat was performed 0, 4, 8 and 12 weeks after sample implantation. FIG. 18 shows tibial CT images 0, 8 and 12 weeks after sample implantation in each osteoporosis model rat. As shown in FIG. 18, in rats to which BV was subcutaneously administered (positive control) or BV-CaP-OxCNH-2, BV-CaP, BV was locally administered (implanted in the tibia), saline, CaP, Good regeneration of the bone matrix was observed inside the cortical bone as compared with rats topically administered OxCNH or CaP-OxCNH-2.
［骨密度評価］
　試料埋入から０、４、８、１２週間後に、実験動物用Ｘ線ＣＴを用いて各骨粗鬆症モデルラット脛骨の骨密度計測を行った。図１９に各骨粗鬆症モデルラット脛骨の全骨密度を、図２０に各骨粗鬆症モデルラット脛骨の皮質骨密度を、図２１に各骨粗鬆症モデルラット脛骨の海綿骨密度を示す。
　図１９に示すように、ＢＶを皮下投与したラットおよびＢＶ－ＣａＰ－ＯｘＣＮＨ－２、ＢＶ－ＣａＰ、ＢＶを局所投与したラットでは、生理食塩水、ＣａＰ、ＯｘＣＮＨ、またはＣａＰ－ＯｘＣＮＨ－２を局所投与したラットと比較して全骨密度の向上が見られた。図２０に示すように、各骨粗鬆症モデルラット脛骨の皮質骨密度では、埋入した試料による大きな差は見られなかった。図２１に示すように、各骨粗鬆症モデルラット脛骨の海綿骨密度では、全骨密度と同じ傾向が見られた。したがって、海綿骨密度が改善することで、全骨密度が改善したと考えられる。
　図２２に、図２１に示すデータを用いて試料埋入直後（０週後）の各骨粗鬆症モデルラット脛骨の海綿骨密度を１としたときの相対値の経時変化を示す。図２２に示すように、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２を局所投与したラットでは、ＢＶを皮下投与し続けたラットと同様に１２週まで海綿骨密度が向上し続けた。一方で、ＢＶ－ＣａＰおよびＢＶを局所投与したラットでは、ＢＶを皮下投与したラットおよびＢＶ－ＣａＰ－ＯｘＣＮＨ－２を局所投与したラットと比較して８週以降の海綿骨密度に大きな向上は見られなかった。なお、各骨粗鬆症モデルラット脛骨の全骨密度でも、海綿骨密度と同じ傾向が見られた。また、生理食塩水、ＣａＰ、ＯｘＣＮＨ、またはＣａＰ－ＯｘＣＮＨ－２を局所投与したラットでは、１２週まで海綿骨密度が減少し続けた。ＢＶ－ＣａＰ－ＯｘＣＮＨ－２は一度の局所投与（患部への埋入）で、１ヵ月毎に複数回投与するＢＶの皮下投与と同等の骨密度向上効果を示した。このことから、ＢＶ－ＣａＰ－ＯｘＣＮＨ－２は単回投与であることから患者への負荷が少なく、効率的に骨形成促進効果を示すことが推察される。
 
  [Bone density evaluation]
Bone mineral density of each osteoporosis model rat tibia was measured using X-ray CT for experimental animals 0, 4, 8 and 12 weeks after sample implantation. FIG. 19 shows the total bone density of each osteoporosis model rat tibia, FIG. 20 shows the cortical bone density of each osteoporosis model rat tibia, and FIG. 21 shows the cancellous bone density of each osteoporosis model rat tibia.
As shown in FIG. 19, in rats subcutaneously administered with BV and rats locally administered with BV-CaP-OxCNH-2, BV-CaP, BV, saline, CaP, OxCNH, or CaP-OxCNH-2 was locally administered. An improvement in total bone mineral density was observed compared to the treated rats. As shown in FIG. 20, the cortical bone mineral density of each osteoporosis model rat tibia did not show a large difference depending on the implanted sample. As shown in FIG. 21, the cancellous bone mineral density of each osteoporosis model rat tibia showed the same tendency as the total bone mineral density. Therefore, it is considered that the total bone density was improved by improving the cancellous bone density.
FIG. 22 shows the time course of the relative value when the cancellous bone mineral density of each osteoporosis model rat tibia immediately after sample implantation (0 week later) is set to 1 using the data shown in FIG. 21. As shown in FIG. 22, in the rats topically administered BV-CaP-OxCNH-2, the cancellous bone mineral density continued to improve until 12 weeks, similar to the rats in which BV was continuously administered subcutaneously. On the other hand, in rats to which BV-CaP and BV were locally administered, there was a significant improvement in cancellous bone mineral density after 8 weeks as compared with rats to which BV was subcutaneously administered and rats to which BV-CaP-OxCNH-2 was locally administered. I couldn't. The total bone mineral density of each osteoporosis model rat tibia showed the same tendency as the cancellous bone mineral density. In addition, in rats topically administered with saline, CaP, OxCNH, or CaP-OxCNH-2, cancellous bone mineral density continued to decrease until 12 weeks. BV-CaP-OxCNH-2 showed the same bone mineral density improving effect as subcutaneous administration of BV administered multiple times every month by one local administration (implantation in the affected area). From this, it is inferred that since BV-CaP-OxCNH-2 is a single dose, the load on the patient is small and the bone formation promoting effect is efficiently exhibited.

Claims (12)

  1. 　リン酸カルシウムとビスホスホネートとカーボン粒子を含有するカーボン粒子複合体。 A carbon particle complex containing calcium phosphate, bisphosphonates and carbon particles.
  2. 　前記カーボン粒子は、カーボンナノホーン、カーボンナノチューブ、ナノグラフェン、ナノダイアモンド、若しくはカーボンファイバー、又はこれらの組み合わせである請求項１に記載のカーボン粒子複合体。 The carbon particle composite according to claim 1, wherein the carbon particles are carbon nanohorns, carbon nanotubes, nanographenes, nanodiamonds, or carbon fibers, or a combination thereof.
  3. 　前記カーボン粒子は、酸化カーボンナノホーンである請求項１に記載のカーボン粒子複合体。 The carbon particle composite according to claim 1, wherein the carbon particles are carbon oxide nanohorns.
  4. 　前記ビスホスホネートは、エチドロン酸、イバンドロン酸、ゾレドロン酸、アレンドロン酸、ミノドロン酸、リセドロン酸、パミドロン酸、インカドロン酸、若しくはこれらの塩、又はこれらの組み合わせである請求項１に記載のカーボン粒子複合体。 The carbon particle composite according to claim 1, wherein the bisphosphonate is etidronic acid, ibandronic acid, zoledronic acid, alendronic acid, minodronic acid, risedronic acid, pamidronic acid, incadronic acid, or a salt thereof, or a combination thereof. ..
  5. 　請求項１に記載のカーボン粒子複合体を含有する破骨細胞をターゲットとした製剤。 A preparation targeting osteoclasts containing the carbon particle complex according to claim 1.
  6. 　ビスホスホネートと、カーボン粒子と、を含むリン酸カルシウム過飽和溶液を調製し、
    　前記ビスホスホネートと、前記カーボン粒子と、リン酸カルシウムと、を共沈させるカーボン粒子複合体の製造方法。     A supersaturated solution of calcium phosphate containing bisphosphonates and carbon particles was prepared.
    A method for producing a carbon particle complex in which the bisphosphonate, the carbon particles, and calcium phosphate are coprecipitated.
  7. 　前記カーボン粒子は、カーボンナノホーン、カーボンナノチューブ、ナノグラフェン、ナノダイアモンド、若しくはカーボンファイバー、又はこれらの組み合わせである請求項６に記載のカーボン粒子複合体の製造方法。 The method for producing a carbon particle composite according to claim 6, wherein the carbon particles are carbon nanohorns, carbon nanotubes, nanographenes, nanodiamonds, or carbon fibers, or a combination thereof.
  8. 　前記カーボン粒子は、酸化カーボンナノホーンである請求項６に記載のカーボン粒子複合体の製造方法。 The method for producing a carbon particle complex according to claim 6, wherein the carbon particles are carbon oxide nanohorns.
  9. 　前記ビスホスホネートは、エチドロン酸、イバンドロン酸、ゾレドロン酸、アレンドロン酸、ミノドロン酸、リセドロン酸、パミドロン酸、インカドロン酸、若しくはこれらの塩、又はこれらの組み合わせである請求項６に記載のカーボン粒子複合体の製造方法。 The carbon particle composite according to claim 6, wherein the bisphosphonate is etidronic acid, ibandronic acid, zoledronic acid, alendronic acid, minodronic acid, risedronic acid, pamidronic acid, incadronic acid, or a salt thereof, or a combination thereof. Manufacturing method.
  10. 　前記カーボン粒子は、官能基としてカルボキシル基を有し、
    　前記リン酸カルシウム過飽和溶液中のカルシウムイオン、あるいは、前記リン酸カルシウム表面のカルシウムイオンが、前記カルボキシル基と静電相互作用で引き合い、前記リン酸カルシウムが前記カーボン粒子と複合化する請求項６に記載のカーボン粒子複合体の製造方法。     The carbon particles have a carboxyl group as a functional group and have a carboxyl group.
    The carbon particle composite according to claim 6, wherein calcium ions in the calcium phosphate supersaturated solution or calcium ions on the surface of the calcium phosphate are attracted to the carboxyl group by electrostatic interaction, and the calcium phosphate is complexed with the carbon particles. Manufacturing method.
  11. 　前記ビスホスホネートはホスホン酸基を有し、前記ホスホン酸基が前記カルシウムイオンと静電相互作用で引き合い、前記ビスホスホネートが前記リン酸カルシウムとともに前記カーボン粒子と複合化する請求項１０に記載のカーボン粒子複合体の製造方法。 The carbon particle composite according to claim 10, wherein the bisphosphonate has a phosphonate group, the phosphonate group attracts the calcium ion by electrostatic interaction, and the bisphosphonate is complexed with the carbon particles together with the calcium phosphate. Production method.
  12. 　前記ビスホスホネートは、前記カーボン粒子の内部空間に内包される、又は、前記カーボン粒子の壁に物理吸着することにより担持される請求項６に記載のカーボン粒子複合体の製造方法。
          The method for producing a carbon particle composite according to claim 6, wherein the bisphosphonate is encapsulated in the internal space of the carbon particles or is supported by physically adsorbing on the wall of the carbon particles.
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