WO2021161987A1 - Composite de particules de carbone contenant du bisphosphonate et son procédé de production - Google Patents

Composite de particules de carbone contenant du bisphosphonate et son procédé de production 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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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

L'invention concerne : un composite de particules de carbone contenant du BP ayant une action de BP améliorée et des effets secondaires supprimés ; et un procédé de production de celui-ci.　L'invention concerne un composite de particules de carbone contenant un phosphate de calcium, un bisphosphonate et des particules de carbone. Les particules de carbone peuvent être des nanocornets de carbone, des nanotubes de carbone, des nanographènes, des nanodiamants et des fibres de carbone, ou une combinaison de ceux-ci. Les particules de carbone peuvent également être des nanocornets d'oxyde de carbone.
PCT/JP2021/004744 2020-02-12 2021-02-09 Composite de particules de carbone contenant du bisphosphonate et son procédé de production WO2021161987A1 (fr)

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Citations (2)

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CN111012948A (zh) * 2019-12-16 2020-04-17 四川大学 具有光热转换性能和功能涂层的骨修复和肿瘤抑制材料及制备方法

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US20180104380A1 (en) * 2015-03-25 2018-04-19 Wichita State University Carbon particulates and composites thereof for musculoskeletal and soft tissue regeneration
CN111012948A (zh) * 2019-12-16 2020-04-17 四川大学 具有光热转换性能和功能涂层的骨修复和肿瘤抑制材料及制备方法

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