Formulations and methods for manufacturing formulations

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

[Technical Field]

[0001] The present invention relates to a bisphosphonate-containing carbon particle composite and a method for producing the same. [Background technology]

[0002] Many cancers frequently metastasize to bone. When cancer metastasizes to the bone, osteoclast-mediated osteolysis progresses, resulting in pain and functional impairment. Furthermore, hypercalcemia due to osteolysis and the re-release of cancer cells from the bone shorten life expectancy. Bisphosphonates (BPs) are prescribed as a treatment to induce apoptosis in osteoclasts. However, oral administration of BPs results in low intestinal absorption (a few percent), and side effects such as esophageal ulcers and esophagitis are problematic. Furthermore, long-term administration of BPs can lead to side effects such as atraumatic subtrochanteric and atypical femoral shaft fractures and BP-related osteonecrosis due to bone metabolism inhibition. Therefore, the safety and efficacy of BPs during long-term use have been questioned overseas. However, no other effective treatments for metastatic bone cancer are available. Therefore, drug delivery systems (DDS) and bone repair scaffolds are being investigated for the treatment of metastatic bone cancer. Although such DDS and scaffolds are currently made using biodegradable materials, the environment of metastatic bone cancer is poorly organized, making it difficult to achieve the goal of sustained drug release and control of bone repair according to the stage of cancer progression.

[0003] On the other hand, the development of DDS using carbon particles, which are resistant to biodegradation and can be chemically modified in a wide variety of ways, is underway. Patent Document 1 describes a method for producing nanostructures such as carbon nanotubes with a BP structure. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 0054154 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a BP-containing carbon particle composite that improves the action of BP and suppresses side effects, and a method for producing the same. [Means for solving the problem]

[0006] To solve the above problems, the inventors determined that carbon particles, which have high biocompatibility and can carry a wide variety of therapeutic drugs and target substances, are suitable among non-biodegradable materials. Therefore, the inventors 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, they determined that by combining BP and CaP with carbon particles, a composite with high bone affinity and capable of inducing osteoclast cell death could be obtained.

[0007] The present invention has been completed based on these findings, and provides a carbon particle composite containing BP, CaP, and carbon particles, and a method for producing the same. [Effects of the Invention]

[0008] The BP-containing carbon particle composite of the present invention can enhance the activity of BP and suppress side effects, and is therefore effective in promoting bone formation in osteoclast-targeting treatments for metastatic bone cancer, osteoporosis, osteogenesis imperfecta, osteitis deformans, and other conditions. [Brief explanation of the drawings]

[0009] [Figure 1] Photographs of BV-CaP-OxCNH and CaP-OxCNH dispersions. [Figure 2] Scanning electron microscope (SEM) images of BV-CaP and BV-CaP-OxCNH. [Figure 3]SEM images of CaP and CaP-OxCNH. [Figure 4] Energy dispersive X-ray spectroscopy (EDX) spectra of BV-CaP-OxCNH, CaP-OxCNH, BV-CaP, and CaP. [Figure 5] Transmission electron microscope (TEM) image of BV-CaP-OxCNH-2 and scanning transmission electron microscope (STEM)-EDX image showing the elemental distribution of carbon (C), calcium (Ca), oxygen (O), and phosphorus (P). [Figure 6] (a) Results of toxicity evaluation of BV-CaP-OxCNH to mouse macrophage-like cells (RAW264.7 cells). (b) Results of toxicity evaluation of BV to RAW264.7 cells. [Figure 7] (a) Cytotoxicity evaluation of BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH in RAW264.7 cells. (b) Cytotoxicity evaluation of BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH in RAW264.7 cells. [Figure 8] Optical micrographs of RAW264.7 cells cultured for 24 hours in medium supplemented with BV-CaP-OxCNH-5, CaP-OxCNH-5, or OxCNH. [Figure 9] Optical micrographs of RAW264.7 cells (a) and osteoclasts differentiated from RAW264.7 cells (b) stained with tartrate-resistant acid phosphatase (TRAP). [Figure 10] Fluorescence micrographs showing the uptake of BV-CaP-OxCNH by osteoclasts. [Figure 11] Results of viability evaluation of osteoclasts treated with OxCNH, CaP-OxCNH, and BV-CaP-OxCNH. [Figure 12] SEM images of BV-CaP-OxCNH, ZO-CaP-OxCNH, and PM-CaP-OxCNH. [Figure 13] EDX spectra of BV-CaP-OxCNH, ZO-CaP-OxCNH, and PM-CaP-OxCNH. [Figure 14] Results of RAW264.7 cytotoxicity evaluation of BV-CaP-OxCNH. [Figure 15] (a) Cytotoxicity evaluation of ZO-CaP-OxCNH in RAW264.7 cells. (b) Cytotoxicity evaluation of ZO in RAW264.7 cells. [Figure 16] (a) Cytotoxicity evaluation of PM-CaP-OxCNH in RAW264.7 cells. (b) Cytotoxicity evaluation of PM in RAW264.7 cells. [Figure 17] Photographs of the uterus of osteoporosis model rats and sham-operated rats (a) and relative uterine weights (b). [Figure 18] CT images of the tibia of an osteoporosis model rat 0, 8, and 12 weeks after sample implantation. [Figure 19] Total bone mineral density of osteoporosis model rats 0, 4, 8, and 12 weeks after sample implantation. [Figure 20] Cortical bone density of osteoporosis model rats 0, 4, 8, and 12 weeks after specimen implantation. [Figure 21] Trabecular bone density of osteoporosis model rats 0, 4, 8, and 12 weeks after sample implantation. [Figure 22] Cancellous bone density ratio of osteoporosis model rats 0, 4, 8, and 12 weeks after sample implantation. DETAILED DESCRIPTION OF THE INVENTION

[0010] The BP-containing carbon particle composite of the present invention and a method for producing the same will be described below, but the present invention should not be construed as being limited to the following embodiments and examples.

[0011] [Carbon particle composite] The carbon particle composite of the present invention contains BP, CaP, and carbon particles. Here, carbon particles include, but are not limited to, CNH, carbon nanotubes, nanographene, nanodiamond, or carbon fiber, or a combination thereof. Carbon particles are suitable for multifunctionalization because they are chemically and physically stable and it is easy to introduce functional groups such as carboxyl groups (-COOH) onto the surface of carbon particles, which are suitable for chemical modification. Among these, CNH is preferred as a carbon particle for use in carbon particle composites due to its advantages, such as size uniformity, dispersion stability in aqueous solutions, the ability to produce high-purity mass production, and the lack of purification. CNH has a spherical shape with a diameter of approximately 100 nm, consisting of thousands of carbon nanotubes with diameters of 2 to 5 nm radially assembled. CNH is robust and does not easily decompose. Furthermore, CNH has a highly irregular surface structure, so it does not strongly aggregate and disperses in isolation in aqueous solutions. OxCNH, obtained by oxidizing CNH with hydrogen peroxide, opens pores in the tube walls due to the cleavage of carbon-carbon bonds, further facilitating the movement of molecules into and out of the internal space, making it a more preferred carbon particle for use in carbon particle composites. OxCNH is hydrophilic due to the introduction of functional groups such as carboxyl groups on the pore edges, making it a more preferred carbon particle for use in carbon particle composites.

[0012] The composition and structure of the CaP contained in the carbon particle composite are not limited. CaP may be a compound containing at least phosphate ions and calcium ions. Furthermore, the CaP may be amorphous. Calcium ions in a CaP supersaturated solution are attracted to the carboxyl groups of the carbon particles through electrostatic interaction, and CaP precipitates from these points, forming a composite of CaP and the carbon particles. Alternatively, calcium ions on the surface of the precipitated CaP are attracted to the carboxyl groups of the carbon particles through electrostatic interaction, forming a composite of CaP and the carbon particles.

[0013] The BP contained in the carbon particle composite is, but is not limited to, etidronic acid, ibandronic acid, zoledronic acid, alendronic acid, minodronic acid, risedronic acid, pamidronic acid, incadronic acid, or a salt thereof, or a combination thereof. The BP may contain at least a phosphonic acid group, inhibit osteoclast activity, and prevent bone resorption. The phosphonic acid group of the BP electrostatically attracts calcium ions in a CaP supersaturated solution or on the surface of precipitated CaP, resulting in the formation of a complex between the CaP and the BP, and further forming a complex with the carbon particle. Furthermore, BP is supported on the carbon particles by being encapsulated in the internal space of the carbon particles or by being physically adsorbed on the surface of the carbon particles.

[0014] The BP-containing carbon particle composite of the present invention can induce osteoclast cell death even with a small amount of BP content.

[0015] [Method of manufacturing carbon particle composite] The carbon particle composite of the present invention can be produced by preparing a CaP supersaturated solution containing BP and carbon particles, and allowing the solution to stand to co-precipitate the three components. The solution used as the raw material for the CaP supersaturated solution is not particularly limited. The raw material for the CaP supersaturated solution may contain, in addition to a solution containing calcium ions (calcium-containing solution) and a solution containing phosphate ions (phosphate-containing solution), a pH adjuster may also be included as appropriate. The raw material for the CaP supersaturated solution may be, for example, a combination of various injectable preparations. When preparing a CaP supersaturated solution by combining various injectable preparations as raw materials for the CaP supersaturated solution, the carbon particle composite of the present invention can also be used as the preparation itself. The CaP supersaturated solution containing BP and carbon particles can be prepared by mixing BP, carbon particles, a calcium-containing solution, and a phosphate-containing solution. During mixing, a pH adjuster or the like may be added as needed. The temperature and time for allowing the CaP supersaturated solution containing BP and carbon particles to stand are not particularly limited and can be appropriately adjusted taking into account the size and dispersibility of the carbon particle composite to be produced.

[0016] [formulation] Since the carbon particle composite of the present invention contains BP, it can be used in preparations targeting osteoclasts. The preparation of the present invention may contain additives, etc., as appropriate, in addition to the BP-containing carbon particle composite. The preparation of the present invention may be administered to a patient by intravenous injection, or may be administered locally to a specific site (affected area), for example.

[0017] The BP-containing carbon particle complex of the present invention can function as a DDS capable of spatially, quantitatively, and temporally controlling drug distribution in the body. The inclusion of CaP in the BP-containing carbon particle complex of the present invention enhances bone affinity, enabling osteoclast cell death with a small amount of BP. Furthermore, the use of carbon particles that are difficult to diffuse in the body allows the locally administered BP-containing carbon particle complex to remain at the affected site for a long time and continue to release BP. Furthermore, since the BP-containing carbon particle complex is localized in lysosomes within osteoclasts, CaP dissolves in the acidic environment, allowing for efficient release of BP. Furthermore, because the carbon particles generate heat upon exposure to light, photothermal therapy is also possible depending on the affected area. On the other hand, because CaP is a component of the bone matrix, it can promote bone repair by osteoblasts after bone resorption by osteoclasts is inhibited.In addition, carbon particles themselves function as an excellent scaffold for bone formation, making them effective in bone formation promotion treatments. Therefore, the BP-containing carbon particle composite of the present invention can efficiently promote bone formation therapy due to the synergistic effect of the DDS and the scaffold. [Example]

[0018] The carbon particle composite and its manufacturing method according to the present invention will be described in more detail below with reference to examples. In these examples, OxCNH was used as an example of carbon particles, and ibandronic acid (Bonviva, BV) was used as an example of BP. BV-CaP-OxCNH was prepared by combining CaP and BV with OxCNH, and its application to bone formation promotion therapy was investigated.

[0019] [Preparation of OxCNH] CNH was prepared by decomposing and vaporizing graphite using a carbon dioxide laser under an argon gas atmosphere (atmospheric pressure) (J. Phys. Chem. C 2008, 112, 1330-1334). The prepared CNH (20 mg) and 30% hydrogen peroxide (20 mL) were placed in a 50 mL vial and ultrasonicated for 5 minutes to disperse the CNH in the hydrogen peroxide. This CNH-hydrogen peroxide dispersion was heated at 70°C for 2 hours while irradiating it with xenon lamp light. After cooling the CNH-hydrogen peroxide dispersion at room temperature, it was filtered and washed with water approximately five times to remove the hydrogen peroxide. The resulting OxCNH aqueous dispersion was freeze-dried to obtain OxCNH powder.

[0020] [Preparation of BV-CaP-OxCNH] OxCNH powder was dispersed in ultrapure water to give concentrations of 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 to obtain sterilized OxCNH dispersions. The BV solution used was Bonviva IV Injection 1 mg Syringe (Chugai Pharmaceutical Co., Ltd.), which contains ibandronate sodium hydrate as the active ingredient. Calcium-containing solution, phosphate-containing solution, and pH adjuster were prepared as raw materials for the CaP supersaturated solution. The calcium-containing solution was prepared by mixing 49.361 mL of Ringer's solution "Otsuka" (Otsuka Pharmaceutical Co., Ltd.) with 0.639 mL of 1 mEq / mL calcium chloride correction solution (Otsuka Pharmaceutical Co., Ltd.). The phosphate-containing solution was prepared by mixing 9.469 mL of CliniSaltz® Infusion (Kyowa Critical Care Co., Ltd.) with 0.531 mL of Terumo Dipotassium Phosphate Injection 20 mEq Kit (Terumo Corporation). The pH adjuster was prepared by mixing 5 mL of Meylon® Intravenous Injection 7% (Otsuka Pharmaceutical Co., Ltd.) with 20 mL of water for injection (Fuso Pharmaceutical Industries, Ltd.). A reaction solution (10 mL) was prepared by mixing sterilized OxCNH aqueous dispersions (0.250 mL each of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL dispersions), BV solution (0.250 mL), calcium-containing solution (7.674 mL), phosphate-containing solution (0.917 mL), and pH adjuster (0.909 mL). The reaction solution was left to stand in an incubator at 25°C for 30 minutes to allow the co-precipitation of BV, CaP, and OxCNH. The resulting BV-CaP-OxCNH sample was collected by centrifugation (6000 rpm, 5 minutes) and washed by repeatedly dispersing the sample in water for injection and centrifuging. The samples prepared using 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL sterile aqueous OxCNH dispersions were named BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, and BV-CaP-OxCNH-6, respectively.

[0021] [Preparation of CaP-OxCNH] As a control without BV, the same procedure as for the preparation of BV-CaP-OxCNH was repeated except that water for injection was used instead of the BV solution to prepare the reaction solution. Samples prepared using 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 5 mg / mL sterile OxCNH aqueous dispersions were designated CaP-OxCNH-1, CaP-OxCNH-2, CaP-OxCNH-3, CaP-OxCNH-4, CaP-OxCNH-5, and CaP-OxCNH-6, respectively.

[0022] [Preparation of BV-CaP and CaP] As a control containing no CNH, a BV and CaP complex (BV-CaP) and CaP particles (CaP) were obtained by the same procedures as for the preparation of BV-CaP-OxCNH or CaP-OxCNH, except that the reaction solution was prepared using water for injection instead of the sterile OxCNH aqueous dispersion.

[0023] [Observation of dispersibility] Figure 1 shows photographs of BV-CaP-OxCNH-1, CaP-OxCNH-1, BV-CaP-OxCNH-2, CaP-OxCNH-2, BV-CaP-OxCNH-3, and CaP-OxCNH-3 dispersed in water for injection. CaP-OxCNH exhibited rapid particle settling and solid-liquid separation. On the other hand, BV-CaP-OxCNH showed no particle settling, suggesting good particle dispersion.

[0024] [Scanning electron microscope observation] The structures of the prepared samples were confirmed using a scanning electron microscope (SEM). For SEM observation, the sample was dropped onto a silicon substrate, dried, and gold was evaporated onto the substrate before observation. SEM images of BV-CaP and BV-CaP-OxCNH are shown in Figure 2. SEM images of CaP and CaP-OxCNH are shown in Figure 3. As shown in Figure 2, nanoparticles with a primary particle diameter of approximately 50 nm were observed in BV-CaP, BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, and BV-CaP-OxCNH-4. In addition, particles with a primary particle diameter of 100 to 150 nm were observed in BV-CaP-OxCNH-5 and BV-CaP-OxCNH-6. Furthermore, cracked particles were prominent in BV-CaP-OxCNH-6. As shown in Figure 3, CaP and CaP-OxCNH were both nanoparticles with a primary particle diameter of approximately 100 to 150 nm. Furthermore, the number of cracked particles increased in CaP-OxCNH-4, CaP-OxCNH-5, and CaP-OxCNH-6 with increasing OxCNH concentration in the reaction solution.

[0025] [Measurement by energy dispersive X-ray spectroscopy] The samples used for SEM observation were subjected to elemental analysis using energy dispersive X-ray spectroscopy (EDX). The results of the elemental analysis are shown in Figure 4. As shown in Figure 4, carbon, oxygen, phosphorus, and calcium peaks were detected in all particles, suggesting that CaP and OxCNH formed complexes. Furthermore, a significant increase in the carbon peak relative to the calcium and phosphorus peaks was confirmed in BV-CaP-OxCNH-5, BV-CaP-OxCNH-6, CaP-OxCNH-5, and CaP-OxCNH-6. This is thought to be due to the increase in the ratio of OxCNH to CaP in the generated particles as the OxCNH concentration in the reaction solution increased.

[0026] [Measurement by inductively coupled plasma optical emission spectroscopy] The obtained samples were subjected to elemental analysis using inductively coupled plasma atomic emission spectroscopy (ICP). The samples were dissolved in acid to prepare solutions for measurement. The calcium content, phosphorus content, and molar ratio of calcium to phosphorus content (Ca / P) in the samples obtained from 10 mL of the reaction solution are shown in Table 1. [Table 1] The results in Table 1 show that the Ca / P molar ratios of BV-CaP-OxCNH and BV-CaP were lower than those of CaP-OxCNH and CaP (BV-CaP-OxCNH / BV-CaP: 1.29–1.36, CaP-OxCNH / CaP: 1.38–1.44). The phosphorus-containing BV loaded in BV-CaP-OxCNH and BV-CaP likely resulted in a higher phosphorus content relative to calcium, resulting in a lower Ca / P molar ratio. Assuming the Ca / P molar ratio of CaP in BV-CaP-OxCNH was the same as that of CaP-OxCNH, the BV content was estimated to be 20–30% (0.16–0.24 μmol) of the amount mixed in the reaction solution. The BV to CaP ratio was also estimated to be 4–7:100. From the above, it was confirmed that BV, CaP, and OxCNH formed a ternary complex in BV-CaP-OxCNH. Furthermore, the calcium and phosphorus contents of BV-CaP-OxCNH and BV-CaP were lower than those of CaP-OxCNH and CaP. This is likely due to the lower pH of the reaction mixture caused by the addition of an acidic BV solution, which inhibited CaP formation. Complexation with BV may also have affected the rate of CaP formation. On the other hand, the concentration of OxCNH in the reaction mixture did not significantly affect the calcium and phosphorus contents. Therefore, the amounts of CaP and BV in BV-CaP-OxCNH do not appear to vary significantly regardless of the OxCNH concentration in the reaction mixture. The small changes in the amounts of CaP and BV with the amount of OxCNH suggest that CaP and BV bind independently of OxCNH. This suggests that BV is primarily supported by CaP, and BV-CaP forms a complex with OxCNH.

[0027] [Observation by transmission electron microscope and scanning transmission electron microscope] The structures of the prepared samples were observed using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM)-EDX. The samples were dropped onto a grid and dried before observation. Figure 5 shows a TEM image of BV-CaP-OxCNH-2 and a STEM-EDX image showing the elemental distribution of carbon (C), calcium (Ca), oxygen (O), and phosphorus (P). As shown in Figure 5, the TEM image of BV-CaP-OxCNH-2 revealed a spherical OxCNH composed of radially arranged tubes, with a substance believed to be BV-CaP attached to the periphery. The elemental distribution revealed that calcium, oxygen, and phosphorus derived from BV-CaP were distributed around the carbon derived from OxCNH. These results confirmed that BV-CaP had attached to the periphery of OxCNH to form a complex.

[0028] [Cytotoxicity evaluation] The cytotoxicity of the prepared samples was evaluated. Phagocytic mouse macrophage-like cells (RAW264.7) were seeded in a 96-well plate and cultured at 37°C. After 24 hours of culture, the medium was removed and medium containing BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, BV-CaP-OxCNH-3, BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, and BV-CaP-OxCNH-6 was added. The concentrations of each sample in the medium were 0.25 μg / mL for BV-CaP-OxCNH-1, 0.5 μg / mL for BV-CaP-OxCNH-2, 1.25 μg / mL for BV-CaP-OxCNH-3, 2.5 μg / mL for BV-CaP-OxCNH-4, 5.0 μg / mL for BV-CaP-OxCNH-5, and 12.5 μg / mL for BV-CaP-OxCNH-6. (Based on the ICP results, the BV concentration in the medium was estimated to be approximately 0.5 μg / mL to 0.75 μg / mL, the CaP-derived Ca concentration was estimated to be approximately 0.089 mM to 0.11 mM, and the P concentration was estimated to be approximately 0.068 mM to 0.081 mM.) As a control, normal medium without BV-CaP-OxCNH was used. After 24 hours of culture, the number of viable cells was determined by measuring absorbance at 450 nm using Cell Counting Kit-8 reagent (Dojindo Laboratories). The cell viability was calculated by setting the absorbance of the control at 100%. Figure 6(a) shows the results of the cytotoxicity evaluation of BV-CaP-OxCNH. BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, and BV-CaP-OxCNH-3 showed almost the same cytotoxicity, while BV-CaP-OxCNH-4, BV-CaP-OxCNH-5, and BV-CaP-OxCNH-6 showed significant cytotoxicity as the OxCNH concentration in the samples increased. The cytotoxicity of BV was evaluated using the same method as described above. The BV concentrations added to the medium were 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL (control: 0 μg / mL). Figure 6(b) shows the results of the BV cytotoxicity evaluation on RAW264.7 cells. As shown in Figure 6(b), BV exhibited concentration-dependent cytotoxicity. The BV concentration in BV-CaP-OxCNH was estimated to be approximately 0.5 μg / mL to 0.75 μg / mL in the medium. Therefore, compared to BV alone, BV-CaP-OxCNH exhibited highly efficient cytotoxicity despite the small amount of BV contained. Cytotoxicity was evaluated for BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH using the same method as above. Each sample was diluted in the medium to a concentration of 0.17 μg / mL, 0.5 μg / mL, or 1.7 μg / mL in terms of OxCNH. Similarly, cytotoxicity was evaluated for BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. Each sample was diluted in the medium to a concentration of 1.7 μg / mL, 5.0 μg / mL, or 16.7 μg / mL in terms of OxCNH. Figure 7(a) shows the results of cytotoxicity evaluation of BV-CaP-OxCNH-2, CaP-OxCNH-2, and OxCNH. As shown in Figure 7(a), CaP-OxCNH-2 and OxCNH had almost the same cytotoxicity, while BV-CaP-OxCNH-2 showed stronger cytotoxicity. Figure 7(b) shows the results of cytotoxicity evaluation of BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH. As shown in Figure 7(b), OxCNH, CaP-OxCNH-5, and BV-CaP-OxCNH-5 showed stronger cytotoxicity in this order. These results suggest that BV-CaP-OxCNH exhibits stronger cytotoxicity due to the loading of BV on CaP-OxCNH. Cells cultured with BV-CaP-OxCNH-5, CaP-OxCNH-5, and OxCNH were observed under an optical microscope. Figure 8 shows optical microscope images of RAW264.7 cells cultured for 24 hours in medium containing BV-CaP-OxCNH-5, CaP-OxCNH-5, or OxCNH. As shown in the bottom panel of Figure 8, OxCNH was presumably internalized into intracellular lysosomes. As shown in the middle panel of Figure 8, CaP-OxCNH formed large aggregates in the medium, presumably making it difficult for the cells to be internalized. As shown in the top panel of Figure 8, BV-CaP-OxCNH was confirmed to be internalized into cells, but most of the cells were apoptotic or necrotic. These results suggest that BV-CaP-OxCNH is internalized into cells and releases BV, causing apoptotic or necrotic death, thereby effectively demonstrating cytotoxicity.

[0029] [Differentiation of RAW264.7 cells into osteoclasts] To evaluate cytotoxicity using osteoclasts, RAW264.7 cells were differentiated into osteoclasts. RAW264.7 cells were seeded into 96-well plates and cultured for 1 day. After 1 day, the cells were replaced with medium containing RANKL (100 ng / mL) and cultured for 5 days to induce osteoclast differentiation. To confirm differentiation, the cells were stained for tartrate-resistant acid phosphatase (TRAP), an osteoclast marker, and observed under an optical microscope. Figure 9 shows optical microscope images of TRAP-stained RAW264.7 cells (a) and osteoclasts differentiated from RAW264.7 cells (b). As shown in Figure 9, TRAP staining was not observed in RAW264.7 cells before differentiation, but was observed in differentiated osteoclasts. These results confirmed that RAW264.7 cells successfully differentiated into osteoclasts.

[0030] [Cytotoxicity evaluation using osteoclasts] The medium from the resulting osteoclast cultures was removed, and medium containing BV-CaP-OxCNH-1, BV-CaP-OxCNH-2, and BV-CaP-OxCNH-3 was added. The OxCNH concentrations in the medium were 2.5 μg / mL for BV-CaP-OxCNH-1, 5.0 μg / mL for BV-CaP-OxCNH-2, and 12.5 μg / mL for BV-CaP-OxCNH-3. As a control, normal medium without BV-CaP-OxCNH was used. After 24 and 48 hours of culture, osteoclast nuclei were stained with Hoechst 33342 and lysosomes with Lysosome Painter Orange to confirm uptake of BV-CaP-OxCNH. The osteoclasts were then observed under a fluorescence microscope. Figure 10 shows fluorescence micrographs showing the uptake of BV-CaP-OxCNH by osteoclasts 24 hours (upper panel) and 48 hours (lower panel). As shown in Figure 10, BV-CaP-OxCNH was taken up into the lysosomes of osteoclasts (arrows). Both mononuclear and multinuclear osteoclasts were observed. The effects of these treatments on osteoclast viability were examined using the alamarBlue assay. Figure 11 shows the viability of osteoclasts treated with OxCNH, CaP-OxCNH, and BV-CaP-OxCNH. In BV-CaP-OxCNH-1 (OxCNH concentration 2.5 μg / mL), BV-CaP-OxCNH-2 (OxCNH concentration 5.0 μg / mL), and BV-CaP-OxCNH-3 (OxCNH concentration 12.5 μg / mL), cell viability decreased with increasing OxCNH concentration. In contrast, OxCNH, CaP-OxCNH-1, CaP-OxCNH-2, and CaP-OxCNH-3 did not show a concentration-dependent decrease in cell viability. [Example]

[0031] In this example, various BP-CaP-OxCNH were prepared by varying the type of BP and the composition of the CaP supersaturated solution. In addition to BV, which was used in Example 1, zoledronic acid (Zometa, ZO) and pamidronic acid (PM) were used as BP. Four concentrations of CaP supersaturated solution were prepared for each of BV, ZO, and PM, and a total of 12 types of BP-CaP-OxCNH were prepared.

[0032] [Preparation of BP-CaP-OxCNH] In the same manner as in Example 1, CNH was oxidized with hydrogen peroxide and washed with water to obtain an OxCNH aqueous dispersion. The obtained OxCNH aqueous dispersion was used as is without freeze-drying. The concentration of the OxCNH aqueous dispersion was calculated by measuring the absorbance at 700 nm with a UV-visible spectrophotometer and using a calibration curve (CNH concentration-absorbance plot) prepared in advance using CNH aqueous dispersions whose concentrations were known. The OxCNH aqueous dispersion was adjusted to a concentration of 2 mg / mL and sterilized by autoclave to obtain a sterilized OxCNH aqueous dispersion. The BV solution was prepared using the Bonviva IV 1 mg syringe used in Example 1. The ZO solution was prepared using Zometa IV infusion 4 mg / 5 mL (Novartis Pharma K.K.), which contains zoledronic acid hydrate as the active ingredient. The PM solution was prepared using Pamidronate Disodium IV Infusion 15 mg "Sawai" (Sawai Pharmaceutical Co., Ltd.), which contains pamidronate disodium hydrate as the active ingredient. These BV, ZO, and PM solutions were prepared to a concentration of 2.94 mM using water for injection. The calcium-containing and phosphate-containing solutions used as raw materials for the CaP supersaturated solution were prepared similarly to those used in Example 1. The calcium-containing solution was prepared by mixing Otsuka Ringer's solution with 1 mEq / mL of calcium chloride correction solution, and the phosphate-containing solution was prepared by mixing CliniSaltz® infusion solution with Terumo dipotassium phosphate injection 20 mEq kit. By varying the amounts of each reagent mixed, four types of calcium-containing and phosphate-containing solutions with different concentrations of Ca and P were prepared. Specifically, calcium-containing and phosphate-containing solutions with Ca and P concentrations of 78%, 67%, and 56%, respectively, were prepared based on the same calcium-containing and phosphate-containing solutions used in Example 1 and the concentrations of Ca or P contained in those solutions. The pH adjuster used was the same as in Example 1. A 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), phosphate-containing solution (0.917 mL), and 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 supersaturated solution were Ca: 6.60 mM, P: 3.30 mM when the same calcium-containing solution and phosphate-containing solution as used in Example 1 were used, and Ca: 5.14 mM, P: 2.56 mM (78%), Ca: 4.40 mM, P: 2.20 mM (67%), Ca: 3.68 mM, P: 1.83 mM (56%) when calcium-containing solution and phosphate-containing solution prepared to have concentrations of 78%, 67%, and 56% were used. These supersaturated solutions were designated CaP supersaturated solutions a, b, c, and d. Immediately after preparation, the reaction solution was placed in a 25°C incubator for 30 minutes to allow the three components, BP, CaP, and OxCNH, to co-precipitate. The resulting sample, BP-CaP-OxCNH, was collected by centrifugation (6000 rpm, 5 minutes) and washed by repeatedly dispersing the sample in water for injection and centrifuging. The samples prepared using CaP supersaturated solutions a, b, c, and d were named BP-CaPa-OxCNH, BP-CaPb-OxCNH, BP-CaPc-OxCNH, and BP-CaPd-OxCNH, respectively. Here, BP corresponds to BV, ZO, or PM.

[0033] [Preparation of CaP-OxCNH] As a control without BP, the same procedure was performed using water for injection instead of BV, ZO, or PM solution to obtain sample CaP-OxCNH. Samples prepared using CaP supersaturated solutions a, b, c, and d were named CaPa-OxCNH, CaPb-OxCNH, CaPc-OxCNH, and CaPd-OxCNH, respectively.

[0034] [SEM observation] The structure of BP-CaP-OxCNH was confirmed by SEM. As in Example 1, the sample was dropped onto a silicon substrate, dried, and gold was evaporated onto it for observation. An SEM image of BP-CaP-OxCNH is shown in Figure 12. In both BP-CaP-OxCNHs, particles with a primary particle diameter of 50 to 100 nm and particles with a primary particle diameter of 100 to 150 nm were observed. Furthermore, the particles with a primary particle diameter of 100 to 150 nm had an uneven structure that was thought to be due to CNH.

[0035] [Measurement by EDX] BP-CaP-OxCNH was dropped onto a silicon substrate, dried, and subjected to elemental analysis using EDX. The results of the elemental analysis are shown in Figure 13. Carbon, oxygen, phosphorus, and calcium peaks were detected in all particles, suggesting that CaP and OxCNH formed a complex. Furthermore, as the Ca and P concentrations in the CaP supersaturated solution decreased (CaP supersaturated solutions a to d), the calcium and phosphorus peaks significantly decreased relative to the carbon peak. This is thought to be due to the fact that the ratio of CaP to OxCNH in the resulting particles decreased as the Ca and P concentrations in the CaP supersaturated solution decreased.

[0036] [Measurement by ICP] Elemental analysis was performed on BP-CaP-OxCNH and CaP-OxCNH using ICP. The samples were dissolved in acid to prepare a solution, and measurements were performed. The calcium content, phosphorus content, and molar ratio of calcium to phosphorus content (Ca / P) in the sample obtained from 10 mL of the reaction solution are shown in Table 2. [Table 2] The results in Table 2 show that the Ca / P molar ratio of BP-CaP-OxCNH was smaller than that of CaP-OxCNH regardless of whether BV, ZO, or PM was used. This is thought to be because the phosphorus-containing BP was loaded in BP-CaP-OxCNH, resulting in a higher phosphorus content relative to calcium, resulting in a lower Ca / P molar ratio. Next, we estimated the BP content in BP-CaP-OxCNH. Here, we assumed that the Ca / P molar ratio of CaP-derived Ca and P in BP-CaP-OxCNH prepared using CaP supersaturated solutions with the same Ca and P concentrations was equal to the Ca / P molar ratio in CaP-OxCNH. Based on this process, we estimated the BP content in BP-CaP-OxCNH to be approximately 30–50% (0.22–0.37 μmol) of the amount mixed in the reaction solution (0.735 μmol) regardless of the CaP supersaturated solution used. These results confirm that BP, CaP, and OxCNH form a ternary complex in BP-CaP-OxCNH. Furthermore, as the Ca and P concentrations in the CaP supersaturated solution decreased (a to d), the calcium and phosphorus contents decreased significantly. This result correlates with the EDX results (Figure 13), and is thought to be due to the fact that as the Ca and P concentrations in the CaP supersaturated solution decreased, the amount of CaP produced decreased, resulting in a decrease in the CaP content in the particles.

[0037] [Cytotoxicity evaluation] The cytotoxicity of BP-CaP-OxCNH against RAW264.7 cells was evaluated using the same method as in Example 1. Each sample was diluted in the medium to a concentration of 1.7 μg / mL, 5.0 μg / mL, or 16.7 μg / mL in terms of OxCNH. The cytotoxicity of ZO alone and PM alone was also evaluated. Figure 14 shows the results of the cytotoxicity evaluation of BV-CaP-OxCNH. BV-CaP-OxCNH showed similar cytotoxicity regardless of the CaP content in the particles. When the OxCNH concentration in the medium was 16.7 μg / mL, the BV concentration in the medium was estimated to be approximately 2.3–3.9 μg / mL. Therefore, compared to BV alone as shown in Figure 6(b), BV-CaP-OxCNH effectively exhibited cytotoxicity despite containing only a small amount of BV. Figure 15(a) shows the cytotoxicity evaluation results of ZO-CaP-OxCNH. Compared to BV-CaP-OxCNH, ZO-CaP-OxCNH exhibited stronger cytotoxicity. As shown in Figure 15(b), ZO alone exhibited cytotoxicity even at a relatively low concentration (2.5 μg / mL), which differs from BV alone, which showed almost no cytotoxicity at the same concentration. This difference in cytotoxicity between ZO alone and BV alone is thought to affect the cytotoxicity of the complex. Furthermore, when the OxCNH concentration in the medium was 16.7 μg / mL in Figure 15(a), the ZO concentration in the medium was estimated to be approximately 2.0–3.4 μg / mL. Therefore, compared to ZO alone, as shown in Figure 15(b), ZO-CaP-OxCNH exhibited efficient cytotoxicity despite containing only a small amount of ZO. Although some differences in cytotoxicity were observed depending on the CaP content in the particles, the differences were not significant. Figure 16(a) shows the results of the cytotoxicity evaluation of PM-CaP-OxCNH. At OxCNH concentrations of 5.0 μg / mL and 16.7 μg / mL in the medium, PM-CaP-OxCNH exhibited stronger cytotoxicity than BV-CaP-OxCNH. On the other hand, at an OxCNH concentration of 1.7 μg / mL in the medium, PM-CaP-OxCNH showed almost no toxicity. Figure 16(b) shows that there was a significant difference in the cytotoxicity of PM alone between 10 μg / mL and 25 μg / mL, which is consistent with the significant difference in the cytotoxicity of PM-CaP-OxCNH between OxCNH concentrations of 1.7 μg / mL and 5.0 μg / mL in the medium. In addition, in Figure 16(a), when the OxCNH concentration in the medium was 16.7 μg / mL, the PM concentration in the medium was estimated to be approximately 2.0-3.4 μg / mL. Therefore, compared to PM alone shown in Figure 16(b), PM-CaP-OxCNH effectively exhibited cytotoxicity despite containing only a small amount of PM. When the OxCNH concentration in the medium was 5 μg / mL, the higher the CaP content in the particles, the stronger the cytotoxicity. [Example]

[0038] In this example, holes were drilled in the tibia of osteoporosis model rats, and samples such as BV-CaP-OxCNH were embedded therein to evaluate the effect of the samples on bone formation.

[0039] [Creation of osteoporosis model rats] Wistar rats (female, 10 weeks old) underwent bilateral ovariectomy and the wounds were sutured. They were then kept under normal conditions for 8 weeks after surgery to create osteoporosis model rats. Sham-operated rats, in which the ovariectomy was not performed, were also prepared for comparison. Weight gain was recorded, and it was confirmed that the osteoporosis model rats had gained weight compared to the sham-operated rats (a notable phenomenon in osteoporosis model rats). Furthermore, bone density measurements were performed using X-ray CT for laboratory animals, and it was confirmed that the osteoporosis model rats had an approximately 20% decrease in trabecular bone density compared to the sham-operated rats, indicating the pathological condition of osteoporosis.

[0040] [Preparation of specimens for embedding] BV-CaP-OxCNH-2, CaP-OxCNH-2, BV-CaP, and CaP were prepared as samples in the same manner as in Example 1. However, the OxCNH dispersion, one of the raw materials used in sample preparation, was prepared in the same manner as in Example 2 (without lyophilization). The obtained samples were suspended in a small amount of water for injection. The OxCNH concentration in the suspension was estimated to be approximately 0.83 mg / mL, and the BV concentration was estimated to be approximately 2.1 mg / mL. In addition to these samples, OxCNH (0.83 mg / mL), BV (1.0 mg / mL as ibandronic acid), and saline (negative control) were also examined for comparison.

[0041] [Tibia reaming and specimen insertion] Three osteoporosis model rats were prepared for each sample, and the left and right tibias of each rat were reamed (the process of drilling a hole in the bone, approximately 1 mm in diameter) using an 18-gauge syringe needle. Each sample was prepared in 1 mL volume, with a total BV dose of 154 μg / kg and a total OxCNH dose of 61.7 μg / kg (approximately 200 g weight per rat). 50 μL of each sample was implanted into the left and right tibias of each osteoporosis model rat, and the wounds were sutured. The osteoporosis model rats were maintained under normal conditions for 12 weeks after implantation, during which CT scans and bone mineral density assessments were performed over time. A positive control group was also administered BV solution subcutaneously. The BV solution was administered subcutaneously to a different site on each rat each time, once daily for one consecutive week every four weeks for a total of 12 weeks (three consecutive weekly administrations at weeks 0, 4, and 8). The BV solution (1.0 mg / mL) was prepared so that the total BV dose per rat was 154 μg / kg of body weight over the 12 weeks. For the 12 weeks following the start of subcutaneous administration, the rats were kept in a normal household while undergoing CT scans and bone mineral density assessments (described below) over time.

[0042] [Uterine observation and weight measurement] After 12 weeks, the sham-operated and osteoporosis model rats were euthanized, and their uteri were observed and removed. Figure 17(a) shows photographs of the laparotomy (top) and the removed uteri (bottom) of a sham-operated rat (left) and an osteoporosis model rat (right). Significant uterine atrophy (a prominent phenomenon in osteoporosis model rats) was observed in the osteoporosis model rats compared to the sham-operated rats. Next, the weights of the uteri removed from the sham-operated and osteoporosis model rats were measured. Figure 17(b) shows the relative weights of the uterines removed from the osteoporosis model rats (right) relative to the weight of the uterines removed from the sham-operated rats (left). The relative weight of the uterines removed from the osteoporosis model rats was approximately 0.3, when the weight of the uterines removed from the sham-operated rats was set to 1. A significant decrease in uterine weight (a prominent phenomenon in osteoporosis model rats) was observed in the osteoporosis model rats compared to the sham-operated rats.

[0043] [CT scan] Computed tomography (CT) images of the tibiae of each osteoporosis model rat were taken 0, 4, 8, and 12 weeks after implantation. Figure 18 shows CT images of the tibiae of each osteoporosis model rat taken 0, 8, and 12 weeks after implantation. As shown in Figure 18, rats receiving subcutaneous BV (positive control) or local administration (implantation into the tibia) of BV-CaP-OxCNH-2, BV-CaP, or BV showed better regeneration of bone matrix inside the cortical bone than rats receiving local administration of saline, CaP, OxCNH, or CaP-OxCNH-2.

[0044] [Bone density evaluation] Bone density measurements were performed on the tibiae of each osteoporosis model rat using an X-ray CT scanner for experimental animals 0, 4, 8, and 12 weeks after sample implantation. Figure 19 shows the total bone density of the tibiae of each osteoporosis model rat, Figure 20 shows the cortical bone density of the tibiae of each osteoporosis model rat, and Figure 21 shows the trabecular bone density of the tibiae of each osteoporosis model rat. As shown in Figure 19, rats receiving subcutaneous BV administration and rats receiving local BV-CaP-OxCNH-2, BV-CaP, or BV administration showed improved total bone mineral density compared with rats receiving local saline, CaP, OxCNH, or CaP-OxCNH-2 administration. As shown in Figure 20, no significant differences were observed in the cortical bone mineral density of the tibias of each osteoporosis model rat among the implanted samples. As shown in Figure 21, the cancellous bone mineral density of the tibias of each osteoporosis model rat showed the same trend as the total bone mineral density. Therefore, it is believed that the improvement in cancellous bone mineral density resulted in the improvement in total bone mineral density. Figure 22 shows the time course of the relative trabecular bone density (TBD) of the tibia of each osteoporosis model rat, calculated using the data shown in Figure 21 and assuming the trabecular bone density (TBD) of the tibia immediately after implantation (week 0) as 1. As shown in Figure 22, rats receiving local BV-CaP-OxCNH-2 administration continued to improve their trabecular bone density up to 12 weeks, similar to rats receiving continuous subcutaneous BV administration. On the other hand, rats receiving local BV-CaP or BV administration did not show significant improvements in TBD after 8 weeks compared with rats receiving subcutaneous BV administration or local BV-CaP-OxCNH-2 administration. The same trend in trabecular bone density was observed in the total BD of the tibia of each osteoporosis model rat. Furthermore, rats receiving local saline, CaP, OxCNH, or CaP-OxCNH-2 administration continued to show a decrease in TBD up to 12 weeks. A single local administration of BV-CaP-OxCNH-2 (implanted into the affected area) showed the same bone mineral density improvement effect as multiple monthly subcutaneous administrations of BV. This suggests that the single administration of BV-CaP-OxCNH-2 places less strain on the patient and efficiently promotes bone formation.

Claims

1. The composition contains calcium phosphate, a bisphosphonate having a phosphonic acid group, and carbon particles which are oxidized carbon nanohorns having a carboxyl group, A preparation targeting osteoclasts, comprising a carbon particle complex in which the phosphonic acid group and the carboxyl group are complexed with calcium ions of calcium phosphate through electrostatic interaction.

2. 2. The formulation of 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.

3. A supersaturated calcium phosphate solution containing a bisphosphonate and carbon particles, which are oxidized carbon nanohorns having carboxyl groups, is prepared; A method for producing a preparation targeting osteoclasts, which contains a carbon particle complex obtained by coprecipitating the bisphosphonate, the carbon particles, and calcium phosphate.

4. 4. The method for producing a formulation according to claim 3, 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. the carbon particles have a carboxyl group as a functional group, The method for producing a formulation according to claim 3, wherein calcium ions in the calcium phosphate supersaturated solution or calcium ions on the surface of the calcium phosphate are attracted to the carboxyl groups through electrostatic interaction, so that the calcium phosphate is composited with the carbon particles.

6. The method for producing a formulation according to claim 5, wherein the bisphosphonate has a phosphonic acid group, and the phosphonic acid group attracts the calcium ion through electrostatic interaction, so that the bisphosphonate is complexed with the carbon particles together with the calcium phosphate.

7. The method for producing a formulation according to claim 3, wherein the bisphosphonate is encapsulated in the internal space of the carbon particles or is supported by physical adsorption on the walls of the carbon particles.

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