Bone graft composition comprising lappaconitine derivative and TCP / ha scaffold

Abstract

The present invention relates to a bone graft composition in which a lappaconitine derivative is surface-adsorbed on a scaffold comprising tricalcium phosphate (TCP) and hydroxyapatite (HA). In the composition of the present invention, the osteogenesis-promoting properties of the lappaconitine derivative are combined with the osteoconductivity of the TCP / HA scaffold, thereby improving problems such as high-concentration adverse effects, unstable adsorption patterns, and localized excessive formation, which occur in existing protein-based osteogenic factors.

Description

Bone graft composition comprising a rapaconitine derivative and a TCP / HA scaffold

[0001] The present invention relates to the field of regenerative medicine and tissue engineering for bone tissue regeneration and treatment of bone defects, and more specifically, to a bone graft composition in which a rapaconitine derivative is adsorbed on the surface of a scaffold composed of tricalcium phosphate (TCP) and hydroxyapatite (HA). The composition of the present invention is useful for improving cell adhesion, bone formation induction, and tissue regeneration efficiency at the bone defect site by combining a biocompatible inorganic scaffold with a physiologically active derivative, and can be applied to various clinical bone treatment applications.

[0002]

[0003] The global market for BMP (bone morphogenic protein)-related bone disease treatments is worth 5 trillion won, and the U.S. FDA has actually approved the use of human recombinant BMP-2 and BMP-7 for orthopedic prescriptions such as open fractures, nonunion fractures, vertebral fusion, and maxillofacial bone enhancement.

[0004] However, despite possessing excellent bone regeneration capabilities, BMP has been associated with significant side effects, including severe pain, bleeding, and death. Consequently, while the Korean Ministry of Food and Drug Safety has approved the use of BMP for alveolar bone regeneration, it has not yet approved it for orthopedic applications, including spinal fusion.

[0005] Consequently, there were limitations in the treatment of bone diseases using conventional BMPs; therefore, the present invention aimed to develop QG3030, a novel bone formation-promoting compound. Since QG3030 promotes bone formation through a mechanism different from that of BMPs, it can eliminate the side effects associated with BMPs.

[0006] Meanwhile, while biomaterials for bone grafts initially relied on their in vivo inertness, their use was significantly restricted by infection and inflammatory responses in surrounding tissues after the procedure. Consequently, with the recent rapid advancement of biomaterial technologies utilizing metals, ceramics, and polymers, materials possessing biocompatibility rather than bioinertness are being designed and developed. This has led to the development of various types of bioactive scaffolds for bone tissue regeneration, tailored to specific sites and purposes.

[0007] As these bioactive scaffolds for bone tissue regeneration require different physical properties depending on the implantation site, must be free from toxic reactions to surrounding tissues, and possess relatively high mechanical properties compared to other artificial organs, they are being marketed and developed as various biomaterials according to the characteristics of the raw materials and their intended use.

[0008] However, in the case of bone graft materials, if the compressive strength and yield value are too low, it is difficult to maintain positional fixation and shape retention characteristics during the suturing or implant placement stages after the injection or dense filling of the bone graft material; additionally, if the adhesion of the bone graft material is too high, it tends to stick to surgical instruments during the procedure, making it difficult to easily fill the bone defect, which has the disadvantage of reduced workability.

[0009] Therefore, there is a need to develop a bone graft composition that possesses biocompatibility and physical properties suitable for implantation into bone defects and maintains its formulation for a certain period after implantation.

[0010] Against this background, the inventors completed the present invention by manufacturing a bone graft material capable of acting as a carrier for QG3030, which is a composition having physical properties suitable for use as a bone graft material.

[0011]

[0012] The technical problem that the present invention aims to solve is to provide a bone graft composition and a method for manufacturing the same that can be applied to various bone defects and bone diseases requiring bone grafting. More specifically, the purpose is to provide a bone graft composition and a method for manufacturing the same that exhibits enhanced bone regeneration ability by combining a scaffold composed of tricalcium phosphate (TCP) and hydroxyapatite (HA) with QG3030, a rapaconitine derivative, as an active ingredient.

[0013]

[0014] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0015]

[0016] As an embodiment for achieving the above technical problem, a bone graft composition is provided, characterized in that a compound represented by Chemical Formula 1 is adsorbed onto the surface of a support comprising tricalcium phosphate (TCP) and hydroxyapatite (HA).

[0017] <Chemical Formula 1>

[0018]

[0019] The rapaconitine derivative used in the present invention may be included in an amount of 0.2 to 30 wt% relative to the weight of the support, which is an amount that can effectively impart biological activity without impairing the composition and bone regeneration function of the support.

[0020] In addition, the above compound contains calcium ions (Ca) present on the surface of the support. 2+ It can be stably adsorbed through ionic bonding and / or coordination bonding with ), thereby forming a surface bonding structure that can be maintained for an appropriate period in an in vivo environment.

[0021] In addition, in the composition according to the present invention, the rapaconitine derivative can be adsorbed in an amount of 0.05 to 5 μg / mm² per unit area (mm²) of the surface of the support.

[0022] In addition, the compound exists in the form of fine coating islands dispersed across the surface of the support, which prevents excessive aggregation of the active ingredient and can provide a surface structure capable of exhibiting synergistic bone regeneration functions.

[0023]

[0024] The bone graft composition according to the present invention has a structure in which a rapaconitine derivative is stably adsorbed on the surface of a scaffold composed of tricalcium phosphate (TCP) and hydroxyapatite (HA), thereby effectively improving problems such as irregular release patterns or insufficient surface binding strength observed in existing protein-based bone formation factors. Calcium ions (Ca on the surface of the scaffold) 2+ The surface binding structure formed by ionic or coordinate bonding between ) and rapaconitine derivatives inhibits the initial acute release of the active ingredient and enables more stable and sustained action in the in vivo environment.

[0025] In addition, the composition according to the present invention allows the rapaconitine derivative to exist in the form of micro-coating islands uniformly dispersed on the surface of a support, thereby enabling the active ingredient to be distributed evenly across the entire surface of the support without excessive aggregation. This surface structure provides an environment in which the active ingredient can act more effectively during successive stages of bone regeneration, such as cell attachment, initial bone matrix formation, and mineralization, and maintains natural osteoconductivity by not interfering with the pore structure of the support.

[0026] Furthermore, the above composition can contribute to improving the regeneration rate and quality at bone defect sites by combining the biocompatibility and osteoconductivity of the TCP / HA scaffold with the bone formation-promoting function of the rapaconitine derivative. In particular, due to the structural characteristics that allow the rapaconitine derivative to be stably maintained on the scaffold surface, the active ingredient can continuously influence biological pathways related to bone formation, such as inducing cell differentiation and increasing cell vitality, thereby exhibiting enhanced bone regeneration efficacy compared to existing graft materials.

[0027]

[0028] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims.

[0029]

[0030] Figure 1 shows the results of Alizarin Red S (ARS) staining performed to confirm the effect of the rapaconitine derivative (QG3030) according to the present invention on promoting bone matrix calcification. Figure 1a shows ARS staining images performed on days 9 and 11 after treating AD-MSCs (human adipose-derived mesenchymal stem cells) with QG3030 at concentrations of 0.1, 1, 10, 100, and 1000 nM. It can be confirmed that while the treatment groups at concentrations of 0.1 to 1000 nM showed an effect on day 9, the group at the 1000 nM concentration showed no effect on day 11. On the other hand, the control group treated with BMP-2 (100 ng / mL) showed a tendency for calcification formation to be inhibited. Figure 1b is a graph quantifying the results of the ARS staining above. QG3030 shows a concentration-dependent increase in calcification in the range of 0.1 to 10 nM, and a tendency for the calcification reaction to decrease when the concentration increases beyond 100 nM. It is confirmed that the BMP-2 treatment group shows a decrease in the calcified area, indicating that bone matrix formation is inhibited.

[0031] Figure 2 shows the effect of QG3030 according to the present invention on increasing the expression of alkaline phosphatase (ALP), an indicator of early osteogenic differentiation. Figure 2a shows the results of ALP staining performed on day 4 after treating AD-MSCs with QG3030 at various concentrations; it is confirmed that ALP expression increases significantly in the range of 0.1 to 10 nM, and ALP expression is clearly visible even up to a concentration of 1000 nM. On the other hand, in the BMP-2 (100 ng / mL) treatment group, the ALP-positive area decreases, indicating that early osteogenic differentiation is not sufficiently induced. Figure 2b is a graph quantifying the ALP staining results in the range of 0.1 to 10 nM; it can be seen that QG3030 significantly increases the ALP-positive area even at low concentrations, whereas the BMP-2 treatment group exhibits relatively low ALP activity.

[0032] Figure 3 shows the results of VK staining performed to confirm the mineralization (Von Kossa, VK) promoting effect of QG3030 according to the present invention. Figure 3a shows the VK staining image performed on day 9 in the AD-MSC culture system, where QG3030 shows an increasing pattern of mineral deposition in the concentration range of 0.1 to 100 nM. On the other hand, in the BMP-2 (100 ng / mL) treatment group, mineralization was observed to be inhibited or decreased. Figure 3b is a graph showing the quantitative analysis of the VK positive area, which confirms that in the QG3030 treatment group, mineralization significantly increases with increasing concentration, mineralization is promoted in a concentration-dependent manner up to about 10 nM, and then the effect decreases, whereas in the BMP-2 treatment group, the level of mineralization actually decreases.

[0033] Figure 4 illustrates the results of an animal model experiment on a skull defect to confirm the in vivo bone regeneration effect of the QG3030 / TCP·HA combination according to the present invention and the optimal range according to the amount of QG3030 adsorbed. Figure 4a shows three-dimensional μCT images taken after treating rat skull defects with Vehicle, TCP / HA alone, TCP / HA + QG3030 (100 ng), and TCP / HA + QG3030 (200 ng). Compared to the TCP / HA alone group, the TCP / HA treatment group with adsorbed QG3030 shows an increase in new bone formation within the defect. In particular, the degree of bone tissue filling up to the center of the defect is most pronounced in the QG3030 (100 ng) treatment group. Although new bone formation is observed in the QG3030 (200 ng) treatment group, the degree of defect filling appears relatively lower compared to the QG3030 (100 ng) treatment group. Figure 4b is a graph quantifying the ratio of the area of ​​new bone formation to the total area of ​​the defect in the Vehicle, TCP / HA alone, TCP / HA + QG3030 (100 ng), and TCP / HA + QG3030 (200 ng) treatment groups. It can be seen that the ratio of new bone formation is highest in the TCP / HA + QG3030 (100 ng) combination group compared to the TCP / HA alone group, whereas the effect is lower in the TCP / HA + QG3030 (200 ng) combination group.

[0034] Figure 5 shows μCT images comparing the bone regeneration pattern of a skull defect when QG3030 is administered alone compared to when QG3030 is used in combination with a TCP / HA scaffold. When comparing the 3D images of the defects of the groups treated with QG3030 alone at various doses (1, 10, 100, 150 ng) based on the vehicle group and the TCP / HA alone group, it was confirmed that a certain effect was observed at 1 to 150 ng, and a particularly high effect was observed at 10 to 100 ng.

[0035]

[0036] The present invention will be explained in more detail below with specific examples. However, the embodiments described below are provided as examples to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0037] Accordingly, the present invention is not limited to the embodiments presented below and may be embodied in other forms, and the embodiments presented below are described merely to clarify the concept of the present invention and are not limited thereto.

[0038]

[0039] definition

[0040] Expressions such as “comprising,” “comprising,” “having,” etc. as used herein should be understood as open-ended terms implying the possibility of including other embodiments in a manner similar to “comprising,” unless otherwise stated in the phrase or sentence containing such expressions.

[0041] The term "and / or" as used herein may mean any one or more of the items, any combination of the items, or all of the items in relation to the term.

[0042]

[0043] In this specification, the term “bone grafting composition” refers to a composition that can be applied to a bone defect or a site at risk of bone defect in vivo to promote the regeneration of bone tissue, mineralization, formation of bone matrix, and formation of bone bridges, and may be composed of a combination of a scaffold and an active ingredient.

[0044] In this specification, the term “support scaffold” means a porous inorganic structure that can serve as a physical and mechanical scaffold for bone formation within a bone defect site and may include calcium phosphate-based materials such as tricalcium phosphate (TCP) and hydroxyapatate (HA).

[0045] In this specification, the term “lappaconitine derivative” refers to a compound of a specific structure obtained by oxidizing and alkoxylating lappaconitine, and may include not only the compound represented by Formula 1 but also all pharmaceutically acceptable salts, hydrates, solvates, and stereoisomers thereof. In this specification, the lappaconitine derivative compound and QG3030 may be used interchangeably.

[0046] In this specification, the term “active ingredient” refers to a compound that can combine with a support to promote cell differentiation, mineralization, and bone matrix formation during the bone regeneration process, and in the present invention, QG3030 may correspond to this.

[0047] In this specification, “surface adsorption” refers to a state in which an active ingredient is attached to the outer surface of a support or the inner surface of a pore by ionic bonding, coordination bonding, or physical interaction. The surface adsorption may include a “coating,” which is a state in which the active ingredient is distributed along the surface of the support in a continuous or discontinuous pattern, and in one example of the present invention, it may appear in the form of microcoating islands.

[0048] In this specification, the term “micro-coating island” refers to a structure in which an active ingredient is not coated on the surface of a support in the form of a continuous film, but is attached in the form of discontinuous micro-clusters at the nano to micrometer level. Although the coating islands individually form a clustered form, they are distributed with a uniform density across the entire surface of the support, thereby preventing surface overconcentration and enabling stable sustained release.

[0049] In this specification, “sustained release” refers to a release characteristic in which an active ingredient is slowly released from the surface of a support or inside a pore over a period of several hours to several days depending on the in vivo environment, exhibiting continuous biological activity.

[0050] In this specification, “over-loading” refers to a condition in which an active ingredient excessively covers the surface and pore structure of a support, which may cause pore occlusion, surface aggregation, or abnormal cell signaling. In the present invention, this may apply when the amount of the active ingredient exceeds about 30 wt% relative to the weight of the support.

[0051] In this specification, “wt% relative to the weight of the support” means the weight ratio of the compound of Formula 1 of the present invention (QG3030) to 100 wt% of the dry weight of the inorganic-based material constituting the support.

[0052]

[0053] Meanwhile, regarding technical and scientific terms used herein, unless otherwise defined, they have the meanings commonly understood by those skilled in the art to which this invention pertains, and are defined considering their functions in the present invention, which may vary depending on the intention or convention of the user or operator. Therefore, definitions of these terms should be based on the content throughout this specification, and descriptions of known functions and configurations that could unnecessarily obscure the essence of the present invention are omitted in the following description.

[0054]

[0055] The present invention will be described in detail below.

[0056]

[0057] The present invention provides a bone graft composition characterized by a compound represented by Chemical Formula 1 being adsorbed onto the surface of a support comprising tricalcium phosphate (TCP) and hydroxyapatite (HA).

[0058] <Chemical Formula 1>

[0059]

[0060]

[0061] Specifically, a support according to one embodiment of the present invention may comprise tricalcium phosphate and hydroxyapatite, and said support may be used as an inorganic-based bone graft material that can be gradually dissolved or absorbed in vivo. The tricalcium phosphate contains calcium ions (Ca 2+ They may have structural characteristics capable of releasing ), and hydroxyapatite may have a crystal structure similar to natural bone tissue in the body. Since the two materials have different solubility and crystal characteristics, mixing them allows for balanced control of initial stability and long-term absorption in vivo.

[0062] The weight ratio of the calcium tricalcium phosphate and hydroxyapatite forming the above support can be adjusted to a range of about 10:90 to about 90:10. If the ratio falls outside this range, the mechanical strength or bioabsorption rate of the support may increase or decrease excessively, which may result in an uneven bone regeneration environment. In particular, if the ratio of calcium tricalcium phosphate becomes excessively high, it is difficult to maintain the compound stably due to rapid dissolution, and if the ratio of hydroxyapatite becomes excessively high, the support may remain excessively in vivo, which may delay the remodeling of surrounding bone tissue.

[0063] The above support may have a pore structure connected to the surface and the interior, and the pores may provide a space where the compound can be adsorbed to the surface or interior region. The pore structure may provide an environment favorable for in vivo fluid exchange and cell infiltration, and if the support contains calcium tricalcium phosphate, the interaction with the rapaconitine derivative compound may be enhanced by the gradual dissolution and release of calcium ions from the pore surface.

[0064] Meanwhile, protein-based osteogenic factors such as BMP-2, which are widely used in existing bone graft technologies, may exhibit various structural and mechanistic limitations during the process of binding to TCP / HA scaffolds. Due to the protein characteristics of the aforementioned BMP-2, electrostatic interactions and Ca on the pore surface 2+ It can be sensitively affected by external variables such as concentration and the wetting state of the scaffold, and due to these characteristics, it may not remain stable on the surface of the scaffold and may aggregate locally in specific areas. This aggregation phenomenon may be further intensified depending on the size or connectivity of the internal pore structure of the scaffold, and an excessive amount may concentrate within specific pores of the scaffold. Such localized concentration can induce excessive initial signal transduction in vivo, thereby leading to abnormal osteoblast differentiation or adipocyte differentiation.

[0065] BMP-2 is a substance with a clear concentration-dependent response threshold; when present at concentrations higher than the optimal level, the Smad1 / 5 / 8 pathway becomes overactivated. This leads to a reflexive increase in inhibitory signals such as Smad6 / 7, which may actually inhibit downstream bone matrix calcification. Additionally, PPARγ signaling, which induces adipocyte differentiation, may operate relatively predominantly at high concentrations of BMP-2, potentially causing so-called fat infiltration where the interior of the scaffold becomes filled with adipocytes. This series of phenomena may lead to a tendency for abnormal tissue to become dominant before mineralization proceeds within the scaffold.

[0066] Furthermore, since the aforementioned BMP-2 is a protein-based material with a large molecular weight, its binding affinity to the scaffold surface is limited, and it can be easily leached out by bodily fluid exchange after adsorption. Therefore, while an excessive localized high concentration state may form initially, a rapid decrease in concentration occurs over time, making it difficult to maintain a continuous and uniform bone formation environment. In particular, regarding the Ca of the TCP / HA scaffold 2+ If the dissolution rate is fast, the chemical environment of the surface to which the BMP-2 was bound changes within a short period of time, which may reduce the structural stability of the BMP-2, and this may be a factor that makes it difficult to promote uniform bone regeneration throughout the support.

[0067] Due to the aforementioned issues, when BMP-2 is applied with TCP / HA-based scaffolds, a so-called non-linear bone formation pattern is frequently observed, characterized by an overlap of excessive initial response followed by a decline in response later on. Consequently, uniform bone regeneration does not occur throughout the pore structure of the scaffold, and mineralization is delayed or inhibited more than expected. Therefore, existing techniques for applying protein-based bone formation factors to TCP / HA scaffolds may have structural limitations in terms of morphological uniformity at the bone defect site and long-term tissue remodeling.

[0068] The above support can be designed to be combined with a rapaconitine derivative compound of Formula 1 of the present invention to overcome the problems of the existing technology. The rapaconitine derivative compound can be maintained in a uniformly dispersed form on the surface of the support without aggregating even at high concentrations, and the support can maintain a stable structure for a long period of time with the compound of the present invention adsorbed thereon and can gradually release the compound in an in vivo environment, thereby providing a more uniform bone formation environment compared to existing support-based bone formation systems.

[0069]

[0070] More specifically, in the present invention, the lapaconitine derivative compound represented by Formula 1 may include the compound represented by Formula 1, its pharmaceutically acceptable salt, as well as its hydrate, solvate, stereoisomer, and radioactive derivative.

[0071] The above-mentioned pharmaceutically acceptable salt refers to a salt that is suitable for use in contact with the tissues of humans and lower animals within the scope of pure medical judgment without causing excessive toxicity, irritation, allergic reactions, etc., and does not adversely affect the biological activity and physicochemical properties of the parent compound. The above-mentioned pharmaceutically acceptable salt is well known in the art. For example, it is described in detail in the literature (SM Berge et al., J. Pharmaceutical Sciences, 66, 1, 1977). The salt may be prepared in the same reaction system during the final separation and purification of the compound of the present invention, or separately by reacting it with an inorganic base or an organic base. Suitable addition salt forms include, for example, alkali metal salts and alkaline earth metal salts such as ammonium salts, lithium, sodium, potassium, magnesium, calcium, etc., salts with organic bases, for example, primary, secondary, and tertiary aliphatic and aromatic amines, for example, methylamine, ethylamine, propylamine, isopropylamine, four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidein, pyridine, quinoline and isoquinoline, benzathine, N-methyl-D-glucarmine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrabamin salts, and salts with amino acids such as arginine and lysine.

[0072] In the present invention, the hydrate or solvate of the compound of Formula 1 can be prepared by conventional methods, for example, by dissolving the base compound of Formula 1 in a solvent such as water, methanol, ethanol, acetone, or 1,4-dioxane, and then adding a free acid or free base, followed by crystallization or recrystallization.

[0073] In addition, the compound of Chemical Formula 1 may have one or more asymmetric centers, and in the case of such a compound, enantiomers or diastereomers may exist.

[0074] In the present invention, the compound of Formula 1 can be prepared by a method comprising the following steps:

[0075] (a) a step of reacting rapaconitine with an oxidizing agent; and

[0076] (b) A step of reacting the product of (a) with an organic solvent in the presence of a base.

[0077] According to one embodiment of the present invention, the rapaconitine of (a) may be rapaconitine hydrogen bromide, in which case step (a) may further include a process of removing hydrogen bromide before reacting the rapaconitine with an oxidizing agent. For example, the process of removing hydrogen bromide from rapaconitine hydrogen bromide may be carried out using dichloromethane (CH2Cl2) in the presence of a base as shown in Reaction Scheme 1 below.

[0078]

[0079] <Reaction Equation 1>

[0080]

[0081]

[0082] Subsequently, the above rapaconitine reacts with an oxidizing agent as shown in Reaction Scheme 2 below and is oxidized to produce a rapaconitine derivative (LAD). The oxidizing agent may be selected from the group consisting of phenyl iodine diacetate (PhI(OAc)2) dissolved in dimethylformamide (DMF), lead(II) acetate (Pb(CH3CO2)2), lead(IV) acetate (Pb(CH3CO2)4), ozone, and HIO4.

[0083]

[0084] <Reaction Equation 2>

[0085]

[0086]

[0087] In the present invention, the synthesized rapaconitine derivative (LAD) may react with an organic solvent in the presence of a base to produce a final product. The base may be selected from the group consisting of sodium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, and potassium hydroxide, and the organic solvent may be an aliphatic alcohol or an alkoxy alcohol.

[0088] The above aliphatic alcohol is CH3(CH2) n It refers to alcohols represented by the general formula OH (where n is 0 or a positive integer), and alkoxy alcohols are CH3(CH2) n O(CH2) n It refers to an alcohol represented by the general formula CH3 (where n is independently 0 or a positive integer). For example, the above aliphatic alcohol may specifically be methanol, ethanol, n-propanol, isopropanol, n-butanol, etc., and the alkoxy alcohol may be methoxymethanol, methoxyethanol, ethoxyethanol, etc.

[0089] The most desirable form of the above-mentioned rapaconitine derivative (LAD) may be a compound of Formula 1 (QG3030) produced by reacting with ethanol in the presence of sodium hydroxide as shown in Reaction Scheme 3 below.

[0090]

[0091] <Reaction Equation 3>

[0092]

[0093]

[0094] In the present invention, the compound of Formula 1 synthesized by the above method can be separated by a general separation and purification process, for example, by diluting and washing with an organic solvent and then concentrating the organic layer under reduced pressure, and if necessary, can be purified by column chromatography and recrystallization using various solvents.

[0095] The compound of Chemical Formula 1 above can promote the differentiation of stem cells into osteoblasts and help bone formation by increasing the expression of a bone formation marker selected from the group consisting of RUNX2 (runt-related transcription factor 2), BMP2 (bone morphogenetic protein 2), and osteocalcin. The stem cells may include mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, bone marrow stem cells, etc.

[0096] In the present invention, the bone-related disease may be any one selected from the group consisting of osteoporosis, osteomyelitis, osteomalacia, bone damage caused by bone metastasis of cancer cells, herniated disc, fibrous dysplasia, Klippel-Feil syndrome, and cystic fibroostomy, but is not limited thereto if it is a disease involving osteoblasts.

[0097] As described above, the rapaconitine derivative (QG3030) used in the present invention can exhibit bone-forming activity suitable for treating bone-related diseases and can be used as an active ingredient in a bone graft composition. As shown in FIGS. 1 to 3, the QG3030 showed results of promoting bone differentiation throughout the entire process, from the early stage to the late stage, in a series of bone differentiation tests using AD-MSC (adipose-derived mesenchymal stem cells).

[0098] Figure 1 shows the results of Alizarin Red S (ARS) staining performed to confirm the bone matrix calcification-promoting effect of QG3030 according to the present invention. According to the results in Figure 1a, when QG3030 was treated at concentrations of 0.1, 1, 10, 100, and 1000 nM, calcium deposition increased across all concentration ranges on day 9 of culture, and a significant increase in calcification was observed particularly in the range of 0.1 to 100 nM. On the other hand, on day 11, the calcification in the high-concentration group of 1000 nM was not maintained and showed a decreasing trend, demonstrating that the effect of QG3030 is optimally expressed within a specific concentration range. Furthermore, in the control group treated with BMP-2 (100 ng / mL), a decrease or inhibition in calcification formation was observed, indicating that unlike traditional protein-based bone formation factors, QG3030 exhibits fewer abnormal adverse effects at high concentrations. In the quantitative analysis of Figure 1b, a concentration-dependent pattern was confirmed in which QG3030 increases calcification in a concentration-dependent manner in the range of 0.1 to 10 nM, and the effect gradually decreases at 100 nM or higher.

[0099] Figure 2 shows an experiment confirming the effect of QG3030 on increasing the expression of alkaline phosphatase (ALP), an indicator of early osteogenic differentiation. According to the results in Figure 2a, when AD-MSCs were treated with QG3030 at concentrations ranging from low (0.1 nM) to high (1000 nM), ALP expression increased across the entire concentration range under culture conditions on day 4. In particular, a distinct increase in ALP expression was already observed at low concentrations of 0.1 to 10 nM, which implies that QG3030 can sufficiently activate the signal for the initiation of early osteogenic differentiation even at low concentrations. Furthermore, ALP expression was maintained even at a relatively high concentration of 1000 nM, which is an important characteristic distinguishing it from BMP-2, where adipocyte differentiation is often activated or ALP decreases at high concentrations. In the quantitative analysis results of Figure 2b, it was confirmed that QG3030 induced a significant increase in ALP activity starting from low concentrations, whereas in the BMP-2 treatment group, the ALP-positive area actually decreased, indicating that it has a higher efficiency in inducing early bone differentiation compared to BMP-2.

[0100] Figure 3 shows the experimental results evaluating the mineralization (Von Kossa, VK) promoting effect of QG3030. The VK staining results in Figure 3a showed that QG3030 increased mineral deposition in all groups treated at concentrations of 0.1, 1, 10, and 100 nM, indicating that QG3030 exerts its effect even in the late stages of calcification and bone matrix formation. On the other hand, in the group treated with BMP-2 (100 ng / mL), a tendency for mineralization to be inhibited or decreased was observed; this result is consistent with reports in the literature that BMP-2 induces adipocyte differentiation or causes abnormal signaling at high concentrations. In the quantitative analysis in Figure 3b, QG3030 also exhibited an optimal activity range that increased mineralization in a concentration-dependent manner up to approximately 10 nM, and although it decreased slightly at higher concentrations thereafter, it showed a significantly more stable response pattern compared to BMP-2.

[0101] As such, QG3030 can consistently induce increased activity across the stage indicators of osteodifferentiation progressing from early (ALP) to mid (VK) to late (ARS) stages, and can function as a stable bone formation factor that exhibits sufficient activity even at low concentrations and has minimal reversal effects even at high concentrations. Therefore, compared to existing protein-based bone formation factors such as BMP-2, QG3030 can demonstrate superior characteristics in terms of concentration-dependent stability, long-term responsiveness, and minimization of abnormal differentiation responses, and can be utilized as an active substance suitable for the bone graft composition of the present invention combined with a TCP / HA scaffold.

[0102] The differentiation stability described above can be an important characteristic that distinguishes it from the concentration-dependent adverse effects of protein-based bone formation factors, and can serve as the basis for the lapaconitine derivative compound to function stably even on the surface of a scaffold where local overconcentration is likely to form when applied as an active ingredient in a scaffold-based bone graft composition. While downstream mineralization processes are inhibited when the Smad signaling pathway is overactivated, as with BMP-2, the inhibitory feedback of the compound does not increase excessively at high concentrations, thereby providing a uniform bone formation environment on the surface of the scaffold or in the pore region.

[0103] As previously discussed, a support according to one embodiment of the present invention is an inorganic-based material composed of tricalcium phosphate and hydroxyapatite, wherein calcium ions (Ca 2+ ) may have the characteristic of being gradually dissolved and released. The rapaconitine derivative compound of the present invention has an oxygen-containing lactone structure characteristic of rapaconitine derivatives or a hydroxyl group that is Ca 2+ Since it can have an electron density distribution capable of forming coordination interactions with, the above Ca 2+It can be stably attached to the surface of a support through chemical and physical adsorption. The above interaction goes beyond simple surface adsorption; by aligning the surface charge of the support and the partial charge of the compound in a mutually complementary manner, it can provide a basis for the rapaconitine derivative compound to be uniformly dispersed in the form of micro-coating islands without aggregating on the surface of the support.

[0104] In addition, when the above-mentioned rapaconitine derivative compound is adsorbed in an amount of about 0.05 μg to about 5 μg per unit surface area (mm²) of the support, the contact area with body fluids in vivo can be sufficiently maintained without excessively occluding the pore structure of the support, and abnormal reactions such as inhibition of bone differentiation may not occur even if the compound is present at a local high concentration on the surface of the support. If the adsorption amount is lower than the above range, it may be difficult to provide a sufficient bone formation signal on the surface of the support, and if the adsorption amount is higher than the above range, the inside of the pores of the support may be excessively filled, and the exchange and inflow of minerals may not proceed smoothly.

[0105] The rapaconitine derivative compound can be attached to the surface of the support via wet-mixing or a solution-based adsorption method, and during this process, physiological saline or PBS solution wets the surface of the support, allowing the compound to penetrate into the micro-irregularities or pores of the surface. This penetration can occur naturally through capillary action, and a monodisperse micro-coating pattern can be formed on the surface of the support at this stage. The coating structure can provide an environment in which the compound can be gradually released during the fluid exchange process after in vivo insertion, and the bone regeneration process can be maintained uniformly by combining with the inorganic dissolution of the support itself.

[0106] Due to the structural characteristics described above, the rapaconitine derivative compound can be maintained more stably on the surface compared to existing protein-based bone formation factors when combined with a tricalcium phosphate and hydroxyapatite scaffold, and can form a uniform concentration profile on the scaffold surface and in the pore region. This combined structure can provide a constant level of bone formation signal over a long period in vivo, thereby contributing to improving the uniformity and reproducibility of scaffold-based bone graft compositions.

[0107]

[0108] Meanwhile, a QG3030 / TCP·HA composition according to one embodiment of the present invention can be prepared by considering the interaction between the rapaconitine derivative compound and the support so that the compound can be stably maintained on the surface of the support or within the pore structure. The composition is prepared considering the surface area of ​​the support, the pore structure, and Ca 2+ Depending on the release characteristics, the process may include several steps so that the above compound can be adsorbed in a uniformly dispersed form. Hereinafter, a method for preparing a composition according to an embodiment of the present invention will be described in detail.

[0109] The method for preparing the above composition may include the steps of preparing a support composed of tricalcium phosphate and hydroxyapatite, and contacting and adsorbing a rapaconitine derivative compound of Formula 1 onto the support. The support may be prepared in any form such as a powder, granule, or molded body, and may be prepared in a state where the compound can naturally diffuse into the surface or internal pores depending on the structural characteristics of the support. The size, shape, or porosity of the support may be selected according to the size of the bone defect site or the method of application.

[0110] In addition, the above-mentioned rapaconitine derivative compound may be in contact with a support in powder form, and the mixing of the compound and the support may be performed in either a dry mixing or a wet mixing manner. The wet mixing may be performed by adding physiological saline, a PBS solution, or a similar neutral buffer solution to the support to wet the surface of the support, and allowing the compound to diffuse and adsorb onto the surface and pore structure of the support through the solution. The mixing method may provide conditions in which the compound can be maintained in a monodispersed form on the surface of the support, and may be controlled so that the composition does not excessively aggregate or clog the pores.

[0111] Meanwhile, the amount of rapaconitine derivative compound that can be adsorbed onto the above-mentioned support can be set within a range of about 0.05 μg to about 5 μg per unit surface area (mm²) of the support. If the amount is lower than the above range, it may be difficult to provide a sufficient bone formation-inducing signal throughout the support, and if the amount is higher than the above range, the pore structure of the support may be partially blocked, thereby reducing fluid exchange. Therefore, the above range is set considering the structural characteristics of the support and the surface distribution characteristics of the compound, and can be understood as a condition in which the compound can be stably maintained in the support-based bone graft composition.

[0112] In addition, the adsorption state can be stabilized by removing the solvent through natural drying, vacuum drying, or storage at room temperature after the above-mentioned rapaconitine derivative compound comes into contact with the support. The drying conditions can be selected within a range that does not affect the structural stability of the compound, and the compound may remain on the surface of the support in a uniform pattern during this process. Since the compound may crystallize and aggregate if the drying process is performed excessively, the process can be controlled within a range that maintains the pore structure of the support.

[0113] In addition, the above-described composition may be provided in a form that can be applied directly to a bone defect site or used in combination with other bone substitutes during surgical procedures. The composition may be processed into various formulations, such as powder, paste, or compression-molded block, and the formulation may be modified depending on the size or shape of the application site. Furthermore, after application to a bone defect site, the compound may be gradually released during contact with body fluids to create a bone regeneration environment, and this process may form a specific bone formation environment in combination with the solubility of tricalcium phosphate and the crystal stability of hydroxyapatite.

[0114] In the preparation process of the above composition, process variables such as the particle size of the rapaconitine derivative compound, mixing time, volume of the added solution, and pH may be closely related to the dispersion state of the compound and its retention on the surface of the support; therefore, these variables can be adjusted according to the requirements of the site where bone grafting is required. For example, if the amount of the solution is excessively large, the compound may migrate to deep pores within the support, which may delay the release time; conversely, if the amount of the solution is insufficient, the compound may not diffuse uniformly on the surface of the support. Accordingly, these variables can be adjusted to a range in which the compound can be adsorbed in a monodispersed state on the surface of the support.

[0115] Specifically, a composition according to one embodiment of the present invention may include a rapaconitine derivative compound in an amount of 0.2 wt% to 30 wt% relative to the weight of the support. If the content of the rapaconitine derivative compound is lower than 0.2 wt%, there is insufficient amount of active ingredient to be dispersed over the entire surface of the support, which may result in a limited bone regeneration effect similar to that observed in the low-concentration QG3030 treatment group as shown in FIG. 5. Conversely, if the content exceeds 30 wt%, the compound may excessively cover the micropores and surface irregularities of the support, causing pore blockage, and a tendency for reduced bone formation may appear due to the excessive application amount, as seen in the 200 mg (40 wt%) treatment group shown in FIG. 4. Therefore, the range of 0.2 wt% to 30 wt% can be understood as a condition for simultaneously ensuring the structural stability of the support and the uniformity of the distribution of the compound.

[0116] Furthermore, according to one embodiment of the present invention, the content can most preferably be controlled to a range of 2 wt% to 20 wt% relative to the weight of the support. This range may be established based on the fact that the treatment group of 10 mg to 100 mg, identified in FIGS. 4 and 5, exhibited the highest bone formation area ratio and defect closure degree in an in vivo skull defect model. If the content is lower than this range, the density of the active ingredient may be insufficient, resulting in insufficient bone differentiation signals; if the content is higher than this range, the bone regeneration efficiency may decrease due to excessive occupancy of the pore structure and increased local concentration. Therefore, the range of 2 wt% to 20 wt% can be understood as a condition in which the compound is stably maintained in the support-based composition while the optimal bone formation effect is expressed.

[0117] As such, the method for preparing the composition of the present invention can provide an environment in which the compound can be stably maintained by considering the physical and chemical properties of the compound and the support, and can provide a configuration advantageous for mitigating the problem of non-uniform distribution or high-concentration reverse reaction of existing protein-based bone formation factors in support-based bone graft compositions.

[0118]

[0119] Due to the above characteristics, the QG3030 / β-TCP·HA-based bone graft composition of the present invention can be used as a pharmaceutical composition for the prevention or treatment of bone-related diseases, and can be used for the treatment, prevention, or improvement of bone-related disorders accompanied by defects, damage, absorption, or reduced regeneration of bone tissue.

[0120] The above bone-related disease may be any one selected from the group consisting of osteoporosis, osteomyelitis, osteomalacia, bone damage caused by bone metastasis of cancer cells, herniated disc, fibrous osteodysplasia, Klippel-Feil syndrome, and cystic fibroostomy. In addition, the bone graft composition of the present invention may be used as a bone regeneration promoter or a bone formation promoter for treating the above bone-related disease.

[0121] The bone graft composition of the present invention can be used for the prevention and / or treatment of bone-related diseases to prevent the occurrence of bone defects or the progression of bone resorption in situations where the occurrence of bone tissue loss or defects is anticipated.

[0122] In addition, the present invention may provide a method for preventing and / or treating the bone-related disease. The method may include (i) a step of preparing a β-TCP·HA scaffold adsorbed with an effective amount of QG3030, and (ii) a step of applying the scaffold to a bone defect or a bone resorption site, and may include a step of directly applying the β-TCP·HA scaffold adsorbed with QG3030 to the bone defect site or inserting it as an implant material during a surgical repair process.

[0123]

[0124] The present invention will be explained in more detail below with reference to examples and comparative examples.

[0125] However, the following examples and comparative examples are merely illustrative of the invention for further detailed explanation, and the invention is not limited to the following examples and comparative examples.

[0126]

[0127] Example 1. Preparation of QG3030 / TCP·HA support composition

[0128] 1-1. Preparation of Rapaconitine Derivatives (LADs) from Rapaconitine

[0129] 400 ml of dimethylformamide was added to 50 g of rapaconitine hydrobromide, followed by the addition of 72.6 g of phenyliodine diacetate. After stirring at 40°C for 10 minutes, 800 ml of ethyl acetate and 160 ml of sodium bicarbonate aqueous solution were added. Subsequently, the mixture was washed twice with 500 ml of water to remove DMF, the organic layer was collected, anhydrous magnesium sulfate was added, and the mixture was filtered under reduced pressure. The organic layer was then subjected to reduced pressure distillation and dried under reduced pressure for 6 hours. The powder obtained through the above process was dissolved in a minimum solution of ethyl acetate / heptane at a ratio of 2:1, loaded onto a silica 60 column equilibrated with the same solution, and eluted. After recovering the main elution peak, 20 g of rapaconitine derivative (LAD) was recovered by reduced pressure distillation.

[0130]

[0131] 1-2. Manufacture of QG3030 from LAD

[0132] 130 ml of ethanol and 9 ml of purified water were added to 20 g of P-1 in a mixed state, cooled to 0°C, and 2.9 g of sodium hydroxide was added. After stirring at room temperature for 12 hours, the ethanol was removed by vacuum distillation, and 9.6 ml of purified water and 36 ml of dichloromethane were added for washing. Subsequently, 2.4 g of ammonium chloride was dissolved in 8.4 ml of purified water to adjust the pH to 9–10, and 54 ml of isopropanol / dichloromethane (85:15) was added for extraction, followed by the addition of 6 ml of sodium chloride aqueous solution. Afterward, 54 ml of isopropanol / dichloromethane (85:15) was added for extraction. Afterwards, 2g of anhydrous magnesium sulfate was added to the organic layer extracted with isopropanol / dichloromethane (85:15), filtered under reduced pressure, and the filtrate was distilled under reduced pressure and dried under reduced pressure for 6 hours to obtain a powdered QG3030 mixture.

[0133]

[0134] 1-3. Preparation of QG3030 / TCP·HA support composition

[0135] The bone graft composition of the present invention was prepared by adsorbing the QG3030 powder prepared in Examples 1-2 above onto a support containing tricalcium calcium phosphate (TCP) and hydroxyapatite (HA). Specifically, FRABONE-I (inobone) support was used as the support. Subsequently, the QG3030 powder prepared in Example 1 above was weighed to concentrations of 0.1 mg, 1 mg, 10 mg, 50 mg, 100 mg, and 150 mg, respectively. For each concentration of QG3030 powder, 100 μL of phosphate-buffered saline (PBS) was added to completely dissolve or disperse the powder, and the QG3030 solution or suspension was added to a tube containing 500 mg of TCP / HA support. The mixture was then gently stirred at room temperature for about 30 minutes to ensure that the QG3030 came into even contact with the surface of the support and the inside of the pores.

[0136] After stirring, the mixture was placed in a freeze-drying device to remove the solvent, thereby preparing powdered bone graft compositions in which QG3030 was adsorbed and immobilized on the surface and pore structure of a TCP / HA support.

[0137]

[0138] Comparative Example 1. TCP / HA single support treatment group

[0139] A TCP / HA single scaffold identical to that in Examples 1-3 was prepared and applied to a bone defect site, except that it did not contain QG3030. The scaffold can be applied under the same conditions by mixing a total amount of 500 mg with 100 μL of physiological saline (PBS).

[0140]

[0141] Comparative Example 2. TCP / HA single support treatment group

[0142] A powdered bone graft composition was prepared in the same manner as in Examples 1-3, except that 200 mg of QG3030 was used, in which QG3030 was adsorbed and fixed to the surface and pore structure of a TCP / HA support.

[0143]

[0144] Experimental Example 1. Evaluation of In Vivo Bone Regeneration Promotion by QG3030 / TCP·HA Combination

[0145] In this experimental example, a Sprague-Dawley (SD) rat skull defect model was constructed to evaluate the in vivo bone regeneration effect of the QG3030 / TCP·HA composition prepared in the example. Male SD rats weighing 300 g or more were acclimatized for one week, and then a circular defect of approximately 8 mm in diameter was formed in the center of the skull under general anesthesia, and the sample corresponding to each experimental group was immediately applied to the created defect.

[0146] First, as Comparative Example 1, only 500 mg of TCP·HA scaffold was applied to the defect to confirm the formation of new bone when the scaffold was administered alone. Subsequently, in the group corresponding to the embodiments of the present invention, QG3030 powder was added to 500 mg of TCP·HA scaffold in adjusted amounts of 1 mg, 10 mg, 50 mg, 100 mg, and 150 mg. Each composition was suspended in 100 μL of PBS, mixed to ensure uniform dispersion throughout the scaffold, and then applied to the defect. The range of 1 to 150 mg was set as the range in which the QG3030 of the present invention can be stably adsorbed onto the surface of the TCP·HA scaffold and inside the pores to form an active density.

[0147] In addition, as Comparative Example 2, QG3030 was added in excess at 200 mg and mixed with the TCP·HA support in the same manner. This was set as a condition to check whether pore occlusion or surface aggregation occurs under conditions where an excessively high proportion of the active ingredient relative to the support is added.

[0148] All experimental groups were maintained for 10 weeks after the procedure, and after euthanasia, the skull was excised and the area of ​​new bone formation relative to the total defect area was analyzed using micro-CT imaging.

[0149] As shown in Fig. 4a, the Vehicle group maintained a distinct bone defect in the center of the defect, and little bone bridge formation was observed. In the TCP / HA-alone treatment group, partial bone formation was observed along the edges of the bone defect, but filling in the center of the defect was weak.

[0150] Meanwhile, among the QG3030 / TCP·HA combination groups of the present invention, in the group with 100 mg of QG3030 applied, bone tissue was observed to extend from the edge of the defect to the center, and the interior of the defect was observed to be filled to a significant extent. This indicates a significantly improved pattern of new bone deposition compared to the TCP / HA alone group.

[0151] On the other hand, in the QG3030 200 mg comparative group, bone tissue expansion was inhibited compared to the TCP / HA alone treatment group, confirming that bone regeneration efficiency decreases when QG3030 is administered at high doses.

[0152] Figure 4b shows the results of a quantitative analysis of the new bone formation rate within the defect for the Vehicle group, TCP / HA alone group, and the QG3030 100 mg combination group. Although the new bone rate increased in the TCP / HA alone group compared to the Vehicle group, a significant portion of the defect still remained unfilled. On the other hand, the QG3030 100 mg combination group exhibited the highest new bone rate, confirming that bone regeneration efficiency can be maximized by adsorbing an appropriate amount of QG3030 onto the TCP / HA scaffold.

[0153] Figure 5 shows a comparison of bone regeneration patterns using μCT images when QG3030 was adsorbed onto a TCP / HA scaffold at concentrations of 1 mg, 10 mg, 100 mg, and 150 mg, respectively.

[0154] Experimental results showed that bone formation around the defect increased in the QG3030 1 mg treatment group, while a more distinct regenerative effect was observed in the 10 mg and 100 mg treatment groups, with new bone extending into the interior of the defect. Additionally, bone bridge formation toward the center of the defect was most active in the 100 mg and 150 mg treatment groups, confirming the tendency for the bone regeneration effect to increase as the concentration of QG3030 increases to a certain level. Meanwhile, in the 150 mg treatment group, bone formation was maintained, but it was observed that the central filling degree tended to decrease slightly compared to the 100 mg group on μCT.

[0155] In other words, the results of the in vivo evaluation of the present invention confirmed that when QG3030 is included in a range of about 0.2 wt% to about 30 wt% relative to the weight of the TCP / HA scaffold, a significant effect of promoting new bone formation can be observed compared to TCP / HA alone. Furthermore, it was confirmed that when the amount is controlled to about 2 wt% to about 30 wt% within the above range, QG3030 can be uniformly distributed across the entire surface of the scaffold while maintaining a porous structure, thereby most effectively inducing the formation of a continuous bone bridge extending to the center of the defect.

[0156]

[0157] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0158] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A bone graft composition characterized by a compound represented by Chemical Formula 1 being adsorbed onto the surface of a support comprising tricalcium phosphate (TCP) and hydroxyapatite (HA); <Chemical Formula 1> .

2. In Paragraph 1, A bone graft composition characterized by the content of the above compound being 0.2 to 30 wt% relative to the weight of the support.

3. In Paragraph 1, The above compound is calcium ions (Ca) on the surface of the support 2+ A bone graft composition characterized by being adsorbed onto a surface through ionic bonding or coordination bonding with ).

4. In Paragraph 1, A bone graft composition characterized by the above compound being adsorbed in an amount of 0.05 to 5 μg / mm² per unit area (mm²) of the surface of a support.

5. In Paragraph 1, A bone graft composition characterized by the above compound being adsorbed onto the surface of a support in the form of micro-coating islands.

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