KR102215165B1 - Composition for promoting bone formation comprising carbon monoxide complex - Google Patents

Abstract

The present invention relates to a composition for promoting bone formation comprising a nitrogen monoxide complex, an agent for promoting bone formation, an implant or a scaffold including the same, wherein the nitrogen monoxide complex can activate human gingival fibroblast-1 (HGF-1). , It has the effect of promoting bone formation.

Description

Composition for promoting bone formation comprising carbon monoxide complex

The present invention relates to a composition for promoting bone formation comprising a nitrogen monoxide complex.

Bone tissue is a dynamic tissue in which bone formation (Bone formation/Osteoblast differentiation) and bone resorption (Bone resorption/Osteoclast differentiation) occur constantly. Osteoblasts absorb bone, while osteoblasts synthesize and fill the bone matrix. Thus, bone mass depends on the relative functions of these cells. In normal adults, the amount of bone resorption and the amount of bone formation are maintained in balance.

In the last 10-20 years, with the prolongation of human lifespan and improvement of the quality of life, the domestic and foreign implant market has grown and become popular. An implant is an artificial tooth or a third tooth, and the volume is increased so that it can be sufficiently wrapped through additional surgery such as bone graft or bone nephrology on the jawbone at the location where the tooth has been removed or removed. It is a dental treatment technique that restores the function of natural teeth by planting a biocompatible implant body. After osteo-integration, which is a morphological, physiological, and direct connection between the jawbone in which normal function is maintained and the surface of the implant body, the process of bone remodeling of the jawbone around the implant is performed. This osseointegration is the most important indicator that determines the success of an implant. If the osseointegration does not occur within the period, the success rate of the implant procedure decreases due to the failure of the initial osseointegration. Therefore, for the success of implant placement, it is necessary to improve the ability of osteosynthesis and bone formation.

The success rate of an implant depends on good initial fusion between the implant material and bone cells. Bioactive substances such as BMP (Bone matrix protein), a protein belonging to the TGF-ß family that promotes the proliferation and differentiation of osteoblasts, are applied directly to the implant surface or distributed around the surgical site during implant placement. Since it was reported that the BMP-based protein plays an important function in bone formation, bone differentiation, and alveolar bone formation, many experiments have been conducted. Not only animals but also humans, BMP-based proteins are involved in bone formation and differentiation. It has been announced that it regenerates and strengthens. However, BMP is not only expensive, but also rapidly decomposed and disappears by enzymes present in the body, and thus effective application is difficult. In addition, the BMP increases the cost of treatment and does not remain effective, so it is difficult to apply, short half-life, and standardization.

Meanwhile, in order to form all new tissues, blood vessels must be generated from surrounding tissues. Specifically, in order to form new bone, the surrounding bones and the bone graft site must be connected with blood vessels. On the other hand, the bone-forming protein such as BMP described above has no consideration for angiogenesis, and thus has a limitation in the case of a large bone defect.

Accordingly, there is a need for a complex or a preparation for promoting bone formation that is effective for angiogenesis and can promote bone formation cells even when the bone defect is large.

Japanese Patent Laid-Open No. 2003-506142

The present invention has been made to solve the above-described problems, and is effective in angiogenesis, and an object of the present invention is to provide a composition for promoting bone formation including a nitrogen monoxide complex capable of promoting bone-forming cells even when the bone defect is large.

To achieve the above object,

The present invention in one embodiment,

It provides a composition for promoting bone formation comprising a nitrogen monoxide complex represented by the following Formula 1:

[Formula 1]

In Formula 1,

a and b represent repeating units, a is 2 to 130, b is 15 to 70,

x, y, and z represent repeating units, x+z is 100, x is 50 to 99, and y is 1 to 10.

In addition, the present invention in one embodiment,

It provides a bone formation promoter comprising a composition for promoting bone formation.

In addition, the present invention in one embodiment,

It provides an implant or scaffold comprising a composition for promoting bone formation.

The nitrogen monoxide complex included in the composition for promoting bone formation according to an embodiment of the present invention can activate HGF-1 (human gingival fibroblast-1) having properties similar to osteoblasts, thereby promoting bone formation. There is an effect that can be made.

The composition may also be used as an additive in a liquid form, and may be coated in a powder form or a bone grafting agent to be applied to an implant or scaffold.

1 is a view showing a skull defect of an experimental animal to which a bone formation promoter is applied in an embodiment of the present invention.
FIG. 2 is a diagram showing a micro-shear tomography image 1 month after implanting the graft materials of groups 1 to 4 in an embodiment of the present invention.
3 is a diagram showing a cell migration assay according to time after dispensing HGF-1 into a well plate and treating the cultured cells with Example 1 in an embodiment of the present invention.

In the present invention, various modifications may be made and various embodiments may be provided, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise.

In the present invention, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance the possibility.

The present invention relates to a composition for promoting bone formation comprising a nitrogen monoxide complex.

Technical features of the present invention, a composition for promoting bone formation comprising a nitrogen monoxide complex capable of promoting osteogenic cells even when the bone defect is large while being effective for angiogenesis, a mixture, an implant or a scaffold comprising the same To provide. More specifically, the nitrogen monoxide complex included in the composition for promoting bone formation can activate HGF-1 ((human gingival fibroblast-1), which has properties similar to osteoblasts, and thus promotes bone formation. It works.

On the other hand, for all new tissue formation, angiogenesis must be performed from surrounding tissues. Specifically, in order to form new bone, the surrounding bones and the bone graft site must be connected with blood vessels. On the other hand, since bone-forming proteins such as BMP, which have been used for conventional bone formation, have no consideration for angiogenesis, there has been a limit to bone formation when the bone defect is large.

Accordingly, the present invention provides a composition for promoting bone formation comprising a nitrogen monoxide complex that is effective in angiogenesis and can promote bone-forming cells even when bone defects are large, and a mixture, implant or scaffold comprising the same is provided. .

Prior to the description, the term "bone formation" among the terms used in the present invention includes promoting bone formation by healing of bone tissue lost due to promotion of osteoblast differentiation and activity. More specifically, in the present invention, it may include promoting bone formation through healing of lost bone tissue by increasing the activity of HGF-1 (human gingival fibroblast-1). In the present invention, facilitation is not limited thereto, but is to increase the amount of bone tissue, bone cells, or bone mattress, or to shorten the time for increasing the amount, and can be confirmed by the therapeutic effect of fracture.

In this respect, bone regeneration or bone formation can be used interchangeably.

In the present invention, "bone" or "bone" may include long bones (long bones), regular bones (fine bones), flat bones (straight bones), alveolar bones, and the like.

Specific symptoms that can be treated by the bone formation promoting composition of the present invention include, but are not limited to, dysplasia in which bone growth or development is abnormal, and various causes of osteopenia, osteoporosis, and bone loss. Representative examples of these symptoms include achondroplasia, clavicle cranial osteosis, incomplete osteoclastosis, weight loss-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal osteodystrophy, invasive bone disorder, oral bone loss, jaw Bone necrosis, mammary osteoblastosis, metabolic bone disease, organ transplant-related bone loss, kidney transplant-related bone loss, ischemic bone disease (e.g., Leg-Calve-Perthes disease, local mobility osteoporosis) ), glucocorticoid-induced bone loss, heparin-induced bone loss, idiopathic osteopenia or osteoporosis, congenital osteopenia or osteoporosis, local osteoporosis, osteomalacia, bone loss associated with artificial joints, HIV-related bone loss, loss of growth hormone and Related bone loss, bone loss related to cystic fibrosis, chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormonal resection bone loss, multiple myeloma, drug-induced bone loss, anorexia nervosa, disease related Includes facial bone loss, disease-related jaw loss, and disease-related skull bone loss. Additional symptoms include aging-related bone loss, such as aging-related facial bone loss, aging-related skull bone loss, aging-related jaw bone loss, and aging-related skull loss.

In addition, the composition of the present invention is useful for improving the results of orthopedic surgery, transplant surgery, joint replacement, bone graft, bone cosmetic surgery and bone repair, such as fracture healing, nonunion healing, delayed union healing and facial reconstruction. can do.

Accordingly, the present invention provides a composition for promoting bone formation comprising a nitrogen monoxide complex represented by the following Formula 1:

[Formula 1]

In Formula 1,

a and b represent repeating units, a is 2 to 130, b is 15 to 70,

x, y, and z represent repeating units, x+z is 100, x is 50 to 99, y is 1 to 10, and z is 1 to 50.

According to an embodiment of the present invention, a may be 78 and b may be 30. In addition, y may be 1. At this time, when x and z represent the mole% of the repeating unit, x+z may be 100, and x may be 50 to 99. This can be estimated based on the fact that at most 50 mol% of z can participate in the formation of NONOates.

Further, the organic molecule used to form the nitrogen monoxide complex according to the present invention may be used without limitation as long as it is a material containing a secondary amine group. For example, pyrrolidine, diethyltriamine, diethylamine, or the like having a secondary amine group may be used. In addition, in the case of polymer materials, it has the form of a polymer containing a secondary amine group in the main chain or side chain of the polymer, and has been improved to have a polymer containing polyethyleneimine or polyethyleneimine, and other secondary amine groups. It may be a polymer or the like. However, it is not limited thereto.

According to an embodiment of the present invention, the nitrogen monoxide complex included in the composition for promoting bone formation can activate HGF-1 (human gingival fibroblast-1) having properties similar to osteoblasts, thereby promoting bone formation. There is an effect that can be made. In a specific example, it was confirmed that the composition according to the present invention exhibits the activity of HGF-1 related to bone formation and that it forms new bone.

The present invention in one embodiment,

It provides a bone formation promoter comprising a composition for promoting bone formation.

More specifically, the composition for promoting bone formation according to an embodiment of the present invention may be formulated and administered in a unit dosage form suitable for intra-body administration of a patient according to a conventional method in the pharmaceutical field. It includes an effective dosage capable of promoting bone formation or bone regeneration by one or several administrations. Formulations suitable for this purpose include injections such as ampoules for injection and sprays such as aerosol formulations as parenteral formulations. Preferably, physiological saline, glucose, mannitol, Ringer's solution, etc. may be used as the preparation of the bone formation accelerator.

When used as a bone formation promoter, the composition for promoting bone formation may be included in the bone formation promoter at a concentration of 5 to 1000 μg/mL, or 10 to 500 μg/mL, 100 to 400 μg/mL, 150 to 300 μg /mL range. However, the concentration should be determined differently depending on the conditions of the implanted site. For example, the concentration may be determined differently depending on the possibility of occurrence of inflammation, open or closed wounds, the ratio of cortical bone and cancellous bone, or the degree of development of peripheral blood vessels. In addition, physiological saline or phosphate buffer saline (PBS) may be used as a solvent. If the composition is less than 5 μg/mL concentration in the mixed preparation, cell activity cannot be improved, and when the concentration exceeds 1000 μg/mL, a problem of cytotoxicity may occur.

In addition to the active ingredient, the accelerator includes one or more pharmaceutically acceptable conventional carriers, such as preservatives, painless agents, solubilizers or stabilizers, etc., in the case of formulations for topical administration, excipients, lubricants, or preservatives Can be included as.

In one embodiment of the present invention, an implant or scaffold comprising a composition for promoting bone formation is provided.

Specifically, as described above, the composition may be used as an injection in a liquid form, and may be coated in a powder form or a bone grafting agent to be applied to an implant or scaffold. Here, the implant or scaffold relates to a medical device for implantation in a living body, and may be implants and scaffolds of all types and shapes used in the art.

The implant or scaffold is a natural polymer composed of collagen, haruronic acid, alginate and chitosan, PLA (poly(lactic acid)), PLGA (poly(glycolic acid)), PGA (poly(glycolic acid)), PCL (polycaprolactone). And a synthetic polymer composed of poly(methyl methacrylate) (PMMA), and one or more components selected from the group consisting of a synthetic polymer composed of titanium, a titanium alloy, and a nickel-cobalt alloy, but are not limited thereto. Preferably it may contain a component of collagen.

Further, the implant or scaffold comprising the composition for promoting bone formation according to the present invention may contain the composition at a concentration of 5 to 1000 μg/mL. Specifically, the composition is characterized in that the formulation containing a concentration of 5 to 1000 μg/mL is coated on the surface of the implant or scaffold. For example, the composition may be mixed with physiological saline, glucose, mannitol, Ringer's solution, and the like. Preferably, a formulation containing a composition for promoting bone formation at a concentration of _5 μg/mL may be coated on an implant or scaffold containing a collagen component.

Hereinafter, the present invention will be described through the following examples. However, the examples are for describing the present invention in detail, and the scope of the present invention is not limited to the following examples.

<Example>

Example 1. Synthesis of F68-BPEI-NO

First, 1.79 g of pNPC (4-nitrophenyl chloroformate) dissolved in 40 mL of DCM (dichloromethane) was added dropwise to 50 mL of DCM containing 10 g of Pluronic F68 (pNPC-F68) to activate the Pluronic F68. After stirring overnight, pNPC was dissolved in 23.2 mL of DCM to produce BPEI (branched polyethyleneimine). Then, 1.76 g of BPEI 1.8k was dissolved in 40 mL of DCM containing 6 mL of TEA, and the mixture was stirred at 0° C. for 15 minutes. After stirring, the F68-pNPC solution was added dropwise to the BPEI containing solution. After the resulting solution was stirred overnight, the temperature was raised to room temperature. After the reaction, the product was purified by precipitation with cold diethyl ether. After removing the remaining BPEI, the final product was freeze-dried and stored.

And, by adding NONOates to the secondary amine portion, py-NO, DETA-NO and F68-BPEI-NO were synthesized from each precursor (pyrrolidine, diethylenetriamine, and F68-BPEI).

Specifically, 1.64 mL of pyrrolidine was mixed with 5 mL of acetonitrile, 5 mL of ether and 3.9 mL of 30% NaOMe. Then, 0.52 mL of diethylene triamine was mixed with 15 mL of acetonitrile and 0.9 mL of NaOMe solution.

Then, F68-BPEI dissolved in 20 mL of methanol was mixed with 3 mL of tetrahydrofuran and 0.2 mL of 30% NaOMe solution.

Each precursor containing solution was placed in a high pressure NO reactor with argon purging. Then, the reactor was immediately filled with 90 psi of NO gas and maintained at this pressure for 3 days to functionalize diazeniumdiolates. Py-NO and DETA-NO were collected by filtration and precipitated with diethyl ether to give the final F68-BPEI-NO.

Finally, 0.22 mg of the prepared F68-BPEI-NO was mixed with 1 mL of saline to prepare a concentration of 1/50 mM (220 μg/mL).

Example 2. Synthesis of F68-BPEI-NO

F68-BPEI-NO was synthesized in the same manner as in Example 1, and 0.11 mg of F68-BPEI-NO was mixed with 1 mL of saline to prepare a concentration of 1/100 mM (110 μg/mL).

<Comparative Example>

Comparative Example 1. Synthesis of F68-BPEI

Except for the addition of NONOates to the secondary amine portion, F68-BPEI was synthesized in the same manner as described in Example 1.

<Experimental Example>

Experimental Example 1. Observation of the formation rate of new bone

Experimental animals and experimental materials

In this experiment, 20 rats weighing 300-500 g over 30 days of age were used, and all experimental procedures including selection and management of experimental animals and surgical treatment were performed according to the guidelines of the Animal Experimental Ethics Committee of Korea University Medical Center. .

As experimental materials, 2% (w/v) lidocaine and 1:100,000 (v/v) epinephrine, fibrinogen, aprotinin, collagen membrane, and bone powder were respectively purchased and used in the following experiments.

Surgical treatment of laboratory animals

First, ketamine (Ketamine-HCl, 35 mg/kg; Yuhan, Seoul, Korea) and xylazine (Xylazine, 5 mg/kg; Bayer Korea) were intramuscularly injected into each rat to induce general anesthesia, and then The hair was removed, and the cranial surgical site was sterilized with povidone iodine. Next, after local invasive anesthesia of the surgical site with 2% lidocaine, a scalp of about 5 cm was incised along the central part from anterior to posterior part of the frontal bone, and the periosteum was removed to remove the frontal bone and parietal bone. Exposed. Using a 6mm trephine bur on the exposed skull, four circular bone defects were formed using a reverse rotation drill motor so as not to damage the meninges, and the graft material was implanted on each skull defect. After the implantation was completed, the periosteum, subcutaneous tissue, and scalp were sutured with absorbable sutures. An antibiotic (gentamicin, 0.1mg/kg) was injected intramuscularly for a week after surgery to minimize the effect of external microbial infection (see FIG. 1).

Setting of experimental group and control group

The skull defects of the experimental animals were divided into 4 groups and treated as follows.

Group 1: control group, group without graft material

Group 2: group containing the graft material of Comparative Example 1 (F68-BPEI)

Group 3: the group containing the graft material of Example 1 (F68-BPEI-NO, concentration 1/50mM)

Group 4: the group containing the graft material of Example 2 (F68-BPEI-NO, concentration 1/100mM)

On the other hand, the graft materials of groups 2, 3, and 4 included the compositions of Comparative Examples 1, 1 and 2, respectively, and each composition was wetted in collagen sponge and bone powder.

Each group was intravenously injected with phentobarbital (100 mg/kg) one month after surgery, and observed, and cranial sections including defects were obtained from each animal after sacrifice. All tissue sections were fixed with 4% paraformaldehyde phosphate buffer solution (paraformaldehyde BPS, pH 7.2) for a week before analysis.

Micro shear tomography and analysis

First, the fixed skull specimens were scanned using a three-dimensional micro focus computed tomography (micro-CT, Sky-Scan 1172TM) system, and then an image image was generated therefrom.

And, the results are shown in FIG. 2.

Referring to FIG. 2, when a collagen sponge was used as a medium, high new bone formation was exhibited in groups 3 and 4 (Examples 1 and 2) containing the nitrogen monoxide complex.

In addition, when bone powder was used as a medium, the shape of the powder was well maintained in groups 3 and 4 (Examples 1 and 2) including the nitrogen monoxide composite. Furthermore, the new bone formation was higher at 1/100 mM than when the concentration of nitrogen monoxide was 1/50 mM (220 μg/mL >110 μg/mL).

On the other hand, it was shown that the amount of new bone was formed more when collagen sponge was used as a media rather than bone powder, which appears to be because it has a structure that better stores the liquid nitrogen monoxide complex.

Experimental Example 2. Cell migration assay

After dispensing 1 Х 10 ⁴ cells/well of human gingival fibroblasts (HGFs) purchased from ATCC (American Type Culture Collection) into a 6-well plate, sterilized to form a gap at regular intervals. The middle portion of the well was scratched using a micropipette tip. The cells were cultured in DMEM for 24 hours.

After treating the cultured cells with Example 1, the migration patterns of the cells over time were confirmed after 24, 36, and 48 hours. Then, it was compared with cells that were not treated with anything (control).

A photograph of the movement of the cells is shown in FIG. 3.

3, it shows that the HGF-1 activity was higher in the nitrogen monoxide treatment group (right) compared to the control (left).

Claims (8)

It includes a nitrogen monoxide complex and collagen represented by the following Formula 1,
The composition is a composition for promoting bone formation, characterized in that to increase the activity of HGF-1 (human gingival fibroblast-1):
[Formula 1]

In Formula 1,
a and b represent repeating units, a is 2 to 130, b is 15 to 70,
x, y and z represent repeating units, x+z is 100, x is 50 to 99, and y is 1 to 10.
delete A bone formation promoter comprising the composition for promoting bone formation according to claim 1.
The method of claim 3,
Bone formation accelerator, the bone formation accelerator composition is contained in a concentration of 5 to 1000 ㎍ / mL for promoting bone formation.
An implant or scaffold comprising the composition for promoting bone formation according to claim 1.
The method of claim 5,
An implant or scaffold, characterized in that a formulation containing a composition for promoting bone formation in a concentration of 5 to 1000 μg/mL is coated on the surface of the implant or scaffold.
The method of claim 5,
An implant or scaffold, characterized in that a formulation containing a composition for promoting bone formation in a concentration of 5 to 1000 μg/mL is supported inside the implant or scaffold.
The method of claim 5,
Implants or scaffolds are: Haruronic acid, alginate, PLA (poly(lactic acid)), PLGA (poly(glycolic acid)), PGA (poly(glycolic acid)), PCL (polycaprolactone), PMMA (poly(methyl methacrylate)). ), an implant or scaffold comprising at least one component selected from the group consisting of titanium, a titanium alloy and a nickel-cobalt alloy.

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