KR102195611B1 - A scaffold for bone regeneration coatd glucosylceramide synthase inhibitor and method for preparing thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor as an active ingredient and a support for bone regeneration using the same, and more specifically, to a support for bone regeneration using the same, more specifically, while inhibiting the differentiation of adipocytes of fibroblasts, A pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor that can help bone regeneration by promoting osteoblast differentiation that can improve bone strength at the same time, and a bone regeneration scaffold using the same will be.

Description

A pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor as an active ingredient, and a support for bone regeneration using the same {A scaffold for bone regeneration coated glucosylceramide synthase inhibitor and method for preparing thereof}

The present invention relates to a pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor as an active ingredient and a support for bone regeneration using the same, and more specifically, to a support for bone regeneration using the same, more specifically, while inhibiting the differentiation of adipocytes of fibroblasts, A pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor that can help bone regeneration by promoting osteoblast differentiation that can improve bone strength at the same time, and a bone regeneration scaffold using the same will be.

With the rapid economic growth and the development of medical technology, the average life expectancy has increased significantly compared to the past. As a result, the prevalence of osteoporosis and other degenerative bone diseases is also increasing due to the rapid increase in the elderly population. Osteoporosis is the cause of various bone defects such as fractures, and bone graft materials such as bone fillers, bone bonding materials, implants and bone supports are required for treatment. However, osteoporosis patients have very poor bone density, bone mass, and bone quality compared to normal people. Therefore, if a bone graft material including a general bone scaffold is inserted, it may cause secondary bone damage due to the difference in physical properties from the graft material. in need. In particular, the support can be used to regenerate the lost tissue through the body's self-healing function, and in the case of a support that acts like a leg connecting the tissue and the tissue, it must be designed to be decomposed according to the regeneration speed of the bone. In other words, if the bone regeneration speed is faster than the decomposition speed of the support, the support becomes a factor that interferes with the regeneration of the bone, and if the regeneration speed of the bone is slower than the decomposition speed of the support, the support is decomposed before the bone regenerates, so use sufficient support. Can't expect effect.

Bone tissue is an important tissue that maintains the skeleton of the human body, and various materials and types of bone tissue replacement and regeneration bone graft materials are being researched and developed for bone tissue regeneration. Bone graft materials can be classified into bone-forming materials, bone conductive materials, and bone-inducing materials according to the bone healing mechanism, and autograft, allograft, or other species depending on the graft material used for transplataion or implantation. It mainly uses methods such as transplantation.

However, until now known pharmaceutical compositions for bone regeneration and compositions used to produce bone regeneration scaffolds using the same are not well fused with cells derived from surrounding bone tissue, have low cell adhesion, and prevent stem cell bone differentiation. There is a drawback of not being able to induce well.

Therefore, in order to compensate for the above-described problems, the present inventors recognized that the development of a regeneration pharmaceutical composition and a bone regeneration support using the same is urgent, and thus completed the present invention.

Korean Patent Publication No. 10-1243956 Korean Patent Publication No. 10-1486314 Korean Patent Publication No. 10-1824521

It is an object of the present invention to contain as an active ingredient a glucosylceramide synthase inhibitor that can help bone regeneration by promoting osteoblast differentiation that can improve bone strength while inhibiting adipocyte differentiation of fibroblasts. It is to provide a pharmaceutical composition for bone regeneration.

Another object of the present invention is that the bone regeneration pharmaceutical composition has a bone regeneration effect even without transplantation with osteoblasts, and there is no fear of side effects such as an immune response during transplantation, and bone cell adhesion is improved and bone differentiation It is to provide a support for bone regeneration that can promote.

In order to achieve the above object, the present invention provides a pharmaceutical composition for bone regeneration comprising a glucosyl ceramide synthase inhibitor as an active ingredient and a support for bone regeneration using the same.

Hereinafter, the present specification will be described in more detail.

The present invention provides a pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor as an active ingredient.

In the present invention, the glucosyl ceramide synthase inhibitor is characterized in that it has the structure of the following [Formula 1].

[Formula 1]

In the present invention, the pharmaceutical composition for bone regeneration is characterized in that it is used for preventing or treating bone diseases.

In the present invention, the bone disease is any one selected from bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone insufficiency, osteomalacia and fractures resulting therefrom, osteogenesis disorders, degenerative bone disease or malocclusion It is characterized by being.

In the present invention, when the pharmaceutical composition for bone regeneration is administered to the rat and measured after 5 weeks by X-ray microcomputed tomography (Micro-CT), 55 to 60% of bone regeneration It is characterized by being promoted.

In addition, the present invention is a first support layer comprising biodegradable collagen; And a second supporting layer comprising a glucosyl ceramide synthase inhibitor as an active ingredient, wherein the second supporting layer surrounds the first supporting layer.

In the present invention, the glucosyl ceramide synthase inhibitor is characterized in that it has the structure of the following [Formula 1].

[Formula 1]

All matters mentioned in the pharmaceutical composition of the present invention and the support using the same are applied equally unless contradictory to each other.

The pharmaceutical composition for bone regeneration of the present invention can help in bone regeneration by promoting osteoblast differentiation that can improve bone strength while inhibiting the differentiation of adipocytes of fibroblasts, and bone defects, osteoporosis, osteoporosis Sex fracture, diabetic fracture, nonunion fracture, bone insufficiency, osteomalacia and fractures resulting therefrom, bone formation disorder, degenerative bone disease, or any one selected from malocclusion, which can prevent or treat bone disease.

In addition, the scaffold for bone regeneration of the present invention has a bone regeneration effect even if it is not transplanted together with osteoblasts, and there is no fear of side effects such as an immune response during transplantation, and bone cell adhesion is improved and bone differentiation can be promoted, It has an excellent effect as a support for bone regeneration.

1 is a (a) X-ray microcomputed tomography (Micro-CT) image and (b) a graph confirming the effect of promoting bone regeneration of the pharmaceutical composition of the present invention.
Figure 2 is a (a) image and (b) graphs confirming the osteoblast differentiation promoting effect of the pharmaceutical composition of the present invention.
3 is a (a) image and (b) a graph confirming the effect of inhibiting adipocyte differentiation of the pharmaceutical composition of the present invention.

Terms used in the present specification have selected general terms that are currently widely used as possible while considering functions in the present invention, but this may vary depending on the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

The numerical range includes the numerical values defined in the above range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were expressly written. All minimum numerical limits given throughout this specification are inclusive of all higher numerical limits as if the higher numerical limits were expressly written. All numerical limits given throughout this specification will include all better numerical ranges within the wider numerical range, as if the narrower numerical limits were expressly written.

Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

Pharmaceutical composition for bone regeneration

The present invention provides a pharmaceutical composition for bone regeneration comprising a glucosylceramide synthase inhibitor as an active ingredient.

The term "bone regeneration" as used in the present invention may mean any phenomenon in which bone damage is treated, alleviated, or improved through processes such as proliferation of damaged bone tissue into bone cells or differentiation of osteoblasts into bone cells. And, but not limited thereto, in particular, the bone regeneration may be by promoting bone differentiation.

In the present specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

The glucosyl ceramide synthase inhibitor of the present invention has the structure of the following [Formula 1].

[Formula 1]

The glucosyl ceramide synthase inhibitor of the present invention may promote the differentiation of osteoblasts to exhibit an effect of promoting bone regeneration, and the pharmaceutical composition for bone regeneration may be used for preventing or treating bone diseases.

The bone disease may be any one selected from bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone insufficiency, osteomalacia and resulting fractures, osteogenesis disorders, degenerative bone disease, or malocclusion, preferably For example, it may be any one selected from osteoporosis, osteogenesis insufficiency, and osteogenic disorders, and more preferably, osteoporosis.

In the present specification, the "prevention" refers to any action that suppresses or delays bone disease by administration of the pharmaceutical composition. In addition, the "treatment" refers to any act of improving or beneficially altering the symptoms of bone disease by administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention does not increase the medicinal efficacy, but may further include an ingredient that is commonly used in pharmaceutical compositions to improve odor, taste, vision, and the like. In addition, the pharmaceutical composition of the present invention may additionally contain a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, Hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol and talc, and the like, but are not limited thereto. In addition, the pharmaceutical composition may be used alone or may contain a substance having a prophylactic or therapeutic activity for bone disease.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and may be formulated for oral or parenteral human or veterinary use. When formulating the pharmaceutical composition of the present invention, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations include at least one excipient, such as starch, calcium carbonate, in the pharmaceutical composition containing the compound of the present invention ( Calcium carbonate), sucrose or lactose, and gelatin can be mixed to prepare. Also, in addition to simple excipients, lubricants such as magnesium, sterate, and talc may be used.

Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, skin external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection It is desirable to select the injection method.

The pharmaceutical composition of the present invention can be administered to an individual to prevent or treat bone disease.

The term "individual" as used in the present invention refers to horses, sheep, pigs, goats, dogs, etc., including humans who have bone diseases and who have diseases that can improve symptoms by administering the pharmaceutical composition of the present invention. It means a mammal, but preferably a human.

The term "administration" as used in the present invention means introducing the pharmaceutical composition of the present invention to a subject by any suitable method. The route of administration can be administered orally or parenterally through any general route as long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention can be administered by any device that allows it to move to target cells.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term used in the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the patient's weight, sex, age, health status, and severity. , Drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. It can be administered single or multiple.

The dosage of the pharmaceutical composition according to the present invention should be a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to prevent or treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the formulation method, the patient's condition and weight, the patient's sex, age, and disease. It can be variously selected by those skilled in the art according to factors such as degree, drug type, route and duration of administration, excretion rate, and response sensitivity. Effective amounts may vary depending on the route of treatment, the use of excipients, and the possibility of use with other agents, as will be appreciated by those of skill in the art.

The dosage or dosage of the pharmaceutical composition containing the glucosylceramide synthase inhibitor according to the present invention as an active ingredient may be varied depending on the patient's age, physical condition, weight, etc., but generally 0.01 to It is preferable to administer within the range of 500 mg/kg (body weight)/day. And, within the range of the effective daily dosage, it can be divided into once a day or several times a day.

When the pharmaceutical composition of the present invention is administered to a rat and measured after 5 weeks by X-ray microcomputed tomography (Micro-CT), 55 to 60% of bone regeneration can be promoted.

In addition, the pharmaceutical composition of the present invention can help in bone regeneration by promoting osteoblast differentiation that can improve bone strength while inhibiting the differentiation of adipocytes of fibroblasts.

Bone regeneration scaffold comprising a pharmaceutical composition for bone regeneration

The present invention includes a first support layer comprising biodegradable collagen; And a second support layer comprising a glucosylceramide synthase inhibitor as an active ingredient; and provides a support for bone regeneration.

The term "bone regeneration support" used in the present invention does not necessarily have to match the shape of the bone damage site to be transplanted, regardless of its shape, and it is sufficient as long as it has been processed into a similar shape. However, if necessary, the shape of the bone damage site of the transplant target may be identified in advance and a suitable shape may be prepared, and may be formed in advance with a structure of a membrane or a structure of a band so as to be generally applied to various situations.

The glucosyl ceramide synthase inhibitor is as defined above in the pharmaceutical composition for bone regeneration.

The bone regeneration support of the present invention may be in a form in which the second support layer surrounds the first support layer, and the glucosylceramide synthase inhibitor has a structure of the following [Formula 1].

[Formula 1]

The bone regeneration support may further include various materials for increasing moldability. That is, the physical properties of the bone regeneration scaffold can be improved by further adding a linear material, a tube material, a particulate material, and an irregular material with excellent biocompatibility.

The bone regeneration scaffold of the present invention has a bone regeneration effect even without transplantation with osteoblasts, and there is no fear of side effects such as an immune response during transplantation, and bone cell adhesion is improved and bone differentiation can be promoted. It has an excellent effect as a regeneration support.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in a variety of different forms, and only the embodiments make the disclosure of the present invention complete, and common knowledge in the technical field to which the present invention pertains. It is provided to inform a person having a complete scope of the invention, and the invention is only defined by the scope of the claims.

The reagents and solvents mentioned below were purchased from Sigma-Aldrich Korea unless otherwise specified.

Experimental Example 1. Confirmation of bone regeneration promoting effect

In order to confirm the bone regeneration promoting effect of the bone regeneration pharmaceutical composition comprising the glucosyl ceramide synthase inhibitor of the present invention as an active ingredient and the scaffold prepared using the same, the following experiment was performed.

A 4 mm diameter trepanation was performed on the calvarias of the rat to form a damaged site, and 100 μg of the pharmaceutical composition of the present invention was treated on the damaged site (experimental group), and the effect of the pharmaceutical composition To confirm, the control group did not perform any treatment. And 5 weeks later, the damaged areas of the experimental group and the control group were confirmed through X-ray-based micro-CT (X-ray microcomputed tomography, Micro-CT), and the results are shown in FIG. 1.

Referring to Figure 1 (a), while the experimental group to which the pharmaceutical composition of the present invention was administered can visually confirm the effect of promoting bone regeneration, it can be seen that bone regeneration hardly occurs in the control group. In addition, referring to Figure 1 (b), the experimental group to which the pharmaceutical composition of the present invention was administered was found to have bone formation in a region corresponding to 58.5% of the total defected volume, whereas the control group had total defects. Bone formation was observed only in the area corresponding to 38.5% of the total defected volume. From the above results, it can be confirmed that the pharmaceutical composition of the present invention has a remarkably excellent effect of promoting bone regeneration.

Experimental Example 2. Confirmation of osteoblast differentiation promoting effect

Alizarin red S staining and PCR analysis were performed to confirm the effect of promoting osteoblast division of the pharmaceutical composition for bone regeneration comprising the glucosylceramide synthase inhibitor of the present invention as an active ingredient. It is shown in Figure 2.

Referring to FIG. 2(a), it can be seen that the experimental group to which the pharmaceutical composition of the present invention was administered has differentiated human mesenchymal stem cells (hMSCs) into osteoblasts. In addition, referring to FIG. 2(b), the experimental group to which the pharmaceutical composition of the present invention was administered is alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP2) compared to the control group. It can be seen that expression of such bone formation genes/proteins is significantly promoted. From the above results, it can be confirmed that the pharmaceutical composition of the present invention promotes the differentiation of osteoblasts.

Experimental Example 3. Adipocyte differentiation inhibition effect confirmation

Alizarin red S staining and PCR analysis were performed to confirm the effect of inhibiting adipocyte distribution of the pharmaceutical composition for bone regeneration containing the glucosylceramide synthase inhibitor of the present invention as an active ingredient. It is shown in Figure 3.

Referring to Figure 3 (a), it can be seen that the differentiation of adipocytes was inhibited in the experimental group to which the pharmaceutical composition of the present invention was administered. In addition, referring to FIG. 3(b), the experimental group to which the pharmaceutical composition of the present invention was administered reduces the expression of major regulating transcription factors such as PPARγ and C/EBPα and targets such as aP2 and LPL compared to the control group. It can be seen that the gene also decreases. From the above results, it can be confirmed that the pharmaceutical composition of the present invention promotes the differentiation of osteoblasts and inhibits the differentiation of adipocytes.

From the above description, it will be understood that those skilled in the art belonging to the present invention can be implemented in other specific forms without changing the technical spirit or essential features of the present invention. In this regard, the embodiments described above are illustrative in all respects, and should be understood as non-limiting.

Claims (7)

Containing glucosylceramide synthase inhibitor as an active ingredient,
When measured after 5 weeks by X-ray microcomputed tomography (Micro-CT), 55 to 60% bone regeneration is promoted for the total defected volume,
The glucosyl ceramide synthase inhibitor is a pharmaceutical composition for bone regeneration, characterized in that it has the structure of the following [Chemical Formula 1].
[Formula 1]

delete The method of claim 1,
The pharmaceutical composition for bone regeneration is a pharmaceutical composition, characterized in that used for preventing or treating bone diseases. The method of claim 1,
The bone disease is any one selected from bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, bone insufficiency, osteomalacia and resulting fractures, osteogenesis disorders, degenerative bone disease, or malocclusion. Pharmaceutical composition. delete delete delete

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