KR102073333B1 - Porous polymer microsphere for the prevention or treatment of bone diseases or cartilage diseases, and preparation method thereof - Google Patents

Abstract

The present invention relates to a porous polymeric microsphere for the prevention or treatment of bone diseases or cartilage diseases and a method of manufacturing the same.
Porous polymer microspheres according to the present invention can be injected directly into the affected area to be treated with drugs effective for the regeneration of damaged bone tissue or cartilage tissue. In addition, since the drug is slowly discharged, it is possible to deliver a desired amount of drug to the affected area for a long time, but can express the excellent regenerative effect directly in the bone tissue, and since it is composed of a biodegradable material, there are no side effects. In addition, the porous polymer microsphere according to the present invention can provide a personalized treatment because it can adjust the rate and amount of the drug is discharged through the control of the porosity and the concentration of the supported drug according to the affected part.

Description

Porous polymer microsphere for the prevention or treatment of bone diseases or cartilage diseases, and preparation method

The present invention relates to a porous polymeric microsphere for the prevention or treatment of bone diseases or cartilage diseases and a method of manufacturing the same.

Bone (bone) is a solid tissue that supports the body to form the internal skeleton and to protect the organs. Bone (bone) tissue is a hard tissue that supports the body, which constitutes the internal skeleton and functions to protect organs. It is physiologically important to maintain homeostasis to maintain a constant blood calcium level in vivo. In order to maintain this homeostasis, bone has to maintain bone formation by osteoblasts and bone resorption by osteoclasts in sequence. If this balance is broken, bone disease is caused. Osteoporosis, one of bone diseases, has been reported to be one of the causes of excessive activity of osteoclasts to increase the risk of fracture due to a decrease in bone mass and weak bone strength. According to the Korean Society of Bone Metabolism (Guideline for Diagnosis and Treatment of Osteoporosis, 2013), approximately 50% of patients after osteoporotic femoral fractures cannot restore pre-fracture maneuverability and independence, and 25% of patients have long-term protection. The average mortality rate in one year is 20%. Therefore, as a treatment for osteoporosis, the Korean Society of Bone Metabolism suggests various treatment methods. First, in the case of female hormone therapy, estrogen was injected by patch, gel, oral administration to prevent osteoporosis caused by postmenopausal estrogen deficiency, and bone mineral density was significantly increased, but edema, breast pain, cholelithiasis, and leg cramps were reported. Adverse reactions causing backs are also reported. Next, bisphosphonate, the most prescribed drug in the world, is injected by oral and intravenous injection. Bisphosphonates are synthetic substances that do not exist in the human body, move to bone and enter osteoclasts to inhibit differentiation and action. However, oral bisphosphonates are known to cause esophagitis, gastric ulcers, prolonged jaw bone necrosis and prolonged headaches, muscle pain and renal dysfunction. Another treatment is parathyroid hormone (PTH). PTH consists of 84 amino acids, but PTH1-34, consisting of 34 amino acids at the amino terminus, has been approved and used in the United States, Europe and Korea. PTH promotes bone formation during intermittent administration at low doses, while maintaining high concentrations requires increased bone resorption. In addition, adverse reactions may result in mild nausea, headache, and leg cramps, and temporary hypercalcemia. The treatment methods reported by the Korean Society of Bone Metabolism have been used as a treatment for osteoporosis, but new treatment methods are still required because they can be accompanied by adverse reactions.

Osteoporosis is caused by bone metastasis of cancer cells in addition to bone loss caused by direct osteoclast activity. Various cancer cells, such as breast cancer, prostate cancer and colon cancer, also metastasize to the bones (bones), and the lungs and livers (bones) are known as the third most common metastases. Bone metastasis of these cancers not only causes metastasis, but also affects osteoblasts involved in bone formation in bone marrow and osteoclasts involved in bone resorption, ultimately leading to excessive bone resorption, which in turn leads to cancer cells. Induces a series of vicious cycles that promote cancer cell proliferation (Yoneda T, Tanaka S, Hata K. Role of RANKL / RANK in primary and secondary breast cancer.World J Orthop 2013 18; 4 (4) Doi: 10.5312 / wjo.v4.i4.178).

Representative osteoporosis therapeutics developed so far are mainly bisphosphonates, which weaken the function of osteoclasts (cells involved in bone resorption) and prevent bone loss, and Merck's 'Posamax' and Procter Gamble Inc.'s 'Actonnell'. However, these therapeutic agents are not inhibitors of bone formation, but degrading inhibitors, which can lead to additional fractures by shattering bones in continuous use, which is why there is an urgent need for alternatives (Datamonitor Research Reports, 2007).

On the other hand, because articular cartilage has no blood vessels, nerves, or lymphatic tissue, damaged joint cartilage is difficult to regenerate spontaneously. Therefore, even in small damage in the articular cartilage, the wound may progress, the degeneration of the joint may occur. Therefore, in order to restore and maintain the function of bone, cartilage tissue, medically, various methods are being planned.

Cartilage is a tissue derived from mesoderm, such as tibial tissue, which forms an endoskeleton with bone. In addition, when cartilage damage is severe, not only cartilage but also bone tissue under the cartilage may be damaged. As described above, cartilage and bone are organic tissues, but conventional tissue and cartilage treatment of cartilage and bone treated them. Specifically, conventional clinical methods for regenerating cartilage include (1) inducing differentiation of stem cells into chondrocytes, and (2) cartilage defects in bone, cartilage tissue, autologous or homogenous cartilage tissue. Implantation method (3) implantation of cartilage-inducing tissue (cartilage, periosteum) or compound on the surface of the cartilage defect site, (4) cartilage cell transplantation to the cartilage defect site by cartilage cell transplantation A method of regenerating only cartilage has been used, such as a method of inducing regeneration.

However, as described above, since the cartilage and the bone are organic tissues, the conventional method of treating cartilage and bone separately has a weak binding force between bone and cartilage, and takes a long time to recover.

As a result, stem cells, which have self-replicating ability, can be differentiated into various tissues and can be easily collected without a large amount of donor dysfunction, have been recognized as an ideal cell source for cell therapy. However, there is still a lack of clear knowledge about factors, environment, etc. for cartilage formation.

Stem cells for articular cartilage regeneration are characterized by self-proliferative capacity and differentiation ability to differentiate into cells forming specific tissues and have recently been proposed as a new cell source for application to articular cartilage treatment. Therefore, theoretically, it is possible to solve the limitations of the conventional cell therapy using chondrocytes and to apply to the degeneration and damage of the articular cartilage as a whole. In addition, adult mesenchymal stem cells and mesenchymal progenitor cells have the advantages of ethical problems and no rejection in vivo upon allogeneic transplantation.

However, not all adult mesenchymal stem cells are completely differentiated into chondrocytes at the same time, and thus, a situation in which a method of inducing differentiation into uniform chondrocytes is required.

As a method for promoting bone / cartilage formation or regeneration of bone / cartilage tissue, there are methods for oral administration of drugs or direct injection of drugs into affected areas, but methods for oral administration of drugs require bone differentiation. The amount of drug delivered to the affected part is only a fraction, so the regenerative effect is not great, and the direct injection of the drug into the affected part has a problem of side effects because a large amount of drug is injected at one time.

As a way to alleviate this problem, a histological approach using biodegradable porous polymeric scaffolds has emerged as an alternative. Biodegradable porous polymeric supports mimic the natural extracellular matrix and are known to be very favorable for the formation and differentiation of osteoblasts and chondrocytes, regeneration of bone and cartilage tissue. They have the advantages of high cell adhesion, but poor mechanical properties, there is a problem that the formation and differentiation rate of osteoblasts and chondrocytes, or the rate of regeneration of bone / cartilage tissue is slow.

Accordingly, there is a need for development of a new therapeutic drug having no problem of side effects while showing osteoblast / chondrocyte formation and differentiation promoting effects, or bone / cartilage tissue regeneration promoting effects.

KR 10-1105285 B

The problem to be solved by the present invention is to provide a porous polymer microsphere loaded with drugs that can prevent or treat bone diseases or cartilage diseases.

Another object of the present invention is to provide a method for producing the porous polymer microspheres.

One aspect of the present invention for achieving the above object is a biodegradable polymer support of a three-dimensional network structure in which pores having a size of 5 to 100 ㎛ are connected to each other; And it provides a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases comprising; and a drug for the treatment of bone diseases or cartilage diseases contained in the network structure of the biodegradable polymer support.

According to the present invention, the porous polymer microspheres may contain 2 to 30 parts by weight of the drug based on 10 parts by weight of the biocompatible polymer.

According to the present invention, the porous polymer microspheres may have a porosity of 10 to 90% and a particle size of 200 to 1000 μm.

According to the present invention, the porous polymer microsphere may have a porosity of 60 to 90%, a pore size of 10 to 40 μm, and a particle size of 200 to 500 μm.

According to the present invention, the drug for the treatment of bone diseases or cartilage diseases is bone forming protein-2, epidermal growth factor (epidermal growth factor), bone forming protein-7, cilastatin, nystatin (nystatin), lovastatin (lovastatin), somatostatin, sovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, cervastatin, ulvastatin, ulstatatin, roschvastatin rosuvastatin, pitavastatin, glucosamine, glucosamine, chondroitin, mevastatin, mevastatin, kartogenin, transforming growth factor-beta1, transformation Growth factor-beta3, 6-bromoindirubin-3-oxime, sulfonamide, permorphamine, mevinolin, genistein, Icariin, melatonin, metmet Metformin, alendronate, zoldronate, etidronate, clodronate, tirudronate, pamidronate, neridro Neridronate, olpadronate, ibandronate, risedronate, prostaglandins, dexamethasone, dexamethasone, bone-forming protein-4, insulin-like growth factor-1 ( insulin-like growth factor-1), fibroblast growth factor-2, fibroblast growth factor-18, fibroblast growth factor, or a mixture thereof.

According to the present invention, the biocompatible polymer is polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyamino acid, polylactite, poly Any one selected from phosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan; Polymers in which two or more of these are blended; And it may be one selected from the group consisting of two or more copolymers.

According to the present invention, the bone disease is a growth stage of development, osteopenia, bone fragility, bone necrosis, fracture, osteoporosis due to bone absorption of excessive osteoclast (osteoporosis), bone dysplasia, bone damage, joint damage due to trauma , Osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, Paget disease, and metastatic bone cancers Cartilage disease is degenerative arthritis, inflammatory arthritis, osteochondromatosis, distortion, bursitis, meniscus, meniscus Plate-shaped meniscal tear, meniscus cyst, collateral ligamentrupture, anterior cruciate ligament injury, posterior Posterior cruciate ligament injury, intra-articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knuckle-knee and pronunciation It may be selected from the group consisting of snapping knee.

According to the present invention, the porous polymer microspheres may be injected into a living body to release a drug for treating the bone disease or cartilage disease.

In addition, the present invention 1) biocompatible polymer; And a drug for treating a bone disease or a cartilage disease; dissolving it in an organic solvent to prepare a polymer solution. 2) preparing an emulsion by adding and homogenizing the polymer solution and gelatin solution; 3) Treatment of biocompatible polymers, bone disease or cartilage disease by using the emulsion as a discontinuous phase and using a polyvinyl alcohol solution as a continuous phase to flow in a fluidic device. Preparing a polymeric microsphere comprising a drug and gelatin; And 4) adding the polymer microspheres in water at 38 to 50 ° C. and stirring to elute gelatin from the polymer microspheres. The method for preparing a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases, including: do.

Porous polymer microspheres according to the present invention can be injected directly into the affected area to be treated with drugs effective for the regeneration of damaged bone tissue or cartilage tissue. In addition, since the drug is slowly discharged, it is possible to deliver a desired amount of drug to the affected area for a long time, but can express the excellent regenerative effect directly in the bone tissue, and since it is composed of a biodegradable material, there are no side effects. In addition, the porous polymer microsphere according to the present invention can provide a personalized treatment because it can adjust the rate and amount of the drug is discharged through the control of the porosity and the concentration of the supported drug according to the affected part.

1 is a scanning electron microscope image of porous polymeric microspheres (SIM / PMSs) of Example 1. FIG.
FIG. 2 shows the drug release behavior test results of porous polymeric microspheres (SIM / PMSs) of Example 1. FIG.
3 is a scanning electron microscope image of microspheres having different pore sizes according to the concentration of gelatin added during the preparation of the porous polymer microspheres.
Figure 4 is a result of measuring the degree of cell proliferation in porous polymer microspheres having different pore size.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

The present invention provides a biodegradable polymer support having a three-dimensional network structure in which pores having a size of 5 to 100 μm are connected to each other; And it provides a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases comprising; and a drug for the treatment of bone diseases or cartilage diseases contained in the network structure of the biodegradable polymer support.

Porous polymer microspheres according to the present invention may be injected into a living body to release the drug for treating the bone disease or cartilage disease. The drug may be released through the pores of the porous polymer microspheres, or may be released while the biodegradable polymer support (hereinafter, referred to as the polymer support) is decomposed in vivo.

The release of the drug may be controlled by the amount and release rate of the drug according to the type of the polymer forming the polymer support, the density of the network structure, porosity and pore size. Therefore, according to the severity of the affected area to be treated, the porosity, pore size, and concentration of the drug contained in the porous polymer microspheres may be appropriately adjusted and applied to the patient.

The porous polymer microsphere according to the present invention may contain 2 to 30 parts by weight of the drug based on 10 parts by weight of the biocompatible polymer. If the amount of the drug is less than the above range, the amount of the drug is too small, and if it exceeds the above range, the amount of the drug to be released in a short time is too high, which is not preferable.

The porous polymer microspheres according to the present invention have a porosity of 10 to 90% and a particle size of 200 to 1000 μm. If the size of the particles is less than the above range, the amount of drug that can be supported is too small to be continuously discharged in vivo, there is the inconvenience of frequent drug administration, and if the size of the particles exceeds the above range, the production of microspheres Is not easy.

In particular, the porous polymer microspheres have a porosity of 60 to 90%, a pore size of 10 to 40 μm, and a particle size of 200 to 500 μm. Within this range, the drug can be easily released in vivo.

Porous polymer microspheres according to the present invention may have a pore size of 5 to 100 ㎛. If the pore size is less than the above range, the pore size is too small to release the drug, and if the pore size exceeds the above range, there is a problem that the drug is released at once. Particularly preferred pore sizes may be 10 to 80 μm.

The drug for treating a bone disease or cartilage disease according to the present invention is bone forming protein-2, epidermal growth factor, bone forming protein-7, cilastatin (cilastatin), nystatin (nystatin), lovastatin (lovastatin), somatostatin (somatostatin) ), Pravastatin, simvastatin, simvastatin, fluvastatin, atorvastatin, cervastatin, cerinastatin, ulinastatin, rosuvastatin, pitavastatin ( pitavastatin, glucosamine, chondroitin, mevastatin, cartogenin, transforming growth factor-beta1, transforming growth factor-beta3, 6-bromoindine-3-oxime, sulfone Amides, permorphamines, mebinolin, genistein, iccarin, melatonin, metformin, allendronate, zoleronate, ethidronate, cladronate, tyrudronate, pamidronate, neridronate, Olpadronate, ibandronate, lizdronate, prostaglandin, dexamethasone, bone morphogenetic protein-4, insulin-like growth factor-1, fibroblast growth factor-2, fibroblast growth factor-18, fibroblast growth factor, or It may be selected from a mixture thereof.

The biocompatible polymer of the porous polymer microsphere according to the present invention is polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyamino acid, Among polylactite, polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan Any one selected; Polymers in which two or more of these are blended; And it may be one selected from the group consisting of two or more copolymers.

Particularly preferred according to the present invention, the drug for treating bone disease or cartilage disease is simvastatin, pravastatin, lovastatin, mevastatin, pitavastatin, bone morphogenic protein-2, fibroblast growth factor, cartogenin, dexamethasone, allendronate, Parmidronate, rezedronate, prostaglandin, or a mixture of two or more thereof.

The bone disease according to the present invention is a growth stage of undergrowth, osteopenia, bone weakness, bone necrosis, fracture, osteoporosis due to bone absorption of excessive osteoclasts (osteoporosis), bone dysplasia, bone damage, joint damage by trauma, From the group consisting of osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, Paget disease and metastatic bone cancers The cartilage disease is selected, degenerative arthritis, inflammatory arthritis, osteochondromatosis, sprain, distortion, bursitis, meniscus, discoid Meniscal tear, meniscus cyst, collateral ligamentrupture, anterior cruciate ligament injury, posterior posterior dorsal Posterior cruciate ligament injury, intra-articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knock-knee and pronunciation It may be selected from the group consisting of snapping knee, but is not limited thereto.

The porous polymeric microspheres according to the present invention may be particularly effective in the prevention or treatment of bone / chondrogenic diseases or bone / cartilage defect diseases by promoting the formation or differentiation of osteoblasts or chondrocytes.

In the present invention, the prevention or treatment of the bone disease or cartilage disease may be to promote the formation of osteoblasts or chondrocytes or to promote the differentiation of osteoblasts or chondrocytes to promote the regeneration of bone or cartilage tissues or to inhibit inflammation. have.

On the other hand, the porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases according to the present invention is 1) a biocompatible polymer; And preparing a polymer solution by dissolving a drug for treating a bone disease or a cartilage disease in an organic solvent. 2) preparing an emulsion by adding and homogenizing the polymer solution and gelatin solution; 3) The emulsion is used as a discontinuous phase and the polyvinyl alcohol solution is used as a continuous phase to flow in a fluidic device, thereby preparing biocompatible polymers, drugs for treating bone diseases, and gelatin. Preparing a polymeric microsphere comprising; And 4) adding the polymer microspheres to water at 38 to 50 ° C. and stirring to elute gelatin from the polymer microspheres.

In the method of preparing the porous polymer microsphere, a polymer solution is prepared by first mixing 2 to 30 parts by weight of the drug with respect to 10 parts by weight of the biocompatible polymer and dissolving it in an organic solvent.

The organic solvent may be one or two or more selected from dimethyl sulfoxide, dichloromethane, tetrahydrofuran and N, N-dimethylformamide, but is not limited thereto.

Next, the prepared polymer solution and gelatin aqueous solution are added and homogenized to prepare an emulsion.

The gelatin aqueous solution may be 1 to 10% by weight gelatin aqueous solution. If the gelatin content is less than the above range, the gelatin content is too low to facilitate the development of pores of the microspheres, and if the gelatin content exceeds the above ranges, the microspheres are not easily prepared, and the pore size is relatively wide, thereby decreasing cell proliferation. Problem occurs.

According to the present invention, the homogenization may further include an emulsion stabilizer. The emulsion stabilizer may be a emulsion stabilizer commonly used, preferably polyvinyl alcohol.

According to the present invention, the homogenization of step 2) may be added to 3 to 15 parts by weight of gelatin based on 10 parts by weight of the biocompatible polymer. If the content of gelatin for the biocompatible polymer is less than the above range, the development of pores is not easy, and it is difficult to achieve the desired porosity. If it exceeds this range, the pores are too large to support the drug.

According to the present invention, 0.5 to 3 parts by weight of the emulsion stabilizer may be added to 10 parts by weight of the biocompatible polymer. When the content of the emulsion stabilizer is out of the above range, it is not easy to form a microsphere of the desired size in the present invention.

In addition, by controlling the content of the gelatin, the drug or both of the content of the biocompatible polymer, it is possible to appropriately control the discharge and discharge rate of the drug.

In addition, the homogenization may be to homogenize for 30 seconds to 10 minutes at 8,000 to 20,000 rpm using a homogenizer, but is not limited thereto.

In the present invention, the biocompatible polymer and the drug for treating bone disease or cartilage disease are as described above.

Next, the desired polymer microspheres can be prepared by using the emulsion in a discontinuous phase and using polyvinyl alcohol in a continuous phase and flowing it in a fluid apparatus. When the prepared microspheres are stirred in water at 38 to 50 ° C., the gelatin components in the microspheres are released from the microspheres, thereby forming pores where gelatin was present, thereby forming a porous polymer microsphere.

At this time, the flow rate of the discontinuous phase and the continuous phase may be 0.2 to 1 mL / min, respectively, preferably 0.5 mL / min. The flow rate is excellent in the degree of dispersion of porosity, it is possible to manufacture a microsphere having a desired size and porosity.

The microspheres prepared in this way may have a porosity of 10 to 90%, a pore size of 5 to 100 μm, a particle size of 200 to 1000 μm, and preferably a porosity of 60 to 90%. It may be a microsphere having a pore size of 10 to 40 ㎛, the particle size of 100 to 400 ㎛.

An effective amount of the porous polymer microspheres according to the present invention can be administered to prevent or treat bone diseases.

As used herein, the term “effective amount” means the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinician, Amounts that induce alleviation of the symptoms of the disease or disorder. The effective amount and frequency of administration for the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimum dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It may be adjusted according to various factors, including the condition, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and drugs used simultaneously. In the prophylactic, therapeutic or ameliorating method of the present invention, it is preferable that an adult is administered at a dose of 0.001 g / kg to 1 g / kg when administered once to several times a day.

The porous polymer microspheres according to the present invention may be prepared using pharmaceutically acceptable and physiologically acceptable auxiliaries in addition to the active ingredient, and the auxiliaries include vitamins, colorants, neutralizing agents, pectic acids and electrolytes. , Alginic acid, organic acid, carbonation agent, excipient, disintegrant, sweetener, binder, coating agent, swelling agent, lubricant, glidant or flavoring agent and the like can be used.

The porous polymeric microspheres may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

The preparation form of the porous polymer microspheres is not particularly limited as long as it is in the form of a conventional pharmaceutical composition, but may preferably be an injectable liquid (liquid solution), and as an acceptable pharmaceutical carrier in a composition formulated into a liquid solution. As sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be mixed and used. Other conventional additives, such as antioxidants, buffers, bacteriostatics, can be added accordingly. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

According to the present invention, the preferred concentration of the drug contained in the porous polymer microspheres may be 1 ng / ml to 500 mg / ml, it may vary depending on the type of drug contained. For example, if the type of drug is for regeneration of cartilage or bone, the preferred drug concentration may be 1 pg / ml to 20 mg / ml, and if the type of drug is for inflammation inhibition, the preferred drug concentration may be It can be 1 μg / ml to 20 mg / ml, but it is obvious that it can easily vary depending on the weight of the affected area.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example

Example  1. Porous Polymer Microsphere  Produce( Simvastatin  contain)

A 2 wt% PLGA solution was prepared by dissolving 140 mg of polylactide-co-glycolide (PLGA) in 7 g of dichloromethane. 750 mg of gelatin was dissolved in 10 g of distilled water to prepare a 7.5 wt% gelatin aqueous solution. PLGA: gelatin is mixed in a 7: 3 weight ratio based on solids and dimethyl so that simvastatin contains 1, 5 and 10 mM (2.94 g, 14.7 g and 29.4 g) (2.1, 10.5 and 21 wt%), respectively An emulsion was prepared by adding simvastatin dissolved in sulfoxide and homogenizing at 13,500 rpm for 1 minute using a homogenizer (homogenizer, Ultra-Turrax T-25 Basic, IKA).

Microspheres were prepared by using the emulsion as a discontinuous phase in a fluid apparatus and flowing it at a flow rate of 0.5 mL / min using 0.5 wt% polyvinyl alcohol in a continuous phase. Distilled water was added to the prepared microspheres and stirred in distilled water at 40 ° C. to elute gelatin in the microspheres, thereby preparing a porous polymer microsphere containing simvastatin.

Example  2. Porous Polymer Microsphere  Produce( Pravastatin  contain)

In the same manner as in Example 1, but instead of simvastatin pravastatin was added to prepare a porous polymeric microsphere.

Example  3. Porous Polymer Microsphere  Manufacture (with mevastatin)

In the same manner as in Example 1, but instead of simvastatin, mevastatin was added to prepare a porous polymeric microsphere.

Example  4. Porous Polymer Microsphere  Manufacture (with Alendronate)

In the same manner as in Example 1, a porous polymer microsphere was prepared by adding allendronate instead of simvastatin.

Example  5. Porous Polymer Microsphere  Manufacture (with dexamethasone)

In the same manner as in Example 1, dexamethasone was added instead of simvastatin to prepare a porous polymer microsphere.

Comparative example . Porous polymer Microsphere  Manufacture Free )

A 2 wt% PLGA solution was prepared by dissolving 140 mg of polylactide-co-glycolide (PLGA) in 7 g of dichloromethane. 750 mg of gelatin was dissolved in 10 g of distilled water to prepare a 7.5 wt% gelatin aqueous solution. The emulsion was prepared by mixing PLGA: gelatin in a 7: 3 weight ratio based on solids and homogenizing for 1 minute at 13,500 rpm using a homogenizer (homogenizer, Ultra-Turrax T-25 Basic, IKA).

Microspheres were prepared by using the emulsion as a discontinuous phase in a fluid apparatus and flowing it at a flow rate of 0.5 mL / min using 0.5 wt% polyvinyl alcohol in a continuous phase. Distilled water was added to the prepared microspheres, and stirred in distilled water at 40 ° C. to elute gelatin in the microspheres, thereby preparing a porous polymer microsphere.

Test Example  1. Morphology Analysis

Test Example  1.1. Scanning electron microscope observation

In order to confirm the morphology of the porous polymer microsphere prepared by the method of Example 1 was observed by a scanning electron microscope, which is shown in Figure 1 below.

FIG. 1 is an image showing a comparison of simvastatin-containing porous polymer microspheres according to Example 1 (hereinafter referred to as “microspheres”) and porous polymer microspheres containing no simvastatin according to a comparative example. The surface and pore size of the microspheres at each concentration showed a similar shape and showed a significant level difference depending on the concentration of the drug.

On the other hand, the microspheres were prepared by increasing the concentration of simvastatin step by step from 1 to 10% in order to confirm the pore size change according to the concentration of the drug, it is shown in Figure 1 and Table 1 measured by a scanning electron microscope.

division Simvastatin concentration in Example 1 (mM) 0 One 5 10 Pore width (㎛) 28.45 ± 10.01 27.11 ± 8.12 26.00 ± 10.37 25.54 ± 8.22 Pore Height (㎛) 24.45 ± 7.03 25.19 ± 6.41 24.15 ± 9.89 24.22 ± 8.05

Referring to Table 1, it was confirmed that the pore size increases with the concentration of the drug.

Test Example  2. Drug Release Behavior

The drug release behavior of the microspheres prepared in Example 1 was confirmed. Specifically, each microsphere was placed in 1 ml of PBS buffer (pH 7.4) and stirred at 100 rpm at 37 ° C. for 1 hour, 3 hours, 6 hours, 10 hours, 1 day, 3 days, 5 days , 7 days, 10 days, 14 days, 21 days and 28 days) was confirmed, and after the measurement was replaced with fresh buffer. The released drug was analyzed by measuring the absorbance at 250 nm, which is shown in Figure 2 below.

Referring to FIG. 2, the amount of drug released increased depending on the concentration of simvastatin.

Test Example  3. Verification of osteoblast differentiation promoting effect

Test Example  3.1. Alkaline Phosphatase  Activity evaluation

The bone fusion efficacy of the porous polymer microspheres containing simvastatin according to Example 1 was evaluated by measuring alkaline phosphatase activity, an early bone differentiation index of osteoblasts.

After dispensing 1 × 10 ⁵ stem cells into the microspheres of each Example and Comparative Example, they were differentiated and cultured for 3 days, 7 days, 10 days and 14 days. After culturing where the stem cells were dispensed, the cells were washed with PBS buffer, 1 × RIPA buffer was injected into the cells and sonicated for 1 minute at 110 W at 4 ° C. Next, the lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C., and then incubated at 37 ° C. for 30 minutes by adding p-nitrophenylphosphate solution to the supernatant, followed by 500 mL of 1 N. The reaction was terminated with NaOH. Absorbance was measured at 405 nm and the results are shown in Table 2 below.

Classification (concentration of drug) Alkaline phosphatase activity (μM / min / μg protein) 3 days later 7 days later 10 days later Control 1.96 1.97 1.97 Comparative example 1.96 1.95 1.96 Example 1 1 mM 2.52 3.25 3.94 5 mM 2.96 3.57 4.12 Example 2 1 mM 2.47 3.13 3.78 5 mM 2.85 3.44 4.01 Example 3 1 mM 2.33 3.11 3.65 5 mM 2.79 3.36 3.94 Example 4 1 mM 2.24 2.87 3.27 5 mM 2.58 3.02 3.54 Example 5 1 mM 2.44 3.10 3.69 5 mM 2.85 3.42 3.97

As shown in Table 2, the alkaline phosphatase activity, which is an initial bone differentiation index for stem cells differentiated into osteoblasts in the porous polymer microspheres according to Examples 1 to 5, was effectively increased as compared with the comparative example.

Test Example 3.2. Calcium Deposition Assessment

The bone fusion efficacy of the porous polymer microspheres containing simvastatin according to Example 1 was evaluated by measuring calcium deposition concentration, which is a late bone differentiation index of osteoblasts.

Specifically, 1 × 10 ⁵ stem cells were dispensed into the microspheres of Example 1 and Comparative Example 1, and then differentiated and cultured for 7 days, 14 days, and 21 days. After incubation, the cells were washed with PBS buffer and then cells were removed from each nanoparticle with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, 0.1% Triton X-100 solution was added to the cells, stored at 65 ° C. for 3 hours, and then ultrasonically applied to crush the membranes of the cells, followed by centrifugation. After separating the supernatant, using a QuantiChrom Calcium Assay kit was measured by comparing the absorbance at 612nm to analyze the calcium deposition degree, it is shown in Table 3 below.

Classification (concentration of drug) Calcium Deposition Concentration (㎍ / mL) 7 days later 21 days later Control 9.7 28.31 Comparative example 9.8 28.42 Example 1 1 mM 10.0 37.74 5 mM 10.6 42.35 Example 2 1 mM 9.9 34.15 5 mM 10.3 41.47 Example 3 1 mM 9.7 34.97 5 mM 10.1 42.12 Example 4 1 mM 9.8 33.14 5 mM 10.1 40.05 Example 5 1 mM 10.0 35.46 5 mM 10.5 41.89

As shown in Table 3, it was evaluated that the calcium deposition concentration for stem cells differentiated into osteoblasts in the porous polymer microspheres of Examples 1 to 5 was increased effectively compared to the comparative example.

Test Example  4. Verification of cartilage cell differentiation promoting effect

Test Example  4.1. GAG  analysis

Glycosaminoglycan (GAG), which is a differentiation marker of chondrocytes, was measured to evaluate chondrocyte differentiation of the porous polymer microspheres containing simvastatin according to Example 1. Specifically, 1 × 10 ⁵ stem cells were dispensed into the microspheres of Example 1 and Comparative Example 1, and then differentiated and cultured for 7 days, 14 days, and 21 days. After incubation, the cells were washed with PBS buffer and then cells were removed from each nanoparticle with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, 0.1% Triton X-100 solution was added to the cells, and ultrasonic membrane was applied at 4 ° C. for 1 minute to crush the membranes of the cells, followed by centrifugation. DNA was isolated using qG-DEX ™ IIc Genomic DNA Extraction Kit and the DNA concentration was measured at 260/280 nm via NanoDrop. In addition, after performing a dimethylmethylene blue (DMMB) assay, the absorbance was measured at 525 nm, and the quantitative analysis of GAG is shown in Table 4 below.

Classification (concentration of drug) GAG / DNA concentration (μg / mL) 21 days later Comparative example 1.1 Example 1 1 mM 1.4 5 mM 1.7 10 mM 2.6 Example 2 1 mM 1.3 5 mM 1.6 10 mM 2.3 Example 3 1 mM 1.2 5 mM 1.6 10 mM 2.2 Example 4 1 mM 1.4 5 mM 1.7 10 mM 2.4 Example 5 1 mM 1.3 5 mM 1.6 10 mM 2.2

As shown in Table 4, the GAG / DNA concentrations of the stem cells differentiated into chondrocytes in the porous polymer microspheres of Examples 1 to 5 were evaluated to increase effectively compared with the comparative example.

Test Example  5.2. Gene expression analysis

Chondrocyte differentiation of the porous polymeric microspheres according to the examples was evaluated through expression analysis of late differentiation marker genes (Aggrecan, COL1A1, COL2A1 and COL10A1) of chondrocytes. Specifically, 1 × 10 ⁵ stem cells were dispensed into the microspheres of Examples and Comparative Examples, and then differentiated and cultured for 21 days. After incubation, the cells were washed with PBS buffer and then cells were removed from each microsphere with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, and then Trizol reagent solution and chloroform were added to the cells, and the cells were separated by ultrasonication at 4 ° C. for 1 minute, followed by centrifugation. After the supernatant was separated, RNA concentration was measured at NanoDrop 260/280 nm using RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila). The isolated RNA was analyzed by cDNA synthesis and real-time polymerase chain reaction (Real-time PCR) using AccuPower RT-PCR PreMix (Bioneer, Korea).

Classification (concentration of drug) Aggrecan /
GAPDH COL1A1 / GAPDH COL2A1 / GAPDH COL10A1 / GAPDH 21 days later 21 days later 21 days later 21 days later Comparative example 1.0 1.0 1.0 1.0 Example 1 1 mM 1.5 1.3 1.3 1.9 5 mM 2.3 1.6 1.8 2.1 10 mM 3.3 2.0 2.2 3.4 Example 2 1 mM 1.4 1.2 1.2 1.8 5 mM 2.2 1.4 1.7 2.0 10 mM 3.1 1.8 2.0 3.2 Example 3 1 mM 1.2 1.3 1.2 1.7 5 mM 2.0 1.7 1.5 1.9 10 mM 3.1 1.9 1.9 2.9 Example 4 1 mM 1.5 1.4 1.3 1.8 5 mM 2.4 1.7 1.9 2.3 10 mM 3.4 2.2 2.4 3.5 Example 5 1 mM 1.4 1.3 1.2 1.8 5 mM 2.0 1.5 1.6 2.2 10 mM 2.9 1.9 1.9 3.0

As shown in Table 5, the comparative example did not have expression of genes related to cartilage differentiation, but cartilage differentiation for stem cells differentiated into chondrocytes in the porous polymer microspheres of Examples 1 to 5 according to the present invention (aggrecan, COL10A1, COL1A1 and COL2A1) increased expression of related late marker genes. Thus, the drug-loaded porous polymer microspheres of the examples according to the present invention proved effective in cartilage differentiation.

Test Example 6 Verification of Inhibitory Effect

Inflammation efficacy of the porous polymer microspheres according to Examples 1 to 5 was evaluated through expression analysis of the inflammatory marker genes.

After dispensing 1 × 10 ⁵ stem cells into microspheres of Examples and Comparative Examples, they were differentiated and cultured for 1 and 3 days. After incubation, Trizol reagent solution and chloroform were added, and the cells were separated by ultrasonication at 4 ° C. for 30 minutes, followed by centrifugation. After the supernatant was separated, RNA concentration was measured at NanoDrop 260/280 nm using RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila). After RNA was isolated, the isolated RNA was analyzed by cDNA synthesis and real-time polymerase chain reaction (Real-time PCR) using AccuPower RT-PCR PreMix (Bioneer, Korea), and COX-2 and IL-6 as inflammatory markers. And TNF-α expression was measured and shown in Table 6 below.

Classification (concentration of drug) TNF-α / GAPDH IL-6 / GAPDH COX-2 / GAPDH 1 day later 3 days later 1 day later 3 days later 1 day later 3 days later Comparative example 4.3 5.0 4.2 4.4 4.0 4.7 Example 1 1 mM 3.8 3.5 3.3 3.0 3.6 3.4 5 mM 3.0 2.5 2.1 1.8 2.2 2.0 Example 2 1 mM 4.0 3.7 3.5 3.1 3.7 3.5 5 mM 3.4 2.6 2.3 2.0 2.4 2.2 Example 3 1 mM 3.9 3.6 3.4 3.2 3.8 3.6 5 mM 3.2 2.7 2.2 2.0 2.5 2.3 Example 4 1 mM 3.7 3.3 3.2 3.1 3.6 3.5 5 mM 3.1 2.6 2.0 1.8 2.1 1.9 Example 5 1 mM 3.8 3.5 3.5 3.1 3.8 3.4 5 mM 3.2 2.6 2.3 2.0 2.4 2.1

Referring to Table 6, while the comparative example is maintained inflammation, the inflammation markers COX-2, IL-6 and TNF-α in the porous polymer microsphere of the embodiment according to the present invention is reduced depending on the concentration of simvastatin It was confirmed.

Test Example 7 Cell Proliferation According to Pore Size of Porous Polymer Microspheres

Porous polymer microspheres of the present invention has the advantage of controlling the pore size and the dose of drug to be loaded according to the concentration of gelatin (gelatin) added during the manufacturing process. Therefore, the degree of cell proliferation in microspheres having different pore sizes was determined according to the gelatin concentration (1 wt%, 7.5 wt% and 15 wt%) in the gelatin aqueous solution added during the preparation of the microspheres. 3 is a scanning electron microscope image of microspheres having different pore sizes depending on the concentration of gelatin (1 wt%, 7.5 wt% and 15 wt%).

To this end, 1 × 10 ⁵ adult stem cells were carefully placed in porous particles having different pore sizes, and then cultured for 1 day, 3 days, and 7 days. After 1, 3 and 7 days of culture, the cells were washed with phosphate buffer solution. After washing, add CCK-8 growth kit reagent (Tetrazolium salt reagent, Cell Counting kit-8, Dojindo, USA) and incubate for 1 hour, and then carefully transfer the culture to a 96-well plate and measure the absorbance at 450 nm. 4 is shown.

Referring to FIG. 4, adult stem cells cultured in porous particles having different pore sizes according to the concentration of gelatin (1% by weight, 7.5% by weight and 15% by weight) are gradually grown after 1 day, 3 days and 7 days. Cells proliferated vigorously, but porous particles made by high concentration of gelatin (15% by weight) compared to other groups (1% by weight and 7.5% by weight of gelatin) showed that the cell proliferation was reduced due to the relatively large pore size. Could.

Hereinafter, the present invention will be described in more detail with reference to preferred examples. However, these examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Claims (23)

delete delete delete delete delete delete delete delete delete 1) biocompatible polymers; And preparing a polymer solution by dissolving simvastatin, pravastatin, mevastatin or dexamethasone in an organic solvent as a drug for treating bone disease or cartilage disease.
2) preparing an emulsion by adding and homogenizing the polymer solution and gelatin solution;
3) The emulsion is used as a discontinuous phase and the polyvinyl alcohol solution is used as a continuous phase to flow in a fluidic device for treating biocompatible polymers, bone diseases or cartilage diseases. Preparing a polymeric microsphere comprising a drug simvastatin, pravastatin, mevastatin or dexamethone, and gelatin; And
4) Method of preparing a porous polymer microsphere for preventing or treating bone diseases or cartilage diseases comprising the step of eluting gelatin from the polymer microspheres by stirring the polymer microspheres in water at 38 to 50 ℃. The method of claim 10,
Method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases, characterized in that it contains 2 to 30 parts by weight of the drug with respect to 10 parts by weight of the biocompatible polymer. The method of claim 10,
Method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases characterized in that the homogenization by adding 3 to 15 parts by weight of the gelatin with respect to 10 parts by weight of the biocompatible polymer. The method of claim 10,
The biocompatible polymer is polyglycolic acid (PGA), polylactic acid (PLA), polylactide-co-glycolide (PLGA), polycaprolactone (PCL), polyamino acid, polylactite, polyphosphazine, polyimino Carbonate, polyphosphoester, polyanhydride, polyorthoester, polyhydroxyvalate, polyhydroxybutyrate, hyaluronic acid, cellulose, heparin, collagen, alginate and chitosan; Polymers in which two or more of these are blended; And a method for producing a porous polymeric microsphere for the prevention or treatment of bone diseases or cartilage diseases, characterized in that selected from the group consisting of two or more copolymers. The method of claim 10,
Step 2) is a method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases, characterized in that further comprises 0.5 to 3 parts by weight of an emulsion stabilizer. The method of claim 10,
Preparation of porous polymeric microspheres for the prevention or treatment of bone diseases or cartilage diseases, characterized in that the porosity is 10 to 90%, the pore size is 5 to 100 ㎛, the particle size is 200 to 1000 ㎛ Way. The method of claim 10,
Preparation of porous polymeric microspheres for the prevention or treatment of bone diseases or cartilage diseases characterized in that the porosity (60 to 90%), the pore size is 10 to 40 ㎛, the particle size is 100 to 400 ㎛ Way. The method of claim 10,
The bone diseases include growth stage development, osteopenia, bone weakness, bone necrosis, fractures, osteoporosis due to excessive absorption of osteoclasts (osteoporosis), bone dysplasia, bone damage, joint damage due to trauma, osteomalacia, rickets, Fibrous osteoarthritis, aplastic bone disease, metabolic bone disease, renal osteodystrophy, periodontal disease, paget disease and metastatic bone cancers,
The cartilage diseases include degenerative arthritis, inflammatory arthritis, osteochondromatosis, sprain, bursitis, meniscus, discoid meniscus tear meniscal tear, meniscus cyst, collateral ligamentrupture, anterior cruciate ligament injury, posterior cruciate ligament injury, intratraarticular vitreous -articular vitreous, exfoliation osteochondritis, plica syndrome, genu varum, knock-knee and pronounced knee Method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases. The method of claim 10,
Step 2) is a method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases, characterized in that homogenizing for 30 seconds to 10 minutes at 8,000 to 20,000 rpm with a homogenizer. The method of claim 10,
Method for producing a porous polymer microsphere for the prevention or treatment of bone diseases or cartilage diseases, characterized in that by controlling the content of the gelatin, drug or the content of the biocompatible polymer to control the discharge and discharge rate of the drug. The method of claim 10,
The gelatin aqueous solution is a method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases, characterized in that 1 to 10% by weight gelatin aqueous solution. The method of claim 10,
The flow rate of the discontinuous phase and the continuous phase is 0.2 to 1 mL / min, respectively, a method for producing a porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases. The method of claim 10,
The organic solvent is one or two or more selected from dimethyl sulfoxide, dichloromethane, tetrahydrofuran, and N, N-dimethylformamide, wherein the porous polymer microspheres for the prevention or treatment of bone diseases or cartilage diseases Manufacturing method. delete

Images