KR20240133027A - Sustained-release particle containing oligopeptide and method for preparing the same - Google Patents

Abstract

The present invention relates to sustained-release particles containing oligopeptides and a method for producing the same, wherein the oligopeptides contained in the sustained-release particles are injected into a joint and continuously released, thereby inducing differentiation of stem cells into chondrocytes, thereby exhibiting an excellent effect in treating arthritis or cartilage damage.
In addition, a method for producing sustained-release particles can be provided in which oligopeptides are uniformly distributed within the sustained-release particles, and thus the release effect of the oligopeptides can be exhibited according to the decomposition of the sustained-release particles in the body.

Description

Sustained-release particle containing oligopeptide and method for preparing the same

The present invention relates to a sustained-release particle comprising an oligopeptide and a method for producing the same.

Cartilage is a structure that exists between the bones of all joints and has both strength and elasticity. The main functions of this cartilage are known to be to prevent friction, maintain elasticity, and distribute the load applied to the knee to provide resistance to pressure. Cartilage, which performs these important functions, can be damaged by traffic accidents, excessive exercise, and especially arthritis.

Arthritis is inflammation of the joints due to various causes, and the representative symptom that appears due to this is joint pain. Arthritis is largely classified into rheumatoid arthritis and osteoarthritis. Rheumatoid arthritis is a type of autoimmune disease caused by inflammation of the tissue called synovium surrounding the joints. It is a chronic disease that affects all joints where synovium exists, and it progresses over several months to several years, and is caused by genetic causes, bacterial or viral infections, and mental and physical stress.

Osteoarthritis, also known as degenerative arthritis, is a type of arthritis that occurs due to degenerative changes in the cartilage and surrounding bones that make up the joint. It mainly causes severe pain and movement disorders in joints that bear a lot of weight, such as the knee joint and hip joint, and if left untreated for a long period of time, it is the most common joint disease that can even lead to joint deformation.

Since this type of cartilage does not have blood vessels within it, once it is damaged, its ability to regenerate on its own is limited, and even if it does regenerate, it is converted into a bone joint and causes pain.

The typical conventional surgical treatment method is to create a small perforation in the subchondral bone to induce bleeding, so that mesenchymal stem cells in the bone marrow can move to the defect area and regenerate cartilage. However, even with this procedure, fibrous cartilage is created instead of normal hyaline cartilage, which not only fails to perform its original function, but also has the problem of easily causing degenerative changes.

In the current degenerative arthritis, the standard treatment is to remove the affected cartilage and bone and perform artificial joint replacement surgery made of metal and polyethylene. However, when the surgery is performed on relatively young patients under the age of 60, the lifespan of the artificial joint becomes an issue.

As the limitations of cartilage tissue regeneration using only cells became known, researchers began to take interest in tissue engineering techniques that introduce cells into a certain support, culture them, and transplant them into the body. Research on cartilage tissue regeneration using support materials made of various materials and stem cells is actively being conducted.

Another treatment method is drug therapy. The drug administration method not only has the possibility of systemic toxicity and various side effects due to the high initial drug concentration, but it is also known that even if the drug concentration in the plasma (in the body) is high, the amount of drug delivered to the affected area is very small.

In contrast, direct topical application of drugs has the advantage of reducing side effects caused by high concentrations of drugs, unlike oral or injectable drugs, as well as showing high efficacy with small amounts of drugs, and can be used by people who are resistant to oral or injectable drugs.

However, since most of the drugs currently in use are water-soluble drugs, rapid initial release (burst) of the drug cannot be avoided. Therefore, although it is a system that can effectively deliver drugs to the affected area, it has the disadvantage of not being easy to deliver drugs over the long term.

There is a need to develop a novel pharmaceutical composition for the treatment of arthritis that can solve the above problems.

KR 10-2020-0087138 A1

The purpose of the present invention is to provide a sustained-release particle comprising an oligopeptide and a method for producing the same.

Another object of the present invention is to provide sustained-release particles which can exhibit excellent effects in the treatment of arthritis or cartilage damage by inducing differentiation of stem cells into chondrocytes by continuously releasing oligopeptides contained in sustained-release particles when injected into a joint.

Another object of the present invention is to provide a method for producing sustained-release particles in which oligopeptides are uniformly distributed within the sustained-release particles, and thus the release effect of the oligopeptides can be exhibited according to the decomposition of the sustained-release particles in the body.

To achieve the above object, the present invention relates to a sustained-release particle comprising an oligopeptide, which may include an oligopeptide composed of 2 to 10 amino acids; and a biodegradable polymer and/or lipid.

The above oligopeptide may be included in an amount of 1 to 10 wt% relative to the weight of the biodegradable polymer.

The above amino acids may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, cystine, methionine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and combinations thereof.

The above oligopeptide may be composed of an amino acid sequence of alanine-alanine-glutamic acid, phenylalanine-phenylalanine-glutamic acid, isoleucine-phenylalanine-glutamic acid, leucine-phenylalanine-glutamic acid, methionine-phenylalanine-glutamic acid, valine-phenylalanine-glutamic acid, tryptophan-phenylalanine-glutamic acid, tyrosine-phenylalanine-glutamic acid, phenylalanine-arginine-asphalic acid, phenylalanine-leucine-glutamic acid, tryptophan-leucine-glutamic acid, tyrosine-isoleucine-asphalic acid, tyrosine-tyrosine-asphalic acid or tyrosine-tyrosine-glutamic acid.

The above biodegradable polymer can be selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polycaprolactone, copolymers thereof, and combinations thereof.

The above lipids are Propylene Glycol Monocaprylate NF, Propylene Glycol Mono and Dicaprylate NF, Glyceryl dibehenate, Behenoyl polyoxyl-8 glycerides, Lauroyl Polyoxyl-32 glycerides, Polyoxyl-32 stearate NF, Stearoyl polyoxyl-32 glycerides, Mixture of Lauroyl Polyoxyl-32 glycerides and PEG 6000, Oleoyl polyoxyl-6 It can be selected from the group consisting of Oleoyl polyoxyl-6 glycerides, Linoleoyl polyoxyl-6 glycerides, Caprylocaproyl polyoxyl-8 glycerides, Glyceryl monooleate, Glyceryl distearate, Diethylene glycol monoethyl ether, and combinations thereof.

The above-mentioned western particles may be a W ₁ (Water)/O (Oil)/W ₂ (Water) emulsion.

According to another embodiment of the present invention, a method for producing sustained-release particles comprising an oligopeptide may include the steps of: dissolving an oligopeptide consisting of 2 to 10 amino acids in water to produce a first aqueous solution; dissolving a biodegradable polymer in an organic solvent to produce an oily solution; dissolving a surfactant in water to produce a second aqueous solution; producing a W ₁ /O emulsion using the first aqueous solution and the oily solution; producing a W ₁ /0/W ₂ emulsion using the first emulsion and the second aqueous solution; and solidifying and evaporating the solvent of the W ₁ /O/W ₂ emulsion.

The above organic solvent may be selected from the group consisting of dichloromethane, dimethyl sulfoxide, ethyl acetate, ether, n-hexane, cyclohexane, tetrahydrofuran, methylene chloride, formamide, dimethyl sulfoxide, isopropanol, chloroform, benzyl alcohol, ethanol, methanol, propanol, acetonitrile, ethyl acetate, acetone, dimethylformamide, tert-butyl alcohol and mixtures thereof.

The above surfactant may be selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, and mixtures thereof.

The present invention provides an oligopeptide contained in a western-type particle that is injected into a joint and continuously released, thereby inducing differentiation of stem cells into chondrocytes, thereby exhibiting an excellent effect in treating arthritis or cartilage damage.

In addition, the present invention provides a method for manufacturing sustained-release particles in which oligopeptides are uniformly distributed within the sustained-release particles, thereby exhibiting a release effect of the oligopeptides according to the decomposition of the sustained-release particles in the body.

FIG. 1 is a drawing of a device for manufacturing nanoparticles according to one embodiment of the present invention.
FIG. 2 is a photograph of an emulsifying device and a porous membrane within the emulsifying device according to one embodiment of the present invention.
Figure 3 shows the results of particle size analysis for Western-type particles according to one embodiment of the present invention.
Figure 4 shows the results of particle size analysis for Western-type particles according to one embodiment of the present invention.
Figure 5 shows the results of a dissolution experiment on Western-type particles according to one embodiment of the present invention.
Figure 6 shows the results of a dissolution experiment on Western-type particles according to one embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Throughout the present invention, "differentiation" may mean a phenomenon in which structures or functions become specialized while cells divide and proliferate and grow, that is, cells, tissues, etc. of a living organism change in form or function in order to perform their respective assigned tasks.

Throughout the present invention, “chondrocytes” may include all chondrocytes induced to differentiate from stem cells, or cells in the process of differentiating into chondrocytes.

Throughout the present invention, "medium" may mean a mixture for culturing or differentiating cells, such as stem cells, in vitro, containing essential elements for cell growth and proliferation, such as sugars, amino acids, various nutrients, serum, growth factors, and minerals.

Stem cells have self-renewal ability and have greater proliferation and regenerative potential than mature cells. In particular, for age-related degenerative diseases such as osteoarthritis, stem cells are expected to be superior to mature cells for treatment due to their rapid differentiation into chondrocytes and lineage differentiation.

Currently, cell therapy for damaged cartilage uses autologous chondrocytes and mesenchymal stem cells in the clinic. Autologous chondrocyte transplantation mainly leads to fibrocartilage tissue, which lacks the mechanical properties of cartilage tissue and degrades over a period of two years.

The sustained-release particles of the present invention are injected into the body to continuously release oligopeptides, and the oligopeptides can promote the regeneration of cartilage tissue in joints by inducing specific differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes.

Specifically, the western-type particles of the present invention may include an oligopeptide consisting of 2 to 10 amino acids; and a biodegradable polymer and/or lipid.

The above biodegradable polymer may be selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polycaprolactone, copolymers thereof, and combinations thereof, and preferably may be selected from the group consisting of polylactic acid, poly(lactic acid-co-glycolic acid), and mixtures thereof, but is not limited to the above examples, and any polymer that is manufactured into sustained-release particles containing the oligopeptide and can continuously release the oligopeptide by decomposition in the body may be used.

The above lipids are Propylene Glycol Monocaprylate NF, Propylene Glycol Mono and Dicaprylate NF, Glyceryl dibehenate, Behenoyl polyoxyl-8 glycerides, Lauroyl Polyoxyl-32 glycerides, Polyoxyl-32 stearate NF, Stearoyl polyoxyl-32 glycerides, Mixture of Lauroyl Polyoxyl-32 glycerides and PEG 6000, Oleoyl polyoxyl-6 The lipid may be selected from the group consisting of Oleoyl polyoxyl-6 glycerides, Linoleoyl polyoxyl-6 glycerides, Caprylocaproyl polyoxyl-8 glycerides, Glyceryl monooleate, Glyceryl distearate, Diethylene glycol monoethyl ether, and combinations thereof. When the lipid is manufactured into nano-sized sustained-release particles, it may be manufactured into an oily solution by dissolving it in an organic solution together with or alone a biodegradable polymer.

In addition, when manufacturing the above-mentioned nano-sized western-type particles, they can be manufactured using only a biodegradable polymer.

The above oligopeptide may be included in an amount of 1 to 10 wt% relative to the weight of the biodegradable polymer. If included in an amount less than the above range, the amount of oligopeptide included in the sustained-release particles may be too small, resulting in insufficient differentiation of stem cells into chondrocytes. If included in an amount exceeding the above range, the problem of difficulty in manufacturing sustained-release particles may occur.

The above-described sustained-release particles may be a W ₁ (Water)/O(Oil)/W ₂ (Water) emulsion. As described below, the sustained-release particles of the present invention may be prepared as an emulsion by dissolving an oligopeptide in a first aqueous solution, dissolving a biodegradable polymer in an oily solution, dissolving a surfactant in a second aqueous solution, and homogeneously mixing the first aqueous solution, the oily solution, and the second aqueous solution using a homogenizer.

The above emulsion can form a W ₁ /O/W ₂ emulsion by uniformly mixing an oligopeptide in a first aqueous solution and a biodegradable polymer in an oily solution, forming an interface by a surfactant in a second aqueous solution.

The above amino acids may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, cystine, methionine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and combinations thereof.

The above oligopeptide is composed of an amino acid sequence of alanine-alanine-glutamic acid, phenylalanine-phenylalanine-glutamic acid, isoleucine-phenylalanine-glutamic acid, leucine-phenylalanine-glutamic acid, methionine-phenylalanine-glutamic acid, valine-phenylalanine-glutamic acid, tryptophan-phenylalanine-glutamic acid, tyrosine-phenylalanine-glutamic acid, phenylalanine-arginine-asphalic acid, phenylalanine-leucine-glutamic acid, tryptophan-leucine-glutamic acid, tyrosine-isoleucine-asphalic acid, tyrosine-tyrosine-asphalic acid or tyrosine-tyrosine-glutamic acid, and preferably may be composed of an amino acid sequence of tyrosine-tyrosine-glutamic acid, but is not limited to the above examples, and any amino acid sequence that has an effect of inducing differentiation of stem cells into chondrocytes may be used without limitation. possible.

The stem cells may include, but are not limited to, those selected from the group consisting of mesenchymal stem cells, induced-pluripotent stem cells, embryonic stem cells, and combinations thereof.

The above mesenchymal stem cells may include, but may not be limited to, those selected from the group consisting of bone marrow-derived stem cells, adipose-derived stem cells, tonsil-derived stem cells, synovial stem cells, and combinations thereof.

The above mesenchymal stem cells may be obtained from bone marrow, tissue, embryo, umbilical cord blood, blood or body fluid, but may not be limited thereto.

The present invention can induce differentiation into chondrocytes by treating stem cells with an oligopeptide consisting of 2 to 10 amino acids.

By treating the stem cells with an oligopeptide, the expression of a gene or protein included in the chondrocytes differentiated from the stem cells may be amplified. For example, the gene or protein may include a biomarker, and specifically, may be selected from the group consisting of collagen type II alpha 1 (COL II), SRY-box 9 (SOX 9), cartilage oligomeric matrix protein (COMP), aggrecan (ACAN), collagen type X alpha 1 (COL X), collagen type I alpha 2 (COL I α2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sulfated glycosaminoglycans (sGAGs), proteoglycans, and combinations thereof, but may not be limited thereto.

In the present invention, the induction of specific differentiation of stem cells into chondrocytes may promote regeneration of cartilage tissue within the joint by inducing specific differentiation of endogenous stem cells or transplanted therapeutic stem cells into chondrocytes, but may not be limited thereto.

The sustained-release particles of the present invention are for the treatment of cartilage damage diseases, wherein the cartilage damage diseases are selected from the group consisting of arthritis, cartilage damage, cartilage defects, degenerative arthritis, rheumatoid arthritis, fractures, plantar fasciitis, humeral ectropion, osteomalacia, and combinations thereof, and may preferably be arthritis or cartilage damage, but are not limited to the above examples.

The above-described western-type particles of the present invention can be mixed with water for injection and used as an injection agent, and in particular, can be used by direct injection into a joint for the treatment of arthritis or cartilage damage.

According to another embodiment of the present invention, a method for producing sustained-release particles comprising an oligopeptide may include the steps of: dissolving an oligopeptide consisting of 2 to 10 amino acids in water to produce a first aqueous solution; dissolving a biodegradable polymer and/or a lipid in an organic solvent to produce an oily solution; dissolving a surfactant in water to produce a second aqueous solution; producing a W ₁ /O emulsion using the first aqueous solution and the oily solution; producing a W ₁ /0/W ₂ emulsion using the first emulsion and the second aqueous solution; and solidifying and evaporating the solvent of the W ₁ /O/W ₂ emulsion.

The above-described western-type particles of the present invention can be produced by a multi-type emulsification method to produce a W ₁ /O/W ₂ emulsion.

Specifically, the step of preparing a first aqueous solution by dissolving an oligopeptide composed of 2 to 10 amino acids in water is to dissolve the oligopeptide in water, and the oligopeptide in the first aqueous solution may be included in an amount of 1 to 10 wt% relative to the weight of the biodegradable polymer described below.

Additionally, the first aqueous solution may further comprise an excipient, and the excipient may be comprised in an amount of 0 to 50 wt% and may be an amphipathic copolymer.

The above amphipathic copolymer may be specifically selected from the group consisting of poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL), poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL), poly(ethylene oxide)-b-poly(butylene oxide) (PEO-b-PBO), poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG), poly(ethylene oxide)-poly(butylene oxide)-poly(ethylene oxide) (PEO-PBO-PEO), polyethylene glycol and mixtures thereof, but is not limited to the above examples. In addition, the excipient may have a weight average molecular weight of 300 to 100,000 Da, but is not limited to the above examples.

The above oily solution is a biodegradable polymer dissolved in an organic solvent, and the biodegradable polymer may be selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polycaprolactone, copolymers thereof, and combinations thereof.

The above biodegradable polymer may be included in an amount of 0.1 to 50 wt% based on the total weight of the organic solvent. The organic solvent may be selected from the group consisting of dichloromethane, dimethyl sulfoxide, ethyl acetate, ether, n-hexane, cyclohexane, tetrahydrofuran, methylene chloride, formamide, dimethyl sulfoxide, isopropanol, chloroform, benzyl alcohol, ethanol, methanol, propanol, acetonitrile, ethyl acetate, acetone, dimethylformamide, tert-butyl alcohol, and mixtures thereof.

The above oil solution may also additionally contain an excipient to ensure the stability of the main ingredient. The excipient may be contained in an amount of 0 to 50 wt% and may be an amphipathic copolymer.

The above amphipathic copolymer may be specifically selected from the group consisting of poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL), poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL), poly(ethylene oxide)-b-poly(butylene oxide) (PEO-b-PBO), poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG), poly(ethylene oxide)-poly(butylene oxide)-poly(ethylene oxide) (PEO-PBO-PEO), polyethylene glycol and mixtures thereof, but is not limited to the above examples. In addition, the excipient may have a weight average molecular weight of 300 to 100,000 Da, but is not limited to the above examples.

In addition, the oily solution may contain lipids. The lipids may be dissolved in the organic solution in place of the biodegradable polymer, and may be mixed with the organic solution together with the biodegradable polymer to produce an oily solution.

The above lipids are Propylene Glycol Monocaprylate NF, Propylene Glycol Mono and Dicaprylate NF, Glyceryl dibehenate, Behenoyl polyoxyl-8 glycerides, Lauroyl Polyoxyl-32 glycerides, Polyoxyl-32 stearate NF, Stearoyl polyoxyl-32 glycerides, Mixture of Lauroyl Polyoxyl-32 glycerides and PEG 6000, Oleoyl polyoxyl-6 It can be selected from the group consisting of Oleoyl polyoxyl-6 glycerides, Linoleoyl polyoxyl-6 glycerides, Caprylocaproyl polyoxyl-8 glycerides, Glyceryl monooleate, Glyceryl distearate, Diethylene glycol monoethyl ether, and combinations thereof.

The above lipid can be dissolved in an organic solvent at a concentration of 0.5 to 10 wt%. In addition, when preparing an oily solution using the above lipid, it should be heated to 20 to 80°C.

The above second aqueous solution can be prepared by dissolving a surfactant in water. The surfactant can be selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, and mixtures thereof, and preferably can be PVA, TPGS, PEG-SA, Tween, Poloxamer, etc., but is not limited to the above examples, and any surfactant that can be used to prepare a W ₁ /O/W ₂ emulsion can be used without limitation.

The above surfactant is added to ensure stability and dispersibility of the multiple emulsion, and may be included in a concentration of 0.01 to 10 wt% in the second aqueous solution.

In addition, the second aqueous solution may additionally include an electrolyte for osmotic control when producing the W ₁ /O/W ₂ emulsion. As described above, if a salt for osmotic control is not additionally included, a problem may arise in which the encapsulation rate of the oligopeptide in the emulsion is reduced. Specifically, the oil (O) membrane of the W ₁ /O/W ₂ emulsion functions as a semi-permeable membrane between the inner and outer aqueous phases, and at this time, an osmotic pressure difference occurs due to the oligopeptide, etc. in the first aqueous solution. When the osmotic pressure of the inner aqueous phase increases, water flows from the outer to the inner phase, and the inner particles become larger due to the incorporation of water, and then the water particles can be released to the outside by coalescence and diffusion phenomena. As described above, the encapsulation rate of the oligopeptide in the finally produced sustained-release particles may be reduced due to the above phenomenon.

The electrolyte included for preparing the above second aqueous solution may be NaCl, but is not limited to the above examples, and any electrolyte that can be used for osmotic control may be used without limitation.

After preparing the first aqueous solution, the oily solution, and the second aqueous solution as described above, an emulsion can be prepared using the solutions.

As a method for producing the above W ₁ /O/W ₂ emulsion, a multiple emulsion can be produced by a two-step emulsification method. Specifically, after mixing the first aqueous solution and the oily solution, a W ₁ /O emulsion can be formed using a homomixer. When forming the W ₁ /O emulsion, the stirring condition was 10,000 rpm to 15,000 rpm for 2 to 5 minutes.

Thereafter, a second phase solution is mixed into the solution in which the W ₁ /O emulsion is formed, and a W ₁ /O/W ₂ emulsion can be prepared using a homomixer or membrane equipment. When preparing the W ₁ /O/W ₂ emulsion, when a homomixer or membrane equipment is used, the injection speed of the solution including the W ₁ /O emulsion may be 0.06 to 200 L/hr, and the injection speed of the second phase solution may be 0.06 to 1,500 L/hr.

The above membrane equipment is as shown in Fig. 1. The emulsifying device (300) can be supplied with W ₁ /0 emulsion and second phase solution through a pipe. The W ₁ /0 emulsion supply unit (100) can supply W ₁ /0 emulsion to the emulsifying device (300) at a constant flow rate using a pump, and the second phase solution supply unit (200) can also supply W 1 /0 emulsion to the emulsifying device (300) at a constant flow rate using a pump.

The above emulsifying device (300) is connected to a second phase solution injection pipe for injecting a second phase solution; a W ₁ /0 emulsion injection pipe for injecting a W ₁ /0 emulsion; and a pipe for supplying the emulsion formed in the emulsifying device to a tank (400), and a porous membrane may be mounted at the interface where the W ₁ /0 emulsion injected through the W ₁ /0 emulsion injection pipe and the second phase solution injected through the second phase solution injection pipe come into contact.

The W ₁ /0 emulsion supplied through the above W ₁ /0 emulsion injection pipe forms a boundary surface in contact with the second phase solution within the emulsifying device. At this time, when the W ₁ /0 emulsion passes through the porous membrane located at the boundary surface, it can be formed in the form of a W ₁ /O/W ₂ emulsion by the second phase solution.

The porous membrane of the present invention may be positioned at a portion where the W ₁ /0 emulsion comes into contact with the second aqueous solution, as shown in FIG. 2. In addition, referring to FIG. 2, the W ₁ /0 emulsion may be injected perpendicular to the flow direction of the second aqueous solution.

The W ₁ /0 emulsion passing through the porous membrane forms a W ₁ /O/W ₂ emulsion on the surface of the membrane, and can be separated from the porous membrane by the flow of the second aqueous solution. The pore diameter of the porous membrane may be 3 to 100 μm.

The W ₁ /O/W ₂ emulsion obtained in the above tank can undergo a solidification and solvent evaporation process. The solidification and solvent evaporation process can be performed under room temperature conditions of 5°C to 40°C, 5°C to 30°C, and 5°C to 20°C for 1 to 24 hours, 1 to 20 hours, and 1 to 15 hours, and the temperature can be raised to evaporate the solvent. The solvent evaporation process can be performed under room temperature conditions of 20°C to 60°C, 30°C to 50°C, and 35°C to 45°C for 1 to 24 hours, 2 to 10 hours, and 2 to 6 hours.

After completing the above solidification and solvent evaporation processes, the temperature of the water bath can be changed to room temperature (5 to 25°C), and the membrane filter can be used to obtain and wash the product. Afterwards, freeze-drying can be performed to finally produce sustained-release particles.

The above freeze-drying conditions may be, under the primary freeze-drying conditions, freeze-drying at -60°C to -20°C for 4 to 8 hours, then freeze-drying at -40°C to -10°C for 8 to 10 hours, and then freeze-drying at -60°C to -20°C for 4 to 8 hours. After the primary freeze-drying, drying may be performed at 5°C to 15°C for 20 to 30 hours, and then drying may be performed at 10 to 30°C for 1 to 3 hours.

The above finally manufactured western-type particles may have a D50 of 0.1 μm to 60 μm.

The above-mentioned sustained-release particles can be manufactured as particles having a D50 of 10 μm to 60 μm, and can also be manufactured as nano-sized particles having a D50 of 50 to 200 nm. The above-mentioned sustained-release particles are spherical particles in which an oligopeptide is uniformly distributed within a biodegradable polymer, and the encapsulation rate of the oligopeptide can exceed 40%. When the encapsulation rate of the oligopeptide is 40% or less, there may be a problem that the effect of inducing specific differentiation of stem cells into chondrocytes is insufficient when injected into a joint.

Manufacturing example

Manufacturing of Western-type particles

Example 1

0.1 g of an oligopeptide consisting of an amino acid sequence of tyrosine-tyrosine-glutamic acid was dissolved in 0.2 mL of DW and 0.2 mL of PEG600 to prepare a first phase solution. 2.0 g of PLA (Resomer 203H) was dissolved in 4.0 mL of DCM to prepare an oily phase solution. The first phase solution and the oily phase solution were mixed and homogenized with a Polytron (12,000 rpm, 3 min.) to prepare a first emulsion. The first emulsion was homogenized with a second phase solution prepared by mixing 800 mL of 0.25% PVA and 1.0 wt% NaCl using a Megatron (5000 rpm, 3 min.). After solidification in a reaction tank at 10°C for 1 hour, solvent evaporation at 40°C for 4 hours, and warming to room temperature for 2 hours, it was obtained using a membrane filter with pores of 5 μm size and freeze-dried.

Example 2

The first aqueous solution was prepared in the same manner as in Example 1, except that the composition of the aqueous solution was a mixture of 0.3 mL of DW and 0.1 mL of PEG600.

Example 3

It was manufactured in the same manner as in Example 1, except that the polymer of the oily solution was changed to PLA (Resomer 202H).

Example 4

It was manufactured in the same manner as in Example 1, except that the polymer of the oily solution was changed to PLGA (Resomer 752H).

Example 5

It was manufactured in the same manner as in Example 1, except that the polymer of the oily solution was changed to a mixture (50:50) of PLGA (Resomer 504H/203H).

Example 6

The second aqueous solution was prepared in the same manner as in Example 1, except that the aqueous solution was changed to a 0.25% PVA solution.

Example 7

Except that the membrane equipment of Fig. 1 was used for the production of the second emulsion, it was produced in the same manner as in Example 1.

Example 8

In the production of an oil solution, 1.0 g of PLA (Resomer 203H) and 1.0 g of biocompatible lipid were mixed, and the process was performed in the same manner as in Example 7, except that ultrafiltration discs and stirred cells were used during the obtaining process.

Experimental method

Experimental Example 1_Particle Size Analysis

Particle size analysis was performed using a Beckman Coulter laser particle size analyzer (LS 13 320 XR particle size analyzer, Beckman Coulter, USA) in the range of 0.01 to 3500 mm in the Universal Liquid Module (ULM) of the suspended solution.

Experimental Example 2_Analysis of Encapsulation Rate

To determine the amount of oligopeptides captured within the microparticles, the freeze-dried microparticles were dissolved in organic solvents (methylene chloride, Acetonitrile) to extract the main components, and then analyzed using liquid chromatography (HPLC, Agilent, 1260 Infinity II system) using the gradient method (A: 0.065% trifluoroacetic in 100% water (v/v), B: 0.050% trifluoroacetic in 100% acetonitrile (v/v)), UV detection wavelength: 220 nm.

Experimental Example 3_Dissolution Analysis

To confirm the drug release behavior in vitro, the lyophilized microparticles were analyzed using the Transwell method under PBS buffer conditions similar to physiological conditions, and the concentration of the eluted drug was analyzed using liquid chromatography (HPLC, Agilent, 1260 Infinity II system) under the gradient method (A: 0.065% trifluoroacetic in 100% water (v/v), B: 0.050% trifluoroacetic in 100% acetonitrile (v/v)), UV detection wavelength: 220 nm.

Experimental Example 4_ Shape Analysis of Microparticles

For morphological analysis of microparticles, the surface of freeze-dried microparticles was coated with platinum and analyzed using a field emission scanning electron microscope (FE-SEM, JSM-6700F, JEOL, Japan).

Experimental Example 5_Shape Analysis of Nanoparticles

For morphological analysis of nanoparticles, freeze-dried nanoparticles were pretreated in a 1.0% phosphotungstic acid hydrate solution, loaded onto a carbon grid, dried, and then analyzed using a transmission electron microscope (FE-TEM, JEM-2100F, JEOL, Japan).

The results of the analysis conducted on the western-type particles of Examples 1 to 8 using the above experimental method are as shown in Table 1 below.

division characteristic SEM Example 1 D50 (um) 45.0 Encapsulation rate (%) 93.0 Dissolution (%, 24hr) 20.7 Example 2 D50 (um) 45.2 Encapsulation rate (%) 80.3 Dissolution (%, 24hr) 21.1 Example 3 D50 (um) 36.8 Encapsulation rate (%) 86.8 Dissolution (%, 24hr) 73.7 Example 4 D50 (um) 40.5 Encapsulation rate (%) 35.9 Dissolution (%, 24hr) 57.1 Example 5 D50 (um) 38.9 Encapsulation rate (%) 66.9 Dissolution (%, 24hr) 22.3 Example 6 D50 (um) 22.2 Encapsulation rate (%) 5.9 Dissolution (%, 24hr) * - Example 7 D50 (um) 22.7 Encapsulation rate (%) 103.8 Dissolution (%, 24hr) 57.4 Example 8 D50 (um) 0.09 Encapsulation rate (%) 96.1 Dissolution (%, 24hr) 64.0

According to the above experimental results, Example 6 did not include an electrolyte in the second aqueous solution, so osmotic control was not performed, and the encapsulation rate of the oligopeptide was very low.

It can be confirmed that Examples 1, 2, 3, 5, 7, and 8 have excellent encapsulation rates. In particular, it can be confirmed that Examples 7 and 8 can be manufactured into long-acting particles with a very uniform diameter, as shown in FIGS. 3 and 4.

In addition, from the results of the dissolution experiment, it can be confirmed that Example 7 (Figure 5) and Example 8 (Figure 6) exhibit a sustained oligopeptide release effect.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims (10)

Oligopeptides consisting of 2 to 10 amino acids; and
Containing biodegradable polymers and/or lipids
Western-type particles containing oligopeptides. In the first paragraph,
The above oligopeptide is contained in an amount of 1 to 10 wt% relative to the weight of the biodegradable polymer.
Western-type particles containing oligopeptides. In the first paragraph,
The above amino acids are selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, cystine, methionine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, and combinations thereof.
Western-type particles containing oligopeptides. In the first paragraph,
The above oligopeptide is composed of an amino acid sequence of alanine-alanine-glutamic acid, phenylalanine-phenylalanine-glutamic acid, isoleucine-phenylalanine-glutamic acid, leucine-phenylalanine-glutamic acid, methionine-phenylalanine-glutamic acid, valine-phenylalanine-glutamic acid, tryptophan-phenylalanine-glutamic acid, tyrosine-phenylalanine-glutamic acid, phenylalanine-arginine-asphalic acid, phenylalanine-leucine-glutamic acid, tryptophan-leucine-glutamic acid, tyrosine-isoleucine-asphalic acid, tyrosine-tyrosine-asphalic acid or tyrosine-tyrosine-glutamic acid.
Western-type particles containing oligopeptides. In the first paragraph,
The above biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polycaprolactone, copolymers thereof, and combinations thereof.
Western-type particles containing oligopeptides. In the first paragraph,
The above lipids are Propylene Glycol Monocaprylate NF, Propylene Glycol Mono and Dicaprylate NF, Glyceryl dibehenate, Behenoyl polyoxyl-8 glycerides, Lauroyl Polyoxyl-32 glycerides, Polyoxyl-32 stearate NF, Stearoyl polyoxyl-32 glycerides, Mixture of Lauroyl Polyoxyl-32 glycerides and PEG 6000, Oleoyl polyoxyl-6 A group selected from the group consisting of Oleoyl polyoxyl-6 glycerides, Linoleoyl polyoxyl-6 glycerides, Caprylocaproyl polyoxyl-8 glycerides, Glyceryl monooleate, Glyceryl distearate, Diethylene glycol monoethyl ether, and combinations thereof.
Western-type particles containing oligopeptides. In the first paragraph,
The above western-type particles are W ₁ (Water)/O (Oil)/W ₂ (Water) emulsions.
Western-type particles containing oligopeptides. A step of preparing a first aqueous solution by dissolving an oligopeptide composed of 2 to 10 amino acids in water;
A step of preparing an oily solution by dissolving a biodegradable polymer and/or lipid in an organic solvent;
A step of preparing a second aqueous solution by dissolving a surfactant in water;
A step of preparing a W ₁ /O emulsion using the first aqueous solution and the oily solution;
A step of preparing a W ₁ /0/W ₂ emulsion using the first emulsion and the second aqueous solution; and
Comprising the steps of solidifying the above W ₁ /O/W ₂ emulsion and evaporating the solvent.
A method for producing a western-type particle comprising an oligopeptide. In Article 8,
The above organic solvent is selected from the group consisting of dichloromethane, dimethyl sulfoxide, ethyl acetate, ether, n-hexane, cyclohexane, tetrahydrofuran, methylene chloride, formamide, dimethyl sulfoxide, isopropanol, chloroform, benzyl alcohol, ethanol, methanol, propanol, acetonitrile, ethyl acetate, acetone, dimethylformamide, tert-butyl alcohol and mixtures thereof.
A method for producing a western-type particle comprising an oligopeptide. In Article 8,
The above surfactant is selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, and mixtures thereof.
A method for producing a western-type particle comprising an oligopeptide.