KR102490400B1 - Composition for treating or prventing osteoarthritis - Google Patents

Abstract

The present invention may provide a pharmaceutical composition for preventing or treating osteoarthritis, including a carrier carrying a type II collagen peptide and rapamycin. The present invention preferably can reduce an inflammatory response caused by inflammatory immune cells occurring in the joint, alleviate cartilage destruction and pain accompanied by the inflammatory response, and further regenerate cartilage. Further, the composition of the present invention can be used as medicine for osteoarthritis, and also inflammatory diseases such as myocardial infarction, cerebral infarction, and spinal cord injury or autoimmune diseases such as atopic dermatitis, rheumatoid arthritis, type 1 diabetes, Crohn's disease, lupus, etc. by carrying other disease-related antigens.

Description

Pharmaceutical composition for preventing or treating osteoarthritis {COMPOSITION FOR TREATING OR PRVENTING OSTEOARTHRITIS}

The present invention relates to a pharmaceutical composition for the prevention or treatment of osteoarthritis, comprising a type 2 collagen peptide and rapamycin-loaded carrier.

Osteoarthritis is known as the most representative joint disease together with rheumatoid arthritis. Osteoarthritis is accompanied by overgrowth of bone around the joints, joint deformity and pain, and progressive movement disorders along with the progression of the disease. Currently, it is known that about 80% of the world's population over the age of 55 and most of the population over the age of 75 have degenerative arthritis. In Korea, the number of people complaining of pain due to osteoarthritis is gradually increasing as society's aging progresses rapidly. As a result, medical expenses are also increasing rapidly.

Osteoarthritis treatment methods known to date include anti-inflammatory drug administration, non-drug therapy for improving lifestyle and eating habits, and outpatient treatment for joint surgery in an invasive manner. Nonsteroidal anti-inflammatory drugs are effective in reducing pain through inflammation, but may cause gastrointestinal side effects, and COX-2 inhibitors, which are drugs used as an alternative, may cause cardiovascular side effects and gastrointestinal damage. In addition, in the case of an outpatient surgical method, it is possible to effectively remove the cause of pain, but there is a burden of possibility of reoperation depending on the condition of the joint and the artificial joint.

In general, osteoarthritis is known to be caused by physical factors, but as the immune system is known to be involved in osteoarthritis through a number of recent papers, understanding of immune cell action in osteoarthritis and immune regulation based on it has become important. The most characteristic features of osteoarthritis are chondrocyte death and cartilage wear. When cartilage wear begins due to physical causes, disease, environment, and genetic factors, etc., the number of inflammatory T cells such as type 1, type 2, and type 17 cells and type 1 macrophages in the synovial membrane increases, while the number of anti-inflammatory regulatory T cells increases. The number of cells (regulatory T cells, Tregs) and type 2 macrophages are reduced. At this time, inflammatory cells secrete inflammatory cytokines such as interleukin 1β (IL-1β), interferon γ (IFN- γ), and tumor necrosis factor α (TNF-α). The secreted cytokines act on the surrounding T cell population to promote the activation of inflammatory cells to secrete more inflammatory cytokines, and at the same time act on chondrocytes to secrete extracellular matrix degrading enzymes such as Matrix Metalloproteinase 13 (MMP-13). promote MMP-13 induces cartilage wear by degrading type 2 collagen protein, which is the most representative extracellular matrix component in cartilage. Inflammatory immune cells and cytokines are relatively dominant over anti-inflammatory immune cells and cytokines, and if the imbalance between these inflammatory and anti-inflammatory immune cells and substances is not corrected, an inflammatory environment is continuously maintained in the joint. Celebrex, a representative anti-inflammatory substance that is currently commercially available, can relieve inflammation, but it only relieves ongoing pain and is difficult to be a fundamental solution. Depending on the condition of the joint, there is a possibility of reoperation in the future, so this method is also difficult to say as a fundamental treatment method.

Since regulatory T cells can suppress the immune response, research and clinical trials are being conducted as a treatment for various inflammatory diseases, and it has been found that they can suppress excessive immune responses in autoimmune diseases, allergic diseases, organ transplant rejection, and the like. However, since adaptive Treg cell transfer requires a step of proliferating/cultivating cells in vitro after isolating cells from a patient, there are disadvantages in that treatment cost is expensive and preparation of the treatment is complicated. In addition, since regulatory T cells of various clones that bind to and act on various antigens are cultured and transplanted into patients, side effects such as infection and cancer may occur due to systemic immunosuppression. In order to solve this problem, it is necessary to develop a therapeutic agent that induces regulatory T cells against a single antigen in the body.

Korea Patent No. 1723265

An object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoarthritis.

Preferably, it is an object of the present invention to provide a composition capable of reducing the inflammatory response by inflammatory immune cells generated in bone joints, alleviating cartilage destruction and pain accompanying the inflammatory reaction, and further regenerating cartilage.

1. Type 2 collagen peptides; and rapamycin or a derivative thereof; A pharmaceutical composition for preventing or treating osteoarthritis comprising a.

2. The pharmaceutical composition for preventing or treating osteoarthritis according to 1 above, wherein the type 2 collagen peptide comprises the amino acid sequence of SEQ ID NO: 1.

3. The pharmaceutical composition for preventing or treating osteoarthritis according to 1 above, wherein the type 2 collagen peptide and rapamycin have a weight ratio of 1 to 0.1 to 10.

4. The pharmaceutical composition for preventing or treating osteoarthritis according to 1 above, further comprising dendritic cells (DC).

5. The method of 1 above, the type 2 collagen peptide; and rapamycin or a derivative thereof is supported in a carrier. A pharmaceutical composition for preventing or treating osteoarthritis.

6. The method of 5 above, wherein the carrier is a virus carrier, virus-like particle (VLP), positively charged polymer, liposome, lipid nanoparticle, gold and semiconductor nanocrystal particles (quantum dot) A pharmaceutical composition for preventing or treating osteoarthritis, which is selected from the group comprising:

7. In the above 5, the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene A pharmaceutical composition for preventing or treating osteoarthritis, which is a lipid nanoparticle containing glycol)] (PEG PE) and cholesterol.

8. The pharmaceutical composition for preventing or treating osteoarthritis according to 5 above, wherein the lipid nanoparticle has a diameter of 50 nm to 1,000 nm.

The present invention may provide a pharmaceutical composition for preventing or treating osteoarthritis, including a carrier containing type 2 collagen peptide and rapamycin. This minimizes the inflammatory reaction caused by type 2 collagen peptides exposed from osteoarthritis articular cartilage, thereby promoting the prevention or treatment of osteoarthritis. Furthermore, not only osteoarthritis, but also other disease-related antigens instead of type 2 collagen peptides can be used to treat inflammatory diseases such as myocardial infarction, cerebral infarction, and spinal injury, or autoimmune diseases such as atopy, rheumatoid arthritis, type 1 diabetes, Crohn's disease, and lupus. can be used as

1 is data analyzing the manufacturing method and characteristics of lipid nanoparticles. It was confirmed that the lipid nanoparticles can normally support type 2 collagen peptides and rapamycin.
FIG. 2 is data confirming that lipid nanoparticles loaded with type 2 collagen peptide and rapamycin can induce differentiation of undifferentiated dendritic cells into tolerogenic dendritic cells.
FIG. 3 is data confirming that lipid nanoparticles carrying type 2 collagen peptide and rapamycin are distributed in the lymph node (LN) near the injection site when injected into an in vivo model.
4 shows that type 2 collagen peptides or peptides having amino acid sequences 259 to 273 of type 2 collagen and lipid nanoparticles carrying rapamycin can activate type 2 collagen-specific regulatory T cells in vivo This is the data confirmed.
5 is data confirming that type 2 collagen peptides or peptides having amino acid sequences 259 to 273 of type 2 collagen and lipid nanoparticles loaded with rapamycin have symptoms alleviated and/or improved in osteoarthritis-induced model mice. to be.
6 is a schematic view of the osteoarthritis treatment mechanism based on lipid nanoparticles of the present invention.

Hereinafter, the present invention will be described in detail. Unless otherwise defined, all terms in this specification are the same as the general meaning of the term understood by a person skilled in the art to which the present invention belongs, and if it conflicts with the meaning of the terms used in this specification, Follow the meaning used in the specification.

The present invention relates to a pharmaceutical composition for preventing or treating osteoarthritis, comprising a type 2 collagen peptide; and rapamycin or a derivative thereof.

The present invention induces differentiation of immature dendritic cells into tolerant dendritic cells by using a type 2 collagen peptide together with rapamycin or a derivative thereof, and induces a type 2 collagen peptide epitope in the MHC molecule of tolerant dendritic cells. can be presented. The dendritic cells induce a large amount of regulatory T cells specific to the type 2 collagen peptide, and the regulatory T cells can suppress inflammatory immune cells in the joint inflammation site to alleviate and/or treat osteoarthritis symptoms.

In the present invention, the type 2 collagen peptide may be a peptide containing at least a partial amino acid sequence of type 2 collagen protein. Type 2 collagen protein has an epitope that binds to MHC molecules of dendritic cells. The at least part may include a sequence that can be recognized as the epitope.

A sequence that can be recognized as an epitope refers to a sequence that operates as an epitope even if it consists of the entire epitope sequence, some amino acids are added to both ends of the sequence, or some amino acids are removed from both ends of the sequence.

The epitope may be the amino acid sequence of SEQ ID NO: 1, and the sequence recognizable as the epitope may include the same or may not include 1 to 3 amino acids at both ends thereof. In addition to the amino acid sequence of SEQ ID NO: 1, any sequence that can be recognized as the epitope may be included without limitation, and a known epitope sequence may be used, or a fragment of type 2 collagen protein cleaved with collagenase may be included. As long as at least part of the above is included, the type/length of the sequence added to both ends is not limited, and the entire type 2 collagen protein may be used.

As mentioned above, the length of the type 2 collagen peptide is not limited as long as it binds to MHC molecules and can be recognized as a type 2 collagen peptide epitope, for example, 0.5 kDa to 120 kDa, 1 kDa to 100 kDa, 1.5 kDa to 50 kDa, 2 kDa to 30 kDa, 3 kDa to 20 kDa, and the like.

The type 2 collagen peptide may fall within the scope of the present invention regardless of which species the collagen peptide is derived from. Type 2 collagen is a gene with a high degree of conservation between species. For example, a type 2 collagen peptide derived from a species selected from the group including human, mouse, cow, goat, horse, cat, and dog is also a gene of the present invention. may fall within the range. Even if some of the sequences that do not match between species are included among the type 2 collagen peptide sequences, the effect of the present invention can be exhibited. For example, even if one or two amino acids are substituted in the amino acid sequence of SEQ ID NO: 1, tolerance The effect of the present invention can be exhibited as long as it is recognized as a type 2 collagen peptide epitope by MHC molecules of dendritic cells. More specifically, the 8th amino acid in SEQ ID NO: 1 may be aspartic acid or glutamic acid, and the 15th amino acid in SEQ ID NO: 1 may be threonine or proline. However, it is not limited thereto.

In the present invention, the rapamycin or a derivative thereof may be included in the scope of the present invention regardless of its type. In the present invention, rapamycin can inhibit the growth of lymphocytes and promote differentiation to induce regulatory T cells specific for type 2 collagen peptides, and any derivative of rapamycin capable of performing these functions is not limited thereto. may be included in the scope of the invention. Examples thereof include benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, pimecrolimus, tacrolimus, and rida pololimus. However, it is not limited thereto.

The composition of the present invention may further include dendritic cells (DC). The dendritic cells may be exposed to a solution containing type 2 collagen peptide and/or rapamycin, and in this case, rapamycin is introduced into the dendritic cell by the type 2 collagen peptide and/or rapamycin, thereby reducing the Type 2 collagen peptides can be presented to MHC molecules on the cell surface. However, it is not limited thereto.

In the present invention, the osteoarthritis is a disease caused by gradual/irreversible degeneration of cartilage tissue, and includes degenerative joint disease, ostoarthrosis, hypertrophic arthritis or degenerative arthritis ( degenerative arthritis), which corresponds to the term referring to the same diseases as the above names. Symptoms of osteoarthritis include: cartilage loses its elasticity and is more easily damaged by injury or use; A stage in which wear of the cartilage causes changes in the underlying bone, in which the bone thickens and may develop cysts, and growths of bone called spurs or osteophytes at the end of the bone in the affected joint. causing itching and pain; loosely floating pieces of bone or cartilage in the joint space; And a step of causing inflammation in the joint membrane or synovial membrane due to cartilage breakdown, which can be classified into four steps as a step of additionally causing cytokines and enzymes that damage cartilage. This corresponds to a disease distinct from rheumatoid arthritis, which causes rapid damage and/or destruction of cartilage tissue due to an autoimmune disease or a systemic inflammatory response.

In the present invention, the composition reduces the number of inflammatory immune cells, such as macrophages and type 1 helper T cells, entering cartilage through the synovial membrane for osteoarthritis, or inflammatory cytokines by inflammatory immune cells, such as For example, by suppressing the secretion of interleukin 1β, TNF-α or IFN-γ, the inflammatory response in cartilage can be alleviated. In addition, it is possible to inhibit the destruction of proteins constituting cartilage tissue and/or extracellular matrix accompanying an inflammatory reaction and alleviate pain.

The weight ratio of the type 2 collagen peptide of the present invention and rapamycin may be included in the scope of the present invention without limitation. For example, it may be 1 to 0.1 to 10, 1 to 0.2 to 8, 1 to 0.3 to 5, 1 to 0.5 to 3. However, it is not limited thereto.

The type 2 collagen peptide of the present invention; And rapamycin or a derivative thereof may be carried in a delivery system and delivered.

The delivery vehicle is a type 2 collagen peptide; and the delivery efficiency of rapamycin or its derivatives to dendritic cells, resulting in the induction efficiency of tolerant dendritic cells presenting type 2 collagen peptides to type 2 MHC molecules and type 2 collagen peptide-specific regulatory T cells. can greatly increase the induction efficiency.

The delivery system has type 2 collagen peptides therein; And rapamycin or a derivative thereof can be loaded and delivered to target cells to release internal substances, which can be selected without limitation by those skilled in the art. For example, selected from the group consisting of virus carriers, virus-like particles (VLPs), positively charged polymers, liposomes, lipid nanoparticles, gold and semiconductor nanocrystal particles (quantum dots) it could be However, it is not limited thereto.

The lipid nanoparticles can be selected and used by those skilled in the art regardless of their components. As the phospholipid molecules constituting the lipid nanoparticles, neutral lipids, anionic and / or cationic phospholipids can all be used, for example, as neutral lipids, L-α-phosphatidylcholine (L-α-phosphatidylcholine, PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn- Glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phophocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoylsn -glycero-3-phophocholine, DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC) It may be at least one selected, and the PC may be PC derived from soybean, egg, hydrogenated soybean or egg, but is not limited thereto.

The anionic lipids include L-α-phosphatidic acid, L-α-phosphatidyl-DL-glycerol, cardiolipin, L -α-phosphatidylinositol (L-α-phosphatidylinositol), L-α-phosphatidylserine (L-α-phosphatidylserine), 1,2-dilauroyl-sn-glycero-3-[phospho-rac-( 1-glycerol)](1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DLPG), 1,2-dilauroyl-sn-glycero-3-[ Phospho-L-serine] (1,2-dilauroyl-sn-glycero-3-[phospho-L-serine], DLPS), 1,2-dilauroyl-sn-glycero-3-phosphate (1 ,2-dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]1,2-dimyristoyl- sn-glycero-3-[phospho-rac-(1-glycerol)], DMPG), 1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] (1,2- dimyristoyl-sn-glycero-3-[phospho-L-serine] (DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (1,2-dimyristoyl-sn-glycero-3-phosphate , DMPA), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1,2-dioleoyl-sn-glycero-3-[phospho-rac-( 1-glycerol)], DOPG), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine](1,2-dioleoyl-sn-glycero-3-[phospho-L- serine], DOPS), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-dipalmitoyl-sn- glycero-3-[phospho-L-three Lin] (1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine], DPPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate (1,2-dipalmitoyl-sn -glycero-3-phosphate, DPPA), 1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1,2-distearoyl-sn-glycero-3- [phospho-rac-(1-glycerol)], DSPG), 1,2-distearoyl-sn-glycero-3-[phospho-L-serine](1,2-distearoyl-sn-glycero-3 -[phospho-L-serine], DSPS), 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), 1,2- Disteroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000](1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000] ), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) 2000]), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP (polyethylene glycol) 2000] (1,2-distearoyl-sn-glycero -3-phosphoethanolamine-N-[PDP (polyethylene glycol) 2000]), 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)](1-palmitoyl -2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], POPG), 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-L-serine ](1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serin e], POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) and oleic acid (oleicacid). It may be at least one selected from the group, but is not limited thereto.

As the cationic lipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), 1,2-dioleoyl- 3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dioleoyl glutamide, distearoylglutamide, dipalmitoyl glutamide) glutamide), dioleoylaspartamide, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), β-[N-(N ',N'-dimethylaminoethane-carbamoyl], (3β-[N-(N',N'-dimethylaminoethane)-carbamoyl], DC-Chol), dimethyldiochtadecylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero -3-phosphoethanolamine (1,2-distearoyl-sn-glycero-3-phophoethanolamine (DSPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoyl -sn-glycero-3-phophoethanolamine, DPPE), but may be at least one selected from the group consisting of, but is not limited thereto.

The lipid particle may be PEGylated on the surface of the phospholipid with additional polyethylene glycol (PEG) to improve the fluidity, loading efficiency and / or stability of the particle, and cholesterol, 1,2-Dioleoyl-3-trimethylammoniumpropane ( DOTAP) or dioleoylphosphatidylethanolamine (DOPE) may be further included. However, it is not limited thereto.

In the present invention, the average diameter of the carrier is included in the scope of the present invention without limitation, and may be selected by those skilled in the art according to conditions such as the amount of peptide and drug to be loaded. The larger the diameter of the carrier, the larger the amount of material can be loaded, but it can affect the blood stability, blood movement speed, etc. of the carrier, so it is possible to select an average diameter within an appropriate range. For example, the average diameter may be 50 nm to 1,000 nm. For example, the lower limit of the average particle diameter may be 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm, and the upper limit may be, for example, 1,000 nm, 900 nm, 800 nm, 700 nm, or 600 nm. there is. However, it is not limited thereto.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in relation to the prevention or treatment of osteoarthritis, or a compound that maintains/increases the solubility and/or absorption of the active ingredient.

A suitable dosage of the composition of the present invention will be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. can However, it is not limited thereto. In addition, the concentration of the active ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentration.

The composition of the present invention is administered in a pharmaceutically effective amount. The effective dose level depends on the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. can be determined The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies widely depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease, etc., and the appropriate dosage is, for example, the patient It may vary depending on the amount of drug accumulated in the body and/or the specific efficacy of the delivery system of the present invention used. For example, it may be 0.01 μg to 1 g per 1 kg of body weight, and may be administered once or several times per unit period per day, week, month, or year, or continuously administered for a long period of time using an infusion pump. It can be. The number of repeated administrations is determined in consideration of the time the drug stays in the body, the concentration of the drug in the body, and the like. The composition may be administered for recurrence even after treatment has been completed according to the course of disease treatment.

Hereinafter, examples will be described in detail to explain the present invention in detail.

Preparation Example 1. Preparation of lipid nanoparticles

Osteoarthritis-specific T cell-inducing lipid nanoparticles were prepared. 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 1,2-dioleoyl-sn-glycero-e-phosphethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- After preparing N-[methoxy(polyehylene glycol)-2000] (ammonium salt) (PEG2000 PE) and cholesterol at concentrations of 10 mg/mL, 25 mg/mL, 10 mg/mL, and 25 mg/mL, respectively, Each lipid component was added to the vial in a molar ratio of 0.475:0.15:0.05:0.12. After adding 70 μg of rapamycin prepared at 10 mg/mL, the glass vial was rotated to blow off the organic solvent to form a thin lipid film, and the organic solvent was further evaporated for 1 hour using a stirrer set at 40 °C. Thereafter, 100 μg of type 2 collagen peptide treated with collagenase III enzyme or type 2 collagen peptide of SEQ ID NO: 1 and 1 mL of phosphate buffered saline were added to a glass vial, and then 2 Time-stirring was conducted to firstly produce type 2 collagen and rapamycin-loaded nanoparticles (LNP-Col II-R or LNP-Col II259-273-R). Thereafter, final lipid nanoparticles having a size of 200 nm were prepared through a continuous extrusion process using an extruder.

Example 1. Confirmation of characteristics of lipid nanoparticles

The size of the nanoparticles prepared by the method of Preparation Example 1 above was measured using a dynamic light scattering device, and the shape was confirmed by transmission electron microscopy (FIG. 1a). The average diameter of the prepared lipid nanoparticles was 215.4 ± 53.8 nm, and it was confirmed that they had a spherical shape. In order to confirm the drug loaded on the nanoparticles, High Performance Liquid Chromatography and Differential Scanning Calorimetry were used, and as a result, it was confirmed that rapamycin was loaded with a loading efficiency of 65.9% ( Fig. 1b, 1c). In addition, it was confirmed through confocal microscopy that type 2 collagen was supported in the nanoparticles (FIG. 1d). To confirm the stability of the lipid nanoparticles, the nanoparticles were placed in a liquid containing 50% (v/v) serum and the size change of the nanoparticles was monitored daily with a dynamic light scattering device (Fig. 1e). As a result, it was confirmed that there was almost no change in the size of the nanoparticles until the third day, which was judged to be sufficient time for the nanoparticles to be absorbed by dendritic cells in the body after nanoparticle injection.

Example 2. Confirmation of anti-inflammatory cell inducing ability of lipid nanoparticles

In order to confirm the induction of anti-inflammatory cells of the nanoparticles prepared in Preparation Example 1 above, dendritic cells were isolated from the bone marrow of 6-week-old C57BL/6J experimental mice ordered from Orient Bio. Different types of lipid nanoparticles were prepared, and the nanoparticles were treated according to the concentration of 10 μg antigen/well to the cells spread at a density of 1×10 ⁶ dendritic cells/well, and 2 days later, 200 ng/ml LPS was treated for 24 hours. Then, the cells were scraped with a cell scraper, and the CD40, CD80, and CD86 phenotypes, known as mature dendritic cell markers, were compared between groups by flow cytometry, and inflammatory (TNF-α) and anti-inflammatory (TGF-β) cytokine expression Levels were compared and analyzed between groups by polymerase chain reaction. As a result, the lipid nanoparticles containing rapamycin effectively induced immature dendritic cells compared to the nanoparticles without rapamycin, and it was confirmed that they mainly secrete anti-inflammatory cytokines ( FIGS. 2a and 2b ). Before proceeding with the above experiments, nanoparticle uptake experiments of dendritic cells were conducted. Cells spread at a density of 1×10 ⁶ dendritic cells/well and nanoparticles prepared in Preparation Example 1 above were labeled with DiI fluorescent material and co-cultured. After 24 hours, the dendritic cells were stained with DAPI and the result was analyzed by confocal microscopy. As a result, about 92% of the dendritic cells absorbed the nanoparticles. Co-cultured with FITC-labeled Col II peptide nanoparticles on dendritic cells spread under the same conditions and analyzed by flow cytometry after 24 hours. As a result, about 93% of dendritic cells carried nanoparticles carrying Col II peptide As a result of confocal microscopy, the absorbed Col II peptide appeared on the surface of dendritic cells. In order to prove that the Col II peptide acts as an antigen and exists on the MHC II molecule of dendritic cells, the nanoparticles prepared in Preparation Example 1 were substituted for the Col II peptide with Eα peptide and co-cultured with dendritic cells. After 24 hours, dendritic cells were stained with an antibody that recognizes Eα peptide and MHC II at the same time and analyzed by flow cytometry and confocal microscopy. As a result, about 82% of dendritic cells absorbed nanoparticles carrying Eα peptide, They displayed the corresponding antigen on the MHC II molecule (Fig. 2c).

Example 3. Lipid Nanoparticles in vivo characterization

In order to check the distribution pattern of the produced lipid nanoparticles in the body, 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR), a fluorescent substance dispersed at a concentration of 2 mg/mL, was added at a ratio of LNP:DiR=500:1 to obtain fluorescence. These labeled lipid nanoparticles were fabricated. 10-week-old C57BL/6 mice were ordered from Orient Bio, and fluorescently labeled nanoparticles were injected into the skin near the right groin of the animals anesthetized with ketamine (200 mg/kg) and lumpoon (10 mg/kg). . 24 hours after the injection, the distribution pattern of the nanoparticles over time was followed and observed using a luminescence-fluorescence preclinical molecular imaging system (IVIS spectrum). As a result, fluorescent substances were found only in the right lymph node near the inguinal region, and no fluorescent substances were found in other organs (heart, lung, liver, spleen, left lymph node near the inguinal region), indicating that the injected nanoparticles migrated to nearby lymph nodes. confirmed (FIG. 3).

Example 4. Confirmation of induction of immune response according to mouse DMM surgery and treatment of lipid nanoparticles

Animal experiments were performed to confirm the possibility of acting as an autoantigen of collagen type 2 in osteoarthritis. C57BL/6 experimental animals were ordered from Orient Bio, and osteoarthritis was induced by medial meniscus destabilization surgery on the right leg of the experimental animals anesthetized with ketamine (200 mg/kg) and lumpoon (10 mg/kg). A stereomicroscope was used for precise surgery. After disinfecting the right leg of the anesthetized experimental animal with ethanol, the lower part of the patellar tendon was opened with a No. 11 blade, and the fat was removed to secure the visual field. After confirming the translucent medial meniscus, the medial meniscus femoral ligament was carefully cut with a blade. In this process, the operation was performed carefully so as not to damage the surrounding cartilage, ligament, and bone tissue. After the medial meniscus destabilization surgery was completed, the internal tissue and the external skin tissue were sutured with an absorbable suture and a non-absorbable suture, respectively, and povidone iodine was applied to the surgical site to disinfect the wound to complete the induction of osteoarthritis.

After osteoarthritis was induced, lipid nanoparticles (LNP-Col II-R) loaded with type 2 collagen peptide and regulatory T cell inducer (rapamycin) were injected intradermally to conduct osteoarthritis treatment experiments. In the control group, nanoparticles loaded with ovalbumin and rapamycin (LNP-OVA-R) were injected. In order to confirm the induction of antigen-specific immunoregulatory T cells, LNP-Antigen-R nanoparticles were injected intradermally into mice, and after a certain period of time, the isolated splenocytes were treated with different antigens to detect CD4+CD25+Foxp3+ by antigen. Stimulation and proliferation of regulatory T cells were compared and analyzed between groups. As a result, it was confirmed that regulatory T cells (of immunotolerance) were activated specifically for collagen type 2 (FIG. 4a). Rheumatism has a more severe degree of inflammation than osteoarthritis, but according to research results that the mechanism that causes the disease and the cells involved in it are not significantly different from osteoarthritis, antigen-specific antigens for type 2 collagen 259-273, known as rheumatism antigen in osteoarthritis The reaction was analyzed further. The experimental method was carried out in the same manner as above, and as a result, it was confirmed that the T cells of the osteoarthritis mice also specifically responded to the type 2 collagen 259-273 antigen, although it was insignificant compared to the type 2 collagen (FIG. 4b). .

Example 5. Confirmation of arthritis treatment effect according to mouse DMM surgery and treatment of lipid nanoparticles

In order to confirm the therapeutic effect of the lipid nanoparticles prepared in Preparation Example 1 on osteoarthritis, the DMM surgery of Example 4 was performed prior to the experimental animals. The prepared nanoparticles were injected intradermally near the right inguinal region according to the experimental plan shown in FIG. 5a, and 8 weeks after surgery, the experimental animals were sacrificed, the right joint area was collected, and a paraffin block was fabricated through fixation and demineralization. Tissues were cut with a thickness of 3 μm using a slice cutter, and the treatment effects of nanoparticles were compared and analyzed between groups using various tissue staining techniques.

As a result of histological examination of paraffin blocks collected by the safranin o fast green staining technique, the articular cartilage tissue was well preserved in the LNP-Col II-R group, but in the other groups, including the LNP-OVA-R group, the articular cartilage tissue was well preserved. It was confirmed that the cartilage tissue was destroyed (Figs. 5a and 5b). In addition, as a result of immunochemical staining analysis of the joint synovium 8 weeks after the start of the experiment, the distribution and activity of IL-10-secreting regulatory T cells were the highest in the LNP-Col II-R group, and type 1 macrophages infiltrated into the synovial membrane. , it was confirmed that the number of inflammatory immune cells, such as CD4+ Th1 cells secreting IFN-γ and CD4+ Th17 cells secreting IL-17, decreased the most (FIG. 5c-g). The decrease in inflammatory cells and substances and the increase in anti-inflammatory cells and substances reduced the pain caused by osteoarthritis, which was confirmed through an allodynia test and an incapacitance test (FIG. 5h). Eight weeks after the end of the experiment, a polymerase chain reaction experiment was conducted with the joint tissue homogenized with a homogenizer, and the expression of type 2 collagen mRNA in the joint tissue injected with LNP-Col II-R was increased compared to other experimental groups. confirmed (Fig. 5i). It was confirmed that the nanoparticles prepared through the process of Preparation Example 1 had a therapeutic effect on osteoarthritis by suppressing joint wear and regenerating the cartilage extracellular matrix through type 2 collagen-specific immunomodulation (immunotolerance). . In addition, in order to confirm the therapeutic effect of collagen type 2 259-273 on osteoarthritis, the lipid nanoparticles (LNP-Col II259-273-R) prepared in Preparation Example 1 were injected intradermally into experimental mice undergoing DMM surgery. As a control, lipid nanoparticles carrying only rapamycin were injected. Eight weeks after the start of the experiment, the collected right leg joints were analyzed with safranin-o-fast green staining, and it was confirmed that the cartilage of the LNP-Col II259-273-R group was better preserved than that of the LNP-R group (Fig. 5j). In addition, as a result of analysis through immunochemical staining, it was confirmed that among the groups that underwent DMM surgery, F4/80+iNOS+ M1 macrophages decreased the most and CD4+Foxp3+ Treg cells increased the most in the LNP-Col II259-273-R group. (Fig. 5k, 5l). When type 2 collagen 259-273 was loaded with rapamycin, it was confirmed that it had a therapeutic effect on osteoarthritis.

Claims (8)

type 2 collagen peptides; and rapamycin or a derivative thereof; including,
The rapamycin derivative is a pharmaceutical composition for preventing or treating osteoarthritis selected from the group consisting of benzoyl rapamycin, temsirolimus, everolimus, zotarolimus, biolimus, pimecrolimus, pimecrolimus, tacrolimus and ridaporolimus .
The pharmaceutical composition for preventing or treating osteoarthritis according to claim 1, wherein the type 2 collagen peptide comprises the amino acid sequence of SEQ ID NO: 1.
The pharmaceutical composition for preventing or treating osteoarthritis according to claim 1, wherein the type 2 collagen peptide and rapamycin have a weight ratio of 1 to 0.1 to 10.
The pharmaceutical composition for preventing or treating osteoarthritis according to claim 1, further comprising dendritic cells (DC).
The method according to claim 1, wherein the type 2 collagen peptide; and rapamycin or a rapamycin derivative is supported in a carrier. A pharmaceutical composition for preventing or treating osteoarthritis.
The method according to claim 5, wherein the carrier comprises a virus carrier, virus-like particle (VLP), positively charged polymer, liposome, lipid nanoparticle, gold and semiconductor nanocrystal particle (quantum dot) A pharmaceutical composition for preventing or treating osteoarthritis that is selected from the group.
The method according to claim 5, wherein the carrier is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000] (PEG2000 PE) and a pharmaceutical composition for preventing or treating osteoarthritis, which is a lipid nanoparticle containing cholesterol.
The pharmaceutical composition for preventing or treating osteoarthritis of claim 6, wherein the lipid nanoparticles have a diameter of 50 nm to 1,000 nm.

Images