Controlled-release local injectable composition containing cyclic peptide and method for producing same

Abstract

The present invention relates to particles comprising a cyclosporine 3-derivative and a biodegradable polymer, a controlled release type topical injection composition comprising the same, and use of the same for preventing hair loss and promoting hair growth.

Description

[Technical Field]

[0001] The present invention relates to a controlled release local injectable composition containing a cyclic peptide and a method for preparing the same. [Background technology]

[0002] Human hair grows and falls out through three phases: anagen, catagen, and telogen. Alopecia occurs when the number of hairs in the anagen phase decreases and the number of hairs in the catagen or telogen phase increases, resulting in an abnormally large number of hairs falling out. Causes of hair loss include excess male hormones, poor blood circulation, excessive sebum secretion, bacterial infection, genetic factors, aging, and stress. Androgenetic alopecia (AGA) is hair loss caused by genetic factors and androgens.

[0003] Cyclosporine A (CsA), an immunosuppressant, has been shown to be more effective than minoxidil and finasteride at inducing hair growth and inhibiting the catagen phase, but when it acts on the whole body, it can cause side effects such as high blood pressure, headaches, and a weakened immune system.

[0004] To overcome the drawbacks of cyclosporine A, studies have been conducted to administer it locally using polymer-based controlled-release drug delivery systems. Polymer-based controlled-release drug delivery systems can control the drug release rate, reduce drug toxicity due to systemic effects, and significantly improve therapeutic efficacy. However, when cyclosporine A is loaded into conventional polymer-based drug delivery systems, an initial burst occurs, making sustained, stable release difficult. The initial burst phenomenon means that a significant amount of the drug is released within a very short period of time, such as within a few hours or one to two days.

[0005] Therefore, there is a need for formulations that provide stable, long-term release of topically administered cyclosporine A family drugs, thereby reducing systemic side effects and reducing the number of doses administered, and that adjust the drug release rate depending on the degree and type of hair loss. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a controlled-release topical injection composition containing a cyclosporin-3 derivative and a biodegradable polymer, and a method for preventing hair loss and promoting hair growth using the same. [Means for solving the problem]

[0007] One embodiment provides controlled-release particles comprising a cyclosporin 3-derivative and a biodegradable polymer.

[0008] Cyclosporine A(C 62 H 111 N 11 O 12 Cyclosporine A is a cyclic compound consisting of 11 amino acids. Seven of these amino acids are N-methylated, while the remaining four are not. The hydrogen attached to the non-N-methylated nitrogen atom forms hydrogen bonds with the carbonyl group within the molecule, making it difficult for it to interact with water molecules. This makes cyclosporine A a poorly soluble drug, with almost no solubility in water. Cyclosporine A has a very large molecular weight (1,202.63 Da), is highly hydrophobic, and has low aqueous solubility (7.3 μg / ml at 37°C), resulting in a low bioavailability of less than 30% when administered orally, resulting in very low absorption.

[0009] Cyclosporin A is represented by the following structural formula 1. TIFF0007804067000001.tif30166 In the above formula, MeBmt is N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine, Abu is L-aminobutyric acid, Sar is sarcosine, MeLeu is N-methyl-L-leucine, Val is L-valine, Ala is L-alanine, DAla is D-alanine, and MeVal is N-methyl-L-valine.

[0010] Unless otherwise specified, the amino acids in cyclosporin A are in the L-configuration, and the amino acid residue numbers are as shown in structural formula 1, with MeBmt being number 1 and the last amino acid residue, MeVal (N-methyl-L-valine), being number 11 in clockwise order. For the nomenclature of cyclosporin A and its derivatives, see Helv. Chim. Acta, 1987;70.

[0011] The cyclosporin 3-position derivative means a derivative in which the sarcosine residue at position 3 is replaced with another residue. For example, the sarcosine residue at position 3 of cyclosporin A is replaced with [2-methylthio-sarcosine 3 ], [2-methylthio-sarcosine 3 ]Cyclosporin A ([2-methylthio-Sar 3 ]Cyclosporine A). 3 ]Cyclosporin A ([D-2-methylthio-Sar 3 ]Cyclosporine A) is called OND-1 and has the chemical formula C 63 H 113 N 11 O 12 S and MW are 1248.70.

[0012] The cyclosporin 3-position derivative is represented by the following chemical formula 1. TIFF0007804067000002.tif26166

[0013] The C is an amino acid represented by -N(R1)-CH(R2)-C(=O)-.

[0014] The R1 can be hydrogen or a methyl group.

[0015] R2 may be hydrogen; or a C1-C6 linear or branched alkyl, alkenyl, or alkynyl group; or an aryl group. Each carbon atom of the alkyl, alkenyl, alkynyl, or aryl group may be substituted or unsubstituted with one to three substituents selected from the group consisting of amino, hydroxy, halo, haloalkyl, ester, alkoxy, cyano, nitro, alkylamino, and dialkylamino. The aryl group may be a phenyl group.

[0016] R2 may be X-R', where X is oxygen (O) or sulfur (S), and R' may be hydrogen or a C1-C6 linear or branched alkyl, alkenyl, alkynyl, or aryl group. In the alkyl, alkenyl, alkynyl, or aryl group, each carbon atom may be substituted or unsubstituted with one to three substituents selected from the group consisting of amino, hydroxy, halo, haloalkyl, ester, alkoxy, cyano, nitro, alkylamino, and dialkylamino.

[0017] X may be sulfur, and R' may be a methyl group or a methyl group substituted with one to three substituents selected from the group consisting of amino, hydroxy, halo, haloalkyl, ester, alkoxy, cyano, nitro, alkylamino, and dialkylamino.

[0018] The C is a D-amino acid or an L-amino acid.

[0019] The cyclosporin 3-derivative is [2-methylthio-sarcosine 3 ]Cyclosporin A ([2-(4-methylbenzenethio)Sar 3 ]Cyclosporine A);[2-dimethylamino-ethylthio-sarcosine 3 ]Cyclosporin A; [N-methyl-aminobutyric acid 3 ]Cyclosporin A; [N-methyl-norvaline 3 ]Cyclosporin A;[2-(methylamino)-hex-4-ynoyl 3 ]Cyclosporin A;[2-(methylamino)-pent-4-ynoyl 3 ]Cyclosporin A; [N-methyl-O-propenyl-serine 3 ]Cyclosporin A; [N-methyl-serine 3 ]Cyclosporin A; [Methylalanine 3 ]Cyclosporin A; [N-methyl-D-norvaline 3 ] cyclosporin A, or a combination thereof.

[0020] The 3-position cyclosporine derivative may be amorphous. The physical state of a drug may be crystalline, amorphous, or a combination thereof, and is an important property that affects the drug release rate of a formulation. In the experimental examples described below, it was confirmed that the 3-position cyclosporine derivative is amorphous and has excellent interaction with polymers.

[0021] The term "controlled-release particles" refers to particles in which the amount or time of drug release within the particles can be controlled by adjusting the ratio of cyclosporine 3-derivative to biodegradable polymer, manufacturing conditions, physical properties of the biodegradable polymer, etc. The controlled-release particles achieve excellent sustained release without an initial burst due to the interaction between the cyclosporine 3-derivative drug and the biodegradable polymer. The interaction may be an intermolecular force such as van der Waals force or hydrogen bond, which can be confirmed by Fourier transform infrared spectroscopy (FT-IR) or differential scanning calorimetry (DSC). In the experimental example described below, the particles exhibit the amide 1 band at 1622 cm due to the binding of the cyclosporine 3-derivative to PLGA. -1 Peak (amide C=O) and ester bond of PLGA at 1746 cm -1 A peak (ester C=O) shift occurred, and the intrinsic melting point or glass transition temperature of the cyclosporine 3-derivative changed. However, no such change occurred in a mixture of the cyclosporine 3-derivative and polymer, which were simply physically mixed. This indicates that the drug and polymer in the controlled-release particles are strongly bound together by intermolecular forces such as van der Waals forces or hydrogen bonds.

[0022] The biodegradable polymer is a synthetic polymer or a natural polymer.

[0023] The synthetic polymers include polyethylene (PEA), polyethylene glycol (PEG), polycaprolactone (PCL), polyalkylcarbonate, polyamino acid, polyhydroxybutyric acid, polyorthoester, polyanhydride, Pluronic (Poly(ethylene oxide) poly(propylene oxide) poly(ethylene oxide)), polylactic acid (Polylactide (PLA)), polyglycolide (Polyglycolide (PGA)), polybutylene succinate (Polybutylene succinate (PBS)), poly(3-hydroxybutyrate-co-3-hydroxyvalerate (Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (Polybutylene terephthal ... The polymer may be one or more selected from the group consisting of poly(D,L-lactic-co-glycolic acid, PBT), polytrimethylene terephthalate (PTT), polyethylene naphthalate (PEN), poly(D,L-lactic-co-glycolic acid, PLGA), (poly(lactic-co-glycolic acid)-glucose (PLGA-glucose), and methoxypolyethylene glycol-(polycaprolactone-co-polylactic acid) (MPEG-(PCL-co-PLA)).

[0024] The synthetic polymer can be polylactic-co-glycolic acid (PLGA), or a double or triple block copolymer with PLGA as the hydrophobic block. PLGA has excellent biocompatibility and is decomposed into water and carbon dioxide, which are harmless to the human body, through the citric acid cycle, a normal metabolic process in the body.

[0025] The biodegradable polymer may form spherical particles in which the hair loss treatment agent is encapsulated.

[0026] By changing the type and composition of the biodegradable polymer, the degradation rate in the body can be changed, thereby controlling the drug release rate. For example, the degradation rate of polylactic acid-co-glycolic acid (PLGA) in the human body varies depending on the ratio of polylactic acid (PLA) to polyglycolic acid (PGA). Therefore, the drug release rate can be controlled by adjusting the ratio of components. Because glycolic acid is less hydrophobic than lactic acid, increasing the ratio of glycolic acid in the PLGA copolymer increases the hydrolysis rate of the polymer, thereby accelerating the drug release rate.

[0027] The polylactic acid-co-glycolic acid (PLGA) has a molar ratio of lactic acid monomer (LA) to glycolic acid monomer (GA) of 90:10 to 40:60 or 85:15 to 50:50, for example, but not limited to, 50:50, 65:35, 75:25, or 85:15.

[0028] The biodegradable polymer may be a single polylactic-co-glycolic acid (PLGA) having a specific LA:GA molar ratio, or a mixture of polylactic-co-glycolic acids (PLGA) each having a different LA:GA molar ratio.

[0029] The biodegradable polymer may be a mixture of PLGA with a 50:50 LA:GA molar ratio and PLGA with a 75:25 LA:GA molar ratio; a mixture of PLGA with a 50:50 LA:GA molar ratio and PLGA with an 85:15 LA:GA molar ratio; a mixture of PLGA with a 75:25 LA:GA molar ratio and PLGA with an 85:15 LA:GA molar ratio; or a mixture of PLGA with a 50:50 LA:GA molar ratio, PLGA with a 75:25 LA:GA molar ratio, and PLGA with an 85:15 LA:GA molar ratio.

[0030] The polylactic-co-glycolic acid (PLGA) may have ester or carboxylic acid end groups.

[0031] The polylactic-co-glycolic acid (PLGA) can have a molecular weight of 4,000 Da to 240,000 Da, 4,000 Da to 75,000 Da, 4,000 Da to 15,000 Da, 7,000 Da to 17,000 Da, or 50,000 Da to 75,000 Da.

[0032] The drug release rate of the particles can vary depending on the molecular weight of the biodegradable polymer. Low-molecular-weight PLGA forms porous particles, resulting in rapid polymer degradation and drug release. High-molecular-weight PLGA forms dense particles with a slow, S-shaped release profile. Increasing the molecular weight and concentration of PLGA used in drug production can achieve high drug encapsulation rates, large particle sizes, and slow drug release rates. In addition to the end groups and molecular weight, other physical properties such as crystallinity and glass transition temperature (Tg) also indirectly affect the degradation rate.

[0033] The PLGA can be enantiomers (poly(D-lactic-co-glycolic acid), or poly(L-lactic-co-glycolic acid)) or racemic (poly(D,L-lactic-co-glycolic acid)).

[0034] As the PLGA, various PLGAs can be used depending on the molar ratio of lactide to glycolide, the terminal group, and the molecular weight. The PLGA may be, for example, 10P001 (50:50); 10P002 (75:25); 10P003 (75:25); 10P007 (90:10); 10P008 (85:15); 10P009 (85:15E); 10P010 (65:35E); 10P016 (50:50E); 10P017 (50:50); 10P019 (50:50E); 10P020 (85:15); 10P022 (50:50); 10P023 (90:10); 10P024 (50:50); 10P025 (90:10) from the Expansorb product group of PCAS, Inc., or Aldrich Evonik Rohm Pharma Resomer GmbH's product range: RG502 (50:50); RG502H (50:50); RG503 (50:50); RG503H (50:50); RG504 (50:50); RG504H (50:50); RG505 (50:50); RG653H (65:35); RG752H (75:25); RG756S (75:25); Purac Examples of such polymers include, but are not limited to, PDLG7502 (75:25); PDLG7502A (75:25); PDLG7507 (75:25); PDLG5002 (50:50); PDLG5002A (50:50); PDLG5004 (50:50); PDLG5004A (50:50); PDLG5010 (50:50) from Biomaterials, and PLGA-7505 (75:25); PLGA-7510 (75:25); PLGA-7515 (75:25); PLGA-7520 (75:25); PLGA-5005 (50:50); PLGA-5010 (50:50); PLGA-5015 (50:50) from Wako Pure Chemical Industries.

[0035] The polylactic acid-co-glycolic acid (PLGA) may be a cationic graft copolymer with polylysine (Poly L-Lysine, PLL) introduced therein. When PLL is introduced or grafted into PLGA, the cationicity increases, thereby increasing the adhesion to cell membranes, which is well known to those skilled in the art.

[0036] The natural polymer may be one or more selected from the group consisting of carboxymethylcellulose (CMC), algin, alginic acid, alginate, hyaluronic acid, polypeptide, protein, gelatin, collagen, albumin, dextran, starch, casein, chitin derivatives, chitosan, and small intestinal submucosa (SIS).

[0037] The weight ratio of the 3-position cyclosporine derivative to the biodegradable polymer may be, but is not limited to, 5:95 to 90:10, 10:90 to 90:10, 20:80 to 80:20, or 20:80 to 70:30, and in one embodiment, 10:90 to 60:40 or 20:80 to 60:40. At these weight ratios, the initial burst phenomenon is suppressed, the drug is released stably over a period of one to four weeks or more, and side effects are reduced.

[0038] The term "particle" as used herein refers to nano- or micro-sized particles. The terms "particle," "microsphere," "nanosphere," "nanoparticle," and "microparticle" are used interchangeably herein. The average diameter of the particles may be 0.01 to 300 μm, 0.1 to 200 μm, 0.1 to 100 μm, or 0.1 to 60 μm.

[0039] The average zeta potential of the particles may be positive or negative. According to one embodiment, the average zeta potential of the particles may be ±2 mV or more, ±10 mV or more, or ±30 mV or more. The average zeta potential of the particles may be +2 mV to -2 mV, +10 mV to -10 mV, or +30 mV to -30 mV. "Zeta potential" is a value calculated indirectly from the thickness of the electric double layer around the particles, and refers to the electrodynamic potential difference resulting from the difference in positive charge density due to repulsive or attractive forces between particles. The thicker the electric double layer, the more electrostatic repulsive forces are generated between particles, making them less likely to aggregate. When the average zeta potential of the particles is within the above range, aggregation between particles is suppressed, ensuring the stability of the dispersion.

[0040] The particle surface may be smooth and spherical. A smooth particle surface means that there are no or very few pores on the surface. A smooth particle surface inhibits drug diffusion and initial burst through pores, and drug release occurs only through the degradation of the biodegradable polymer. Therefore, a smooth particle surface means that the particle has suitable morphological characteristics for a controlled-release agent. According to the experimental examples described below, the particles contained in the PLGA controlled-release composition prepared in accordance with the present invention have a smooth, spherical surface, which inhibits initial burst and provides excellent controlled release.

[0041] The particles may contain 5 to 60% (w / w) or 15 to 40% (w / w) of a cyclosporine 3-derivative, which suppresses the initial burst of the drug and allows for stable release over the intended period.

[0042] Another embodiment provides a composition for preventing hair loss or promoting hair growth comprising the controlled-release particles.

[0043] The composition may be an injectable formulation for topical administration.

[0044] The composition may be a composition for treating or preventing hair loss.

[0045] When the composition is topically administered, the cyclosporine 3-position derivative is released continuously for at least one week, for example, for 1 to 4 weeks, 2 to 4 weeks, or 3 to 4 weeks.

[0046] In order to achieve sufficient efficacy when a drug is administered locally to the site of hair loss, it is important that the drug does not diffuse throughout the body. According to the experimental examples described below, the composition of the present invention has a high amount remaining on the skin after topical administration, and therefore has excellent effects in preventing hair loss or promoting hair growth.

[0047] The hair loss may be any one selected from the group consisting of androgenetic alopecia (AGA), female-pattern hair loss, alopecia areata, telogen effluvium, and cicatricial alopecia.

[0048] The term "prevention" refers to any action in which the administration of a composition according to the present invention inhibits or delays the onset of a disease or abnormal condition.

[0049] The composition can be administered intralesionally. Specifically, it can be administered intradermally or via mesotherapy into the scalp where hair growth is desired to be promoted, or into the subcutaneous fat layer near the hair follicles. The administration can be dermal or subcutaneous administration near the hair follicles.

[0050] The composition may be for mesotherapy administration. Mesotherapy is an injection therapy in which a drug is directly injected into a target area, and refers to an injection therapy in which a small amount of drug is injected into the middle layer of the skin (dermis layer) using a fine syringe, thereby enhancing the effect, increasing the duration of the effect, and minimizing side effects.

[0051] The composition may further comprise one or more selected from the group consisting of water for injection, an isotonic agent, and a suspending agent. According to one embodiment, the controlled-release composition for local injection of the present invention is a suspension in which PLGA particles containing a cyclosporine 3-derivative are uniformly suspended. The suspension can be prepared by mixing the composition with one or more selected from the group consisting of water for injection, an isotonic agent, and a suspending agent.

[0052] The water for injection may be, for example, one or more selected from the group consisting of saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and ethanol, but is not limited thereto.

[0053] The isotonic agent is, for example, one or more selected from the group consisting of D-mannitol, maltitol, sorbitol, lactitol, xylitol, and sodium chloride, but is not limited thereto.

[0054] The suspending agent may be one or more selected from the group consisting of sodium carboxymethylcellulose, polysorbate 80, starch, starch derivatives, polyhydric alcohols, chitosan, chitosan derivatives, cellulose, collagen, gelatin, hyaluronic acid (HA), alginic acid, algin, pectin, carrageenan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, polylysine, titin, fibrin, agarose, fluran, and xanthan gum, but is not limited thereto.

[0055] The composition may be formulated for local injection, depot injection, or skin implant.

[0056] Another embodiment provides a method for producing controlled-release particles loaded with a cyclosporin-3-derivative, comprising the steps of:

[0057] a) dissolving a cyclosporin 3-derivative and a biodegradable polymer in a solvent to prepare a drug-polymer dispersed phase;

[0058] b) mixing the dispersed phase with a continuous phase containing a surfactant (emulsifier) ​​to form an emulsion; and

[0059] c) mixing and stirring the prepared emulsion with a quenching medium to remove the solvent from the dispersed phase, thereby forming particles carrying the cyclosporin-3-derivative;

[0060] In the above production method, the details regarding the cyclosporin 3-derivative and the biodegradable polymer are the same as those described above.

[0061] The solvent in step a) may be a dispersion medium. The dispersion medium is not particularly limited as long as it can dissolve the biodegradable polymer. According to one embodiment, the solvent for the dispersed phase may be an organic solvent with a low boiling point, and the boiling point may be 25 to 85°C, 25 to 70°C, 25 to 60°C, or 25 to 50°C. When the boiling point of the organic solvent is within this range, evaporation of the solvent for the dispersed phase and drying of the particles after particle formation are advantageous.

[0062] The dispersion medium is, for example, one or more selected from the group consisting of dichloromethane, chloroform, acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (dimethylformaldehyde), ethyl acetate (EA), butyl acetate, ethyl formate, isopropyl acetate, isopropyl formate, diethyl ether, and glycofurol, but is not limited to these.

[0063] In step a), the solvent may further contain a cosolvent. The cosolvent can improve drug release parameters and encapsulation efficiency. When ethanol is used as a cosolvent, the ethanol concentration can affect the release profile of hydrophobic drugs. For example, it has been shown that as the ethanol concentration increases, the interfacial tension (IFT) decreases, causing the drug to leach from the core of the PLGA particles and crystallize on the surface of the microparticles. The cosolvent can be selected to dissolve biodegradable polymers, be miscible with the dispersion medium, have low drug solubility, and have a boiling point lower than that of the dispersion medium. For example, the cosolvent may be one or more selected from the group consisting of methanol, ethanol, acetone, isopropanol, diethyl ether, chloroform, ethyl acetate, DMSO, and acetonitrile, but is not limited thereto.

[0064] In the above preparation method, the dispersed phase in step a) may further include a release control agent. The release control agent prevents the microparticles from swelling upon contact with an aqueous solution and delays drug release even after the microparticles swell and disintegrate. The release control agent may be a hydrophilic release control agent, such as one or more selected from the group consisting of retinoic acid, polyoxyethylene sorbitan fatty acid esters, glyceryl monooleate, sorbitan fatty acid esters, polyvinyl alcohol, poloxamer, polyethylene glycol, glyceryl palmitostearate, benzyl benzoate, ethyl oleate, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl β-cyclodextrin, Tween 80, Span 20, PEG 400, Mrij 52, Brij 58, and poloxamer P1.

[0065] The release-controlling agent may be a vegetable oil, such as lecithin, castor oil, MCT oil (Medium Chin Triglyceride oil), corn oil, cottonseed oil, sesame oil, soybean oil, cinnamon oil, almond oil, arachis oil, linseed oil, olive oil, caraway oil, rosemary oil, peppermint oil, sunflower oil, eucalyptus oil, lavender oil, coconut oil, orange oil, anise oil, clove oil, concentrated borage oil, or SEFOL 860. 860, Miglyol, ATMOS 300, GM Orphic 80, DL-α-Tocopheryl acetate, Labrafil, Imwitor 742, Myvacet, Myvaplex, Myverol, Generol, Myritol, Captex, Capmul, Neobee M5, Mazol 1400, Maisine 35-1, Crossential GLO E50 (concentrated borage oil ethyl ester), Nikkol EOO (concentrated borage oil ethyl ester),Examples of suitable glycerides include, but are not limited to, ethyl olive oleate, ethyl oleate, oleic acid or linoleic acid, ethyl linoleate, isopropyl palmitate, isopropyl myristate, polyglyceryl oleate, polyglyceryl palmitostearate, diglyceryl monooleate, tetraglyceryl monooleate, hexaglyn, and decaglyn;

[0066] The controlled-release agent may be an animal oil or its derivatives, such as, but not limited to, squalene, hydrogenated squalene, omega-3 essential fatty acids (EFAs), eicosapentaenoic acid, docosahexaenoic acid, and the ethyl-esterified form of the oil, Incromega, available from Croda Co.

[0067] The surfactant (or emulsifier) ​​of the continuous phase may be one or more selected from the group consisting of nonionic surfactants, anionic surfactants and cationic surfactants.

[0068] Examples of the surfactant include polymer surfactants such as polyethylene glycol (PEG), hydroxypropylmethylcellulose (HPMC), methylcellulose, polyvinylpyrrolidone (PVP), gelatin, chitosan, Eudragit, and polyvinyl alcohol (PVA); and hydrophilic low-molecular surfactants such as lecithin, Tween (polyoxyethylene sorbitan fatty acid ester), poloxamer, polyoxyethylene castor oil derivatives, Cremophor, cyclodexterins, sodium lauryl sulfate (SLS), sodium stearate, esteramines, ethylenediamine, Myrj52, Brij58, and fatty amines. amine); the lipophilic low molecular weight surfactant may be one or more selected from the group consisting of span, labrasol, rheodol MO-60, glyceryl monooleate, and triacetin. In one embodiment, the surfactant is polyvinyl alcohol, but is not limited to these.

[0069] The surfactant may be contained in the continuous phase at 0.1 to 20% (w / v), 0.1 to 10% (w / v), or 1 to 3% (w / v), and may be appropriately adjusted within a range that helps form a stable emulsion of the biodegradable polymer solution.

[0070] In step b), the dispersed phase and the continuous phase may be mixed at a volume ratio of 1:5 to 1:50, or 1:10 to 1:25. When the volume ratio of the dispersed phase to the continuous phase is within this range, the spacing between the microdroplets in the O / W emulsion after homogenization is appropriate, which is advantageous for the formation of microparticles of uniform size.

[0071] In step c), the emulsion is mixed and stirred with a large amount of quenching medium, causing the dispersion medium and cosolvent to diffuse into the continuous phase and evaporate, inducing initial polymer hardening and encapsulating the drug particles within the particles. That is, the dispersed phase solvent (dispersion medium (organic solvent) or cosolvent) in the microdroplets dispersed in the O / W emulsion rapidly diffuses and is extracted into the continuous phase, while the biodegradable polymer rapidly solidifies, forming drug-loaded particles.

[0072] The quenching medium may be water, methanol, ethanol, propanol, ethyl acetate, PVA (polyvinyl alcohol), or a combination thereof. The quenching medium may be a solution of the same components as the continuous phase.

[0073] The amount of the quenching medium mixed with the emulsion is, for example, 1 to 100 times, 2 to 100 times, 3 to 100 times, 4 to 100 times, 5 to 100 times, 6 to 100 times, 7 to 100 times, 8 to 100 times, 9 to 100 times, 10 to 100 times, 20 to 100 times, 30 to 100 times, 1 to 50 times, 2 to 50 times, 3 to 5 ...50 times, 5 to 50 times, 6 to 100 times, 7 to 100 times, 8 to 100 times, 9 to 100 times, 10 to 100 times, 20 to 100 times, 30 to 100 times, 1 to 50 times, 2 to 50 times, 3 to 50 times, 4 to 50 times, 5 to 50 times, 6 to 100 times, 7 to 100 times, 8 to 100 times, 9 to 100 times, 10 to 100 times, 20 to 100 times, 30 to 100 times, 1 to 50 times, 2 to 50 times, 3 to 50 times, 4 to 50 times, 5 to 50 times, 6 to 100 times, 7 to 100 times, 8 to The preferred ranges are, but are not limited to, up to 50 times, 4 to 50 times, 5 to 50 times, 6 to 50 times, 7 to 50 times, 8 to 50 times, 9 to 50 times, 10 to 50 times, 20 to 50 times, 30 to 50 times, 1 to 20 times, 2 to 20 times, 3 to 20 times, 4 to 20 times, 5 to 20 times, 6 to 20 times, 7 to 20 times, 8 to 20 times, 9 to 20 times, and 10 to 20 times.

[0074] Step c) can be performed using methods such as solvent extraction, solvent evaporation, and spray drying. The manufacturing method affects the properties of the drug delivery composition, such as particle size, drug release characteristics, and drug encapsulation rate, so an appropriate manufacturing method should be selected as needed. For example, step c) can be performed using solvent evaporation, phase separation, self-healing encapsulation, or mechanical processes such as membrane emulsification, spray drying, supercritical fluid, and microfluidics. Step c) can be performed using emulsification methods such as mechanical stirring, high-pressure emulsification, fluid bed, static motor, ultrasound, membrane, nozzle spray, or dripping. Step c) can be performed using strong stirring with a magnetic stirrer to prevent the droplets from agglomerating together.

[0075] The step c) can be carried out for 4 to 12 hours, but the carrying out time can be adjusted so as to suppress particle aggregation and ensure proper encapsulation of the drug.

[0076] In one embodiment, the production method may further comprise, after step c), a step d) of separating the produced particles carrying the cyclosporin 3-derivative. In one embodiment, step d) comprises centrifuging the drug-loaded particles produced in step c).

[0077] The manufacturing method may further comprise, after step d), a step e) of vacuum drying or freeze-drying the separated particles.

[0078] In step e), residual dispersion medium (organic solvent) or cosolvent in the particles can be removed by vacuum drying at room temperature or freeze-drying at -40°C, which induces secondary polymer hardening. The drying process solidifies the structure of the drug-loaded particles, allowing the drug to be completely encapsulated.

[0079] The vacuum drying can be carried out at a temperature of, for example, 30 to 40°C, and the freeze drying can be carried out at -30 to -40°C.

[0080] The step e) can be carried out for, for example, 24 to 48 hours.

[0081] The production method of the present invention may further comprise a step f) of vacuum drying or freeze-drying after the step e).

[0082] By the step f), the solvent remaining in the particles is completely removed, the particle structure becomes more solid, and the drug can be completely encapsulated.

[0083] The additional vacuum drying or freeze-drying conditions in step f) can be carried out in the same manner as in step e).

[0084] If the vacuum drying is performed at a temperature below 30°C, the residual solvent may not be completely removed. On the other hand, if the vacuum drying temperature exceeds 40°C, the glass transition temperature (Tg) of the biodegradable polymer may be exceeded, and the particle structure may be deformed during the drying process, resulting in a decrease in the drug release control effect.

[0085] The freeze-drying process may be carried out under vacuum conditions.

[0086] The step f) is carried out for 12 to 24 hours, but is not limited thereto.

[0087] If the additional drying time is less than 12 hours, the residual solvent may not be completely removed, whereas if the drying time exceeds 24 hours, productivity may decrease. [Effects of the Invention]

[0088] The composition for preventing hair loss or promoting hair growth containing the controlled-release particles of the present invention can be administered locally to the area where hair loss is progressing by methods such as mesotherapy, and can provide a sufficient amount of cyclosporine 3-position derivatives, which are difficult to penetrate through the skin, and since the drug does not increase rapidly in the systemic blood upon local administration, systemic side effects can be minimized.

[0089] The composition for preventing hair loss or promoting hair growth of the present invention is excellent in terms of patient compliance and convenience, since it can maintain a constant drug concentration at the hair loss site after topical administration for a long period of time.

[0090] The controlled-release particles of the present invention can adjust the drug release rate by changing the degree of biodegradation such as the molecular weight of PLGA, the release control agent, solvent, polymer, surfactant, drug content, stirring speed in the emulsification process, high-speed emulsification pressure, ultrasonic irradiation time, continuous phase / separated phase ratio, and drying method, and particles with an appropriate release rate can be produced depending on the type and severity of hair loss disease. [Brief explanation of the drawings]

[0091] [Figure 1]1 shows an SEM image of Example 1-A (#125). [Figure 2] 1 shows an SEM image of Example 1-B (#126). [Figure 3] 1 shows an SEM image of Example 1-C (#127). [Figure 4] 1 shows an SEM image of Example 2-A (#109). [Figure 5] 1 shows an SEM image of Example 2-B (#110). [Figure 6] 1 shows an SEM image of Example 2-C (#111). [Figure 7] 1 shows an SEM image of Example 4-1-A (#122). [Figure 8] 1 shows an SEM image of Example 4-1-B (#108). [Figure 9] 1 shows an SEM image of Example 4-1-C (#73). [Figure 10] 1 shows an SEM image of Example 4-1-D (#73-1). [Figure 11] FIG. 1 shows SEM images of Examples 31-B to 31-D. [Figure 12-13] SEM images of Examples 39-B, 40-B, 41-B, and 42-B are shown. [Figure 14-16] The results of particle size analysis of Examples 1-A to 1-C are shown below. [Figure 17-19] The results of particle size analysis of Examples 31-B to 31-D are shown below. [Figure 20-26] 1 shows experimental results of drug release patterns. [Figure 28-31] The results of XRD and DSC measurements are shown below. [Figure 32-35] The results of FT-IR measurements are shown below. [Figure 36-38] The results of DSC measurements are shown below. [Figure 39] The results of confirming whether or not the composition of the present invention has a hair growth effect when administered by injection are shown below. [Figure 40] The results of confirming whether the composition of the present invention was eliminated into the systemic blood when administered by local injection (intradermal (intralesional) injection) are shown below. [Figure 41] 1 shows the results of confirming the amount of the composition remaining on the skin when the composition of the present invention was administered by local injection (intradermal (intralesional) injection). [Figure 42] FIG. 1 is a graph showing a comparison of the remaining drug amount (%) over time after cutaneous administration of three types of drug formulations using PLGA polymers with different composition ratios. DETAILED DESCRIPTION OF THE INVENTION

[0092] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited by these examples. Furthermore, terms specifically defined in this specification should be understood to have the meanings commonly used in the technical field to which the present invention pertains.

[0093] <Production Example: Production of 3-Cyclosporin Derivatives> The general method for alkylating cyclosporine A is as follows: First, diisopropylamine ((i-Pr)NH) is added to tetrahydrofuran (THF) under nitrogen, and butyllithium (BuLi) dissolved in tetrahydrofuran (THF) is added at 78°C, followed by stirring for 30 minutes. Cyclosporine A dissolved in tetrahydrofuran (THF) is added to the resulting LDA solution, and after stirring for 1 hour, an electrophile is added.

[0094] Production Example 1. N-methyl-D-aminobutyric acid 3 ]Cyclosporin A ([N-methyl-D-Abu 3 ]Synthesis of Cyclosporine A) Using the standard method, 1.0 g of cyclosporine A dissolved in 50 ml of tetrahydrofuran (THF) was added to 10 equivalents of LDA solution at 78°C. The reaction mixture was stirred at 78°C for 2 hours, and then 0.4 ml of ethyl iodide was added. After the mixture was brought to room temperature and stirred for an additional 24 hours, 20 ml of water was added and the mixture was concentrated. Ether (EtO) was added to the residue, which was then washed successively with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (100 g of silica gel, dichloromethane:methyl alcohol = 96:4) and then by HPLC to give the title compound (0.1 g).

[0095] Production Example 2: N-methyl-D-norvaline 3 ]Cyclosporin A ([N-methyl-D-norval 3 ]Synthesis of Cyclosporine A) Using the standard method, 1.0 g of cyclosporine A dissolved in 50 ml of tetrahydrofuran (THF) was added to 10 equivalents of LDA solution at 78°C. The reaction mixture was stirred at 78°C for 2 hours, and then 0.41 ml of propyl iodide was added. After the mixture was brought to room temperature and stirred for an additional 24 hours, 20 ml of water was added and the mixture was concentrated. Ether (EtO) was added to the residue, which was then washed successively with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (100 g of silica gel, dichloromethane:methyl alcohol = 96:4) and then by HPLC to give the title compound (0.12 g).

[0096] Preparation Example 3: [D-2-(methylamino)hex-4-ynoyl 3 ]Cyclosporin A ([D-2-(Methylamino)hexa-4-ynoyl 3 ]Synthesis of Cyclosporine A) Using the standard method, 1.0 g of cyclosporine A dissolved in 50 ml of tetrahydrofuran (THF) was added to 10 equivalents of LDA at 78°C. The reaction mixture was stirred at 78°C for 2 hours, and then 0.73 ml of 1-bromo-2-butyne was added. The mixture was allowed to warm to room temperature and stirred for an additional 24 hours. 20 ml of water was added and the mixture was concentrated. The residue was then diluted with ether (EtO), washed sequentially with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (100 g of silica gel, dichloromethane:methyl alcohol = 96:4) and then by HPLC to give the title compound (0.13 g).

[0097] Preparation Example 4: [D-2-(methylamino)pent-4-ynoyl 3 ]Cyclosporin A ([D-2-(Methylamino)pent-4-ynoyl 3 ]Synthesis of Cyclosporine A) Using standard procedures, tetrahydrofuran (THF) (200 ml), diisopropylamine ((i-Pr)NH) (3.2 ml), butyllithium (BuLi) (8 ml), cyclosporine A (3.76 g) dissolved in tetrahydrofuran (THF) (50 ml), and propargyl bromide (3.57 g) were used. After allowing the mixture to reach room temperature, it was stirred for an additional 2 hours, 40 ml of water was added, and the mixture was concentrated. Ether (EtO) was added to the residue, which was then washed successively with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (silica gel 100 g, dichloromethane:methyl alcohol = 96:4) and then by HPLC to obtain the title compound.

[0098] Preparation Example 5: D-2-methylthio-sarcosine 3 ]Cyclosporin A ([D-2-methylthio-Sar 3 ]Synthesis of Cyclosporine A) Using standard procedures, tetrahydrofuran (THF) (100 ml), diisopropylamine ((i-Pr)NH) (1.6 ml), butyllithium (BuLi) (4.0 ml), cyclosporine A (1.0 g) dissolved in tetrahydrofuran (THF) (30 ml), and dimethyl disulfide (MeS) (1.5 ml) were used. After stirring at 0°C for 14 hours, 20 ml of water was added and the mixture was concentrated. Ether was added to the residue, which was then washed successively with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (silica gel 100 g, dichloromethane:methyl alcohol = 50:1 to 96:4) and then purified by HPLC to obtain the title compound (OND-1).

[0099] Preparation Example 6: [D-2-(4-methylbenzenethio)sarcosine 3 ]Cyclosporin A ([D-2-(4-methylbenzenethio)Sar 3 ]Synthesis of Cyclosporine A). Similar to Preparation 5, in this case di(tol-4-yl)disulfide was utilized as the electrophile to give the title compound.

[0100] Preparation Example 7: D-2-Dimethylamino-ethylthio-sarcosine 3 ]Cyclosporin A ([D-2-Dimethylamino-ethylthio-Sar 3 ]Synthesis of Cyclosporine A) Similar to Preparation 5, in this case Bis(2-dimethylaminoethyl)disulfide was utilized as the electrophile to afford the title compound.

[0101] Preparation Example 8: N-methyl-D-serine 3 ]Cyclosporin A ([N-methyl-D-Ser 3 ]Synthesis of Cyclosporine A) Using the standard method, 1.0 g of cyclosporine A dissolved in 50 ml of tetrahydrofuran (THF) was added to 10 equivalents of LDA solution at 78°C. The reaction mixture was stirred at 78°C for 2 hours, followed by the addition of 2.0 g of paraformaldehyde. The mixture was allowed to warm to room temperature and stirred for an additional 24 hours. 20 ml of water was added and the mixture was concentrated. Ether (EtO) was added to the residue, which was then washed successively with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (100 g of silica gel, dichloromethane:methyl alcohol = 96:4) and then by HPLC to give the title compound (0.3 g).

[0102] Preparation Example 9: N-methyl-O-propyl-D-serine 3 ]Cyclosporin A (N-methyl-O-Propenyl-D-Ser 3 Synthesis of Cyclosporine A Using a standard method, [D-methylserine 3

[0023] -Cyclosporin A (0.62 g, 0.5 mmol), tetrabutylammonium chloride (0.11 g, 0.5 mmol), and allyl bromide (0.24 g, mmol) were dissolved in dichloromethane (50 mL) and stirred with 30% sodium hydroxide (NaOH) (1.5 mL) for 2 hours. 50 mL of dichloromethane was added, followed by washing with water and saturated aqueous sodium chloride, drying over anhydrous sodium sulfate (MgSO), and concentration. The residue was purified by silica gel column chromatography (silica gel 100 g, dichloromethane:methyl alcohol = 97:3) and then by HPLC to give the title compound (0.4 g).

[0103] Preparation Example 10: N-methyl-D-alanine 3 ]Cyclosporin A ([N-methyl-D-Ala 3 ]Synthesis of Cyclosporine A) Using standard procedures, cyclosporine A (2.0 g) dissolved in tetrahydrofuran (THF) (120 ml), diisopropylamine ((i-Pr)NH) (1.74 ml), butyllithium (BuLi) (4.6 ml), tetrahydrofuran (THF) (30 ml), and methyl iodide (MeI) (0.51 ml) were used. After stirring for 1 hour at room temperature, 10 ml of water was added and the mixture was concentrated. The residue was added with ether, washed sequentially with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate (MgSO), and concentrated. The residue was purified by silica gel column chromatography (silica gel 100 g, dichloromethane:methyl alcohol = 50:1 to 96:4) and then purified by HPLC to give the title compound (0.26 g). [Example]

[0104] PLGA-based sustained release particles with various LA:GA molar ratios were manufactured (vacuum drying method).

[0105] The controlled-release PLGA particles of Examples 1-A to 1-C were produced by the following method. The components and conditions used in the production process are shown in Table 1 below.

[0106] Step 1: OND-1 (40 mg) and PLGA (160 mg) were dissolved in DCM (dichloromethane) (2 ml) to prepare a dispersed phase. (See Production Example 5 for OND-1.)

[0107] Step 2: The prepared dispersed phase was added to a continuous phase of 1% PVA aqueous solution (500 mg PVA, 50 ml DW), and emulsified using a homogenizer to prepare an emulsion.

[0108] Step 3: The prepared emulsion was placed in a quenching medium, 1% PVA aqueous solution (2500 mg PVA, 250 ml DW), to stabilize it, and then the DCM was evaporated while stirring with a magnetic stirrer for at least 4 to 24 hours to form PLGA particles.

[0109] Step 4: The solution in which the PLGA particles had been formed was centrifuged (13,000 rpm, 10 minutes, 4°C) to precipitate the PLGA particles, and the PVA present in the supernatant was then removed.

[0110] Step 5: The PLGA particle precipitate was resuspended in 10 ml of distilled water (DW) and centrifuged (13,000 rpm, 10 minutes, 4°C), and this process was repeated four times.

[0111] Step 6. After washing was completed, the precipitate was vacuum dried and the resulting PLGA particles were stored. [Table 1]

[0112] 1) [D-2-methylthio-sarcosine 3 ]Cyclosporin A ([D-2-methylthio-Sar 3 ]Cyclosporin A)

[0113] 2) The numbers in parentheses represent the LA:GA molar ratio, terminal group, and molecular weight, respectively. For example, "(PLGA 50:50, acid, 7000-17000)" represents PLGA with an LA:GA molar ratio of 50:50, a carboxylic acid terminal group, and a molecular weight of 7000-17000 (the same applies below). [Example]

[0114] PLGA-based sustained release particles with various LA:GA molar ratios were manufactured (freeze-drying method).

[0115] The controlled-release PLGA particles of Examples 2-A to 2-C were prepared in the same manner as in Example 1, except that the drying method in step 6 was changed to freeze-drying (freeze-drying after dispersion in a 1% mannitol aqueous solution). The ingredients and conditions used in the manufacturing process are shown in Table 2 below. [Table 2] [Example]

[0116] Production of sustained-release particles based on PLGA with a 50:50 LA:GA molar ratio and different end groups and molecular weights

[0117] The controlled-release particles of Examples 3-A to 3-C were prepared in the same manner as in Example 1, except that PLGA with a 50:50 LA:GA molar ratio and different end groups or molecular weights was used in the dispersed phase of Example 1. The components and conditions used in the preparation process are shown in Table 3 below. [Table 3] [Example]

[0118] Production of sustained release particles based on PLGA with different end groups and molecular weights and a LA:GA molar ratio of 75:25

[0119] The controlled-release particles of Examples 4-A to 4-C were prepared in the same manner as in Example 1, except that PLGA with a LA:GA molar ratio of 75:25, which had different end groups or molecular weights, was used in the dispersed phase of Example 1. The components and conditions used in the preparation process are shown in Table 4 below. [Table 4]

[0120] [Example 4-1] PLGA-based sustained-release particles were manufactured under different emulsification conditions.

[0121] The controlled-release particles of Examples 4-1-A to 4-1-D were prepared in the same manner as in Example 1-B or Example 2-B, except that the emulsification conditions were changed. The ingredients and conditions used in the preparation process are shown in Table 5 below. [Table 5] [Example]

[0122] We produced sustained-release particles based on PLGA with different molecular weights and a LA:GA molar ratio of 85:15.

[0123] The controlled-release particles of Example 5-A were prepared in the same manner as in Example 1, except that the dispersed phase was changed to PLGA. The ingredients and conditions used in the manufacturing process are shown in Table 6 below. [Table 6] [Example]

[0124] PLGA-based sustained-release particles were prepared using solvents with different interfacial tensions (IFT) with respect to water as the dispersed phase solvent.

[0125] The PLGA controlled-release particles of Examples 6-A to 6-J were prepared in the same manner as in Example 1-B, except that the solvent for the dispersed phase was changed to a solvent with a different interfacial tension (IFT) value relative to water. The ingredients and conditions used in the manufacturing process are shown in Tables 7 and 8 below. The terms used in Tables 7 and 8 below are as follows: (DCM = dichloromethane; EA = ethyl acetate; DMSO = dimethyl sulfoxide; ACN = acetonitrile) [Table 7] [Table 8] [Example]

[0126] Preparation of PLGA-based sustained release particles using solvents with different interfacial tensions (IFT) with water

[0127] The controlled-release PLGA particles of Examples 7-A to 7-J were prepared in the same manner as in Example 1-A, except that the solvent for the dispersed phase was changed to a solvent with a different interfacial tension (IFT) value relative to water. The ingredients and conditions used in the preparation process are shown in Tables 9 and 10 below. [Table 9] [Table 10] [Example]

[0128] Preparation of PLGA-based sustained release particles using solvents with different interfacial tensions (IFT) with water

[0129] The controlled-release PLGA particles of Examples 8-A to 8-J were prepared in the same manner as in Example 1-C, except that the dispersed phase solvent was changed to a solvent with a different interfacial tension (IFT) value relative to water. The ingredients and conditions used in the preparation process are shown in Tables 11 and 12 below. [Table 11] [Table 12] [Example]

[0130] PLGA-based sustained release particles are produced by mixing a cosolvent with the dispersed phase solvent.

[0131] The PLGA controlled-release particles of Examples 9-A to 9-I were prepared in the same manner as in Example 1-B, except that the dispersed phase solvent was DCM, chloroform, ethyl acetate (EA), ethyl formate (Ethyl Formate), DMSO, ACN, butyl acetate, isopropyl formate, or glycofurol, and the co-solvent was methanol, ethanol, acetone, isopropanol, diethyl ether, chloroform, ethyl acetate (EA), DMSO, or CAN. The ingredients and conditions used in the manufacturing process are listed in Tables 13 and 14 below. [Table 13] [Table 14] [Example]

[0132] Manufacturing natural polymer-based sustained release particles

[0133] PLGA controlled-release particles of Examples 10-A to 10-I were prepared in the same manner as in Example 1, except that a natural polymer was used instead of PLGA. The ingredients and conditions used in the preparation process are listed in Table 15 below. CMC is carboxymethylcellulose. [Table 15] [Example]

[0134] Manufacture of sustained-release particles made from synthetic biodegradable polymers (Aliphatic Homopolymer)

[0135] PLGA controlled-release particles of Examples 11-A to 11-I were prepared in the same manner as in Example 1, except that a synthetic biodegradable polymer (aliphatic homopolymer) was used instead of PLGA. The ingredients and conditions used in the preparation process are listed in Table 16 below. PCL is poly-ε-caprolactone; PLA is poly(lactic acid); PGA is polyglycolic acid; PHA is polyhydroxyalkanoate; PHB is polyhydroxybutyrate; and PEG is polyethyleneglycol. [Table 16] [Example]

[0136] Preparation of sustained release particles based on synthetic biodegradable polymer (Aliphatic copolymer)

[0137] The PLGA controlled-release particles of Examples 12-A to 12-I were prepared in the same manner as in Example 1, except that a synthetic biodegradable polymer (aliphatic homopolymer) was used instead of PLGA. The ingredients and conditions used in the preparation process are listed in Table 17 below. The terms used in Table 17 are as follows: PEA=polyethylene adipate;PBS=polybutylene succinate;PHBV=poly(3-hydroxybutyrate-co-3-hydroxyvalerate);PET=polyethylene terephthalate;PLGA=polylactide-co-glycolide;PLGA-glucose=polylactide-co-glycolide-glucose;MPEG-(PCL-co-PLA)=methoxy polyethylene glycol-(polycaprolactone-co-polylactide);mPEG-b-PLGA=Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide);PLGA-b-PEG-b-PLGA=Poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide). [Table 17] [Example]

[0138] Manufacture of sustained release particles made from synthetic biodegradable polymers (semi-aromatic or aromatic copolymers)

[0139] The PLGA controlled-release particles of Examples 13-A to 13-D were prepared using the same method as in Example 1, except that a synthetic biodegradable polymer (semi-aromatic or aromatic copolymer) was used. The ingredients and conditions used in the preparation process are listed in Table 18 below. The terms used in Table 18 are as follows: PBT = polybutylene terephthalate; PTT = polytrimethylene terephthalate; PEN = polyethylene naphthalate. [Table 18] [Example]

[0140] PLGA-based sustained-release particles were fabricated by changing the surfactant in the continuous phase.

[0141] The PLGA controlled-release particles of Examples 14-A to 14-H were prepared in the same manner as in Example 1-B, except that the continuous phase was changed to a 1% (w / v) aqueous solution of PVA, PVP, HPMC, PEG, Eudragit, Chitosan, Methyl cellulose, or Gelatin (500 mg of each surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are shown in Tables 19 and 20 below. [Table 19] [Table 20] [Example]

[0142] PLGA-based sustained-release particles were fabricated by changing the surfactant in the continuous phase.

[0143] The PLGA controlled-release particles of Examples 15-A to 15-H were prepared in the same manner as in Example 1-A, except that the continuous phase was changed to a 1% (w / v) aqueous solution of PVA, PVP, HPMC, PEG, Eudragit, Chitosan, Methyl cellulose, or Gelatin (500 mg of each surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are shown in Table 21 below. [Table 21] [Example]

[0144] PLGA-based sustained-release particles were fabricated by changing the surfactant in the continuous phase.

[0145] The PLGA controlled-release particles of Examples 16-A to 16-H were prepared in the same manner as in Example 1-C, except that the continuous phase was changed to a 1% (w / v) aqueous solution of PVA, PVP, HPMC, PEG, Eudragit, Chitosan, Methyl cellulose, or Gelatin (500 mg of each surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are shown in Table 22 below. [Table 22] [Example]

[0146] PLGA-based sustained-release particles are prepared using hydrophilic low-molecular-weight surfactants (emulsifiers or emulsion stabilizers) in the continuous phase.

[0147] The PLGA controlled-release particles of Examples 17-A to 17-J were prepared in the same manner as in Example 1-B, except that in step 2, the continuous phase surfactant was changed to 1% (w / v) lecithin, poloxamer, Tween, cremophor, SLS, sodium stearate, ethylenediamine, Mrij 52, Brij 58, or cyclodextrin aqueous solution (500 mg of each hydrophilic low molecular weight surfactant and 50 ml of DW). The ingredients and conditions used in the preparation are listed in Tables 23 and 24 below. In Table 23, SLS is sodium lauryl sulfate. [Table 23] [Table 24] [Example]

[0148] PLGA-based sustained-release particles were prepared using a hydrophilic low-molecular-weight surfactant (emulsifier or emulsion stabilizer) in the continuous phase.

[0149] The PLGA controlled-release particles of Examples 18-A to 18-J were prepared in the same manner as in Example 1-A, except that in step 2, the continuous phase surfactant was replaced with 1% (w / v) lecithin, poloxamer, tween, cremophor, SLS, sodium stearate, ethylenediamine, Mrij 52, Brij 58, or cyclodextrin aqueous solution (500 mg of each hydrophilic low molecular weight surfactant and 50 ml of DW). The ingredients and conditions used in the preparation process are listed in Table 25 below. [Table 25] [Example]

[0150] PLGA-based sustained-release particles were prepared using a hydrophilic low-molecular-weight surfactant (emulsifier or emulsion stabilizer) in the continuous phase.

[0151] The PLGA controlled-release formulations of Examples 19-A to 19-J were prepared in the same manner as in Example 1-C, except that in step 2, the continuous phase surfactant was replaced with 1% (w / v) lecithin, poloxamer, tween, cremophor, SLS, sodium stearate, ethylenediamine, Mrij 52, Brij 58, or cyclodextrin aqueous solution (500 mg of each hydrophilic low molecular weight surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are listed in Table 26 below. [Table 26] [Example]

[0152] Preparation of PLGA-based sustained-release formulations using lipophilic low-molecular-weight surfactants (emulsifiers or emulsion stabilizers) in the continuous phase

[0153] The PLGA controlled-release formulations of Examples 20-A to 20-E were prepared in the same manner as in Example 1-B, except that in step 2, the continuous phase surfactant was changed to 1% Span, Labrasol, Rheodol MO-60, glycerol monooleate, or triacetin aqueous solution (500 mg of each lipophilic low molecular weight surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are listed in Table 27 below. [Table 27] [Example]

[0154] PLGA-based sustained-release particles were prepared using lipophilic low-molecular-weight surfactants (emulsifiers or emulsion stabilizers) in the continuous phase.

[0155] The PLGA controlled-release particles of Examples 21-A to 21-E were prepared in the same manner as in Example 1-A, except that in step 2, the continuous phase surfactant was changed to 1% Span, Labrasol, Rheodol MO-60, glycerol monooleate, or triacetin aqueous solution (500 mg of each lipophilic low-molecular-weight surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are shown in Table 28 below. [Table 28] [Example]

[0156] PLGA-based sustained-release particles were prepared using lipophilic low-molecular-weight surfactants (emulsifiers or emulsion stabilizers) in the continuous phase.

[0157] The PLGA controlled-release particles of Examples 22-A to 22-E were prepared in the same manner as in Example 1-C, except that in step 2, the continuous phase surfactant was changed to 1% Span, Labrasol, Rheodol MO-60, glycerol monooleate, or triacetin aqueous solution (500 mg of each lipophilic low-molecular-weight surfactant and 50 ml of DW). The ingredients and conditions used in the manufacturing process are listed in Table 29 below. [Table 29] [Example]

[0158] PLGA-based sustained-release particles are manufactured by adding a modifier (additive, low molecular weight surfactant) to the dispersed phase.

[0159] The PLGA controlled-release formulations of Examples 23-A to 23-G were prepared in the same manner as in Example 1-B, except that 100 mg of Tween 80, Span 20, PEG 400, Mrij 52, Brij 58, Poloxamer P124, or SLS was further added to the dispersed phase as a modifier (additive) in step 2. The ingredients and conditions used in the manufacturing process are shown in Tables 30 and 31 below. [Table 30] [Table 31] [Example]

[0160] PLGA-based sustained release particles are manufactured by adding modifiers (additives, low molecular weight surfactants) to the dispersed phase.

[0161] The PLGA controlled-release particles of Examples 24-A to 24-G were prepared in the same manner as in Example 1-A, except that 100 mg of Tween 80, Span 20, PEG 400, Mrij 52, Brij 58, Poloxamer P124, or SLS was further added as a modifier to the dispersed phase in step 2. The ingredients and conditions used in the manufacturing process are shown in Tables 32 and 33 below. [Table 32] [Table 33] [Example]

[0162] PLGA-based sustained release particles are manufactured by adding modifiers (additives, low molecular weight surfactants) to the dispersed phase.

[0163] The PLGA controlled-release particles of Examples 25-A to 25-G were prepared in the same manner as in Examples 1 to C, except that 100 mg of Tween 80, Span 20, PEG 400, Mrij 52, Brij 58, Poloxamer P124, or SLS was further added as a modifier to the dispersed phase in step 2. The ingredients and conditions used in the manufacturing process are shown in Tables 34 and 35 below. [Table 34] [Table 35] [Example]

[0164] Preparation of PLGA-based sustained release particles by varying the drug content relative to the polymer

[0165] The controlled-release PLGA particles of Examples 26-A to 26-F were prepared in the same manner as in Example 1-B, except that the drug and polymer contents were changed. The ingredients and conditions used in the preparation process are shown in Table 36 below. [Table 36] [Example]

[0166] Preparation of PLGA-based sustained release particles by varying the drug content relative to the polymer

[0167] The controlled-release PLGA particles of Examples 27-A to 27-F were prepared in the same manner as in Example 1-A, except that the drug and polymer contents were changed. The ingredients and conditions used in the preparation process are shown in Table 37 below. [Table 37] [Example]

[0168] Preparation of PLGA-based sustained release particles by varying the drug content relative to the polymer

[0169] The controlled-release PLGA particles of Examples 28-A to 28-F were prepared in the same manner as in Example 1-C, except that the drug and polymer contents were changed. The ingredients and conditions used in the preparation process are shown in Table 38 below. [Table 38] [Example]

[0170] PLGA-based sustained release particles were fabricated by varying the dispersed / continuous phase ratio.

[0171] The controlled-release PLGA particles of Examples 29-A to 29-F were prepared in the same manner as in Example 1-B, except that the content of the continuous phase was changed. The ingredients and conditions used in the preparation process are shown in Table 39 below. [Table 39] [Example]

[0172] PLGA-based sustained release particles were fabricated by varying the dispersed / continuous phase ratio.

[0173] The PLGA controlled-release formulations of Examples 30-A to 30-F were prepared in the same manner as in Example 1-A, except that the content of the continuous phase was changed. The ingredients and conditions used in the preparation process are shown in Table 40 below. [Table 40] [Example]

[0174] Changing the emulsification method to produce PLGA-based sustained-release particles

[0175] The controlled-release PLGA particles of Examples 31-A to 31-D were prepared in the same manner as in Example 1-C, except that the homogenizer conditions were changed in step 2. The ingredients and conditions used in the preparation process are shown in Table 41 below. [Table 41] [Example]

[0176] PLGA-based sustained release particles are manufactured using ultrasonic irradiation.

[0177] The controlled-release PLGA particles of Examples 32-A to 32-B were prepared in the same manner as in Example 1-B, except that an ultrasonicator was used instead of a homogenizer in step 2. The ingredients and conditions used in the preparation process are shown in Table 32 below. [Table 42] [Example]

[0178] PLGA-based sustained-release particles were produced by changing the vacuum drying conditions.

[0179] Except for changing the vacuum drying conditions in Example 1-B, the controlled-release particles of Examples 33-A to 33-D were prepared in the same manner as in Example 1. The components and conditions used in the preparation process are shown in Table 43 below. [Table 43] [Example]

[0180] PLGA-based sustained-release particles were produced by changing the vacuum drying conditions.

[0181] The controlled-release particles of Examples 34-A to 34-D were prepared in the same manner as in Example 1-A, except that the vacuum drying conditions were changed. The ingredients and conditions used in the preparation process are shown in Table 44 below. [Table 44] [Example]

[0182] PLGA-based sustained-release particles were produced by changing the vacuum drying conditions.

[0183] The controlled-release particles of Examples 35-A to 35-D were prepared in the same manner as in Example 1-C, except that the vacuum drying conditions were changed. The ingredients and conditions used in the preparation process are shown in Table 45 below. [Table 45] [Example]

[0184] PLGA-based sustained-release particles were produced by varying the emulsification and freeze-drying conditions.

[0185] The controlled-release particles of Examples 36-A to 36-D were prepared in the same manner as in Example 2-B, except that the homogenizer or freeze-drying conditions were changed. The ingredients and conditions used in the preparation process are shown in Table 46 below. [Table 46] [Example]

[0186] PLGA-based sustained-release particles were produced by varying the emulsification and freeze-drying conditions.

[0187] The controlled-release particles of Examples 37-A to 37-D were prepared in the same manner as in Example 2-A, except that the homogenizer or freeze-drying conditions were changed. The ingredients and conditions used in the preparation process are shown in Table 47 below. [Table 47] [Example]

[0188] PLGA-based sustained-release particles were produced by varying the emulsification and freeze-drying conditions.

[0189] The controlled-release particles of Examples 38-A to 38-D were prepared in the same manner as in Example 2-C, except that the homogenizer or freeze-drying conditions were changed. The ingredients and conditions used in the preparation process are shown in Table 48 below. [Table 48] [Example]

[0190] [D-2-(4-methylbenzenethio)sarcosine 3 ]Production of PLGA-based sustained-release particles containing cyclosporine A

[0191] In Example 1, the drug was converted from OND-1 to [D-2-(4-methylbenzenethio)sarcosine 3 The controlled-release particles of Examples 39-A to 39-C were prepared in the same manner, except that the ingredients and conditions used in the manufacturing process were shown in Table 49 below. [Table 49] [Example]

[0192] [N-methyl-aminobutyric acid 3 ]Production of PLGA-based sustained-release particles containing cyclosporine A

[0193] In Example 1, the drug was extracted from OND-1 with [N-methyl-aminobutyric acid 3 ]([N-methyl-Abu 3 The controlled-release particles of Examples 40-A to 40-C were prepared in the same manner, except that Cyclosporine A was used instead. The ingredients and conditions used in the manufacturing process are shown in Table 50 below. [Table 50] [Example]

[0194] [N-methyl-norvaline 3 ]Production of PLGA-based sustained-release particles containing cyclosporine A

[0195] In Example 1, the drug was extracted from OND-1 with [N-methyl-norvaline 3 ]Cyclosporin A ([N-methyl-Nva 3 The controlled-release particles of Examples 41-A to 41-C were prepared in the same manner, except that Cyclosporine A was used instead. The ingredients and conditions used in the manufacturing process are shown in Table 51 below. [Table 51] [Example]

[0196] [D-methylalanine 3 ]Production of PLGA-based sustained-release particles containing cyclosporine A

[0197] In Example 1, the drug was converted from OND-1 to [D-methylalanine 3 ]Cyclosporin A ([D-MeAla 3 The controlled-release particles of Examples 42-A to 42-C were prepared in the same manner, except that Cyclosporine A was used instead. The ingredients and conditions used in the manufacturing process are shown in Table 52 below. [Table 52]

[0198] Experimental Example 1: Evaluating the size, morphology, and surface condition of PLGA particles In this experiment, the size, morphology, and surface condition of the PLGA particles prepared in the examples were analyzed using SEM images to confirm whether they had morphological characteristics favorable for sustained release. The PLGA particles prepared in each example were dispersed in distilled water, centrifuged, and the precipitated PLGA particles were vacuum-dried again and observed under SEM.

[0199] 1 to 3 show SEM images of Examples 1-A to 1-C produced under vacuum drying conditions.

[0200] According to FIG. 1, Example 1-A exhibits a relatively uniform particle size distribution of about 5 to 30 μm, and exhibits a spherical shape with a smooth surface.

[0201] According to FIG. 2, Example 1-B exhibits a relatively uniform particle size distribution of about 20 to 40 μm, and exhibits a spherical shape with a smooth surface.

[0202] According to FIG. 3, Example 1-C exhibits a relatively uniform particle size distribution of about 10 to 30 μm, and exhibits a spherical shape with a smooth surface.

[0203] 4 to 6 show SEM images of Examples 2-A to 2-C produced under freeze-drying conditions.

[0204] According to FIG. 4, Example 2-A exhibits a relatively uniform particle size distribution of about 5 to 10 μm, and exhibits a spherical shape with a smooth surface.

[0205] According to FIG. 5, Example 2-B exhibits a polydisperse particle size distribution of about 2 to 20 μm, and exhibits a spherical shape with a smooth surface.

[0206] According to FIG. 6, Example 2-C exhibits a polydisperse particle size distribution of about 5 to 60 μm, and exhibits a spherical shape with a smooth surface.

[0207] 7 to 10 show SEM images of Examples 4-1-A to 4-1-D.

[0208] According to FIG. 7, Example 4-1-A exhibits a relatively uniform particle size distribution of about 3 to 8 μm, and exhibits a spherical shape with a smooth surface.

[0209] According to FIG. 8, Example 4-1-B exhibits a polydisperse particle size distribution of about 0.2 to 2 μm and exhibits a spherical shape.

[0210] 9, Example 4-1-C exhibits a particle size distribution of approximately 5 to 25 μm and a spherical shape. Furthermore, after one month of in-vitro release, it exhibits a particle size distribution of 3 to 10 μm and a spherical shape.

[0211] 10, Example 4-1-D (#73-1) exhibits a relatively uniform particle size distribution of approximately 2 to 8 μm, a smooth spherical shape, and after one month of in vitro release, the surface has dissolved and the interior has become hollow.

[0212] It can be seen from FIG. 11 that Examples 31-B to 31-D exhibited spherical shapes with smooth surfaces despite the changes in emulsification conditions.

[0213] 12 and 13, it can be seen that Examples 39-B, 40-B, 41-B, and 42-B, which used different cyclosporin 3-derivatives, exhibited spherical shapes with smooth surfaces.

[0214] 1 to 13, it can be seen that the PLGA particles containing the cyclosporine 3-derivative have a smooth surface shape, are free from the initial burst phenomenon due to external pores, and exhibit morphological characteristics that enable stable sustained drug release.

[0215] Experimental Example 2: Particle size analysis of PLGA controlled release formulation The sample was photographed at random positions using an SEM, and particle size distribution was confirmed by analyzing particle size using an image analysis program. The number of particles measured was at least 300, and particle size was measured by diameter.

[0216] 2.1. Particle size analysis of vacuum-dried PLGA controlled-release formulations with different LA:GA ratios

[0217] 14 to 16 show the results of particle size analysis for Examples 1-A to 1-C.

[0218] The average particle sizes of Example 1-A (#125), Example 1-B (#126) and Example 1-C (#127) were 16.8 μm, 15.4 μm and 9.44 μm, respectively.

[0219] 2.2. Particle size analysis of vacuum-dried PLGA controlled-release formulations based on emulsification rate

[0220] 17 to 19 show the results of particle size analysis of Examples 31-B to 31-D.

[0221] The average particle sizes of Examples 31-B (#120), 31-C (#121), and 31-D (#122) were 20.9 μm, 0.74 μm, and 0.61 μm, respectively. The average particle size tended to decrease as the homogenizer stirring speed increased to 5,000, 10,000, and 15,000 rpm. In particular, the average particle size decreased rapidly when the stirring speed increased from 5,000 rpm to 10,000 rpm. For example, the preferred homogenizer stirring speed for producing particles of approximately 20 μm in size is 5,000 rpm, while the preferred homogenizer stirring speed for producing particles of 1 μm or less is 10,000 rpm or higher.

[0222] Experimental Example 3. Measurement of drug encapsulation rate and yield of PLGA controlled release formulation The drug-loading capacity is the amount of drug encapsulated divided by the amount of recovered microspheres. The encapsulation efficiency can be expressed as the amount of drug encapsulated relative to the amount of drug input. The amount of encapsulated drug was measured by HPLC analysis (HPLC pump & column oven method). The HPLC conditions and analytical method are as follows: [Table 53]

[0223] [Analysis method] 1 mg of PLGA microspheres was dissolved in 1 ml of 75% ACN, vortexed for 30 seconds, and then sonicated for 3 minutes. The supernatant was then filtered through a 0.22 μm syringe filter and analyzed by HPLC. The drug content, encapsulation rate, and yield of microspheres were calculated using Equations 1 to 3.

[0224] [Formula 1] Drug loading capacity (%) = weight of drug determined in microsphere (mg) ÷ weight of microsphere recovered (mg) × 100

[0225] [Formula 2] Encapsulation rate (%) = Amount of encapsulated drug (weight of drug determined in microsphere) (mg) ÷ Amount of drug injected (weight of the feeding drug) (mg) × 100

[0226] [Formula 3] Yield of microspheres (%) = weight of microspheres (mg) ÷ (weight of the feeding drug + polymer) (mg) × 100

[0227] The encapsulation rate and yield of the OND-1-containing PLGA controlled-release particles are shown in Table 54 below. [Table 54]

[0228] In addition, the experimental results of measuring the drug content, encapsulation rate and yield for PLGA controlled-release particles containing cyclosporin A derivatives substituted with various 3-position derivatives are shown in Tables 55 and 56 below. [Table 55] [Table 56]

[0229] Experimental Example 4: Measuring the zeta potential of PLGA controlled release formulations The zeta potential was measured to analyze whether or not there was interparticle aggregation. If the zeta potential value was ±30mV or higher, repulsive forces would be generated and particles would not aggregate, but if it was ±10mV or lower, particles would aggregate. Therefore, a zeta potential value of -30mV or lower and +30mV or higher can be considered a stable dispersion.

[0230] In this experiment, a zetasizer (model name: Nano ZS 90, Malvern; dispersant: DW; temperature: 25°C, equilibration time: 120 seconds) was used to measure the surface charge of vacuum-dried PLGA sustained-release preparations of Examples 1, 3, 4, 5, 14, 17, and 20. The measurement conditions for the zetasizer were as follows, and measurements were taken at three locations, with the average values ​​shown in the following table. [Table 57] [Table 58]

[0231] As can be seen from the table, both Example 1 and Example 4 exhibit zeta potential values ​​of -30 mV or less, which indicates that they have excellent dispersibility.

[0232] Experimental Example 5. In-vitro release test (1) Experimental method The delayed drug release effect depends on the time it takes for PLGA to decompose. When PBS with 0.2% Tween 80 (pH 7.4), which is commonly used in in vitro drug release tests, is used as the release medium, the drug is not released for at least 2 weeks and up to 4 weeks. This differs from the in vivo release results.

[0233] Therefore, rat plasma was used as a release medium to perform an in vivo release test similar to in vivo conditions and calculate the optimal IV-IVC (in vitro-in vivo correlation). Rat plasma was prepared by dispensing blood obtained from rats into heparin-coated tubes and centrifuging the supernatant at 3,000 rpm for 10 minutes. The particles prepared in each Example were added to 2 ml of the obtained rat plasma at a concentration of 50 μg / ml (based on OND-1) and then stored in an incubator at 37°C with shaking at 90 rpm. Samples were then taken at regular intervals and analyzed by HPLC to measure the drug release amount. The HPLC conditions are as shown in Table 52. The HPLC analysis method is as follows:

[0234] [HPLC analysis method] The vial to be sampled was vortexed for 30 seconds, after which 100 μl was dispensed from the vial and centrifuged at 13,000 rpm for 30 minutes. 90 μl of the separated supernatant was dispensed, and 270 μl of ACN (Sample A) was added. 360 μl of 75% ACN (Sample B) was added to the remaining pellet. Samples A and B were sonicated for 3 minutes, and then centrifuged at 13,000 rpm for 30 minutes. 300 μl of the separated supernatant from Samples A and B was dispensed, filtered through a 0.22 μm syringe filter, and then analyzed by HPLC. The experimental results of the drug release patterns are shown in Figures 20-27.

[0235] The above results demonstrate that the PLGA release control agent of the present invention exhibits sustained release. Furthermore, based on the experimental results, drugs with appropriate drug release patterns can be prepared as needed. For example, for patients with severe hair loss, drugs with high initial drug release can be prepared, for patients with mild hair loss or those requiring prevention, drugs with gradual drug release can be prepared, and drugs with different release patterns can be prepared for women.

[0236] The specific results and details of each experiment are as follows:

[0237] 5.1. Comparison of drug release patterns depending on the LA:GA ratio of PLGA The OND-1 drug release patterns of Examples 1-A to 1-C are shown in Figure 20. As shown in Figure 20, the drug release rate was fastest for PLGA with an LA:GA ratio of 50:50, 75:25, and 85:15, with 38%, 17%, and 0% released, respectively, on day 7, and 53%, 32%, and 9%, respectively, released on day 14. Examples 1-A, 1-B, and 1-C all showed no initial burst phenomenon and exhibited excellent drug release patterns as sustained-release formulations.

[0238] SEM images of particles taken 7 days after drug release were also taken and are shown in Figure 21. As shown in Figure 21, Example 1-A, in which 38% of the drug was released, lost most of its spherical shape and showed aggregated particles. Example 1-B, in which 17% of the drug was released, maintained its spherical shape, but the surface became rough and many holes were observed. On the other hand, Example 1-C, in which 0% of the drug was released, showed spherical particles with smooth surfaces. The in-vitro release results and the morphological observation results are consistent with each other.

[0239] Previous literature has shown that the in vitro drug release profile of cyclosporine A (CsA)-containing PLGA microspheres exhibits a biphasic drug release pattern, characterized by an initial rapid burst followed by a slower release pattern. For example, one paper (J. Nanotechnology 2014, Article ID 683153) reported that approximately 70%–92% of the drug was released within 24 hours, while another paper (J. of Con Release 151 (2011) 286–294) reported that 75%–90% of the drug was released within 24 hours. Numerous papers have pointed out the initial burst phenomenon of cyclosporine A-containing PLGA microspheres.

[0240] The causes of biphasic release, which is a gradual release pattern after an initial burst release, have not been clearly elucidated, but may be due to the internal structure of the nanoparticles formed by freeze-drying, CsA remaining on the particle surface during the manufacturing process, a sudden increase in solubility due to the transformation of CsA from a crystalline structure to an amorphous structure during the freeze-drying process, or a sudden increase in solubility due to the formation of an amorphous solid dispersion between CsA and PLGA. The inventors determined that the most important causes of biphasic release are the weak binding strength between CsA and PLGA and their relatively high hydrophilicity.

[0241] CsA has an aqueous solubility of 27 μg / mL and a partition coefficient (logP) of 2.92 (Ismailos et al. 1991, Czogalla et al. 2009). Meanwhile, the CsA derivative OND-1 has an aqueous solubility of 10 μg / mL and a partition coefficient (logP) of 3.84. OND-1 is more hydrophobic than CsA and has a higher partition coefficient, which delays drug release from microspheres, allowing for sustained release and release rate control. Therefore, PLGA microspheres containing CsA, which has a lower partition coefficient than OND-1, exhibit an initial burst phenomenon, whereas PLGA microspheres containing OND-1, a more hydrophobic molecule, exhibit no initial burst and enable sustained drug release.

[0242] In conclusion, the OND-1-containing PLGA microspheres of the present invention differ from conventional CsA-containing PLGA microspheres in that they have high in vivo stability, excellent release control, and no initial burst in vivo, making them excellent in controlling drug release in vivo.

[0243] Additionally, the FT-IR results in Experimental Example 7 (described below) showed that OND-1, unlike CsA, shifted the amide I peak of OND-1, indicating a strong interaction between OND-1 and PLGA. Experimental Example 8 showed that OND-1, being more hydrophobic than CsA, strongly bound to PLGA, resulting in the Tg of OND-1 microspheres being neutralized by PLGA, resulting in a stable morphology. Therefore, the stable drug release pattern of OND-1-loaded PLGA microspheres may be due to the difference in the chemical properties of CsA and its 3-position derivative.

[0244] 5.2. Comparison of drug release patterns depending on the end group and molecular weight of PLGA (LA:GA = 50:50) The drug release patterns of Examples 1-A and 3-A to 3-C are shown in Figure 22. As shown in Figure 22, the drug release rates were fastest in Examples 1-A, 3-A, 3-C, and 3-B, namely, acid-7K, acid-38K, ester-38K, and ester-7K, in that order. On day 8, 37%, 32%, 21%, and 12%, respectively, were released, and on day 14, 54%, 45%, 29%, and 19% were released. All Examples showed an excellent sustained release pattern without an initial burst phenomenon.

[0245] 5.3. Comparison of drug release patterns depending on the end group and molecular weight of PLGA (LA:GA = 75:25) The drug release patterns of Examples 1-B and 4-A to 4-C are shown in Figure 23. As shown in Figure 23, the drug release rates were fastest in Examples 1-B, 4-B, 4-A, and 4-C, i.e., acid-4K, ester-4K, acid-38K, and ester-38K, with 17%, 11%, 0%, and 0% released, respectively, on day 8, and 33%, 17%, 9%, and 4%, respectively, released on day 14. All Examples showed no initial burst phenomenon and exhibited excellent drug release patterns as sustained-release formulations.

[0246] 5.4. Comparison of drug release patterns depending on the end group and molecular weight of PLGA (LA:GA = 85:15) The drug release patterns of Examples 1-C and 5-A are shown in Figure 24. As shown in Figure 24, the drug release rates were fastest in Examples 1-C and 5-A, i.e., ester-50K and ester-190K, with 9% and 1% released, respectively, on day 14. Neither Examples 1-C nor 5-A exhibited an initial burst phenomenon, demonstrating an excellent drug release pattern for sustained-release formulations.

[0247] 5.5. Confirmation of drug release pattern depending on drug content The drug release patterns of Examples 26-A to 26-C are shown in Figure 25. As shown in Figure 25, the drug release rates were fastest in Examples 26-C, 26-B, and 26-A, i.e., in the order of OND-1 contents of 60%, 40%, and 20%, with 36%, 24%, and 15% released, respectively, on day 14. All Examples showed no initial burst phenomenon and exhibited excellent drug release patterns as sustained-release formulations.

[0248] 5.6. Confirmation of drug release pattern by regulator The drug release patterns of Examples 23-A to 23-F are shown in Figure 26. According to Figure 26, the release rates of Examples 23-B, C, F, E, A, and D, i.e., the modifiers Span20, Poloxamer P124, Mrij52, Brij58, Tween80, and PEG400, were 96%, 53%, 48%, 39%, 34%, and 12%, respectively. All Examples showed no initial burst phenomenon, and demonstrated excellent drug release patterns as sustained-release formulations.

[0249] 5.7.3 Release patterns of drugs containing derivatives According to FIG. 27, the 3-position derivative of cyclosporin [D-2-(4-methylbenzenethio)Sar 3 ]Cyclosporine A, [N-methyl-D-Abu 3 ]Cyclosporine A, [N-methyl-D-Nva 3 ]Cyclosporine A and [D-MeAla 3 ]Cyclosporine A, like OND-1, showed no initial burst and showed an excellent drug release pattern as a sustained-release formulation.

[0250] Experimental Example 6. Difference in crystallinity between OND-1 and CsA: Results of XRD and DSC experiments Drug crystallinity can exist in crystalline, amorphous, or a combination of these forms, and is one of the important physicochemical properties that affect the rate of drug release.

[0251] 6.1. Confirmation of crystallinity by XRD (X-ray diffraction) The XRD measurement conditions and crystallization method used in this experiment are as follows: The measurement results are shown in Figures 28 and 29.

[0252] [Measurement conditions] -X-ray diffractometer, model name: MiniFlex, Rigaku, Tokyo, Japan / -X-ray generator: graphite monochromatized Cu Kα radiation(λ=1.5418Å / -2θrange:from 10° to 30° with a step size of 0.02° / -Scanning rate:1.0° / min

[0253] [Crystallization method] - Evaporation crystallization: 20 mg of CsA or OND-1 is dissolved in 5 ml of ACN, and the ACN is slowly evaporated at room temperature for 24 hours or more to obtain crystals.

[0254] Drowning-out crystallization: 20 mg of CsA or OND-1 dissolved in 5 ml of ACN was added to 50 ml of DW to form crystals, which were then filtered to obtain the crystals.

[0255] As shown in Figure 28, cyclosporine A showed a crystalline XRD pattern regardless of the manufacturing conditions. The XRD measurement results for raw cyclosporine A showed strong peaks characteristic of crystalline forms, confirming that the raw cyclosporine A powder was crystalline.

[0256] However, as shown in Figure 29, OND-1, a 3-position derivative of cyclosporine, always exhibited an amorphous XRD pattern regardless of the manufacturing conditions. It also exhibited an amorphous form when recrystallized by evaporation or drowning to induce a crystalline form. This difference suggests that OND-1, a 3-position derivative of cyclosporine, may exhibit release characteristics completely different from those of cyclosporine A.

[0257] 6.2. Confirmation of crystallinity by DSC (Differential scanning calorimetry) The melting point, glass transition temperature, and presence or absence of recrystallization of the drugs were determined by differential scanning calorimetry (DSC), and the difference in crystallinity between cyclosporine A and OND-1 was confirmed. The conditions for DSC measurement were as follows. The results of the DSC measurement are shown in Figures 30 and 31.

[0258] [Measurement conditions] DSC model: DSC Q2000, TA instruments / Purging gas: N2 Gas (flow: 70 ml / min) / Heating rate: 5°C / Isothermal temperature: 10°C / min) / Cycle number: 1 (from 20°C to 200°C)

[0259] According to FIG. 30, the raw powder of cyclosporin A exhibits a melting temperature (Tm) of about 112° C. in the first-order temperature rise curve, which indicates that the cyclosporin A is in a crystalline form.

[0260] On the other hand, as shown in Figure 31, the raw powder of OND-1 exhibited a phase transition due to the formation of a thermotropic liquid crystal or a glass-transition temperature (Tg) of crystalline cyclosporine A (112°C) that disappeared in the first and second heating curves, and was confirmed to be amorphous, at approximately 125°C.

[0261] Experimental Example 7: Evaluation of the interaction between drug and polymer within particles of PLGA controlled release formulation (FT-IR) Stable particles capable of controlled drug release must not undergo phase separation or recrystallization during storage and must suppress the initial burst. This requires a close interaction between the drug and PLGA. To study the interaction between the drug and PLGA, FT-IR spectroscopy was performed in the amide I region, which is the absorption region of the four amide groups in the cyclosporine A molecule, and the PLGA ester wavelength region.

[0262] In particular, the cyclosporin A amide I region (1600-1700 cm -1 ) is a region that shows the structural change of four amide bonds in the molecule, and is an important indicator of how the secondary structure of cyclosporin A changes when it is bound to the surrounding solvent or polymer. For example, in crystallized cyclosporin A, the four amide groups are involved in the formation of intramolecular hydrogen bonds, but when cyclosporin A comes into contact with a polar solvent or polymer, the amide groups form hydrogen bonds with the external solvent or polymer instead of the internal hydrogen bonds. Therefore, crystallized cyclosporin A shows a 1624 cm peak, which is characteristic of intramolecular hydrogen bonds. -1 However, when the compound is bound to a polar solvent or polymer, it exhibits a peak at 1630 cm , which is characteristic of intermolecular hydrogen bonds. -1 For OND-1, the shift of the amide I band due to the polarity of the solvent was confirmed. As a result, as in CsA, the peak shifted to 1622 cm -1 From 1634cm -1 It was found that it would move to.

[0263] Based on these FT-IR peak shifts, the shifts in the spectroscopic absorption peaks of the amide I region and the PLGA ester region can be used as evidence of chemical interactions to evaluate the chemical interactions between CsA and OND-1 and the PLGA network (Quimica Nova 35.4(2012):723-727; Current Drug Delivery 2006, 3, 343-349; The AAPS Journal 2007; 9(2)).

[0264] In this experiment, to evaluate the interaction between the cyclosporine 3-derivative and the PLGA polymer within the PLGA particles, the formulation of Example 26 was analyzed using Fourier-transform infrared spectroscopy (FT-IR). Furthermore, groups in which OND-1 and PLGA were physically mixed at weight ratios of 2:8, 3:7, 4:6, 5:5, and 8:2 (Physical Mixing, PM) were used as comparison groups. The FT-IR measurement conditions were as follows. The measurement results are shown in Figures 32 to 35.

[0265] [FT-IR measurement conditions] FT-IR Model: Nicolet is 5, Thermo Fisher / Range: 800-3000cm -1 / Interval:4cm -1 / Crystal type:Diamond(pH 1~12)

[0266] According to Figures 32 and 33, OND-1 has an amide I band peak (amide C=O) at 1622 cm -1 The ester bond peak of PLGA (ester C=O) is 1746 cm -1 It was expressed as:

[0267] As shown in Figure 34, the comparison groups in which OND-1 and PLGA were physically mixed at weight ratios of 2:8, 3:7, 4:6, 5:5, and 8:2 (OND-1:PLGA) showed the amide 1 band peak of OND-1 at 1622 cm without any peak shift. -1 This indicates that no mutual bonding occurs between the two materials.

[0268] However, according to Figure 35, the nanoparticles of the present invention do not show the amide I band of OND-1 at 1622 cm -1 1633-1634cm from the peak -1 A significant shift to the OND-1 band was observed. In particular, in nanoparticles with an OND-1:PLGA weight ratio of 2:8 (Example 26-A) and 3:7 (Example 26-B), the OND-1 amide I band peak shifted significantly, indicating strong interconnection between OND-1 and PLGA. A consistent trend in peak shift was also observed in particles with an OND-1:PLGA weight ratio of 4:6, 5:5, and 8:2. As the OND-1 weight ratio increased, the amount of OND-1 bound to PLGA decreased, resulting in a decrease in the peak shift.

[0269] Previous studies have shown that no shift in the amide I region was observed in the FT-IR spectra of PLGA microsphere formulations containing cyclosporine (CsA). This is thought to be due to the absence or weak chemical interaction between CsA and PLGA (Quimica Nova 35.4 (2012): 723-727, Current Drug Delivery, 2006, 3, 343-349, Materials Research, Vol. 11, No. 2, 207-211, 2008, International Journal of Pharmaceutics 389 (2010) 186-194). Therefore, the initial burst and poor formulation stability of PLGA microspheres containing CsA are thought to be due to the weak chemical interaction between CsA and PLGA.

[0270] Previous research results did not predict that when a cyclosporine 3-derivative and PLGA formed particles, the intramolecular amide group would strongly bind to PLGA. This may be because OND-1 is a cyclosporine A derivative, but the addition of a methylthio group at the 3-position changes its properties. Experimental examples of the present invention showed that, unlike CsA, which is mostly crystalline, OND-1 remained amorphous despite various crystallization processes. Therefore, OND-1-containing PLGA nanoparticles can exhibit properties completely different from existing CsA-based PLGA nanoparticles.

[0271] In conclusion, PLGA microspheres containing OND-1 differ from PLGA microspheres containing CsA in that the OND-1 amide I peak shift indicates a strong interaction between the two. These experimental results support the idea that the drug-controlled formulation of the present invention is highly stable, has no initial burst in vivo, and has excellent controlled-release properties.

[0272] Experimental Example 8. Evaluation of the stability of drugs and polymers within particles of PLGA controlled release formulations (DSC) In order to prevent phase separation or recrystallization of drug-loaded particles during storage, intermolecular interactions between the drug and the polymer carrier are necessary. In this experiment, the melting point or glass transition temperature of the drug and the presence or absence of recrystallization were confirmed by differential scanning calorimetry (DSC). The DSC measurement conditions were the same as those in Example 6-2. The measurement results are shown in Figures 36 to 38.

[0273] As shown in Figure 36, the glass transition temperature (Tg) of OND-1 or the phase transition due to the formation of thermotropic liquid crystal was observed at 125°C, and the Tg of PLGA was observed at 51°C.

[0274] Figures 37 and 38 show the results for the nanoparticle particles of Example 26-A. Figure 37 shows that the Tg (or phase transition due to the formation of thermotropic liquid crystals) of OND-1 is 125°C, whereas the Tg of OND-1 did not appear at 20% NP. Figure 38 also shows that the Tg values ​​at 50% and 80% NP were lower than the Tg of OND-1. This may be because the interaction between OND-1 and PLGA prevented the OND-1 molecules from undergoing phase change or molecular fluidity due to temperature rise within the particles, and the PLGA may have stably maintained OND-1 in an amorphous state.

[0275] Experimental Example 9: In vitro skin penetration experiment of cyclosporin 3-derivatives The percutaneous absorption and diffusion test (Franz Diffusion Cell) was used to confirm whether or not cyclosporine 3-derivatives penetrated the skin after topical application.

[0276] Specifically, pig skin was debrided to remove subcutaneous fat from the dermis and cut into 3 cm diameter disks. The stratum corneum was placed in the upper donor chamber and the dermis in the lower receptor chamber, and the Franz diffusion device (5 ml receptor volume, 0.785 cm diffusion area) was used. 2 ) was attached. 0.2 ml of olive oil containing 200 μg of OND-1 was placed in the donor chamber and incubated at 37°C and 600 rpm. Every hour, 500 μl of aqueous solution (PBS containing 0.2% Tween 80, pH 7.4) was sampled and the same amount of solution was replenished.

[0277] The 3-position cyclosporine derivative in the sampled solution was quantitatively analyzed by HPLC, and the average value of five repeated experiments was calculated to determine the amount of permeated 3-position cyclosporine derivative, which is shown in Table 58 below.

[0278] The skin samples used for 24 hours were separated from the skin permeation device, washed several times with ethanol, dried, and tape-stripped (10 times with Scotch tape) to completely remove any remaining drug on the surface. The collected skin samples were dissolved by incubating them in a collagenase type I (Gibco) solution at 37°C for 24 hours. Samples were then extracted using a liquid-liquid phase extraction method using methyl tert-butyl ether (MTBE). The 3-position cyclosporine derivatives were quantitatively analyzed by HPLC, and the amount of drug deposited in the skin was calculated.

[0279] The specific method for the liquid-liquid extraction method is as follows. 200-400 μl of each skin dissolution sample was dispensed, and 1.2 ml of MTBE was added. After high-speed shaking for 10 minutes, the sample was centrifuged (13,000 rpm, 3 minutes) to separate the organic solvent layer (1 ml) and dried under a nitrogen stream for 10 minutes. The sample was dissolved in a reconstitution solvent (75% acetonitrile) and filtered through a syringe filter to obtain the sample to be analyzed.

[0280] [HPLC analysis conditions] Analytical system: Agilent 1100 series, CSA 4000 / Analytical column: Kromasil 100-5-C18, 4.6 x 150 at 70°C / Mobile phase: Mobile phase A is water, mobile phase B is acetonitrile / Mobile phase flow rate: ml / min / Sample injection volume: 20 μl [Table 59]

[0281] As shown in Table 59, the transdermal and systemic absorption of cyclosporine 3-derivatives was confirmed to be extremely small. The deposition of cyclosporine 3-derivatives in the skin was approximately 0.3 to 1.1 μg / cm. 2 The experimental results showed that the hair growth effect of cyclosporine 3-derivatives requires drug delivery by intralesional mesotherapy or other methods.

[0282] Experimental Example 10: Confirmation of hair growth effect by local drug delivery (intralesional mesotherapy)

[0283] 10-1. Confirmation of hair growth effect by local injection administration It was confirmed whether or not a cyclosporin 3-derivative, when administered locally by injection, would have the effect of promoting hair follicle growth and hair growth.

[0284] Specifically, the hair on the backs of 42-49 day-old mice (C57BL / 6, female) was removed, and each mouse was weighed and divided into groups so that the weights were evenly distributed. A depot solution containing the particles of Example 1-B was administered locally by intralesional mesotherapy (0.25 mm (31 G) × 8 mm insulin syringe), and the hair growth effect was confirmed.

[0285] As shown in Figure 39, hair follicle formation and hair growth were prominent at the site of local administration, but no hair follicle formation was observed in the surrounding area. Therefore, it was confirmed that local administration of cyclosporine 3-derivatives exhibits a strong hair growth effect.

[0286] 10-2. Confirmation of blood concentration of drugs administered by local injection In this experiment, it was confirmed whether the hair growth effect of cyclosporin administered topically in Experimental Example 10-1 was due to a systemic effect or a local effect.

[0287] Specifically, the drug concentration in blood was measured using liquid-liquid extraction with MTBE (methyl tert-butyl ether) and LC / MS analysis. The preparation of skin samples from the hair growth and non-administered areas and the liquid-liquid extraction with MTBE were the same as in Example 9. The LC / MS analysis conditions were as follows: The HPLC analysis system used was an Agilent 1100 series CSA 4000, the column was a Unison UK-C8 (3 μm), 2.0 × 50, and the temperature was 70°C. Mobile phase A was water and formic acid (100:0.1, v / v), and mobile phase B was methanol and formic acid (100:0.1, v / v). Mobile phase B was initially increased to 20% for 3 minutes, and after equilibration, the mobile phase flow rate was 0.4 mL / min, and the sample injection volume was 3 μL. The gravimetric analyzer used was a 4000 Q TRAP Mass Spectrometer system manufactured by Applied Bioscience.

[0288] The remaining amounts of drug at the administration site and non-administration site as determined by LC / MS analysis are shown in Table 60 below. [Table 60]

[0289] According to Table 60, OND-1 was detected in large amounts in the skin at the site of topical administration, whereas OND-1 was hardly detected in the surrounding skin and systemic blood. Therefore, it was confirmed that the hair growth effect of cyclosporine 3-derivatives was due to a local, rather than a systemic, action, and that the hair growth effect can be obtained by topically administering cyclosporine 3-derivatives to the hair loss site.

[0290] Experimental Example 11: When PLGA Depot was administered by local injection (intradermal (intralesional) injection), drug disappearance into the systemic blood was confirmed. If a locally administered drug is easily lost into the systemic blood, there is a risk of systemic toxicity, low efficacy, etc. In this experiment, the degree of drug loss due to diffusion into the systemic blood was confirmed when the PLGA depot formulation of the present invention was administered by local injection.

[0291] Four- to five-week-old Sprague-Dawley female rats were prepared. Prior to drug administration, the animals were stripped of hair from their backs and weighed to ensure even distribution of individual weights. After a one-day adaptation period, the OND-1-containing drug formulation (#126) from Example 1-B and a control OND-1 solution (PG:EtOH:DW (=6:2:2)) were administered intradermally (ID) at six sites on the shaved backs. Approximately 50 μl of the injection solution was administered using a 0.25 mm (31 G) x 8 mm insulin syringe.

[0292] After drug administration, approximately 300-400 μl of blood was collected from the subclavian vein of the experimental animals at predetermined intervals (1, 3, 6, 9, 12, 15, 18, 21, 24, 28, 32, 36, 40, 44, 48, 54, 72, 78, 144, 168, and 192 hours after drug administration). The collected blood samples were processed using liquid-liquid phase extraction with MTBE (methyl tert-butyl ether), and the drug concentration in the blood was measured by LC / MS analysis. (See Experimental Example 10 above for details on the MTBE liquid-liquid phase extraction method and LC / MS analysis.)

[0293] The results of the experiment are shown in Table 61 and FIG. [Table 61]

[0294] As shown in Table 61 and Figure 40, the experimental animal group administered the drug-encapsulated PLGA particle formulation of the present invention showed lower systemic exposure than the group administered the general solution formulation. Quantitative comparisons of the AUC (area under the curve) values ​​confirmed that the blood drug concentration in the group administered the general solution was approximately 8 times higher than that in the group administered the PLGA particle formulation.

[0295] The results of this experiment show that when PLGA particles containing a cyclosporine 3-derivative are administered topically to the skin, they suppress the systemic side effects caused by increased blood levels of the drug that can occur with general injectable solution formulations, while maintaining a gradual and long-lasting therapeutic window for drug concentration in the hair loss area, resulting in sustained hair loss and hair growth effects.Comparing the condition of the experimental animals during the experiment, no specific clinical symptoms were observed in any of the treatment groups.

[0296] Experimental Example 12: Confirmation of the amount of PLGA Depot remaining on the skin after local injection (intradermal (intralesional) injection) In order for a topically administered drug to be effective, it must remain at the site of topical administration for a certain period of time. In this experiment, we investigated the amount of drug remaining over time after topical administration to the skin.

[0297] Four to five week old Sprague Dawley female rats were prepared and their back hair was removed. The experimental animals were weighed and divided so that the weight of each individual was evenly distributed, and a one-day adaptation period was allowed. An injectable formulation containing Example 1-B particles was injected transdermally (ID) into the hairless back (using a 0.25 mm (31 G) x 8 mm insulin syringe).

[0298] After the observations were completed, skin samples were collected from the backs of the experimental animals and subjected to liquid-liquid extraction using MTBE (methyl tert-butyl ether) and HPLC for quantitative analysis of residual drugs (see Experimental Example 9 for specific methods).

[0299] The results of the HPLC analysis are shown in Figure 41. Figure 41 shows that the PLGA Depot had an AUC four times higher than that of the standard solution, demonstrating a significantly longer and sustained skin retention. This indicates that when a cyclosporine 3-derivative is delivered to a local skin site in the form of PLGA particles, it minimizes the rapid increase in systemic blood drug concentration and resulting systemic side effects that can occur with simple injectable solution formulations, while maintaining a long-lasting, long-lasting therapeutic window for the drug concentration in the hair loss site, resulting in sustained hair loss and hair growth effects.

[0300] Experimental Example 13. Evaluation of remaining drug in the skin based on the LA:GA ratio of PLGA Four- to five-week-old Sprague-Dawley rats (female) were prepared, their dorsal hair removed, and the experimental animals were weighed and divided to ensure even weight distribution. After a one-day adaptation period, OND-1-containing PLGA particles with different LA:GA ratios (Examples 1-A (#125), 1-B (#126), and 1-C (#127)) were intradermally injected (ID, injection solution: approximately 50 μl of saline) into the lower back of the hairless rats at specified intervals (1, 4, 8, 10, 15, 16, and 17 days before the end point, a total of seven injections) using a 0.25 mm (31 G) x 8 mm insulin syringe.

[0301] After the observation was completed, skin samples of similar size were collected from the dorsum of the experimental animals and quantitatively analyzed for drugs by HPLC using liquid-liquid phase extraction with MTBE (see Experimental Example 9).

[0302] The results of the experiment are shown in Figure 42. As shown in Figure 42, the three formulations with different LA:GA ratios showed differences in the drug disappearance rate at the administration site over time.

[0303] To confirm this numerically, the AUC (area under curve) values ​​of the drug residual curve over time were plotted in a graph, and the AUC values ​​of the three formulations with different LA:GA ratios showed statistical significance with each other (t-test, *p<0.05, **p<0.01, ***p<0.001).

[0304] These results demonstrate that PLGA particles loaded with cyclosporine 3-derivative drugs exhibit significant differences in the degree of drug disappearance at the administration site depending on the type of PLGA polymer (PLA:PGA = 50:50, 75:25, 85:15). This indicates that it is possible to manufacture sustained-release formulations with controlled drug release rates by changing the type of PLGA polymer as needed. Comparison of the condition of the experimental animals during the experiment revealed no specific clinical symptoms in any of the treatment groups.

Claims

1. Use of a cyclosporin 3-derivative for producing a pharmaceutical composition for preventing hair loss or promoting hair growth in a topical administration form, the composition comprising controlled-release particles comprising a cyclosporin 3-derivative and a biodegradable polymer: The cyclosporin 3-derivative is [D-2-methylthio-sarcosine 3 ]cyclosporin A, [D-2-(4-methylbenzenethio)sarcosine 3 ]cyclosporin A, [N-methyl-aminobutyric acid 3 ]cyclosporin A, [N-methyl-norvaline 3 ]cyclosporin A, and [D-methyl-alanine 3 ]cyclosporin A; The biodegradable polymer is Use of poly(D,L-lactic-co-glycolic acid), PLGA.

2. The cyclosporin 3-derivative is [2-methylthio-sarcosine ３ ] cyclosporin A, 2. The use according to claim 1.

3. The poly(lactic-co-glycolic acid) (PLGA) has a molar ratio of lactide to glycolic acid monomers of 90:10 to 40:

60.

2. The use according to claim 1.

4. The end groups of the polylactic acid-co-glycolic acid are ester (neutral charge) or carboxylic acid (negative charge).

2. The use according to claim 1.

5. The polylactic acid-co-glycolic acid has a molecular weight of 4,000 to 240,000 Da.

2. The use according to claim 1.

6. The polylactic acid-co-glycolic acid is a cationic graft copolymer into which poly-L-lysine has been introduced.

2. The use according to claim 1.

7. the weight ratio of the cyclosporin 3-derivative to the biodegradable polymer is 5:95 to 90:10; 2. The use according to claim 1.

8. The average diameter of the particles is 0.01 to 300 μm.

2. The use according to claim 1.

9. The particles have an average zeta potential of +2 mV or more or -2 mV or less; 2. The use according to claim 1.

10. The particles contain 5 to 40% (w / w) of a cyclosporin 3-derivative.

2. The use according to claim 1.

11. The hair loss is one selected from the group consisting of androgenetic alopecia (AGA), female-pattern hair loss, autoimmune disease alopecia areata, telogen effluvium, and cicatricial alopecia; 2. The use according to claim 1.

12. The composition is for topical administration to the skin intralesionally, intradermally, or subcutaneously.

2. The use according to claim 1.

13. The composition is in the form of a local injection formulation, a depot injection, or a skin implant.

2. The use according to claim 1.

14. The composition further comprises one or more selected from the group consisting of water for injection, an isotonic agent, and a suspending agent.

2. The use according to claim 1.

15. The water for injection is at least one selected from the group consisting of saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and ethanol; 15. The use according to claim 14.

16. The isotonicity agent is at least one selected from the group consisting of D-mannitol, maltitol, sorbitol, lactitol, xylitol, and sodium chloride.

15. The use according to claim 14.

17. The suspending agent may be sodium carboxymethylcellulose, polysorbate 80, starch, starch derivatives, polyhydric alcohol, chitosan, chitosan derivatives, cellulose, collagen, gelatin, hyaluronic acid (HA), alginic acid, pectin, carrageenan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, or the like. sulfate), polylysine, titin, fibrin, agarose, fluran, and xanthan gum; 15. The use according to claim 14.

18. A method for producing controlled-release particles loaded with a cyclosporin 3-derivative according to claim 1, comprising the following steps: a) dissolving a cyclosporin 3-derivative and a biodegradable polymer in a solvent to prepare a drug-polymer dispersed phase; wherein the cyclosporin 3-derivative is [D-2-methylthio-sarcosine 3 ]cyclosporin A, [D-2-(4-methylbenzenethio)sarcosine 3 ]cyclosporin A, [N-methyl-aminobutyric acid 3 ]cyclosporin A, [N-methyl-norvaline 3 ]cyclosporin A, and [D-methyl-alanine 3 ]cyclosporin A; The biodegradable polymer is Polylactic acid-co-glycolic acid (poly(D,L-lactic-co-glycolic acid), PLGA), b) mixing the dispersed phase with a continuous phase containing a surfactant to form an emulsion; and c) mixing and stirring the prepared emulsion with a quenching medium to remove the solvent from the dispersed phase, thereby producing particles carrying the cyclosporin-3-derivative;

19. The solvent for the dispersed phase is at least one selected from the group consisting of dichloromethane, chloroform, acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide, ethyl acetate (EA), butyl acetate, ethyl formate, isopropyl acetate, isopropyl formate, diethyl ether, and glycofurol; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

20. The solvent of the dispersed phase further comprises one or more co-solvents selected from the group consisting of methanol, ethanol, acetone, isopropanol, diethyl ether, chloroform, ethyl acetate, DMSO, and acetonitrile; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

21. The continuous phase surfactant is one or more selected from the group consisting of polyethylene glycol, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, lecithin, gelatin, chitosan, Eudragit, polyvinyl alcohol, polyoxyethylene sorbitan fatty acid esters, poloxamers, spans, polyoxyethylene castor oil derivatives, Cremophor, Myrj 52, Brij 58, cyclodextrins, sodium lauryl sulfate, sodium stearate, esteramines, linear diamines, and aliphatic amines; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

22. the continuous phase surfactant is polyvinyl alcohol; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

23. The continuous phase surfactant is one or more selected from the group consisting of span, labrasol, rheodol MO-60, glyceryl monooleate, and triacetin; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

24. The continuous phase surfactant is contained in the continuous phase at 0.1 to 20% (w / v). A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

25. The mixing ratio of the dispersed phase to the continuous phase in step b) is 1:5 to 1:50 by volume; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

26. The quenching medium is water, methanol, ethanol, propanol, ethyl acetate, PVA (polyvinyl alcohol), or a combination thereof. A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

27. The quenching medium is mixed in an amount of 1 to 100 times the emulsion. A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

28. The step c) is carried out by an emulsification method selected from mechanical stirring, high-pressure emulsification, fluid bed, static motor, ultrasonic, membrane, nozzle injection, and dripping; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

29. After step c), d) further comprising a step of separating the obtained particles carrying the cyclosporin 3-position derivative; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 18.

30. After step d), e) vacuum drying or freeze drying the separated particles; A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 29.

31. The vacuum drying is carried out at a temperature of 30 to 40°C, and the freeze-drying is carried out at -30 to -40°C. A method for producing controlled-release particles carrying the cyclosporin 3-derivative according to claim 30.

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