KR20200032409A - Composition for preventing, improving or treating inflammatory disease comprising pH sensitive block copolymer comprising curcumin derivative - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating inflammatory diseases containing a pH-sensitive block copolymer, which contains a curcumin derivative, as an active component. The pH-sensitive block copolymer, which contains the curcumin derivative according to the present invention forms micelles so that it is possible to load a large amount of curcumin and allow the curcumin derivative to last longer in the body. In addition, since micelles collapse at a weakly acidic pH of the inflammatory site and an acetal bond to which the curcumin derivative is linked is broken, the curcumin derivative is generated and released to act specifically on the inflammatory site to have less toxicity to normal cells, thereby having an anti-inflammatory effect by itself.

Description

Composition for preventing, improving or treating inflammatory disease comprising ｐＨ sensitive block copolymer comprising curcumin derivative}

The present invention relates to a composition for preventing, improving or treating an inflammatory disease comprising a curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

Many natural compounds have been used to develop new drugs. More than 50% of newly approved drugs were developed with natural inspiration, such as biological macromolecules, unmodified natural compounds and natural product derivatives, which account for 45% of the best-selling drugs.

Among natural compounds, curcumin has been widely used in the field of traditional medicine due to its strong anti-inflammatory action and has been steadily studied in the field of new drug development. In the field of new drug development, natural drugs have many advantages such as chemical diversity, but many of the natural compounds are poorly soluble in the blood and thus have low bioavailability. About 30% of natural compounds, and about 40% of marketable drugs are poorly water soluble, and these drugs must be dissolved in a water miscible solvent that can cause toxicity. On the other hand, polymer prodrug nanoparticles have the advantage of being able to exclude the use of toxic organic solvents because they can be very stably dispersed in water.

Meanwhile, the existing pharmaceutical nanotechnology field has extensively studied various drug delivery systems such as liposomes, micelles, and solid nanoparticles. In the case of the conventional drug delivery system, the therapeutic efficacy of the drug is maximized, but the content of the drug loaded is very limited. It is well known that the drug content of common polymer prodrug nanoparticles is less than 20% by weight. In addition, the loading efficiency of the recently developed curcumin-drug delivery system does not exceed 20% by weight. When the therapeutic efficacy of the drug is weak or the loading efficiency of the drug is low, a large amount of the drug carrier should be used to achieve sufficient therapeutic efficacy. When the drug carrier is used excessively, there is a risk of causing toxicity.

Accordingly, there is a need for a study capable of effectively suppressing inflammatory cytokines by preparing curcumin in a micellar form and loading a large amount of curcumin.

The present inventors are trying to increase the bioavailability of the curcumin derivative, keep it in the body for a long time, and search for a method of specifically delivering it to the inflammatory site. When a block copolymer containing the curcumin derivative is formed of micelles, a large amount of curcumin is loaded. It can be, the residence time in the living body is increased, it was confirmed that the fact that it effectively inhibits inflammatory cytokines by acting specifically on the inflammatory site, and completed the present invention.

KR 10-0773078 B

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

In addition, another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases comprising a curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

In addition, another object of the present invention is to provide a composition for the diagnosis of inflammatory diseases comprising a curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

In addition, another object of the present invention is to provide a method for preparing a pH-sensitive block copolymer containing curcumin derivatives.

In order to achieve the above object, the present invention is a polyethylene glycol compound (A); And a curcumin derivative-substituted poly (β-aminoester) or poly (amidoamine) compound (B) copolymerized with a curcumin derivative-containing pH-sensitive block copolymer represented by any one of the following Chemical Formulas 1-1 to 1-3 Provided is a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the coal as an active ingredient:

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,

X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,

X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,

R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,

R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,

m and n are each independently an integer of 15 to 10000,

p is an integer from 1 to 10.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising a polymer micelle containing the curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases comprising the curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

In addition, the present invention provides a composition for diagnosing an inflammatory disease comprising the curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

In addition, the present invention provides a method for preparing the pH-sensitive block copolymer containing curcumin derivatives.

The pH-sensitive block copolymer containing curcumin derivatives according to the present invention forms micelles so that a large amount of curcumin can be loaded and the curcumin derivatives last longer in the body. In addition, at the weakly acidic pH of the inflammatory site, micelles collapse and the acetal bond to which the curcumin derivative is linked is broken, and curcumin derivative is generated and released to act specifically on the inflammatory site and has less toxicity to normal cells, so it has an anti-inflammatory effect by itself. .

1 is a reaction scheme showing the process of synthesizing the AICP of the present invention.
2A is a diagram showing the chemical structure of Compound 1 and the results of ¹ H NMR according to an embodiment of the present invention, and FIG. 2B is a diagram showing the chemical structure of AICP and the results of ¹ H NMR.
Figure 3a is a result of measuring the UV-vis absorbance of AICP,
Figure 3b is a result showing the fluorescence emission spectrum of AICP.
Figure 3c is a diagram showing the measurement results of the critical micelle concentration of AICP.
Figure 3d is a view showing the observation result of a transmission electron microscope (TEM) for the size analysis of AICP.
3E shows a TEM image of AICP micelles at pH 6.6.
3F is a graph showing the release kinetics of curcumin from AICP micelles at pH 6.0, pH 6.6 and pH 7.4.
3G is a diagram showing the AICP fluorescence spectrum in water and THF.
Figure 3h is shown by measuring the fluorescence spectrum of AICP micelles at various pH.
3i shows the fluorescence signal intensity of AICP micelles at 560 nm.
4 shows the ¹ H NMR spectrum of AICP micelles formulated in D ₂ O.
FIG. 5 is a view showing the diameter of micelles in various pH environments for 48 hours in order to investigate deamicellation of AICP micelles.
6 is a graph comparing curcumin and AICP micelles with H ₂ O ₂ scavenging activity.
7A is a diagram showing the results of the cytotoxicity evaluation of AICP micelles.
Figure 7b is a graph showing a comparison of the apoptosis inhibitory effect of curcumin and AICP micelles in LPS-stimulated RAW264.7 cells, Figure 7c is a comparison of the apoptosis inhibitory effect of curcumin and AICP micelles in H ₂ O ₂ stimulated RAW264.7 cells It is a graph shown.
7D is a result showing the antioxidant effect of AICP micelles in LPS-stimulated RAW264.7 cells.
7E and 7F are results showing the anti-inflammatory effect of AICP micelles on the expression of IL-1β (e) and TNF-α (f) in LPS-stimulated RAW264.7 cells.
8A shows an overview of an experiment injecting AICP micelles into a joint with osteoarthritis induced by MIA.
Figure 8b shows the fluorescence image in the incision-induced inflammation-induced joint after AICP micelle injection, Figure 8c is a result of quantitative analysis of the fluorescence signal by the AICP micelle in the inflammation-induced joint.
8D shows the H ₂ O ₂ level in the inflammation-inducing joint as a fluorescence signal intensity.
8E and 8F are graphs showing the therapeutic effect of AICP micelles on the expression of IL-1β (e) and TNF-α (f) in inflammation-inducing joints.
9 is a histological examination results of MIA-induced inflammation.
10A is a result of ALT activity evaluation for confirming in vivo toxicity of AICP micelles, and FIG. 10B is a result of performing histological examination of each organ after administration of AICP micelles.

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

Pharmaceutical compositions for the prevention or treatment of inflammatory diseases of the present invention,

Polyethylene glycol-based compounds (A); And

One or more compounds or copolymers thereof selected from the group consisting of poly (β-aminoester) substituted with curcumin derivatives represented by the following formula 1 and poly (amido amines) substituted with curcumin derivatives represented by the following formula ( B);

It contains the curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

[Formula 1]

In Formula 1, X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,

X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group.

Preferably, in Chemical Formula 1, X ₁ to X ₄ are each H, and X ₅ and X ₆ are each independently H or OH.

The curcumin derivative-containing pH-sensitive block copolymer includes a polyethylene glycol compound (A); And a curcumin derivative substituted poly (β-aminoester) or poly (amidoamine) compound (B), which may be represented by any one of the following Chemical Formulas 1-1 to 1-3.

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,

X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,

X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,

R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,

R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,

m and n are each independently an integer of 15 to 10000,

p is an integer from 1 to 10.

Preferably, R ₄ in the above formula is -CH ₂ -CH ₂ -COO-CH ₂ -CH ₂ -in the formulas 1-1 to 1-3 (= N-CH ₂ -CH ₂ -COO-CH _2- CH ₂ -O-), or -CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -(= N-CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -O- Connected to)

In Chemical Formula 1-1, it is not -CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -(= N-CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -O-connected in the direction).

The pH-sensitive block copolymer containing curcumin derivatives according to the present invention has a hydrophilic property with a poly (β-aminoester) substituted curcumin derivative, a poly (amido amine) substituted curcumin derivative, or a copolymer thereof, which is sensitive to pH. The curcumin derivative is substituted by copolymerizing the polyethylene glycol-based compound, and since micelles collapse at weakly acidic inflammatory sites because the micelle formation is possible and sensitive to changes in pH, the curcumin derivative enclosed inside the micelle is released and has an anti-inflammatory effect. It is characterized by having.

The curcumin derivative-containing pH-sensitive block copolymer is capable of forming or disintegrating micelles according to pH change due to amphiphilicity and pH sensitivity, and preferably forms micelles when the pH is in the range of 6.7 to 7.4, and the pH is In the case of 6.6 or less, the micelles will collapse. In particular, the block copolymer has an advantage of exhibiting excellent sensitivity within a pH ± 0.2 range.

The curcumin derivative-containing pH-sensitive block copolymer is a poly (β-aminoester) or poly (amido amine) in which the hydrophilic polyethylene glycol-based compound and the curcumin derivative are substituted as long as the pH sensitivity is maintained and the properties of forming micelles are maintained. ) In addition to the compound, it may further include other conventional monomers in the art, which also falls within the scope of the present invention.

The molecular weight range of the block copolymer is not particularly limited, but is preferably in the range of 1000 to 20,000. When the molecular weight is less than 1000, not only is it difficult to form block copolymer micelles at a specific pH, but even if it is formed, it is soluble in water and easy to disintegrate. In addition, when the molecular weight exceeds 20000, the balance of hydrophilicity / hydrophobicity is broken, and it is possible to precipitate without forming micelles at a specific pH.

Polyethylene glycol-based compound (A), which is one of the constituents of the block copolymer according to the present invention, can be used without limitation, a biodegradable compound having conventional hydrophilicity known in the art, but is acrylate or at the end of the polyethylene glycol-based compound. It is particularly preferable to have a monofunctional group such as methacrylate or to include polyethylene glycol in the main chain. In the case of the polyethylene glycol-based compound (A) containing polyethylene glycol in the main chain, the polyethylene glycol is contained in the main chain rather than the terminal, thereby forming a come-like block copolymer, which makes nano more stable than micelles. Particles can form. The molecular weight (Mn) of the polyethylene glycol-based compound is not particularly limited, but is preferably in the range of 500 to 5000. When the molecular weight (Mn) of the polyethylene glycol-based compound is outside the above range, for example, if it is less than 500 and exceeds 5000, it is difficult to control the molecular weight of the final block copolymer and it is easy to form micelles using the block copolymer. Do not. In other words, when the molecular weight is less than 500, the length of the hydrophilic block is too short at a specific pH, so that self-assembly due to hydrophilicity / hydrophobicity does not occur, and it is difficult to form micelles and is likely to dissolve and disintegrate even if formed. In addition, when the molecular weight exceeds 5000, the block length becomes too large compared to the molecular weight of the poly (β-aminoester) or poly (amido amine) in which the curcumin derivative which is hydrophobic is substituted, thereby breaking the balance of hydrophilicity / hydrophobicity and causing micelles at a specific pH. It may not form and may precipitate.

The content of the polyethylene glycol (A) -based block in the pH-sensitive block copolymer containing curcumin derivatives according to the present invention is not particularly limited, but is preferably 5 to 95 parts by weight, preferably 5 to 40 parts by weight. Polyethylene glycol is a monoacrylate and can act as a terminator that reacts at only one end in the polymerization process. Therefore, when the content of the polyethylene glycol-based block is less than 5 parts by weight, the molecular weight of the polymer of the block copolymer is high, but the yield of the copolymer to which the polyethylene glycol-based block is connected decreases, and when it exceeds 95 parts by weight, the termination reaction is too high. Polyethylene glycol-based blocks may be connected a lot due to a lot of occurrence, but the molecular weight is lowered because polymerization does not occur much. In addition, the block copolymer is a double block copolymer of type AB by adjusting the reaction molar ratio of a poly (β-aminoester) or poly (amido amine) substituted with a polyethylene glycol-based compound and a curcumin derivative; ABA or BAB type triple block copolymers or more can form a variety of block shapes.

One or more compounds selected from the group consisting of poly (β-aminoester) substituted with curcumin derivatives and poly (amido amine) substituted with curcumin derivatives, which are other components of the block copolymer according to the present invention, or The copolymer (B) is a poly (β-aminoester) or poly (amido amine) compound substituted with curcumin derivatives having both hydrophobicity and pH sensitivity, and non-limiting examples thereof include poly (β- substituted curcumin derivatives) Aminoesters), curcumin derivative substituted poly (amido amines), or mixed copolymers thereof. They have ionization properties that change their solubility in water depending on pH due to the tertiary amine groups present in themselves, and thus can form and / or decay micelle structures according to changes in pH in the body. Specifically, they may include tertiary amine groups that are ionized at pH 6.6 or lower. The compounds may be prepared according to conventional methods known in the art, for example, a curcumin-containing bisacrylate compound or curcumin by first reacting an acryloyl group-containing compound or acrylamide compound with a curcumin derivative. After obtaining the -containing bisacrylamide compound, the curcumin derivative is substituted poly (β-aminoester) through the Michael polymerization of the curcumin-containing bisacrylate compound or curcumin-containing bisacrylamide compound, and an amine-based compound, or Poly (amido amine) compounds can be obtained.

Non-limiting examples of acryloyl group-containing compounds used herein include acryloyl chloride, chloromethylaclylate, chloroethylaclylate, chloropropylaclylate, chlorobutylacrylate, chloropentylaclylate or chloro And phenyl acrylate.

In addition, non-limiting examples of acrylamide-based compounds include acrylamide, chloromethylacrylamide, chloroethylacrylamide, chloropropylacrylamide, chlorobutylacrylamide, chloropentylacrylamide or chlorophenylacrylamide. have.

Further, the amine-based compound can be used without limitation as long as it has an amine group, and primary amines, secondary amine-containing diamine compounds, tertiary amines, or mixtures thereof are preferred.

Non-limiting examples of the primary amine compound include 3-methyl-4- (3-methylphenyl) piperazine, 3-methylpiperazine, 4- (bis- (fluorophenyl) methyl) piperazine, 4- (e Methoxycarbonylmethyl) piperazine, 4- (phenylmethyl) piperazine, 4- (1-phenylethyl) piperazine, 4- (1,1-dimethoxycarbonyl) piperazine, 4- (2- (bis -(2-propenyl) amino) ethyl) piperazine, methylamine, ethylamine, butylamine, hexylamine, 2-ethyl hexyl amine, 2-piperidine-1-ethylamine, C-aziridine-1- Mono-methylamine, 1- (2-aminoethyl) piperazine, 4- (aminomethyl) piperazine, N-methylethylenediamine (N -methylethylenediamine), N-ethylethylenediamine, N-hexylethylenediamine, picoliamine, adenine, etc.

Non-limiting examples of the secondary amine-containing diamine compound include piperazine, piperidine, pyrrolidine, 3,3-dimethylpiperidine, 4,4 ' -Trimethylene dipiperidine, N, N'-dimethylethylene diamine, N, N'-diethyl ethylene diamine, imidazolidine or diazephan, etc.

Non-limiting examples of the tertiary amine include triethylamine, N, N-diisopropylethylamine, N-methylporporine, N-methylpiperidine, 4-dimethylaminopyridine, N, N-dimethylaniline, 2 , 6-lutidine or pyridine.

When preparing the poly (β-aminoester) or poly (amido amine) in which the curcumin derivative showing the pH sensitivity is substituted, the reaction molar ratio of the acryloyl group-containing compound or acrylamide compound, and the amine-based compound is 1: 0.5. The ~ 2.0 range is preferred. When the molar ratio of the amine-based compound is less than 0.5 or more than 2.0, the molecular weight of the polymer to be polymerized becomes 1000 or less, making it difficult to form micelles.

Preferably, the polyethylene glycol-based compound (A); And one or more compounds or copolymers thereof (B) selected from the group consisting of poly (β-aminoesters) substituted with curcumin derivatives and poly (amido amines) substituted with curcumin derivatives. The curcumin derivative-containing pH-sensitive block copolymer may be represented by any one of the following Chemical Formulas 2 to 6.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

In Chemical Formulas 2 to 6,

X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,

X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,

R ₁ to R ₃ , R ₅ and R ₆ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,

R ₄ and R _{7 are-} (CH ₂ ) _1-10 -alkylene groups,

m and n are each independently an integer of 15 to 10000,

p is an integer from 1 to 10.

Preferably, the pH-sensitive block copolymer containing curcumin derivatives of the present invention may be represented by the following Chemical Formulas 7 or 8.

[Formula 7]

[Formula 8]

In the above formula 7 or 8,

m and n are each independently an integer of 15 to 10000.

Most preferably, the pH-sensitive block copolymer containing curcumin derivatives of the present invention may be represented by the following Chemical Formula 9.

[Formula 9]

In Chemical Formula 9,

m and n are each independently an integer of 15 to 10000.

In this specification, "AICP (Anti-Inflammatory polymeric curcumin)" refers to the pH-sensitive block copolymer containing curcumin derivatives described above.

The curcumin derivative-containing pH-sensitive block copolymer may contain 25% by weight or more of curcumin, and specifically, curcumin may be loaded at 28% by weight.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a polymer micelle containing a pH-sensitive block copolymer containing curcumin derivatives as an active ingredient.

The polymer micelles contain curcumin derivatives in the polymer itself, and when the pH is reached, the micelles collapse to release curcumin derivatives to suppress inflammatory cytokines such as TNF-α and IL-1β.

The polymer micelles form micelles when injected into the body, and then, when they reach a region where the pH is low, such as an inflamed region, the micelles collapse, thereby obtaining a target-oriented therapeutic effect through release of curcumin.

The method for producing a polymer micelle according to the present invention can be used alone or in combination with methods such as stirring, heating, ultrasonic injection, solvent evaporation using an emulsification method, matrix formation or dialysis using an organic solvent.

The diameter of the polymer micelle is not particularly limited, but is preferably in the range of 10 to 1000 nm.

The pH-sensitive micelles of the present invention form stable micelles at a specific pH, for example, the pH range of normal cells in the body, pH 6.7 to 7.4, and when the pH range of 6.6 or less of abnormal cells such as inflammatory sites is below 6.6, the micelle structure collapses, resulting in inflammatory It can be used as a carrier for drug release targeted to the site. That is, at low pH (pH 6.6 or lower), the degree of ionization of the poly (β-aminoester) substituted with curcumin derivatives, the poly (amido amine) substituted with curcumin derivative derivatives, or a mixed copolymer thereof is increased. Due to this, the entire copolymer becomes water-soluble and cannot form micelles. At pH 7.4, the degree of ionization thereof decreases to show hydrophobic characteristics, thereby forming micelles by self-assembly.

'Inflammatory disease' of the present invention is a concept including all diseases that cause inflammation, and inflammation occurs when immune-eligible cells are activated in response to an external individual or antigenic protein. The inflammatory process can be advantageous as in the case of causing the invading individual to be phagocytosed or neutralized, but it can also be detrimental, as in the case of arthritis when causing bone and cartilage destruction and consequent limitations of joint function. The inflammatory response is usually induced by trauma or antigens, such as viral, bacterial, protozoan, or fungal antigens.

Inflammatory diseases suitable for prevention or treatment according to the present invention include osteoarthritis, diabetes, autoimmune myositis, arteriosclerosis, stroke, cirrhosis, meningitis, retinitis, tonsillitis, sore throat, bronchitis, pneumonia, inflammatory gastric ulcer, sinusitis, rhinitis, conjunctivitis, asthma, Inflammatory bowel disease, rheumatoid inflammatory disease, inflammatory collagen vascular disease, pelvic inflammatory disease, glomerulonephritis, inflammatory skin disease and sarcoidosis, but are not limited thereto. Preferably, the inflammatory disease of the present invention is a) rheumatoid inflammatory disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, Behcet's disease, autoimmune myositis mysitis), b) ulcerative colitis (UC), Crohn's disease, c) psoriasis, atopic dermatitis, contact dermatitis. Eczematous dermatitis, seborrheic dermatitis, lichen planus, lichen simplex chronicus, pemphigus, bolus pemphigus, epidermolysis bullosa, urticaria, angioedema Diseases of the sebaceous glands, such as vasculitis, erythema or skin eosinophilia, pneumococcal dermatitis, systemic deprivation dermatitis, stagnant dermatitis, hair follicles and acne, pericarditis, beard pseudo folliculitis and drugs Inflammatory responses such as rash, erythema multiforme, Erythema nodosum, and Granuloma annulare, and inflammatory celiac disease, encephalitis, pelvic inflammatory disease (PID) other than d) : Pelvic Inflammatory Disease), but is not limited thereto.

The composition of the present invention may further contain one or more known active ingredients having an effect of preventing or treating against inflammatory diseases together with the pH-sensitive block copolymer containing curcumin derivatives.

In the present invention, "prevention" refers to all actions to suppress or delay the development of inflammatory diseases by administering the pharmaceutical composition of the present invention to an individual, and "treatment" means administering the pharmaceutical composition of the present invention to an individual It means all actions that improve or benefit the symptoms of an inflammatory disease.

In the pharmaceutical composition of the present invention, the curcumin derivative-containing pH-sensitive block copolymer may preferably be contained in an amount of 0.001 to 1000 µg / ml based on the total volume of the pharmaceutical composition, and more preferably 1 to 500 Μg / ml. When the pH-sensitive block copolymer containing the curcumin derivative is used in an appropriate amount within the above range, it is economical, and the anti-inflammatory effect of the curcumin derivative-containing pH-sensitive block copolymer is sufficiently exhibited, making it more suitable to achieve the object of the present invention. There is this.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent.

In the present invention, the term "pharmaceutically acceptable" means that it is conventionally used in the pharmaceutical field, without stimulating the organism upon administration and without inhibiting the biological activity and properties of the compound to be administered.

In the present invention, the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. You can. These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , It may be used by additionally adding a binder or a lubricant.

The pharmaceutical composition of the present invention may be formulated and used in various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of the formulation for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, preparation powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc. Can be mentioned.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as Dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearte, sodium stearyl fumarate or polyethylene glycol wax can be used, and sweeteners, fragrances, syrups and the like can also be used. You can. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil, etc. may be additionally used.

Non-limiting examples of the parenteral preparations include injection solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, and the like.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, external preparations, etc. may be used, and the non-aqueous solvents, suspensions are propylene glycol, polyethylene Vegetable oils such as glycol and olive oil, and injectable esters such as ethyl oleate may be used.

In addition, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, and it is used for the preparation of ampoules or vials. It can be formulated for unit administration. In addition, when the pharmaceutical composition of the present invention is formulated as an aerosol agent, a propellant or the like may be combined with an additive so that the water-dispersed concentrate or wet powder is dispersed.

In addition, when formulating the pharmaceutical composition of the present invention with ointment, cream, etc., animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide Etc. can be formulated as a carrier.

The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may be varied by a method of formulating the pharmaceutical composition, a method of administration, an administration time and / or a route of administration, and the like to achieve the reaction to be achieved by administration of the pharmaceutical composition. Type and extent, type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, components of the drug or other composition used simultaneously or simultaneously with the subject. It may vary depending on various factors and similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.

By “administration” in the present invention is meant providing the subject with the composition of the invention in any suitable way.

The dosage for the more preferred effect of the pharmaceutical composition of the present invention may be 1 mg / kg to 10,000 mg / kg per day, more preferably 1 mg / kg to 1000 mg / kg per day. Administration of the pharmaceutical composition of the present invention may be administered once a day, it may be divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way.

The route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in the manner, and as long as the pharmaceutical composition can reach the targeted site of interest in any route and mode of administration. Can follow. The pharmaceutical composition may be administered by oral administration or parenteral administration.

The parenteral administration method may be administered by, for example, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, intracranial injection, or intrathoracic injection, A method of applying the composition to a disease site, spraying, or inhaling may also be used, but is not limited thereto.

The pharmaceutical composition of the present invention may preferably be administered orally or injected, and more preferably, administered orally.

In addition, the pharmaceutical composition may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy, and biological response modifiers.

In addition, the pharmaceutical composition of the present invention can be administered for human or animal use.

In addition, the present invention provides a method of preventing or treating inflammatory diseases, comprising administering the pharmaceutical composition to an individual other than a human.

In the present invention, "individual" means all animals, including mammals, including rats, livestock, and humans.

In the method of preventing or treating the inflammatory disease of the present invention, the dosage, route of administration, administration method, etc. of the pharmaceutical composition are as described above in connection with the pharmaceutical composition of the present invention.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases comprising the curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

By "improvement" in the present invention is meant any action that at least reduces the severity of parameters associated with the condition being treated, for example symptoms.

When the food composition of the present invention is used as a food additive, the composition may be added as it is or used with other foods or food ingredients, and may be suitably used according to a conventional method.

The type of the food is not particularly limited, and includes all foods in the ordinary sense. Non-limiting examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other dairy products including noodles, gums, ice cream, various soups, beverages, And tea, drink, alcoholic beverages, or vitamin complexes.

When the food composition of the present invention is a beverage composition, it may contain various flavoring agents or natural carbohydrates, etc., as additional components, like a conventional beverage. Non-limiting examples of the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; And synthetic sweeteners such as saccharin and aspartame. The proportion of the additional component added can be appropriately determined by the choice of those skilled in the art.

The food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginsam and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonated agents used in carbonated beverages. The health functional food composition of the present invention in the foil may contain flesh for the preparation of natural fruit juice, fruit drink or vegetable drink. These ingredients may be used independently or in combination of two or more. The proportions of these additives can also be appropriately selected by those skilled in the art.

In addition, the present invention provides a composition for diagnosing an inflammatory disease comprising a polymer micelle containing the curcumin derivative-containing pH-sensitive block copolymer as an active ingredient.

The term "diagnosis" in this specification means to confirm the presence or characteristic of a pathological condition. In the present invention, the "diagnosis" can be interpreted as observing and confirming whether or not the development of an inflammatory disease.

Polymer micelles containing a pH-sensitive block copolymer containing curcumin derivatives of the present invention exhibit a disappearance-recovery phenomenon of a pH-dependent fluorescent signal.

Specifically, the polymer micelle of the present invention forms a stable micelle at a specific pH, for example, pH 6.7 to 7.4, which is the pH range of normal cells in the body, and exhibits the disappearance of the fluorescent signal because curcumin approaches the hydrophobic core. In addition, when the pH range of the abnormal cells, such as inflammatory sites, is 6.6 or less, the micelle structure collapses due to the rapid hydrophobic-hydrophilic transition of the proton amine group, thereby recovering the fluorescence signal. The pH-dependent fluorescence signal extinction-recovery action of the polymer micelles of the present invention was specifically confirmed through an optical image of MIA-induced osteoarthritis of an embodiment of the present invention.

That is, the polymer micelle may exhibit a stronger fluorescent signal in the pH 6.6 or lower range compared to the pH 6.7 to 7.4 range.

Due to this pH-dependent fluorescence signal extinction-recovery action, the polymer micelle can be usefully used as a composition for diagnosing inflammatory diseases.

In addition, the present invention

1) reacting the acryloyl group-containing compound and curcumin derivative to obtain a curcumin derivative-containing bisacrylate compound; And

2) reacting the compound prepared in step 1) with an amine compound and an ethylene glycol compound to obtain a curcumin derivative-containing block copolymer; wherein the compound is represented by any one of Formulas 1-1 to 1-3 , It provides a method for preparing a pH-sensitive block copolymer containing curcumin derivatives.

Hereinafter, a method of preparing the pH-sensitive block copolymer containing the curcumin derivative will be described in detail.

The step 1) is a step of preparing a bisacrylate compound containing a curcumin derivative, first dissolving the curcumin derivative, an acryloyl group-containing compound, and trimethylamine in an organic solvent, and 0 to 10 ° C, preferably 2 to 6 ° C Reacts in cancer. Thereafter, the temperature was returned to room temperature to terminate the reaction, and then purified to obtain a product.

The 2) step is a step of preparing a curcumin derivative-containing block copolymer, and the curcumin derivative-containing bisacrylate compound and the amine-based compound prepared in step 1) are dissolved in an organic solvent and mixed. Ethylene glycol-based compound is added to the mixture, and the temperature is heated to 40 to 60 ° C. and stirred to initiate polymer polymerization. After 36 to 96 hours of polymer polymerization, the reaction mixture is precipitated in a cold organic solvent to prepare a pH sensitive block copolymer containing curcumin derivatives. In the reaction, it is also possible to react by adding the curcumin derivative-containing bisacrylate compound prepared in step 1) after dissolving and mixing the amine-based compound and the ethylene glycol-based compound in an organic solvent.

The acryloyl group-containing compound in step 1) is preferably acryloyl chloride, the amine-based compound in step 2) is preferably 4.4'-trimethylene-dipiperidine, and the ethylene glycol-based compound is poly (ethylene It is preferred that it is glycol) acrylate, ie methoxy polyethylene glycol monoacrylate.

The organic solvent used in each step may be chloroform, tetrahydrofuran, dimethyl sulfoxide, dimethyl formamide, methylene chloride, benzene, hexane, etc., but is not limited thereto.

The pH-sensitive block copolymer containing curcumin derivatives according to the present invention forms micelles so that a large amount of curcumin can be loaded and the curcumin derivatives last longer in the body. In addition, at the weakly acidic pH of the inflammatory site, micelles collapse and the acetal bond to which the curcumin derivative is linked is broken, and curcumin derivative is generated and released to act specifically on the inflammatory site and has less toxicity to normal cells, so it has an anti-inflammatory effect by itself. .

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

Example

Curcumin was purchased from Alfa Aesar (Haverhill, MA, USA). Acryloyl chloride, poly (ethylene glycol) acrylate and 4,4'-trimethylene dipiperidine are Sigma-Aldrich ( St.Louis, MO, USA).

Preparation of AICP

Curcumin (5.42 mmol), acryloyl chloride (10.84 mmol) and trimethylamine (TEA) (10.84 mmol) were dissolved in 100 mL of dry tetrahydrofuran (THF) at 4 ° C. The reaction was carried out in dark conditions at 4 ° C. for 12 hours, and compound 1 was obtained from silica gel chromatography (hexane / ethyl acetate = 60/40).

AICP was obtained by Michael addition polymerization of Compound 1. 4,4'-trimethylene dipiperidine (4.4 mmol) and poly (ethylene glycol) acrylate (0.22 mmol) were dissolved in dry dichloromethane. Polymerization was initiated by adding Compound 1 (4.18 mmol) to the reaction mixture and increasing the temperature to 50 ° C. After 48 hours of reaction, AICP was obtained by extraction using dichloromethane and isolation by precipitation in cold hexane. Figure 1 shows the process of synthesizing the above-described AICP as a reaction scheme. As shown in Fig. 1, curcumin was reacted with acryloyl chloride to obtain diacrylate compound 1, and AICP was obtained from compound 1, trimethylene dipiperidine and poly (ethylene glycol) (PEG) -acrylate. Michael addition was synthesized from polymerization.

The chemical structures of Compound 1 and AICP were confirmed by ¹ H NMR (400 MHz, CDCl3) (FIGS. 2A and 2B), and the molecular weight of AICP was gel permeation chromatography (1515 isocratic HPLC with 2415 refractive index detector, Waters , Milford, MA, USA). In addition, the content of curcumin in AICP was determined to be 28 wt% by spectroscopy.

Preparation of AICP micelles

The AICP (1 mg) was dissolved in 1 mL of tetrahydrofuran (THF). The solution was added to 10 mL of phosphate buffered saline (PBS, pH 7.4). After THF was completely evaporated, AICP micelles were obtained.

Experimental Example 1. Synthesis and properties of AICP

AICP was developed as an anti-inflammatory treatment to maximize curcumin's therapeutic potential. Curcumin has excellent antioxidant and anti-inflammatory effects, but its application is limited due to its low bioavailability. Biodegradable poly (β-aminoester) (PAE) is considered a powerful strategy for the development of biocompatible materials due to its excellent biocompatibility and acid-triggered hydrophilic-hydrophobic transfer activity. By using the PAE chemistry, curcumin incorporates a PEG segment into the PAE backbone, thereby increasing the therapeutic effect and expanding clinical application.

AICP photochemical properties

The photochemical properties of AICP were investigated using UV-spectroscopy and fluorescence spectroscopy. Figure 3a is a result of measuring the UV-vis absorbance of AICP, Figure 3b is a result showing the fluorescence emission spectrum of AICP. Curcumin showed light-absorption peaks at 270 nm and 420 nm, while AICP had no absorption range between 230-300 nm and an orange absorption peak at 405 nm in a concentration dependent manner (FIG. 3A). The blue shift observed in the absorption spectrum of AICP is a result of the acrylation of curcumin. The fluorescence spectrum of AICP was observed at 570 nm (Figure 3b).

AICP's critical micelle concentration

AICP is designed to form micelles from curcumin-based hydrophobic and hydrophilic PEG segments. AICP micelles can be self-assembled to generate micelles in aqueous conditions.

The critical micelle concentration of AICP in PBS at pH 7.4 was measured using pyrene as a fluorescent probe. Specifically, AICP micelles were prepared at various concentrations in a phosphate buffer solution (PBS) containing 10% serum, and pyrene dissolved in methanol was added thereto at the same concentration. The fluorescence emission spectrum of AICP micelles in which pyrene was enclosed was set to an excitation wavelength of 337 nm, and the critical micelle concentration was measured using a fluorescent spectrophotometer (FP-6500, JASCO Corp., Japan). The ratio of emission intensity at 373 nm and 384 nm was plotted against the logarithmic concentration of AICP. The measurement results of the critical micelle concentration of the AICP are shown in FIG. 3C.

As shown in Figure 3c, it can be seen that AICP forms a thermodynamically stable micelle by encapsulating a fluorescent probe within the hydrophobic center of the micelle at a concentration of 7 μg / mL or more.

AICP micelle size analysis and transmission electron microscopy (TEM) observation

The size of the AICP micelles was determined by dynamic light scattering using a particle size analyzer (Brookhaven Instrument Corp., Hotsvile, NY, USA) at critical micelle concentrations. In addition, AICP micelles were observed using a transmission electron microscope (Bio-TEM., Hitach Corp., Japan) after staining with phospotungstic acid staining.

AICP is self-assembled in a phosphate buffer solution (PBS) containing 10% serum at a concentration of 7 μg / mL or higher, and stable micelles with an average diameter of about 170 nm as confirmed by dynamic light scattering and electron microscopy (TEM) Can be formed (FIG. 3D). 4 shows the ¹ H NMR spectrum of AICP micelles formulated in D ₂ O. As shown in Fig. 4, micelle formation was confirmed by ¹ H NMR. As a result of self-assembly of AIPC micelles at D ₂ O, strong peaks of PEG corona were observed on the surface due to the structural properties of the micelles.

Size change of AICP micelles in various pH environments

AICP micelles are designed to be pH reactive by protonic tertiary amine groups in the backbone. To investigate the demicellation of AICP micelles, the diameters of micelles in various pH environments for 48 hours were observed and are shown in FIG. 5. 5 shows the size change of AICP micelles at pH 6.0 and pH 7.4 in PBS with 10% FBS.

3E shows a TEM image of AICP micelles at pH 6.6. Referring to Figure 3e, it can be seen that the size of the AICP micelles increased at pH 6.6.

Measurement of the release rate of curcumin from AICP micelles

Based on the instability of AICP micelles in an acidic environment, curcumin release profiles from AICP micelles were determined at various pHs. AICP micelles (1 mg / ml) were placed in a dialysis tube (Molecular Weight Blocking 2000, Membrane Filtration Product, Inc., Seguin, TX, USA), and the tube contained 1% tween 80 solution (pH 7.4, 6.6, 6.0) Immersed in 15 mL of buffer. The tube was mechanically stirred at 37 ° C. for 10 days. 100 μL of solution was collected at appropriate time intervals and replaced with a fresh solution. The amount of curcumin released from micelles was determined by a microplate reader (Biotek Instruments, Winooski, VT, USA) with an absorbance of 430 nm compared to the curcumin standard solution.

3F is a graph showing the release kinetics of curcumin from AICP micelles at pH 6.0, pH 6.6 and pH 7.4. Referring to FIG. 3F, at pH 6.0, AICP micelles released more than 95% of curcumin within 8 days due to acid-catalyzed hydrolysis of ester bonds by abundant protons in acidic buffer. Thus, it can be seen that curcumin is released faster at lower pH.

Measuring the sensitivity of AICP micelles to pH

To use AICP micelles as pH-sensitive fluorescent materials, the AICP fluorescence spectra in water and THF were recorded and shown in FIG. 3G. 3G shows the fluorescence spectrum and images of AICP micelles in water and THF. Looking at Figure 3g, AICP micelles in water, the fluorescence signal disappeared, it can be seen that the fluorescence signal intensity increased in THF.

To evaluate the pH dependent change of the fluorescence properties of AICP micelles, the fluorescence spectrum was recorded for a wide range of pH values and is shown in Figure 3h. Figure 3h is shown by measuring the fluorescence spectrum of AICP micelles at various pH. Looking at Figure 3h, peak fluorescence at 560 nm showed a value that increases with decreasing pH. 3i shows the fluorescence signal intensity of AICP micelles at 560 nm. Referring to Figure 3i, the fluorescence signal intensity of the AICP micelles at 560 nm was found to increase 5 times when the pH is lowered from pH 8 to pH 2. These results indicate that the fluorescence signal intensity of curcumin in the main chain of AICP is pH dependent.

AICP Michelle H ₂ O ₂ Erasing ability measurement

The ability to remove the H ₂ O ₂ in the AICP is then incubated with the AICP was evaluated by measuring the concentration of H ₂ O _2. After adding AICP or curcumin to 1 mL of H ₂ O ₂ solution (2 μM) for 1 hour, the concentration of H ₂ O ₂ is measured according to the manufacturer's protocol using an Amplex Red analyzer (Invitrogen, Carlsbad, CA). It is shown in Fig. 6.

6 is a graph showing H ₂ O ₂ scavenging ability of curcumin and AICP micelles. As shown in FIG. 6, AICP micelles significantly reduced the amount of H ₂ O ₂ , and 100 μg of AICP micelles reduced the concentration of H ₂ O ₂ by 40% due to the intrinsic antioxidant effect of curcumin. These results suggest that the micelle system improves the solubility of curcumin in solvent-free water.

Experimental Example 2. AICP micelle activity

Cytotoxicity of AICP micelles

Cytotoxicity of AICP micelles was evaluated using a mouse macrophage cell line (RAW264.7). To evaluate the cytotoxicity of AICP micelles, 3- (4,5-dimethylthiazyl-2-yl) -2,5-diphenyltetrazolium bromide (MTT) analysis was performed. RAW264.7 cells were cultured in 24-well plates for 24 hours to reach ~ 80% confluency. Cells were treated with varying amounts of AICP micelles for 24 hours. 100 μL of MTT solution was added to each well and incubated for 4 hours. After incubation, the resulting formazan crystals were dissolved with 200 μl of dimethyl sulfoxide. After 10 minutes of incubation, absorbance at 570 nm was measured using a microplate reader. Cell viability was determined by comparing the absorbance of cells treated with AICP micelles with that of the control cells.

After adding various amounts of curcumin or AICP micelles to 1 mL of cell culture medium, cytotoxicity was measured and shown in FIG. 7A. Figure 7a is a result showing the cytotoxicity of AICP micelles. Looking at Figure 7a, AICP micelles were found to be non-toxic or negligible at doses less than 100 μg / mL.

Antioxidant effect of AICP micelle

Next, we tried to compare and evaluate the activity of AICP micelles against 28% by weight of free curcumin.

The antioxidant activity of AICP micelles was determined by measuring the H ₂ O ₂ concentration of LPS-stimulated RAW264.7 cells. Cells were cultured with various concentrations of AICP micelles and curcumin. After 24 hours, the medium was collected and centrifuged (8000 g, 10 minutes). The H ₂ O ₂ concentration of the supernatant was measured using Amplex red analysis and is shown in FIGS. 7B and 7C.

Figure 7b is a graph showing a comparison of the apoptosis inhibitory effect of curcumin and AICP micelles in LPS-stimulated RAW264.7 cells, Figure 7c is a comparison of the apoptosis inhibitory effect of curcumin and AICP micelles in H ₂ O ₂ stimulated RAW264.7 cells It is a graph shown.

7B and 7C, toxicity induced by LPS and H ₂ O ₂ was observed, but AICP micelles significantly inhibited cell death.

In addition, the antioxidant effect of AICP micelles in LPS-stimulated cells was evaluated. Activated macrophages produce ROS, which are known to activate inflammatory transcription factors. 7D is a result showing the antioxidant effect of AICP micelles in LPS-stimulated RAW264.7 cells. As can be seen in Figure 7d, both curcumin and AICP micelles inhibit the level of ROS, but AICP micelles showed a stronger antioxidant effect than curcumin.

Anti-inflammatory effect of AICP micelle

To evaluate the anti-inflammatory effect of AICP micelles, RAW264.7 cells were cultured with 200 μM H2O2 or 1 μg / mL LPS. Cells were incubated for 24 hours with various concentrations of curcumin or AICP micelles. 100 μL of MTT solution was added to each well and incubated for 4 hours. After incubation, the resulting formazan crystals were dissolved with 200 μl of dimethyl sulfoxide. After 10 minutes of incubation, absorbance at 570 nm was measured using a microplate reader. Cell viability was determined by comparing the absorbance of AICP-treated cells with that of the control cells.

7E and 7F are results showing the anti-inflammatory effect of AICP micelles on the expression of IL-1β (e) and TNF-α (f) in LPS-stimulated RAW264.7 cells.

7E and 7F, the levels of the inflammatory cytokines TNF-α and IL-1β were significantly decreased in a concentration-dependent manner by AICP micelles. On the other hand, curcumin showed only moderate anti-inflammatory effect because curcumin solid was dispersed in PBS to prevent strong toxicity in vitro.

These results suggest that AICP micelles have potential as antioxidants and anti-inflammatory agents.

Experimental Example 3. Inflammation-responsive fluorescent switch of AICP micelle

Based on the light absorption properties of curcumin and the pH-reactive chemical reaction of poly (β-aminoester), the fluorescence activity of AICP micelles in the inflamed joint was observed. Inflammation was caused by MIA injections that could immediately interfere with the metabolism of chondrocytes.

Monoidoacetic acid (Sigma-Aldrich, St.Louis, MO) was dissolved in PBS at a concentration of 10 mg / mL. Mice (8 weeks of age) were anesthetized by intraperitoneal injection with a mixed solution of ketamine and xylazine (8: 1 ratio). To induce osteoarthritis (OA), 10 μL of MIA solution was injected intraarticularly through the patellar tendon perpendicular to the tibia of the mouse. On the 5th day after the MIA injection, mice were injected intramuscularly with the following solution near the osteoarthritis joint: (a) physiological saline, (b) curcumin (1.4 mg / kg), (c) curcumin (0.7 mg / kg), (d) AICP micelles (5 mg / kg) and (e) AICP micelles (2.5 mg / mL). Each group was given intravenously for 28 days once every 3 days. For imaging diagnosis of inflammation for MIA injection, 10 μl of MIA (10 mg / mL) was injected into the knee and injected into the synovial cavity of the knee. After 3 hours, 50 μL of AICP micelles (1 mg / mL) were administered to the knee joint. Fluorescence images of the inflamed knee were obtained using IVIS (Lumina2, PerkinElmer, Waltham, MA).

8A shows an overview of an experiment injecting AICP micelles into a joint with osteoarthritis induced by MIA. AICP micelles (5 mg / kg) were injected directly 3 hours after MIA treatment, and fluorescence images were obtained 1 hour after AICP micelle injection (FIG. 8A). Figure 8b shows the fluorescence image in the incision-induced inflammation-induced joint after AICP micelle injection, Figure 8c is a result of quantitative analysis of the fluorescence signal by the AICP micelle in the inflammation-induced joint. 8B and 8C, a background signal was observed in the patellar tendon, and a remarkably strong fluorescence signal was observed in the inflammation-induced joint. These results suggest that AICP micelles can be used as a diagnostic agent for targeted treatment of inflammatory diseases as an inflammation-specific fluorescence switch.

Experimental Example 4. Therapeutic activity of AICP micelles

In the development of osteoarthritis, ROS plays a critical role in articular cartilage degeneration. The antioxidant activity of AICP micelles was measured by Amplex red assay. 8D shows the level of H ₂ O ₂ in the joint as a fluorescence signal intensity. Referring to FIG. 8D, the level of H ₂ O ₂ was higher in the inflammation-induced joint than in the non-inflammatory joint. In addition, AICP micelles inhibited the level of H ₂ O ₂ in inflammation-inducing joints to a level similar to that of non-inflammatory joints, but curcumin showed only moderate levels of antioxidant effects.

The amount of TNF-α and IL-1β was measured to evaluate the therapeutic activity of AICP micelles against MIA-induced osteoarthritis. The inflammation-inducing joint specimens of mice were weighed, homogenized in cold PBS, and centrifuged (10,000 g) for 10 minutes at 4 ° C. The level of cytokines was measured with the mouse TNF-α ELISA kit (eBioscience, San Diego, CA, USA) and the IL-1β kit (eBioscience, San Diego, CA, USA). 8E and 8F are graphs showing the therapeutic effect of AICP micelles on the expression of IL-1β (e) and TNF-α (f) in inflammation-inducing joints. MIA treatment significantly increased the level of TNF-α. Curcumin showed a non-significant effect on the expression of TNF-α, but AICP micelles significantly inhibited the level of TNF-α (FIG. 8f). The level of IL-1β was also significantly increased by treatment with MIA. Treatment of AICP micelles showed a stronger inhibitory effect on the expression of Il-1β than curcumin (FIG. 8E).

The main symptoms of osteoarthritis including loss of ECM, proteoglycan, collagen and aggrecan are degeneration and loss of cartilage. MIA-induced inflammatory joint specimens were harvested from the mice for histological examination. The joint samples of the mice were fixed in 4% paraformaldehyde and then decalcified using Decalcifying Solution-Lite (Sigma-Aldrich, St. Louis, MO) for 6 hours. The tissue was placed in paraffin and sections were made. The tissue sections were stained with H & E, Masson's Trichrome, safranin O and aggrecan (sc-33695, Santacruz, dallas, TX).

9 is a histological examination results of MIA-induced inflammation. In the histological examination, cartilage tissue was stained with H & E, safranin-O, masson's trichrome and aggrecan. Referring to FIG. 9, intact articular cartilage has a regular and smooth surface with evenly distributed agrican, collagen and proteoglycan. Treatment of MIA caused irregular surfaces of soft tissue and loss of ECM components. In contrast, the inflammation-inducing joint treated with AICP micelles showed strong expression of proteoglycans, agricans and collagen, structural integrity of cartilage and smooth surfaces. These results indicate that AICP micelles exhibit high anti-inflammatory activity.

On the other hand, in case of inflammation-induced joints treated with an equivalent amount of curcumin, there was a limiting effect on the expression of collagen, agrican and proteoglycan. This may be because curcumin is low in solubility and is quickly removed. On the other hand, AICP micelles can enhance the therapeutic activity with the release of curcumin caused by inflammation and long lasting residence time in the joints inducing osteoarthritis. These results indicate that AICP micelles have very strong anti-inflammatory activity.

Experimental Example 5. Toxicity evaluation of AICP micelles

To study the toxicity of AICP micelles in vivo, normal mice were intravenously injected with AICP micelles (10 mg / kg and 20 mg / kg). After 7 days, serum was collected to measure serum ALT activity in mice, and the results are shown in FIG. 10A. The level of ALT was measured with an enzyme assay kit (Asan Pharma, Seoul, Korea) using a microplate reader (SynergyMX, BioTek instruments, Inc.).

As shown in Figure 10A, AICP micelles did not induce changes in ALT activity compared to untreated mice.

In addition, the results of histological examination of each organ after AICP micelle administration as shown above are shown in FIG. 10B. As shown in Figure 10B, AICP micelles did not induce histological changes to the accumulated toxicity in each organ compared to untreated mice.

These results indicate that AICP micelles in an amount exhibiting potential anti-inflammatory activity do not cause chronic damage to the organs of the mouse.

The pH-sensitive block copolymer containing curcumin derivatives of the present invention was developed as a polymer prodrug that releases curcumin and can be formed into hydrodynamically stable micelles by self-assembly in aqueous solutions. The polymer micelle of the present invention exhibited pH-dependent fluorescence and showed very strong antioxidant and anti-inflammatory effects in LPS or H ₂ O ₂ stimulated cells. In a mouse model of osteoarthritis induced by MIA, the polymer micelles emitted a clearly perceptible fluorescence signal in the osteoarthritis knee joint and inhibited the expression of inflammatory TNF-α and IL-1β. In addition, the AICP micelles significantly inhibited cartilage degradation, which was demonstrated by maintaining the structural integrity of the articular cartilage and extracellular matrix.

That is, considering the excellent biocompatibility, high drug loading efficiency, pathological stimulation-induced therapeutic action, and very strong therapeutic efficacy, the AICP micelle of the present invention has a surprising potential for use in managing various inflammatory diseases.

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

Claims (14)

Polyethylene glycol compound (A); And a curcumin derivative-substituted poly (β-aminoester) or poly (amidoamine) compound (B) copolymerized with a curcumin derivative-containing pH-sensitive block copolymer represented by any one of the following Chemical Formulas 1-1 to 1-3 Pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a coal as an active ingredient:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,
X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,
X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,
R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,
R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,
m and n are each independently an integer of 15 to 10000,
p is an integer from 1 to 10. The method of claim 1, wherein R ₄ of the formula
In the formulas 1-1 to 1-3, -CH ₂ -CH ₂ -COO-CH ₂ -CH ₂ -(= N-CH ₂ -CH ₂ -COO-CH ₂ -CH ₂ -O- is connected in the direction of ), Or -CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -(= connected to the direction of N-CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -O-),
In the above Chemical Formula 1-1, it is not -CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -(= N-CH ₂ -CH ₂ -CONH-CH ₂ -CH ₂ -O-connected in the direction) Characteristic, pharmaceutical composition for the prevention or treatment of inflammatory diseases. According to claim 1,
The curcumin derivative-containing pH-sensitive block copolymer forms micelles in the pH range of 6.7 to 7.4, and micelles formed in the pH range of 6.6 or less collapse, thereby preventing or treating inflammatory diseases. According to claim 1,
The curcumin derivative-containing pH-sensitive block copolymer is characterized in that the curcumin content is 25% by weight or more, pharmaceutical composition for the prevention or treatment of inflammatory diseases. According to claim 1,
The curcumin derivative-containing pH-sensitive block copolymer is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it is at least one block copolymer selected from the group consisting of block copolymers represented by the following Chemical Formulas 2 to 6:
[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

In Chemical Formulas 2 to 6,
X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,
X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,
R ₁ to R ₃ , R ₅ and R ₆ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,
R ₄ and R _{7 are-} (CH ₂ ) _1-10 -alkylene groups,
m and n are each independently an integer of 15 to 10000,
p is an integer from 1 to 10. The method of claim 5,
The curcumin derivative-containing pH-sensitive block copolymer is characterized in that the block copolymer represented by the following formula (7) or (8), pharmaceutical composition for the prevention or treatment of inflammatory diseases:
[Formula 7]

[Formula 8]

In Chemical Formulas 7 and 8,
m and n are each independently an integer of 15 to 10000. According to claim 1,
The curcumin derivative-containing pH-sensitive block copolymer is characterized in that the block copolymer represented by the formula (9), pharmaceutical composition for the prevention or treatment of inflammatory diseases:
[Formula 9]

In Chemical Formula 9,
m and n are each independently an integer of 15 to 10000. Polyethylene glycol compound (A); And a curcumin derivative-substituted poly (β-aminoester) or poly (amidoamine) compound (B) copolymerized with a curcumin derivative-containing pH-sensitive block copolymer represented by any one of the following Chemical Formulas 1-1 to 1-3 Pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising polymer micelles containing coal as an active ingredient:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,
X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,
X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,
R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,
R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,
m and n are each independently an integer of 15 to 10000,
p is an integer from 1 to 10. The method according to any one of claims 1 to 8,
The inflammatory diseases include osteoarthritis, diabetes, autoimmune myositis, atherosclerosis, stroke, cirrhosis, meningitis, retinitis, tonsillitis, pharyngitis, bronchitis, pneumonia, inflammatory gastric ulcer, sinusitis, rhinitis, conjunctivitis, asthma, inflammatory bowel disease, rheumatoid inflammatory disease, Pharmaceutical composition for the prevention or treatment of inflammatory diseases characterized in that it is selected from the group consisting of inflammatory collagen vascular disease, pelvic inflammatory disease, glomerulonephritis, inflammatory skin disease and sarcoidosis. Polyethylene glycol compound (A); And a curcumin derivative-substituted poly (β-aminoester) or poly (amidoamine) compound (B) copolymerized with a curcumin derivative-containing pH-sensitive block copolymer represented by any one of the following Chemical Formulas 1-1 to 1-3 Food composition for preventing or improving an inflammatory disease comprising a coal as an active ingredient:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,
X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,
X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,
R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,
R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,
m and n are each independently an integer of 15 to 10000,
p is an integer from 1 to 10. Polyethylene glycol compound (A); And a curcumin derivative-substituted poly (β-aminoester) or poly (amidoamine) compound (B) copolymerized with a curcumin derivative-containing pH-sensitive block copolymer represented by any one of the following Chemical Formulas 1-1 to 1-3 Composition for the diagnosis of inflammatory diseases comprising polymer micelles containing coal as an active ingredient:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,
X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,
X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,
R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,
R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,
m and n are each independently an integer of 15 to 10000,
p is an integer from 1 to 10. The method of claim 11,
The polymer micelle is a composition for the diagnosis of inflammatory diseases, characterized in that it exhibits a stronger fluorescence signal in the pH 6.6 or less range compared to the pH 6.7 to 7.4 range. 1) reacting the acryloyl group-containing compound and curcumin derivative to obtain a curcumin derivative-containing bisacrylate compound; And
2) Reacting the compound prepared in step 1) with an amine compound and an ethylene glycol compound to obtain a curcumin derivative-containing block copolymer; which includes one of the following Chemical Formulas 1-1 to 1-3 , Curcumin derivative-containing pH-sensitive block copolymer production method:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

In Chemical Formulas 1-1 to 1-3,
X ₁ to X ₄ are each independently an H or C ₁ -C ₅ alkyl group,
X ₅ and X ₆ are each independently H, OH or a C ₁ -C ₅ alkoxy group,
R ₁ to R ₃ are C ₁ -C ₃₀ straight chain or C ₄ -C ₁₀ branched alkyl groups,
R ₄ is -CH ₂ -CH ₂ -COO-R ₅ -or -CH ₂ -CH ₂ -CONH-R _5- , R ₅ is an alkyl group of C ₁ -C ₃ ,
m and n are each independently an integer of 15 to 10000,
p is an integer from 1 to 10. In claim 13,
The acryloyl group-containing compound in step 1) is acryloyl chloride, the amine compound in step 2) is 4,4'-trimethylene dipiperidine, and the ethylene glycol compound is poly (ethylene glycol) acrylate Characterized in that, the method for producing a pH-sensitive block copolymer containing curcumin derivatives.

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