CN101422615B - Oridonin polymer micelle administration preparation and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine and provides an oridonin A polymer micelle drug delivery preparation and a preparation method. The polymer is an amphiphilic block copolymer, the hydrophilic segment is polyethylene glycol monomethyl ether, and the hydrophobic segment is selected from biodegradable poly (D, L-lactic acid), poly (L-lactic acid), poly (lactide-glycolide), polycaprolactone, polyhexadecyl cyanoacrylate or mixtures thereof. The drug in the polymer drug micelle is encapsulated in the above-mentioned one or two polymers in physical form or covalently combined with the polymer through a chemical reaction and then prepared into a micelle. The prepared micelles exist in the state of water dispersion or freeze-dried powder. The oridonin nanometer polymer micelle has a micelle particle size distribution range of 5-500nm and a drug load of 0.01-40%. The Rubescensin A polymer micelle prepared by the present invention prolongs the circulation time of the drug in the body, and the AUC _0-∞ value is 2.19 times that of ordinary injections, making it easier for the drug to accumulate in the tumor site, improving the curative effect and reducing the toxicity .

Description

A kind of oridonin A polymer micelle drug delivery preparation and preparation method

technical field

The invention belongs to the technical field of medicine, and relates to a polymer micelle drug delivery preparation of oridonin and a preparation method thereof, specifically, directly physically encapsulating oridonin into a synthesized amphiphilic block copolymer Oridonin and the synthesized amphiphilic block copolymer are further covalently bonded to form an amphiphilic block copolymer-oridonin A bonded drug, and then prepared into micelles.

Background technique

Oridonin is a natural organic compound of ent-kau-rene-diterpenoid isolated from Labtea plants of the genus Rabdosia. The ingredients have strong anti-tumor activity and are effective against a variety of transplanted tumors. They are mainly used clinically for the treatment of liver cancer, esophageal cancer, and pancreatic cancer. Oridonin has poor water solubility and cannot meet the needs of directly making an aqueous solution for injection. In order to make an aqueous solution, surfactants such as Tween-80 need to be added, but intravenous injection of such ingredients is prone to hemolysis. In developed countries disabled. In order to solve this problem, some people made it into preparations such as cyclodextrin inclusion complex, emulsion and liposome, which solved certain problems, but there were also many deficiencies. The present invention prepares it into a biodegradable polymer micellar aqueous solution or freeze-dried powder, which greatly improves its water solubility and drug loading capacity, and is non-irritating to the human body, has no hemolysis, and has a good target The tropism improves the efficacy of the drug and reduces the toxicity.

Polymer micelles refer to a type of micelles composed of amphiphilic block copolymers, the amphiphilic copolymers are composed of hydrophobic segments and hydrophilic segments, and the volume of the hydrophilic segment is generally larger than that of the hydrophobic segment, which can be spontaneously arranged in water Nucleation-shell structure, the hydrophobic segment forms the micelle core, and the hydrophilic segment forms the micelle shell. The low-molecular-weight surfactants used in pharmacy to solubilize insoluble drugs are either unsatisfactory in solubilization effect or relatively toxic. For example, polyoxyethylene castor oil EL, which is used to solubilize paclitaxel and cyclosporine A, can cause side effects such as allergic reactions, hyperlipidemia, neurotoxicity, and P-glycoprotein reversal. As a new type of carrier, polymer micelles basically overcome the above shortcomings, and have a wide range of drug loading, stable structure, excellent tissue permeability, and long residence time in the body, enabling drugs to reach the target effectively. Moreover, it has a lower CMC and is more stable in the systemic circulation. Based on the above advantages, polymer micelles have been extensively studied in the past few years. Among them, biodegradable polymer micelles have become a research hotspot in recent years because they can be degraded in vivo and excreted from the body without accumulation. Physically encapsulating drugs with biodegradable amphiphilic block copolymers is a method of physically preparing polymer micelles. Recently, another form of polymer micelles preparation—polymer-bonded drug micelles has been prepared and studied. received great attention from everyone. "Polymer-bonded drug micelle" means that the drug is covalently bonded to the amphiphilic block copolymer polymer polymer, and then prepared into a micelle. After injection into the body, the drug is dissociated from the polymer under physiological conditions to play a therapeutic or diagnostic role. After the drug is bonded to the polymer, the hydrophilicity of the drug is improved. Under the protection of the polymer, the drug can be protected from the attack of enzymes and the immune system in the physiological environment, achieving the purpose of slow release and long-term effect. Amphiphilic block copolymers are used to bond drugs to self-assemble into polymer micelles. The drugs are in the inner core of the micelles. Because they are covalently bound to the copolymers, they are relatively stable. Diffusion escapes from the bundle core. Therefore, this kind of micelles not only has the advantages of traditional physical entrapped micelles, but also avoids the sudden release caused by kinetic instability, thereby improving the efficacy of drugs and reducing toxicity. The studies on polymer micelle-bonded drugs have been reported, including doxorubicin, paclitaxel, and docetaxel.

Contents of the invention

The object of the present invention is to provide a kind of oridonin A polymer micelle drug delivery preparation and preparation method, and this polymer micelle includes two forms, one is that oridonin A is encapsulated in the polymer micelle in physical form The other is that Rubescensin A is covalently bonded to the polymer to form an amphiphilic block copolymer-Oridbescensine A bonded drug, and then prepared into micelles .

The polymer described in the present invention refers to an amphiphilic block copolymer, wherein the hydrophilic segment is polyethylene glycol monomethyl ether (mPEG), and the hydrophobic segment is selected from biodegradable hexadecanoic acid, poly(D, L - Lactic acid) (PDLLA), poly(L-lactic acid) (PLLA), poly(lactide, glycolide) (PLGA), polyε-caprolactone (PCL), poly(lactide, ε-caprolactone lactone) (PCLA), poly(lactide, glycolide, ε-caprolactone) (PCLGA) or mixtures thereof. Wherein the hydrophobic block is a copolymer of lactide and glycolide, the mass percentage of lactide and glycolide is 99:1 ~ 1:99; the hydrophobic block is lactide and ε-hexyl Copolymer of lactone, the mass percentage of lactide and ε-caprolactone is 99:1～1:99; the hydrophobic block is a ternary copolymerization of lactide, glycolide and ε-caprolactone The mass percentage of lactide, glycolide and ε-caprolactone is 98～50:1～49:1～49. Wherein oridonin accounts for 0.01-40% of the total weight of the drug and the polymer; the molecular weight of the amphiphilic block copolymer is 500-100000, wherein the number average molecular weight of the hydrophilic block is 200-50000, and the hydrophobicity The number average molecular weight of the block is 50-50000; the total amount of the hydrophilic block accounts for 10-98% of the total weight of the copolymer;

The prepared oridonin polymer micelles, wherein the amphiphilic block copolymer is selected from biodegradable polyethylene glycol monomethyl ether-hexadecanoic acid, polyethylene glycol monomethyl ether-poly(D , L-lactic acid) (mPEG-PDLLA), polyethylene glycol monomethyl ether - poly (L-lactic acid) (mPEG-PLLA), polyethylene glycol monomethyl ether - poly (lactide, glycolide) ( mPEG-PLGA), polyethylene glycol monomethyl ether-polycaprolactone (mPEG-PCL), polyethylene glycol monomethyl ether-poly(lactide, ε-caprolactone) (mPEG-PCLA) and poly One or more of ethylene glycol monomethyl ether-poly(lactide, glycolide, ε-caprolactone) (mPEG-PCLGA). Among them, mPEG-PDLLA, mPEG-PLLA and mPEG-PLGA are preferred. Wherein the molecular weight distribution of polyethylene glycol monomethyl ether (mPEG) is 200-30000, preferably 1000-5000, and particularly preferably 2000 and 5000.

The present invention provides the preparation method of two kinds of amphiphilic block copolymers-oridonin A bonded drug micelle, and its steps and conditions are as follows:

method 1:

Step (1): Preparation of block copolymers of polyethylene glycol monomethyl ether and various aliphatic cyclic esters

In the presence of polyethylene glycol monomethyl ether and a catalyst, the ring-opening polymerization reaction is carried out with the aliphatic cyclic ester monomer. Aliphatic cyclic ester monomer refers to lactide, ε-caprolactone, mixture of lactide and glycolide, mixture of lactide and ε-caprolactone, lactide, glycolide and ε- Ternary mixture of caprolactone. In the monomer mixture of lactide and glycolide, the mass percentage of lactide and glycolide is 99:1~1:99; in the mixture of lactide and ε-caprolactone, the ratio of lactide and ε -The mass percentage of caprolactone is 99:1～1:99; in the ternary mixture of lactide, glycolide and ε-caprolactone, the mass of lactide, glycolide and ε-caprolactone The percentage is 98～50:1～49:1～49. The catalyst is stannous octoate, the dosage is 0.01-1% of the mass of polymerized monomers, the polymerization temperature is 110-180°C, and the polymerization time is 3-24 hours;

Step (2): Convert the terminal hydroxyl group of polyethylene glycol monomethyl ether and aliphatic polyester block copolymer to terminal carboxyl group

The block copolymer synthesized in step (1) is in the presence of catalyst pyridine, triethylamine or 4-N, N-dimethylaminopyridine, in anhydrous dichloromethane or 1,4-dioxane solution, Carry out esterification reaction with acid anhydride at 0-25°C, the amount of catalyst used is 0.05-2 times the molar mass of the terminal hydroxyl group of the block copolymer, the amount of acid anhydride is 1-3 times the molar mass of the terminal hydroxyl group of the block copolymer, and the reaction time is 24 ~48h, the terminal hydroxyl group of the block copolymer is converted to the terminal carboxyl group.

Step (3): reacting the carboxyl group of polyethylene glycol monomethyl ether and the aliphatic polyester block copolymer with the hydroxyl group of Rubescensin A to prepare an amphiphilic block copolymer-Oridonin A bonded medicine

In the presence of catalyst 4-N, N-dimethylaminopyridine and condensing agent dicyclohexylcarbodiimide, react in dichloromethane, dimethylsulfoxide or dimethylformamide solution, the reaction temperature is 0～ 25°C, reaction time 12-48h, wherein, the molar amount of 4-N,N-dimethylaminopyridine is 0.05-2 times of the carboxyl group of the block copolymer, and the molar amount of dicyclohexylcarbodiimide is the block copolymer The molar amount of oridonin is 1 to 3 times that of the carboxyl group at the end of the copolymer, and the molar amount of oridonin is 1 to 2 times that of the carboxyl group at the end of the block copolymer, and the amphiphilic block copolymer-oridonin bonded drug is obtained through the reaction.

Method 2:

Step (1): Preparation of block copolymers of polyethylene glycol monomethyl ether and various aliphatic cyclic esters

In the presence of polyethylene glycol monomethyl ether and a catalyst, the ring-opening polymerization reaction is carried out with the aliphatic cyclic ester monomer. Aliphatic cyclic ester monomer refers to lactide, ε-caprolactone, mixture of lactide and glycolide, mixture of lactide and ε-caprolactone, lactide, glycolide and ε- Ternary mixture of caprolactone. In the monomer mixture of lactide and glycolide, the mass percentage of lactide and glycolide is 99:1~1:99; in the mixture of lactide and ε-caprolactone, the ratio of lactide and ε -The mass percentage of caprolactone is 99:1～1:99; in the ternary mixture of lactide, glycolide and ε-caprolactone, the mass of lactide, glycolide and ε-caprolactone The percentage is 98～50:1～49:1～49. The catalyst is stannous octoate, the dosage is 0.01-1% of the mass of polymerized monomers, the polymerization temperature is 110-180°C, and the polymerization time is 3-24 hours;

Step (2): Preparation of Oridonin A Carboxyl Derivatives

Put oridonin in the presence of catalyst pyridine, triethylamine or 4-N, N-dimethylaminopyridine, in anhydrous chloroform or 1,4-dioxane solution, and acid anhydride at 0～ The esterification reaction is carried out at 25°C, the dosage of the catalyst is 0.05-2 times the molar mass of oridonin, the dosage of the acid anhydride is 1-2 times the molar mass of the terminal hydroxyl group of the block copolymer, the reaction time is 24-48h, and the carboxyl group-containing Oridonin A derivatives.

Step (3): The amphiphilic block copolymer synthesized in step (1) is covalently bonded to the carboxyl-containing oridonin derivative to prepare the amphiphilic block copolymer-oridonin Bonding drug

In the presence of catalyst 4-N, N-dimethylaminopyridine and condensing agent dicyclohexylcarbodiimide, react in dichloromethane, dimethylsulfoxide or dimethylformamide solution, the reaction temperature is 0～ 25°C, reaction time 12-48h, wherein, the molar dosage of 4-N,N-dimethylaminopyridine is 0.05-2 times that of carboxyl-containing oridonin derivatives, and the molar dosage of dicyclohexylcarbodiimide The dosage is 1 to 3 times that of the carboxyl group-containing oridonin derivative, and the molar dosage of the block copolymer is 0.5 to 1 time that of the carboxyl group-containing oridonin derivative. The reaction results in amphiphilic block copolymerization Substance-Oridonin A bonded drug.

The preparation of Rubescensin A polymer micelle preparation of the present invention, its steps are as follows:

Method 1: Co-solvent evaporation method

(1), prepare polymer micelle aqueous solution, at first dissolve amphiphilic block copolymer and oridonin or both with organic solvent acetone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide or their mixture The hydrophilic block copolymer-oridonin A bonded drug is slowly added into the water phase under stirring, and the organic solvent is evaporated to dryness with open stirring at room temperature or the organic solvent is evaporated to dryness at room temperature under reduced pressure to obtain a micellar aqueous solution.

(2) The micellar aqueous solution is centrifuged at 5000-20000r/min for 5-30 minutes to remove large aggregated particles, and the clear liquid is obtained by filtration.

(3) After centrifugation, the filtered supernatant can also be directly freeze-dried to make freeze-dried powder, or add certain freeze-drying protective agents such as one or more of sucrose, glucose, mannitol, lactose, trehalose, dextran, etc. Then freeze-dry to make freeze-dried powder. The dosage of the lyoprotectant is 5-30% of the total amount of the preparation.

Method 2: Dialysis

(1) To prepare an aqueous solution of polymer micelles, first dissolve the amphiphilic block copolymer and Rubescensine A or amphiphilic Sexual block copolymer-oridonin A bonded drug, and then slowly added to the water phase under stirring to obtain a micellar dispersion, which was put into a semipermeable membrane dialysis bag with a certain molecular weight cut-off, and the dialysis bag Add a large amount of water outside, and dialyze for 24-48 hours to remove organic solvents and small molecule free drugs to obtain dialysate.

(2) After dialysis, the micellar aqueous solution is centrifuged at 5000-20000 r/min for 5-30 minutes to remove large aggregated particles, and the clear liquid is obtained by filtration.

(3) After centrifugation, the filtered supernatant can also be directly freeze-dried to make freeze-dried powder, or add certain freeze-drying protective agents such as one or more of sucrose, glucose, mannitol, lactose, trehalose, dextran, etc. Then freeze-dry to make freeze-dried powder. The dosage of the lyoprotectant is 5-30% of the total amount of the preparation.

The oridonin A polymer micelle prepared by the invention prolongs the circulation time of the drug in the body, makes the drug more likely to accumulate in the tumor site, improves the curative effect and reduces the toxicity.

Description of drawings:

Figure 1: Oridonin structural formula

Figure 2: Release curve of mPEG-PDLLA ₈₀₀₀ oridonin A polymer micelles in vitro

Figure 3: Pharmacokinetic study of Rubescensin A ordinary injection and polymer micellar solution in rats ■——Oridonin A ordinary injection; ◆——◆Oridbescensin A block copolymerization micelles

Detailed ways

Embodiment 1: Preparation of amphiphilic block copolymer mPEG-PDLLA

(1) Add 2.5g D, L-lactide and 4g polyethylene glycol monomethyl ether (molecular weight 5000) into a three-necked bottle equipped with a magnetic stirrer, and heat the oil bath to 140°C for melting (decompression vacuum ), add a mass percentage of 0.05% stannous octoate catalyst, react for 8h, stop the reaction, cool to room temperature, and dissolve the product with 10ml of dichloromethane or chloroform, precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hour, the product mPEG-PDLLA was obtained, and the molecular weight of the PDLLA block was calculated to be 3000 by NMR. The mPEG-PDLLA ₈₀₀₀ with molecular weight of 8000 was obtained

(2) Add 4.5g D, L-lactide and 4g polyethylene glycol monomethyl ether (molecular weight 5000) into a three-necked bottle equipped with a magnetic stirrer, and heat the oil bath to 140°C for melting (decompression vacuum ), add a mass percentage of 0.05% stannous octoate catalyst, react for 10h, stop the reaction, cool to room temperature, and dissolve the product with 10ml of dichloromethane or chloroform, precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hour, the product mPEG-PDLLA was obtained, and the molecular weight of the PDLLA block was calculated to be 5000 by NMR. The mPEG-PDLLA ₁₀₀₀₀ with a molecular weight of 10000 was obtained

(3) Add 9g of D, L-lactide and 4g of polyethylene glycol monomethyl ether (molecular weight: 5000) into a three-necked bottle equipped with a magnetic stirrer, and heat the oil bath to 140°C to melt (decompression and vacuum) Finally, add a stannous octoate catalyst with a mass percentage of 0.05%, heat up to 160°C, continue the reaction for 10h, stop the reaction, cool to room temperature, dissolve the product with 15ml of dichloromethane or chloroform, precipitate with 300ml of cold ether, filter, and 30°C After vacuum drying for 24-48 hours, the product mPEG-PDLLA was obtained, and the molecular weight of the PDLLA block was calculated to be 10,000 by NMR. The mPEG-PDLLA ₁₅₀₀₀ with a molecular weight of 15000 was obtained

Embodiment 2: Preparation of amphiphilic block copolymer mPEG-PLLA

Add 4.5g of L-lactide and 4g of polyethylene glycol monomethyl ether (molecular weight: 5000) into a three-necked bottle equipped with a magnetic stirrer, heat the oil bath to 140°C for melting (decompression and vacuum), then add mass The percentage is 0.05% stannous octoate catalyst, react for 10h, stop the reaction, cool to room temperature, dissolve the product with 10ml of dichloromethane or chloroform, precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hours to obtain the product mPEG - PLLA, the molecular weight of the PLLA block is calculated to be 5000 by nuclear magnetic spectrum. The mPEG-PLLA ₁₀₀₀₀ with a molecular weight of 10000 was obtained

Embodiment 3: Preparation of amphiphilic block copolymer mPEG-PLGA

(1) Add 4.5g of D, L-lactide and glycolide mixture (LA/GA=50/50) and 4g of polyethylene glycol monomethyl ether (molecular weight 5000) to a three-necked magnetic stirrer In the bottle, after the oil bath is heated to 140°C for melting (decompression and vacuum), add the stannous octoate catalyst that is 0.05% by mass percentage, react for 10h, stop the reaction, cool to room temperature, and the product is dissolved with 10ml of dichloromethane or chloroform. Precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hours to obtain the product mPEG-PLGA. The molecular weight of the PLGA block is calculated to be 5000 by NMR. The mPEG-PLGA _10000(50/50) with a molecular weight of 10000 was obtained

(2) Add 4.5g of D, L-lactide and glycolide mixture (LA/GA=75/25) and 4g of polyethylene glycol monomethyl ether (molecular weight 5000) to a three-necked three-necked stirrer equipped with a magnetic stirrer. In the bottle, after the oil bath is heated to 140°C for melting (decompression and vacuum), add the stannous octoate catalyst that is 0.05% by mass percentage, react for 10h, stop the reaction, cool to room temperature, and the product is dissolved with 10ml of dichloromethane or chloroform. Precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hours to obtain the product mPEG-PLGA. The molecular weight of the PLGA block is calculated to be 5000 by NMR. The mPEG-PLGA _10000(75/25) with a molecular weight of 10000 was obtained

Embodiment 4: Preparation of amphiphilic block copolymer mPEG-PDLLA

(1) Add 2.5g D, L-lactide and 4g polyethylene glycol monomethyl ether (molecular weight 2000) into a three-necked bottle equipped with a magnetic stirrer, and heat the oil bath to 140°C for melting (decompression vacuum ), add a mass percentage of 0.05% stannous octoate catalyst, react for 8h, stop the reaction, cool to room temperature, and dissolve the product with 10ml of dichloromethane or chloroform, precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hours, the product mPEG-PDLLA was obtained, and the molecular weight of the PDLLA block was calculated to be 1250 by NMR. The mPEG-PDLLA ₃₂₅₀ with a molecular weight of 3250 was obtained

(2) Add 4g of D, L-lactide and 4g of polyethylene glycol monomethyl ether (molecular weight: 2000) into a three-necked bottle equipped with a magnetic stirrer, and heat the oil bath to 140°C to melt (decompression and vacuum) Finally, add a stannous octoate catalyst with a mass percentage of 0.05%, react for 10 hours, stop the reaction, cool to room temperature, dissolve the product in 10ml of dichloromethane or chloroform, precipitate with 200ml of cold ether, filter, and vacuum dry at 30°C for 24-48 hours , the product mPEG-PDLLA was obtained, and the molecular weight of the PDLLA block was calculated to be 2000 by nuclear magnetic spectrum. The mPEG-PDLLA ₄₀₀₀ with a molecular weight of 4000 was obtained

Example 5

Weigh 100mg of mPEG-PDLLA ₈₀₀₀ and dissolve it in 2ml of acetone solution, and let it stand. After the polymer is completely dissolved, dissolve 10mg of Rubescensin A in the polymer acetone solution as the oil phase. Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. The resulting solution was evaporated under reduced pressure to remove acetone, the temperature was controlled below 30°C, the resulting suspension was centrifuged at 10,000r/min for 15min, the supernatant was taken to obtain a micellar solution, 0.2g trehalose was added to dissolve, and the mixture was filtered through a 0.22μm microporous membrane Filtration, freeze-drying to obtain oridonin A polymer micelles freeze-dried powder. The average particle size of micelles is 139nm.

Example 6

Weigh 150mg of mPEG-PDLLA ₈₀₀₀ and dissolve it in 2ml of acetone solution, and let it stand. After the polymer is completely dissolved, dissolve 20mg of Rubescensin A in the polymer acetone solution as the oil phase. Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. The obtained solution was rotary evaporated under reduced pressure to remove acetone, the temperature was controlled below 30° C., the obtained suspension was centrifuged at 10,000 r/min for 15 minutes, and the supernatant was collected to obtain a micellar solution. Add 0.1 g of mannose and 0.05 g of sucrose to dissolve, filter through a 0.22 μm microporous membrane, and freeze-dry to obtain oridonin A polymer micelle freeze-dried powder. The average particle size of micelles is 144nm.

Example 7

Weigh 200mg of mPEG-PDLLA ₁₅₀₀₀ and dissolve it in 2ml of dimethylformamide solution, and let it stand until the polymer is completely swollen, then dissolve 40mg of Rubescensin A in the polymer dimethylformamide solution as the oil phase. Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. Add the obtained solution into a dialysis bag with a molecular weight cut-off of 10,000, dialyze the bag with 2-4L double-distilled water for 12 hours, take the solution in the dialysis bag to obtain a micellar solution, add 0.2g mannose to dissolve, and filter through a 0.22μm microporous membrane , and freeze-dried to obtain oridonin A polymer micelles freeze-dried powder. The average particle size of micelles is 187nm.

Example 8

Weigh 200 mg of mPEG-PLLA ₁₀₀₀₀ and dissolve it in 2 ml of tetrahydrofuran solution, and let it stand. After the polymer is completely dissolved, dissolve 30 mg of oridonin in the polymer solution of tetrahydrofuran as the oil phase. Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. The resulting solution was evaporated under reduced pressure to remove tetrahydrofuran, the temperature was controlled below 30°C, the resulting suspension was centrifuged at 10,000r/min for 15min, and the supernatant was taken to obtain a micellar solution, which was dissolved by adding 0.3g of glucose and filtered through a 0.22μm microporous membrane , and freeze-dried to obtain oridonin A polymer micelles freeze-dried powder. The average particle size of micelles is 167nm.

Example 9

Weigh 200 mg of mPEG-PLLA ₁₀₀₀₀ and 20 mg of egg phospholipid, dissolve it in 3 ml of acetone solution, and place it until the polymer is completely dissolved, then dissolve 50 mg of Rubescensin A in the polymer acetone solution as the oil phase. Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. The resulting solution was evaporated under reduced pressure to remove acetone, the temperature was controlled below 30°C, the resulting suspension was centrifuged at 10,000r/min for 15min, and the supernatant was taken to obtain a micellar solution, which was dissolved by adding 0.3g of glucose and filtered through a 0.22μm microporous membrane , and freeze-dried to obtain oridonin A polymer micelles freeze-dried powder. The average particle size of micelles is 167nm.

Example 10

Weigh 200 mg of mPEG-PLGA _{10000 (75/25)} and 25 mg of soybean lecithin, dissolve it in 3 ml of acetone solution, and place it until the polymer is completely dissolved, then dissolve 50 mg of Rubescensin A in the polymer acetone solution as the oil phase . Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. The resulting solution was evaporated under reduced pressure to remove acetone, and the temperature was controlled below 50°C. The resulting suspension was centrifuged at 10,000 r/min for 15 minutes, and the supernatant was taken to obtain a micellar solution, which was dissolved by adding 0.2g mannose and 0.1g sucrose, and then subjected to 0.22 μm microporous membrane filtration, and freeze-drying to obtain oridonin A polymer micelles freeze-dried powder. The average particle size of micelles is 154nm.

Example 11

Weigh 100mg of mPEG-PDLLA ₄₀₀₀ and dissolve it in 2ml of acetone solution, and let it stand. After the polymer is completely dissolved, dissolve 10mg of Rubescensin A in the polymer acetone solution as the oil phase. Under magnetic stirring, slowly add into 10ml of water phase, and continue to stir and balance for 2h. The obtained solution was evaporated under reduced pressure to remove acetone, and the temperature was controlled below 30°C. The obtained suspension was centrifuged at 10,000 r/min for 15 minutes, and the supernatant was taken to obtain the micellar solution. Pore membrane filtration, freeze-drying to obtain oridonin A polymer micelles freeze-dried powder. The average particle size of micelles is 118nm.

Example 12: Conversion of amphiphilic block copolymer terminal hydroxyl groups to carboxyl terminal groups

(1) Weigh 1.6g of mPEG-PDLLA ₈₀₀₀ and dissolve it in 20ml of anhydrous 1,4-dioxane solution, and add 0.04g of succinic anhydride, 0.02g of DMAP, and 0.1ml of TEA in sequence at 0°C. Stir the reaction at room temperature for 24 h, filter, settle the filtrate with 300 ml of cold diethyl ether, filter, and vacuum-dry the precipitate at 30°C for 24 h to obtain mPEG-PDLLA ₈₀₀₀ -COOH.

(2) Weigh 0.65g of mPEG-PDLLA ₃₂₅₀ and dissolve it in 20ml of anhydrous 1,4-dioxane solution, add 0.04g of succinic anhydride, 0.02g of DMAP, and 0.1ml of TEA in sequence at 0°C. Stir the reaction at room temperature for 24 hours, filter, settle the filtrate with 200 ml of cold diethyl ether, filter, and vacuum-dry the precipitate at 30°C for 24 hours to obtain mPEG-PDLLA ₃₂₅₀ -COOH.

(3) Weigh 0.8g of mPEG-PDLLA ₃₂₅₀ and dissolve it in 20ml of anhydrous 1,4-dioxane solution, and add 0.04g of succinic anhydride, 0.02g of DMAP, and 0.1ml of TEA in sequence at 0°C. Stir the reaction at room temperature for 24 hours, filter, settle the filtrate with 200 ml of cold diethyl ether, filter, and vacuum-dry the precipitate at 30°C for 24 hours to obtain mPEG-PDLLA ₃₂₅₀ -COOH.

(4) Weigh 2 g of mPEG-PLGA _{10000 (75/25)} and dissolve it in 20 ml of anhydrous 1,4-dioxane solution, and add 0.04 g of succinic anhydride, 0.02 g of DMAP, and 0.1 ml of TEA in sequence at 0°C. Stir the reaction at room temperature for 24 h, filter, settle the filtrate with 300 ml of cold diethyl ether, filter, and vacuum-dry the precipitate at 30°C for 24 h to obtain mPEG-PLGA _10000(75/25) -COOH.

Example 13: Preparation of Amphiphilic Block Copolymer-Oridonin A Bonded Drug

(1) Weigh 1g of mPEG-PDLLA ₈₀₀₀ -COOH and dissolve it in 20ml of anhydrous dichloromethane. After the polymer dissolves, cool it in an ice bath to 0°C, add 60mg of oridonin, 25mg of DCC, and 15mg of DMAP, and react at 0°C for 24h , filter off the generated precipitate, wash the filtrate with 0.1mol/l dilute hydrochloric acid solution, dry over anhydrous sodium sulfate, filter, concentrate the filtrate, precipitate a large amount of ether, and dry in vacuo to obtain the bonded drug. The drug loading was 4.3%.

(2) Weigh 0.325g of mPEG-PDLLA ₃₂₅₀ -COOH, dissolve it in 20ml of anhydrous dichloromethane, after the polymer dissolves, cool it in an ice bath to 0°C, add 45mg oridonin, 21mgDCC, 10mgDMAP, React for 24 hours, filter out the formed precipitate, wash the filtrate with 0.1 mol/l dilute hydrochloric acid solution, dry over anhydrous sodium sulfate, filter, concentrate the filtrate, precipitate a large amount of ether, and dry in vacuo to obtain the bonded drug. The drug loading was 10.5%.

(3) Weigh 0.4g of mPEG-PDLLA ₄₀₀₀ -COOH and dissolve it in 20ml of anhydrous dichloromethane. After the polymer dissolves, cool it in an ice bath to 0°C, add 45mg oridonin, 21mgDCC, 10mgDMAP, and React for 36 hours, filter out the formed precipitate, wash the filtrate with 0.1 mol/l dilute hydrochloric acid solution, dry over anhydrous sodium sulfate, filter, concentrate the filtrate, precipitate a large amount of ether, and dry in vacuum to obtain the bonded drug. The drug loading was 8.3%.

(4) Weigh 1g of mPEG-PLGA ₁₀₀₀₀ -COOH and dissolve it in 20ml of anhydrous dichloromethane. After the polymer dissolves, cool it in an ice bath to 0°C, add 45mg of oridonin, 21mg of DCC, and 10mg of DMAP, and react at 0°C for 24h , filter off the generated precipitate, wash the filtrate with 0.1mol/l dilute hydrochloric acid solution, dry over anhydrous sodium sulfate, filter, concentrate the filtrate, precipitate a large amount of ether, and dry in vacuo to obtain the bonded drug. The drug loading was 3.5%.

Example 14: Preparation method 1 of amphiphilic block copolymer-oridonin A-bonded drug micelles freeze-dried powder:

(1) Take 100mg of mPEG-PDLLA ₈₀₀₀ -Oridonin A bonded drug, dissolve it in 2ml of acetone solution, place it, and after the polymer is completely dissolved, slowly add it into 10ml of the water phase under magnetic stirring, and continue to stir to balance 2h. The obtained solution was evaporated under reduced pressure to remove acetone, the temperature was controlled below 30°C, the obtained suspension was centrifuged at 10000r/min for 15min, the supernatant was taken to obtain the micellar solution, 0.2g of mannose was added, and filtered through a 0.22μm microporous membrane , and the filtrate was freeze-dried to obtain oridonin A polymer micelles freeze-dried powder. The particle size was measured to be 156 nm.

(2) Take 100mg of mPEG-PDLLA ₃₂₅₀ - oridonin A bonded drug, dissolve it in 2ml of acetone solution, place it, and after the polymer is completely dissolved, slowly add it into 10ml of the water phase under magnetic stirring, and continue to stir to balance 2h. The resulting solution was evaporated under reduced pressure to remove acetone, and the temperature was controlled below 30°C. The resulting suspension was centrifuged at 10,000 r/min for 15 minutes, and the supernatant was taken to obtain a micellar solution. Add 0.1 g of mannose and 0.1 g of sucrose, and micro filter through a pore filter, and freeze-dry the filtrate to obtain the freeze-dried powder of oridonin A polymer micelles. The particle size was measured to be 113 nm.

(3) Take 100mg of mPEG-PDLLA ₄₀₀₀ - oridonin A bonded drug, dissolve it in 2ml of acetone solution, place it, and after the polymer is completely dissolved, slowly add it into 10ml of the water phase under magnetic stirring, and continue to stir to balance 2h. The resulting solution was evaporated under reduced pressure to remove acetone, and the temperature was controlled below 30°C. The resulting suspension was centrifuged at 10,000 r/min for 15 minutes, and the supernatant was taken to obtain a micellar solution. Add 0.1 g of mannose and 0.1 g of sucrose, and micro filter through a pore filter, and freeze-dry the filtrate to obtain the freeze-dried powder of oridonin A polymer micelles. The particle size was measured to be 126 nm.

(4) Take 100mg of mPEG-PLGA ₁₀₀₀₀ -Oridonin A bonded drug, dissolve it in 2ml of acetone solution, place it, and after the polymer is completely dissolved, slowly add it into 10ml of the water phase under magnetic stirring, and continue to stir to balance 2h. The resulting solution was evaporated under reduced pressure to remove acetone, the temperature was controlled below 30°C, the resulting suspension was centrifuged at 10,000r/min for 15min, the supernatant was taken to obtain a micellar solution, 0.2g of trehalose was added, and filtered through a 0.22μm microporous membrane , and the filtrate was freeze-dried to obtain oridonin A polymer micelles freeze-dried powder. The particle size was measured to be 178 nm.

Method Two:

(1) Take 0.364g of Rubescensine A and dissolve it in 20ml of anhydrous 1,4-dioxane solution. After the polymer dissolves, cool it in an ice bath to 0°C, and add 0.2g of succinic anhydride and 0.1g of DMAP in turn. , reacted at room temperature for 24h, filtered off the resulting precipitate, concentrated the filtrate and settled it with 200ml of cold ether, filtered, and dried the precipitate under vacuum at 40°C for 24h, oridonin-COOH.

(2) Take 0.4g of mPEG-PDLLA ₄₀₀₀ and dissolve it in 20ml of anhydrous dichloromethane. After the polymer dissolves, cool it in an ice bath to 0°C, add 40mg of oridonin-COOH, 25mg of DCC, and 15mg of DMAP, and react at 0°C After 24 hours, the formed precipitate was filtered off, the filtrate was washed with 0.1 mol/l dilute hydrochloric acid solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, a large amount of ether was precipitated, and the bonded drug was obtained by vacuum drying.

(3) Take 100mg of mPEG-PDLLA ₄₀₀₀ - oridonin A bonded drug, dissolve it in 2ml of acetone solution, place it, and after the polymer is completely dissolved, slowly add it into 10ml of the water phase under magnetic stirring, and continue to stir to balance 2h. The resulting solution was evaporated under reduced pressure to remove acetone, and the temperature was controlled below 30°C. The resulting suspension was centrifuged at 10,000 r/min for 15 minutes, and the supernatant was taken to obtain a micellar solution. Add 0.1 g of mannose and 0.1 g of sucrose, and micro filter through a pore filter, and freeze-dry the filtrate to obtain the freeze-dried powder of oridonin A polymer micelles. The particle size was measured to be 122 nm.

Example 15: 10 healthy Wistar rats were randomly divided into 2 groups with 5 rats in each group. Before the experiment, they were fasted overnight, and Oridonin A common injection and the micellar solution prepared in Example 7 were administered respectively through the tail vein, and the dosage was 20 mg/kg. At 0.0167, 0.0833, 0.167, 0.333, 0.5, 1, 2, 4, 8, 12, 24 hours after administration, take 0.5ml of blood from the orbit of the rat and put it in a 1.5ml dry centrifuge tube anticoagulated with 2% heparin, 5000r/min After centrifugation for 5 min, 0.1 ml of plasma was taken for processing and HPLC was used to determine the drug concentration, and the 3P87 program was used for data processing to calculate the pharmacokinetic parameters. The results are shown in Tables 1 and 2.

Table 1 Pharmacokinetic parameters of Oridonin A injection

Table 2 Oridonin A block copolymer micelles pharmacokinetic parameters

From the comparison of Table 1 and 2, it can be seen that the pharmacokinetic parameters of Rubescensin A common injection are t _1/2 π=0.158h, t _1/2 α=1.179h, t _1/2 β=8.404h, AUC _0-∞ =7.686 (mg/L)·h, MRT _0-∞ =9.261h. The pharmacokinetic parameters of Rubescensin A block copolymer micelles are t _1/2 π=0.095h, t _1/2 α=3.097h, t ₁ /2=48.68h, AUC _0-∞ =16.75(mg /L)·h, MRT _0-∞ = 17.37h. The analysis results show that the pharmacokinetic behavior of Rubescensin A block copolymer micelles in rats has an obvious sustained-release effect, and the AUC _0-∞ value is 2.19 times that of the injection, which improves the effect of Rubescensin A. of bioavailability.

Claims (6)

1. Oridonin polymer micelle, it is characterized in that: this micelle be encapsulated in one or both polymer by rubescensine A with physical form or form amphipathic nature block polymer-rubescensine A bonding medicine by chemical reaction and polymer covalent bond after form, wherein rubescensine A accounts for the 0.01-40% of gross weight, described polymer refer to amphipathic nature block polymer or with the mixture of phospholipid, wherein phospholipid is selected from soybean phospholipid, egg phosphatide, saturated soybean phospholipid, in the saturated egg phosphatide one or more, amphipathic nature block polymer is selected from biodegradable poly glycol monomethyl ether-hexadecanoic acid, poly glycol monomethyl ether-PDLLA, poly glycol monomethyl ether-poly (l-lactic acid), poly glycol monomethyl ether-polylactide-co-glycolide, poly glycol monomethyl ether-polycaprolactone, in poly glycol monomethyl ether-polylactide-6-caprolactone and poly glycol monomethyl ether-polylactide-co-glycolide-6-caprolactone one or more, the hydrophilic section of described amphipathic nature block polymer is poly glycol monomethyl ether, and hydrophobic section is selected from biodegradable poly-cetyl cyanoacrylate, PDLLA, poly (l-lactic acid), polylactide-co-glycolide, poly-epsilon-caprolactone, polylactide-6-caprolactone, polylactide-co-glycolide-6-caprolactone or their mixture; In the wherein said polylactide-co-glycolide, the mass percent of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 99: 1～1: 99; The mass percent of lactide and 6-caprolactone is 99: 1～1: 99 in polylactide-6-caprolactone; In polylactide-co-glycolide-6-caprolactone, the mass percent of lactide, Acetic acid, hydroxy-, bimol. cyclic ester and 6-caprolactone is 98～50: 1～49: 1～49; The molecular weight of described amphipathic nature block polymer is 500-100000, and wherein hydrophilic block number-average molecular weight is 200-50000, and hydrophobicity block number-average molecular weight is 50-50000; Wherein the hydrophilic block total amount accounts for the 10-98% of copolymer gross weight.

2. Oridonin polymer micelle according to claim 1, it is characterized in that: described amphipathic nature block polymer-rubescensine A bonding medicated bag is drawn together poly glycol monomethyl ether-PDLLA-rubescensine A bonding medicine, poly glycol monomethyl ether-poly (l-lactic acid)-rubescensine A bonding medicine, poly glycol monomethyl ether-polylactide-co-glycolide-rubescensine A bonding medicine, poly glycol monomethyl ether-polylactide-6-caprolactone-rubescensine A bonding medicine, poly glycol monomethyl ether-polycaprolactone-rubescensine A bonding medicine, poly glycol monomethyl ether-polylactide-co-glycolide-6-caprolactone-rubescensine A bonding medicine, its bonding position is at 1 or 14 hydroxyls of rubescensine A.

3. Oridonin polymer micelle according to claim 1, wherein the hydrophilic section of amphipathic nature block polymer is poly glycol monomethyl ether, its molecular weight distribution is 200-30000.

4. the preparation method of an Oridonin polymer micelle as claimed in claim 1 is characterized in that, its step is as follows:

(1) block copolymer of preparation poly glycol monomethyl ether and various aliphatic cyclic esters

In the presence of poly glycol monomethyl ether, catalyst, carry out ring-opening polymerization with the aliphatic cyclic ester monomer, catalyst is stannous octoate, and consumption is the 0.01-1% of polymerization single polymerization monomer quality, and 110～180 ℃ of polymerization temperatures, polymerization time are 3-24 hour;

(2) terminal hydroxy group with poly glycol monomethyl ether and aliphatic poly ester block copolymer changes the end carboxyl into;

The block copolymer that (1) is synthetic is at catalyst pyridine, triethylamine or 4-N, the N-dimethylamino naphthyridine exists lower, in anhydrous methylene chloride or 1,4-dioxane solution, under 0～25 ℃, carry out esterification with anhydride, catalyst amount is 0.05～2 times of block copolymer terminal hydroxy group molal weight, the anhydride consumption is 1～3 times of block copolymer terminal hydroxy group molal weight, and response time 24～48h changes the terminal hydroxy group of block copolymer into the end carboxyl;

(3) with end carboxyl and the rubescensine A hydroxyl reaction of poly glycol monomethyl ether and aliphatic poly ester block copolymer, prepare amphipathic nature block polymer-rubescensine A bonding medicine

At catalyst 4-N, N-dimethylamino naphthyridine and condensing agent dicyclohexylcarbodiimide exist lower, at dichloromethane, react in dimethyl sulfoxide or the dimethyl formamide solution, 0～25 ℃ of reaction temperature, response time 12-48h, wherein, 4-N, the mole dosage of N-dimethylamino naphthyridine is 0.05～2 times of block copolymer end carboxyl, the mole dosage of dicyclohexylcarbodiimide is 1～3 times of block copolymer end carboxyl, the mole dosage of rubescensine A is 1～2 times of block copolymer end carboxyl, and reaction obtains amphipathic nature block polymer-rubescensine A bonding medicine.

5. the preparation method of an Oridonin polymer micelle as claimed in claim 1, it is characterized in that: its preparation method is as follows:

(1) block copolymer of preparation poly glycol monomethyl ether and various aliphatic cyclic esters

In the presence of poly glycol monomethyl ether, catalyst, carry out ring-opening polymerization with the aliphatic cyclic ester monomer, catalyst is stannous octoate, and consumption is the 0.01-1% of polymerization single polymerization monomer quality, and 110～180 ℃ of polymerization temperatures, polymerization time are 3-24 hour;

(2) rubescensine A contains the preparation of carboxy derivatives

With rubescensine A at catalyst pyridine, triethylamine or 4-N, the N-dimethylamino naphthyridine exists lower, in anhydrous chloroform or 1,4-dioxane solution, under 0～25 ℃, carry out esterification with anhydride, catalyst amount is 0.05-2 times of rubescensine A molal weight, the anhydride consumption is 1～2 times of block copolymer terminal hydroxy group molal weight, and response time 24～48h obtains carboxylic Oridonin derivative;

(3) amphipathic nature block polymer and the carboxylic Oridonin derivative that (1) is synthetic carries out covalent bonding, preparation amphipathic nature block polymer-rubescensine A bonding medicine

At catalyst 4-N, N-dimethylamino naphthyridine and condensing agent dicyclohexylcarbodiimide exist lower, at dichloromethane, react in dimethyl sulfoxide or the dimethyl formamide solution, 0～25 ℃ of reaction temperature, response time 12-48h, wherein, 4-N, the mole dosage of N-dimethylamino naphthyridine is 0.05～2 times of carboxylic Oridonin derivative, the mole dosage of dicyclohexylcarbodiimide is 1～3 times of carboxylic Oridonin derivative, the mole dosage of block copolymer is 0.5～1 times of carboxylic Oridonin derivative, and reaction obtains amphipathic nature block polymer-rubescensine A bonding medicine.

6. according to claim 4 or the preparation method of 5 described Oridonin polymer micelles, it is characterized in that: described aliphatic cyclic ester monomer refers to lactide, 6-caprolactone, the mixture of lactide and Acetic acid, hydroxy-, bimol. cyclic ester, the mixture of lactide and 6-caprolactone, the ternary mixture of lactide, Acetic acid, hydroxy-, bimol. cyclic ester and 6-caprolactone; In the monomer mixture of lactide and Acetic acid, hydroxy-, bimol. cyclic ester, the mass percent of lactide and Acetic acid, hydroxy-, bimol. cyclic ester is 99: 1～1: 99; In the mixture of lactide and 6-caprolactone, the mass percent of lactide and 6-caprolactone is 99: 1～1: 99; In the ternary mixture of lactide, Acetic acid, hydroxy-, bimol. cyclic ester and 6-caprolactone, the mass percent of lactide, Acetic acid, hydroxy-, bimol. cyclic ester and 6-caprolactone is 98～50: 1～49: 1～49; For the synthesis of anhydride refer to a kind of in acetic anhydride, succinic anhydride or the maleic anhydride.

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