WO2024002385A1 - Echinomycin antibiotic micelle, preparation method therefor, and use - Google Patents

Echinomycin antibiotic micelle, preparation method therefor, and use Download PDF

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Publication number
WO2024002385A1
WO2024002385A1 PCT/CN2023/111047 CN2023111047W WO2024002385A1 WO 2024002385 A1 WO2024002385 A1 WO 2024002385A1 CN 2023111047 W CN2023111047 W CN 2023111047W WO 2024002385 A1 WO2024002385 A1 WO 2024002385A1
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Prior art keywords
echinomycin
solution
micelle
antibiotic
present
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PCT/CN2023/111047
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French (fr)
Chinese (zh)
Inventor
唐莉
张川
徐靖
张�成
邱海棠
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成都华昊中天药业有限公司
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Publication of WO2024002385A1 publication Critical patent/WO2024002385A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention relates to the field of medical technology, and more specifically to micelles containing echinomycin antibiotics and amphiphilic copolymers and their preparation methods and uses.
  • Echinomycin antibiotics are quinoxaline HIF1 ⁇ (hypoxia-inducible factor 1 ⁇ subunit) inhibitors that competitively inhibit the ability of HIF1 ⁇ to bind to specific DNA sequences. Echinomycin antibiotics have poor water solubility and short half-life in the body. Multiple phase 1 and phase 2 trials have been conducted clinically to demonstrate the ability of echinomycin antibiotics to retain solid tumors. Some of the phase 2 trials administered 1200 ⁇ g/m 2 echinomycin, but the results showed that there was no significant gain. beneficial. Some clinical trial studies have used echinomycin formulations that included polyoxyethylene castor oil, resulting in significant dose-limiting toxicities.
  • CN108495619A uses a thin film hydration method to prepare echinomycin liposomes, but the preparation process used is not conducive to industrial scale-up production and the drug loading capacity is low.
  • the present invention provides a new type of echinomycin antibiotic micelle preparation, which has higher solubility of echinomycin antibiotics and smaller particle size, and helps to improve the condition of echinobacteriaceae.
  • the in vivo release behavior and tissue distribution of echinomycin antibiotics allow echinomycin antibiotics to achieve higher efficacy at smaller doses while reducing the toxicity of echinomycin antibiotics.
  • the present invention also provides a technology for preparing novel echinomycin antibiotic micellar preparations, which is conducive to industrial scale-up production.
  • the invention provides a micelle comprising an echinomycin antibiotic and one or more amphiphilic copolymers.
  • the echinomycin antibiotic is selected from the group consisting of echinomycin, actinoleukin, triostin, F-43, 59266, and 6270 and echinoserine etc.
  • the echinomycin antibiotic is echinomycin.
  • the amphiphilic copolymer is selected from one or more of block copolymers, graft copolymers, and dendrimers.
  • the amphiphilic copolymer is a block copolymer.
  • the hydrophilic segment of the block copolymer is selected from polyethylene glycol, methoxypolyethylene glycol, polyacrylic acid, polymethacrylic acid, polyacrylic acid derivatives, polymethyl One or more of acrylic acid derivatives and polyamino acids
  • the hydrophobic segment of the block copolymer is selected from polylactic acid, lactide-glycolide copolymer, polycaprolactone, polycarbonate, polycarbonate derivatives, polybutylene One or more of acid esters, polyacrylic acid derivatives, polymethacrylic acid derivatives, and polyamino acids.
  • polyacrylic acid derivatives in the hydrophilic segment of the block copolymer include, but are not limited to, polyhydroxyethyl acrylate, polyacrylic acid-N,N-dimethylaminoethyl ester, polyacrylic acid-N,N-diethylaminoethyl ester , polyethylene glycol diacrylate, polyglyceryl acrylate, etc.
  • polymethacrylic acid derivatives in the hydrophilic segment of the block copolymer include, but are not limited to, polyhydroxyethyl methacrylate, polymethacrylic acid-N,N-dimethylaminomethyl ester, polymethacrylic acid-N , N-diethylaminoethyl ester, polyethylene glycol dimethacrylate, polyglyceryl methacrylate and poly(2-methacryloyloxyethylphosphocholine), etc.
  • polycarbonate derivatives in the hydrophobic segment of the block copolymer include, but are not limited to, poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylic acid ethyl ester), poly(5- Methyl-2-oxo-1,3-dioxane-5-carboxylic acid benzyl ester), poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylic acid propyl ester) ) and poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylic acid isopropyl ester), etc.
  • polyacrylic acid derivatives in the hydrophobic segment of the block copolymer include, but are not limited to, polymethyl acrylate, polyethyl acrylate, polypropyl acrylate, polybutyl acrylate, polybenzyl acrylate, and the like.
  • polymethacrylic acid derivatives in the hydrophobic segment of the block copolymer include, but are not limited to, polymethyl methacrylate, polyethyl methacrylate, polypropyl methacrylate, polybutyl methacrylate, and polymethacrylate. Benzyl acrylate, etc.
  • the amphiphilic copolymer is a methoxy polyethylene glycol-polylactic acid block copolymer.
  • the weight average molecular weight of the hydrophilic segment of the block copolymer ranges from 100 Da to 50,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophilic segment of the block copolymer ranges from 1,000 Da to 10,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophilic segment of the block copolymer ranges from 2000 Da to 5000 Da.
  • the weight average molecular weight of the hydrophobic segment of the block copolymer ranges from 500 Da to 100,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophobic segment of the block copolymer ranges from 2,000 Da to 50,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophobic segment of the block copolymer ranges from 5000 Da to 25000 Da.
  • the amphiphilic copolymer is methoxypoly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA), wherein the amphiphilic copolymer is The weight average molecular weight of the water segment mPEG segment is 2000Da-5000Da, and the weight average molecular weight of the hydrophobic segment PDLLA segment is 5000Da-25000Da.
  • the weight average molecular weight of the hydrophilic and hydrophobic segments of the block copolymer affects the release, in vivo and in vitro stability, and in vivo tissue distribution of the micelle preparation.
  • the block copolymer has a polydispersity index of less than 2.0.
  • the ratio of the amphiphilic copolymer to the echinomycin antibiotic in the micelle ranges from about 0.1:1 to 200:1 by weight, such as 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 6:1, 8:1, 9:1, 10:1, 12:1, 15:1, 18:1, 20: 1. 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190: 1 or 200:1, or any range in between.
  • the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 0.5:1 to 100:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 1:1 to 100:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 6:1 to 25:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 8:1 to 20:1 by weight.
  • the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle is 9:1 by weight.
  • the weight ratio of amphiphilic copolymer to echinomycin antibiotics in micelles is related to the stability and release of micelle preparations.
  • the micelles further include one or more of a pH adjuster, a tension adjuster, an antioxidant, a preservative, a buffer, a metal chelating agent, and an inert gas.
  • the pH adjuster is selected from sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium carbonate, Tris, sodium linoleate, sodium oleate, potassium carbonate, potassium linoleate, One or more of potassium oleate and lactic acid.
  • the tonicity adjusting agent is selected from sodium deoxycholate, sodium chloride, glycerin, sorbitol, xylitol, mannitol, glucose, trehalose, maltose, sucrose, raffinose , one or more of lactose, dextran, polyethylene glycol and propylene glycol.
  • the tonicity adjusting agent is sodium deoxycholate.
  • the preservative is selected from one or more of hydroxyphenyl alkyl esters, benzoic acid, sodium benzoate, sorbic acid, chlorethidine acetate, and benzalkonium bromide.
  • the antioxidant is selected from the group consisting of sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, ascorbic acid, tert-butyl-p-hydroxyanisole, 2,6-di-tert-butylated hydroxyl One or more of toluene, vitamin E, and ascorbyl palmitate.
  • the buffer is selected from the group consisting of phosphate buffer, citrate buffer, Tris buffer, carbonate buffer, succinate buffer, maleate buffer, and boron buffer. One or more salt buffers.
  • the metal chelating agent is selected from one or more of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, and calcium sodium ethylenediaminetetraacetate.
  • the inert gas is selected from one or both of nitrogen and argon.
  • the concentration of echinomycin antibiotics in the micelles is 0.1 mg/ml-20 mg/ml, such as 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml. ml, 3mg/ml, 4mg/ml, 5mg/ml, 8mg/ml, 10mg/ml, 12mg/ml, 15mg/ml, 18mg/ml or 20mg/ml.
  • concentrations are beneficial for clinical use and production.
  • the particle size of the micelles is 10 nm-150 nm. Such a particle size is conducive to the enhanced penetration and retention effect (EPR effect) of drug-loaded micelles.
  • the micelles have a polydispersity index (PDI) of less than 0.3, such as less than 0.2.
  • PDI polydispersity index characterizes the size distribution of particle size. The smaller the value, the more uniform the particle size distribution.
  • the micelles are in liquid or lyophilized powder form.
  • the micelles are in liquid form, containing water, such as deionized or purified water.
  • the micelles are in the form of a lyophilized powder that does not contain a lyoprotectant. In one or more embodiments, the micelles are in the form of a lyophilized powder containing a lyoprotectant. In one or more embodiments, the lyoprotectant is selected from the group consisting of mannitol, glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, lysine, proline, arginine, hydroxypropyl One or more of ⁇ -cyclodextrin, dextran, polyethylene glycol, and polyvinyl alcohol.
  • the micelles in liquid form comprise or consist of echinomycin, mPEG-PDLLA, sodium deoxycholate, and purified water.
  • micelles in the form of lyophilized powder comprise or consist of echinomycin, mPEG-PDLLA and sodium deoxycholate.
  • the present invention provides a method for preparing micelles of the present invention.
  • the micelle preparation method is selected from the group consisting of direct dispersion method, solid phase dispersion method, organic solvent injection method, emulsification method, membrane emulsification method, microfluidic method, ultrasonic method, and homogenization method. one or more of them.
  • the micelle preparation method is selected from the group consisting of organic solvent injection method, microfluidic method and emulsification method.
  • the micelle preparation method is an organic solvent injection method.
  • the organic solvent injection method includes the following steps:
  • step (1) pour the organic solution of echinomycin antibiotics containing the amphiphilic copolymer obtained in step (1) into the stirring water.
  • the drug-loaded micelle solution containing organic solvent is obtained from the phase solution;
  • the organic solvent is selected from the group consisting of chloroform, dichloromethane, tert-butyl alcohol, isopropyl alcohol, ethyl acetate, ethanol , one or more of methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
  • the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution and surfactant solution.
  • the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait.
  • the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions.
  • the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions.
  • the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions.
  • the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions.
  • the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions .
  • the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, polyethylene glycol 15-hydroxystearate (HS15) and other solutions.
  • the aqueous solution in step (2) of the above organic solvent injection method, contains 0.1%-2% sodium deoxycholate, 1%-10% acetic acid Ethyl ester, 0.5%-5% benzyl alcohol aqueous solution. In one or more embodiments of the present invention, in step (2) of the above organic solvent injection method, the aqueous solution is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and Composed of deionized or purified water.
  • the obtained drug-loaded micelle solution in step (2) of the above-mentioned organic solvent injection method, can be diluted to a certain extent if necessary according to the purpose of the process.
  • the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
  • the micelle solution after removing the organic solvent may be concentrated if necessary according to the purpose of the process.
  • step (4) of the above-mentioned organic solvent injection method sterilization is performed by filtration.
  • the micelle preparation method is a microfluidic method.
  • the microfluidic method includes the following steps:
  • step (1) Inject the organic solution of echinomycin antibiotics containing the amphiphilic copolymer and the aqueous phase solution obtained in step (1) into the micromixer simultaneously from different liquid inlets to obtain a drug-loaded micellar solution containing an organic solvent. ;
  • the organic solvent is selected from chloroform, dichloromethane, tert-butyl alcohol, isopropyl alcohol, ethyl acetate, ethanol , one or more of methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
  • the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution, surfactant solution, aqueous solution containing low concentration organic solvent.
  • the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait.
  • the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions.
  • the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions.
  • the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions.
  • the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions.
  • the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions .
  • the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, HS15 and other solutions.
  • the aqueous solution containing a low concentration of organic solvent is selected from ethanol, methanol, acetone, tetrahydrofuran, dimethylformamide (DMF), dimethyl, etc. with an organic solvent concentration ranging from 0.1% to 10%.
  • Aqueous solutions such as methyl sulfoxide (DMSO).
  • each phase solution in step (2) of the above-mentioned microfluidic method, is simultaneously injected into the micromixer from different liquid inlets through compressed air or a pump.
  • the total flow rate of each phase solution is 1ml/min-1L/min, such as 1ml/min, 2ml/min, 3ml/min, 5ml/min, 10ml/min, 15ml/min, 20ml/min, 25ml/min, 30ml/min, 50ml/min, 80ml/min, 100ml/min, 150ml/min, 200ml/min, 300ml/min, 400ml/min, 500ml/min, 600ml/min, 800ml/ min or 1L/min, or any range in between.
  • the total flow rate of each phase solution is 20 ml/min to 50 ml/min. In one or more embodiments, the total flow rate of each phase solution is 25 ml/min to 30 ml/min. In one or more embodiments, the flow rate ratio of the organic phase solution and the aqueous phase solution is 1:1-1:300, such as 1:1, 1:2, 1:3, 1:5, 1:6, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1:150, 1: 200, 1:250 or 1:300, or any range in between. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:8-1:15.
  • the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:9-1:12.
  • the micromixer may be selected from a T-type tee, an x-type mixer, and a multi-inlet vortex mixer (MIVM).
  • MIVM multi-inlet vortex mixer
  • the micromixer is a T-shaped tee.
  • the inner diameters of the inlet and outlet of the micromixer can be 0.1mm-5mm respectively, and they can be the same or different.
  • the material of the micromixer can be selected from polyetheretherketone (PEEK), stainless steel, polytetrafluoroethylene (PTFE), etc.
  • the obtained drug-loaded micelle solution containing an organic solvent in step (2) of the above-mentioned microfluidic method, can be diluted to a certain extent if necessary according to the purpose of the process.
  • the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
  • the micellar solution after removing the organic solvent in step (3) of the above-mentioned microfluidic method, can be concentrated if necessary according to the purpose of the process.
  • step (4) of the above-mentioned microfluidic method sterilization is performed by filtration.
  • the microfluidic method includes the following steps:
  • step (3) Inject the echinomycin antibiotic organic solution obtained in step (1), the polymer organic solution and the aqueous phase solution obtained in step (2) simultaneously into the micromixer through different liquid inlets to obtain a carrier containing organic solvents.
  • Pharmaceutical micelle solution
  • the organic solvent is used to dissolve echinomycin antibiotics, which can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics which can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics which can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics which can be selected from chloroform, dichloromethane, tert.
  • butanol, isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone is selected from chloro
  • the organic solvent is used to dissolve the amphiphilic copolymer, which can be selected from chloroform, dichloromethane, tert-butyl One or more of alcohol, isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
  • the amphiphilic copolymer which can be selected from chloroform, dichloromethane, tert-butyl
  • alcohol isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
  • the organic solvent used to dissolve echinomycin antibiotics in step (1) and the organic solvent used to dissolve the amphiphilic copolymer in step (2) can be the same, or Can be different.
  • the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution, surfactant solution, aqueous solution containing low concentration organic solvent.
  • the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait.
  • the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions.
  • the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions.
  • the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions.
  • the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions.
  • the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions .
  • the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, HS15 and other solutions.
  • the aqueous solution containing a low-concentration organic solvent is selected from aqueous solutions such as ethanol, methanol, acetone, tetrahydrofuran, DMF, DMSO, etc. with an organic solvent concentration ranging from 0.1% to 10%.
  • each phase solution in step (3) of the above-mentioned microfluidic method, is simultaneously injected into the micromixer from different liquid inlets through compressed air or a pump.
  • the total flow rate of each phase solution is 1ml/min-1L/min, such as 1ml/min, 2ml/min, 3ml/min, 5ml/min, 10ml/min, 15ml/min, 20ml/min, 25ml/min, 30ml/min, 50ml/min, 80ml/min, 100ml/min, 150ml/min, 200ml/min, 300ml/min, 400ml/min, 500ml/min, 600ml/ min, 800ml/min or 1L/min, or any range in between.
  • the total flow rate of each phase solution is 20 ml/min to 50 ml/min. In one or more embodiments, the total flow rate of each phase solution is 25 ml/min to 30 ml/min. In one or more embodiments, the flow rate ratio of the total organic phase solution to the aqueous phase solution is 1:1-1:300, such as 1:1, 1:2, 1:3, 1:5, 1:6 , 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1:150, 1 :200, 1:250, or 1:300, or any range in between.
  • the flow rate ratio of the echinomycin antibiotic organic solution and the polymer organic solution is 1:1-1:300, for example, 1:1, 1:2, 1:3, 1:5 , 1:6, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1 :150, 1:200, 1:250 or 1:300, or any range in between.
  • the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:8-1:15.
  • the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:9-1:12.
  • the micromixer may be an x-type mixer and a multi-inlet vortex mixer (MIVM).
  • MIVM multi-inlet vortex mixer
  • the inner diameters of the inlet and outlet of the micromixer can be 0.1mm-5mm respectively, and they can be the same or different.
  • the material of the micromixer can be selected from polyetheretherketone (PEEK), stainless steel, polytetrafluoroethylene (PTFE), etc.
  • the obtained drug-loaded micelle solution containing an organic solvent in step (3) of the above-mentioned microfluidic method, can be diluted to a certain extent if necessary according to the purpose of the process.
  • step (4) of the above-mentioned microfluidic method the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
  • the micelle solution after removing the organic solvent in step (4) of the above-mentioned microfluidic method, can be concentrated if necessary according to the purpose of the process.
  • step (5) of the above-mentioned microfluidic method sterilization is performed by filtration.
  • the microfluidic method includes the following steps:
  • step (3) Inject the echinomycin antibiotic organic solution obtained in step (1) and the polymer aqueous phase solution obtained in step (2) simultaneously into the micromixer through different liquid inlets to obtain drug-loaded micelles containing organic solvents. solution;
  • the organic solvent is used to dissolve echinomycin antibiotics, which can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics which can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics which can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics can be selected from chloroform, dichloromethane, tert.
  • echinomycin antibiotics which can be selected from chloroform, dichloromethane, tert.
  • butanol, isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone is selected from chloro
  • the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution, surfactant solution, aqueous solution containing low concentration organic solvent.
  • the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait.
  • the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions.
  • the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions.
  • the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions.
  • the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions.
  • the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions .
  • the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, HS15 and other solutions.
  • the aqueous solution containing a low-concentration organic solvent is selected from aqueous solutions such as ethanol, methanol, acetone, tetrahydrofuran, DMF, DMSO, etc. with an organic solvent concentration ranging from 0.1% to 10%.
  • each phase solution in step (3) of the above-mentioned microfluidic method, is simultaneously injected into the micromixer from different liquid inlets through compressed air or a pump.
  • the total flow rate of each phase solution is 1ml/min-1L/min, such as 1ml/min, 2ml/min, 3ml/min, 5ml/min, 10ml/min, 15ml/min, 20ml/min, 25ml/min, 30ml/min, 50ml/min, 80ml/min, 100ml/min, 150ml/min, 200ml/min, 300ml/min, 400ml/min, 500ml/min, 600ml/min, 800ml/ min or 1L/min, or any range in between.
  • the total flow rate of each phase solution is 20 ml/min to 50 ml/min. In one or more embodiments, the total flow rate of each phase solution is 25 ml/min to 30 ml/min. In one or more embodiments, the flow rate ratio of the organic phase solution and the aqueous phase solution is 1:1-1:300, such as 1:1, 1:2, 1:3, 1:5, 1:6, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1:150, 1:200, 1:250 or 1:300, or any range in between. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:8-1:15.
  • the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:9-1:12.
  • the micromixer may be selected from a T-type tee, an x-type mixer, and a multi-inlet vortex mixer (MIVM).
  • MIVM multi-inlet vortex mixer
  • the micromixer is a T-shaped tee.
  • the inner diameters of the inlet and outlet of the micromixer can be 0.1mm-5mm respectively, and they can be the same or different.
  • the material of the micromixer can be selected from polyetheretherketone (PEEK), stainless steel, polytetrafluoroethylene (PTFE), etc.
  • the obtained drug-loaded micelle solution containing an organic solvent in step (3) of the above-mentioned microfluidic method, can be diluted to a certain extent if necessary according to the purpose of the process.
  • step (4) of the above-mentioned microfluidic method the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
  • the micelle solution after removing the organic solvent in step (4) of the above-mentioned microfluidic method, can be concentrated if necessary according to the purpose of the process.
  • step (5) of the above-mentioned microfluidic method sterilization is performed by filtration.
  • the micelle preparation method is an emulsification method.
  • the emulsification method includes the following steps:
  • the organic solvent is selected from one or more of chloroform, dichloromethane, and ethyl acetate.
  • the solid proportion in the echinomycin antibiotic organic solution is 0.1%-50% (weight/volume ratio), for example 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 18%, 20%, 25% ,30%,35%,40%, 45% or 50%, or any range in between.
  • the solid proportion in the echinomycin antibiotic organic solution is 10% (weight/volume ratio).
  • the aqueous solution contains 0.1%-2% sodium deoxycholate, 1%-10% ethyl acetate Ester, 0.5%-5% benzyl alcohol aqueous solution.
  • the aqueous phase solution is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and deoxycholate. Composed of ionized water or purified water.
  • step (2) of the emulsification method the echinomycin antibiotic organic solution and the aqueous phase solution are mixed in a ratio of 1:1 to 1:9 (volume ratio). ratio to mix. In a specific embodiment, the echinomycin antibiotic organic solution and the aqueous phase solution are mixed at a ratio of 1:5 (volume ratio).
  • step (2) of the above-mentioned emulsification method the echinomycin antibiotic organic solution and the aqueous phase solution are mixed evenly through high-speed shearing to form a colostrum.
  • the cold aqueous medium refers to an aqueous medium with a temperature lower than 5°C.
  • the aqueous medium is deionized or purified water.
  • the organic solvent is removed by one or more means selected from volatilization, dialysis and ultrafiltration. In a specific embodiment, the organic solvent is removed by ultrafiltration.
  • step (5) of the emulsification method sterilization is performed by filtration.
  • the micelle preparation method further includes the step of freeze-drying the obtained drug micelles.
  • the micelles obtained by the micelle preparation method of the present invention have better stability and are convenient for scale-up research and production.
  • the present invention provides pharmaceutical formulations made from the echinomycin antibiotic micelles.
  • the echinomycin antibiotic micelles of the present invention can be prepared parenterally, by inhalation, intraperitoneally, intravesically, intramuscularly, intravenously, or intratracheally. , pharmaceutical preparations for subcutaneous, intraocular, intrathecal, transdermal, rectal or vaginal administration.
  • the echinomycin antibiotic micelles can be prepared into pharmaceutical preparations for intravenous administration.
  • the pharmaceutical formulation may be a solid formulation, a liquid formulation, or a gas formulation.
  • the pharmaceutical formulation may be an injectable liquid formulation.
  • the pharmaceutical preparation may be a micellar sterile freeze-dried powder preparation, which is prepared before use. Reconstitute with appropriate media such as water for injection, physiological saline, glucose solution, etc.
  • the pharmaceutical formulation further contains other drugs.
  • the other drugs include additional anti-cancer drugs.
  • the additional anti-cancer drug is a chemotherapy drug, a targeted anti-tumor drug, or/and an immunotherapy drug.
  • the present invention provides the use of the echinomycin antibiotic micelles in the preparation of medicaments for preventing or treating cancer.
  • the present invention provides the echinomycin antibiotic micelles or pharmaceutical preparations containing the echinomycin antibiotic micelles according to the present invention for preventing or treating cancer.
  • the present invention provides a method for preventing or treating cancer in a subject, the method comprising administering to the patient a therapeutically effective amount of an echinomycin antibiotic micelle of the present invention or a micelle containing the echinobacterium Pharmaceutical preparations of antibiotic micelles.
  • subject includes mammals and non-mammals.
  • mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (eg, chimpanzees and other apes and monkeys); domestic animals, such as cattle, horses, sheep, goats, pigs; , such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs.
  • the subject is a human.
  • treatment includes alleviating, alleviating or ameliorating symptoms of a disease or condition, inhibiting a disease or condition, such as preventing the progression of a disease or condition, ameliorating a disease or condition, making a disease or condition better, alleviating symptoms caused by a disease or condition , or to abort symptoms of a disease or condition, prevent other symptoms, ameliorate or prevent underlying metabolic causes of symptoms, and further, the term includes preventive purposes.
  • the term also includes obtaining therapeutic and/or prophylactic effects. The therapeutic effect refers to the cure or improvement of the underlying disease being treated.
  • cure or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, such as a subject's condition being observed to improve although the subject may still be affected by the underlying disease.
  • the echinomycin antibiotic micelles or pharmaceutical formulations of the present invention can be administered to subjects who are at risk of developing a specific disease, such as cancer, or even if a diagnosis of the disease has not yet been made, to a subject who has developed the disease.
  • subjects with multiple physiological symptoms are administered the echinomycin antibiotic micelles or pharmaceutical preparations of the present invention.
  • terapéuticaally effective amount refers to at least one active substance (such as the echinomycin antibiotics of the present invention) that is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. micelles). The result may be a reduction and/or alleviation of signs, symptoms or causes, or any other desired change in the biological system.
  • a “therapeutically effective amount” is the amount of the Aspergillus species of the invention required to provide clinically significant symptom relief.
  • the amount of antibiotic micelles in the composition The therapeutically effective amount appropriate in any individual case can be determined using techniques such as dose escalation trials.
  • the present invention makes the echinomycin antibiotics into micelles, which improves the solubility of the echinomycin antibiotics and makes them more conducive to clinical use.
  • the prepared micelles have the characteristics of small particle size, uniform particle size distribution, large drug loading capacity and The characteristics of high stability, compared with the injection, reduce the elimination rate in the plasma, prolong the action time of the drug in the body, improve the anti-tumor effect of echinomycin antibiotics in vitro and in vivo, and also reduce the echinomycin In vivo toxicity of antibiotics.
  • the technology for preparing echinomycin antibiotic drug-loaded micelles provided by the invention has good reproducibility, high drug loading capacity, and is conducive to scale-up and industrial production.
  • Figure 1 is a hydration particle size diagram of echinomycin drug-loaded micelles prepared according to Example 5.
  • DLS dynamic light scattering
  • instrument Malvern Nanoparticle Size Analyzer, model: NANO ZS90
  • Detection mode automatic; detect the refractive index of the sample to be tested: 1.340; dispersion medium: ultrapure water; temperature 25°C; viscosity: 0.8872cp; refractive index of the dispersion medium: 1.330, and obtain the particle size diagram.
  • Preparation of reference substance solution Precisely weigh about 20 mg of the echinomycin antibiotic reference substance into a 100 ml measuring flask, add an appropriate amount of acetonitrile to dissolve, and dilute to the mark, shake well, accurately measure 1 ml and place it into a 10 ml measuring flask, add acetonitrile and dilute to Scale and shake well.
  • test solution Precisely measure 0.5 ml of the echinomycin antibiotic micellar solution into a 5 ml volumetric flask, add acetonitrile and dilute to the mark, shake well. Filter, take the filtrate for testing (Waters ACQUITY Arc 2998PDA), and obtain the echinomycin antibiotic content in the sample.
  • the filtered echinomycin antibiotic micelle solution is divided into vials and freeze-dried using a freeze-drying machine. A small amount of the freeze-dried product is weighed and the content of the echinomycin antibiotics in the freeze-dried product is measured according to the method in Test Example 2. (w/w%), which is the drug loading capacity of echinomycin antibiotic micelles.
  • Test Example 4 Pharmacokinetic test of echinomycin antibiotic micelles.
  • a pharmacokinetic test was conducted on the echinomycin antibiotic micelles of the present invention.
  • Three SD rats of each gender were used as experimental animals, and administration was carried out using a cross-administration method.
  • male SD rats were given echinomycin injection (its preparation method is as follows: weigh For 7.5 mg of echinomycin raw material, add about 8 g of a mixed solution of Kolliphor EL and ethanol (1:1, v/v) and dissolve it.
  • a syringe pump (Baoding Leifu, TYB01-01) to inject the echinomycin organic phase solution into the aqueous phase at an injection rate of 0.03 ml/s. After the injection is completed, shear for 3 minutes. After shearing is completed, purified water is added to dilute the solution to reduce the acetone content in the micellar solution to less than 2% (v/w%). The diluted micellar solution was dialyzed using a dialysis bag (Soleba, molecular weight cutoff of 8000 daltons). The dialysate, that is, purified water, was replaced at regular intervals.
  • a 0.22 ⁇ m polyethersulfone filter membrane Millipore, syringe filter
  • DLS dynamic light scattering
  • a syringe pump (Baoding Leifu, TYB01-01) to inject the echinomycin organic phase solution into the aqueous phase at an injection rate of 0.03 ml/s. After the injection is completed, shear for 3 minutes. After shearing is completed, purified water is added to dilute the solution to reduce the acetone content in the micellar solution to less than 2% (v/w%). The diluted micellar solution was dialyzed using a dialysis bag (Soleba, molecular weight cutoff of 8000 daltons). The dialysate, that is, purified water, was replaced at regular intervals.
  • a 0.22 ⁇ m polyethersulfone filter membrane Millipore, syringe filter
  • DLS dynamic light scattering
  • echinomycin weigh 30 mg of echinomycin and 270 mg of methoxy polyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 5000, PDLLA segment weight average molecular weight 5000) and add it to the vial, then add 3 ml acetone Dissolve to obtain an organic phase solution of echinomycin.
  • the echinomycin organic phase solution and purified water were respectively injected into the T-shaped tee (inner diameter 0.5mm, Cole Parmer, HV-06365-77) through a syringe pump (Baoding Reif, TYB01-01).
  • micellar solution flows out from the outlet of the T-shaped tee.
  • purified water is added immediately to dilute it to reduce the acetone content in the micellar solution to 2 % (v/w%) or less.
  • the diluted micellar solution was dialyzed using a dialysis bag (Soleba, molecular weight cutoff of 8000 daltons). The dialysate, that is, purified water, was replaced at regular intervals.
  • a 0.22 ⁇ m polyethersulfone filter membrane Millipore, syringe filter
  • DLS dynamic light scattering
  • the echinomycin organic phase solution and purified water were respectively injected into the liquid inlet of the T-shaped tee (inner diameter 0.5mm, Cole Parmer, HV-06365-77) through a syringe pump (Baoding Leifer, TYB01-01), with a total flow rate of 30ml/min, the flow rate ratio is 1:12, the micellar solution flows out from the outlet of the T-shaped tee, and purified water is added immediately after preparation to dilute it to reduce the acetone content in the micellar solution to 2% (v/w%) the following.
  • micellar solution Use a hollow fiber ultrafiltration column (MidiKros) to ultrafiltrate the micellar solution, ultrafiltrate 6 times the volume and then concentrate to 1/3 of the original solution volume.
  • a 0.22 ⁇ m polyethersulfone filter membrane (Millipore, syringe filter) was used to filter the micellar solution, and dynamic light scattering (DLS) was used to detect the hydrated particle size of the micelles.
  • the average particle size is 60.1nm and PDI is 0.156.
  • the echinomycin organic phase solution and purified water were respectively injected into the liquid inlet of the T-shaped tee (inner diameter 0.5mm, Cole Parmer, HV-06365-77) through a syringe pump (Baoding Leifer, TYB01-01), with a total flow rate of 25ml/min, the flow rate ratio is 1:9, the micellar solution flows out from the outlet of the T-shaped tee, and purified water is added immediately after preparation to dilute it to reduce the acetone content in the micellar solution to 1.5% (v/w%) the following.
  • a hollow fiber ultrafiltration column (MidiKros) to ultrafiltrate the micellar solution, ultrafiltrate 8 times the volume and then concentrate to 1/10 of the volume of the original solution.
  • a 0.22 ⁇ m polyethersulfone filter membrane (Millipore, syringe filter) to filter the micellar solution, and use dynamic light scattering (DLS) to detect the micelle hydration particle size as described in Test Example 1.
  • the average particle size is 66.6nm and the PDI is 0.121.
  • the echinomycin content was measured as described in Test Example 2 and was 0.21 mg/ml.
  • the aqueous phase is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and deionized water, a total of 25ml
  • the aqueous phase is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and deionized water, a total of 25ml
  • organic phase After mixing at the ratio of water phase), it is sheared by a high-speed shearing machine (IKA, Germany, model: T25DS25) at a shear rate of 5000 rpm to mix the two phases to form colostrum.
  • the nanoemulsion formed after high-pressure homogenization of the colostrum by a microjet homogenizer (Nuoze Fluid Technology (Shanghai) Co., Ltd., model: Nano) at a pressure of 5000 psi was added to about 50 times the volume of 5°C deionized water, and then cold. It was then concentrated by ultrafiltration using a regenerated cellulose membrane (Hangzhou Cobetter Filtration Equipment Co., Ltd.) with a molecular weight cutoff (MWCO) of 300 to obtain a nanosuspension.
  • a microjet homogenizer Nuoze Fluid Technology (Shanghai) Co., Ltd., model: Nano
  • the nanosuspension was sterilized and filtered through a 0.22 ⁇ m polyethersulfone filter membrane (Millipore, syringe filter), then divided into vials and freeze-dried to obtain echinomycin nanomicelles.
  • echinomycin nanomicelles were reconstituted with 0.9% sodium chloride solution, dynamic light scattering (DLS) was used to detect the hydrated particle size of the micelles as described in Test Example 1.
  • the average particle size was 78.42 nm and the PDI was 0.043.
  • a pharmacokinetic test was carried out on the echinomycin nanomicelle freeze-dried powder prepared in this example, and the results are as shown in the following table:

Abstract

A micelle used for echinomycin antibiotic in vivo delivery. The micelle consists of an echinomycin antibiotic and an amphiphilic copolymer, wherein the echinomycin antibiotic is encapsulated in the micelle. Also provided are a preparation method for the micelle, and a use.

Description

棘霉素类抗生素胶束及其制备方法和用途Echinomycin antibiotic micelles and preparation methods and uses thereof 技术领域Technical field
本发明涉及医药技术领域，更具体而言涉及含棘霉素类抗生素和两亲性共聚物的胶束及其制备方法和用途。The present invention relates to the field of medical technology, and more specifically to micelles containing echinomycin antibiotics and amphiphilic copolymers and their preparation methods and uses.
背景技术Background technique
棘霉素类抗生素是一种喹喔啉类HIF1α(缺氧诱导因子1α亚基)抑制剂，具有竞争性抑制HIF1α与特定DNA序列结合的能力。棘霉素类抗生素水溶性差，体内半衰期短。临床上已经进行多个1期和2期试验来证明棘霉素类抗生素滞留实体瘤的能力，其中一些2期试验以1200μg/m2棘霉素进行给药，但是结果表明并没有显著的获益。一些临床试验研究所使用的棘霉素制剂包括聚氧乙烯蓖麻油，由此造成了明显的剂量限制性毒性。因此需要一种制剂来提高棘霉素类抗生素溶解度，降低棘霉素类抗生素毒性，提高棘霉素类抗生素疗效。CN108495619A使用薄膜水化法制备了棘霉素脂质体，但是其采用的制备工艺不利于产业化放大生产，载药量较低。Echinomycin antibiotics are quinoxaline HIF1α (hypoxia-inducible factor 1α subunit) inhibitors that competitively inhibit the ability of HIF1α to bind to specific DNA sequences. Echinomycin antibiotics have poor water solubility and short half-life in the body. Multiple phase 1 and phase 2 trials have been conducted clinically to demonstrate the ability of echinomycin antibiotics to retain solid tumors. Some of the phase 2 trials administered 1200 μg/m 2 echinomycin, but the results showed that there was no significant gain. beneficial. Some clinical trial studies have used echinomycin formulations that included polyoxyethylene castor oil, resulting in significant dose-limiting toxicities. Therefore, a preparation is needed to improve the solubility of echinomycin antibiotics, reduce the toxicity of echinomycin antibiotics, and improve the efficacy of echinomycin antibiotics. CN108495619A uses a thin film hydration method to prepare echinomycin liposomes, but the preparation process used is not conducive to industrial scale-up production and the drug loading capacity is low.
发明内容Contents of the invention
为了解决现有技术中存在的问题，本发明提供了一种新型棘霉素类抗生素胶束制剂，其具有较高的棘霉素类抗生素溶解度和较小的粒径，有助于改善棘霉素类抗生素的体内释放行为和组织分布，从而允许棘霉素类抗生素以较小的剂量实现较高的疗效，同时降低棘霉素类抗生素的毒性。此外，本发明还提供了一种制备新型棘霉素类抗生素胶束制剂的技术，该制备技术有利于产业化放大生产。In order to solve the problems existing in the prior art, the present invention provides a new type of echinomycin antibiotic micelle preparation, which has higher solubility of echinomycin antibiotics and smaller particle size, and helps to improve the condition of echinobacteriaceae. The in vivo release behavior and tissue distribution of echinomycin antibiotics allow echinomycin antibiotics to achieve higher efficacy at smaller doses while reducing the toxicity of echinomycin antibiotics. In addition, the present invention also provides a technology for preparing novel echinomycin antibiotic micellar preparations, which is conducive to industrial scale-up production.
一方面，本发明提供了一种胶束，其包含棘霉素类抗生素和一种或多种两亲性共聚物。In one aspect, the invention provides a micelle comprising an echinomycin antibiotic and one or more amphiphilic copolymers.
在本发明的一个或多个实施方式中，棘霉素类抗生素选自棘霉素(echinomycin)、放线菌白素(actinoleukin)、三骨菌素(triostin)、F-43、59266、6270和echinoserine等。In one or more embodiments of the invention, the echinomycin antibiotic is selected from the group consisting of echinomycin, actinoleukin, triostin, F-43, 59266, and 6270 and echinoserine etc.
在本发明的一个或多个实施方式中，棘霉素类抗生素为棘霉素。In one or more embodiments of the invention, the echinomycin antibiotic is echinomycin.
在本发明的一个或多个实施方式中，两亲性共聚物选自嵌段共聚物、接枝共聚物、树枝状大分子中的一种或多种。 In one or more embodiments of the present invention, the amphiphilic copolymer is selected from one or more of block copolymers, graft copolymers, and dendrimers.
在本发明的一个或多个实施方式中，两亲性共聚物为嵌段共聚物。In one or more embodiments of the invention, the amphiphilic copolymer is a block copolymer.
在本发明的一个或多个实施方式中，嵌段共聚物亲水段选自聚乙二醇、甲氧基聚乙二醇、聚丙烯酸、聚甲基丙烯酸、聚丙烯酸衍生物、聚甲基丙烯酸衍生物、聚氨基酸中的一种或多种，嵌段共聚物疏水段选自聚乳酸、丙交酯乙交酯共聚物、聚己内酯、聚碳酸酯、聚碳酸衍生物、聚丁酸酯、聚丙烯酸衍生物、聚甲基丙烯酸衍生物、聚氨基酸中的一种或多种。嵌段共聚物亲水段中的聚丙烯酸衍生物的实例包括但不限于聚丙烯酸羟乙酯、聚丙烯酸-N,N-二甲氨基乙酯、聚丙烯酸-N,N-二乙氨基乙酯、聚乙二醇二丙烯酸酯聚丙烯酸甘油酯等。嵌段共聚物亲水段中的聚甲基丙烯酸衍生物的实例包括但不限于聚甲基丙烯酸羟乙酯、聚甲基丙烯酸-N,N-二甲氨基甲酯、聚甲基丙烯酸-N,N-二乙氨基乙酯、聚乙二醇二甲基丙烯酸酯、聚甲基丙烯酸甘油酯和聚(2-甲基丙烯酰氧基乙基磷酸胆碱)等。嵌段共聚物疏水段中的聚碳酸衍生物的实例包括但不限于聚(5-甲基-2-氧代-1,3-二恶烷-5-羧酸乙酯)、聚(5-甲基-2-氧代-1,3-二恶烷-5-羧酸苄酯)、聚(5-甲基-2-氧代-1,3-二恶烷-5-羧酸丙酯)和聚(5-甲基-2-氧代-1,3-二恶烷-5-羧酸异丙酯)等。嵌段共聚物疏水段中的聚丙烯酸衍生物的实例包括但不限于聚丙烯酸甲酯、聚丙烯酸乙酯、聚丙烯酸丙酯、聚丙烯酸丁酯和聚丙烯酸苄酯等。嵌段共聚物疏水段中的聚甲基丙烯酸衍生物的实例包括但不限于聚甲基丙烯酸甲酯、聚甲基丙烯酸乙酯、聚甲基丙烯酸丙酯、聚甲基丙烯酸丁酯和聚甲基丙烯酸苄酯等。In one or more embodiments of the invention, the hydrophilic segment of the block copolymer is selected from polyethylene glycol, methoxypolyethylene glycol, polyacrylic acid, polymethacrylic acid, polyacrylic acid derivatives, polymethyl One or more of acrylic acid derivatives and polyamino acids, and the hydrophobic segment of the block copolymer is selected from polylactic acid, lactide-glycolide copolymer, polycaprolactone, polycarbonate, polycarbonate derivatives, polybutylene One or more of acid esters, polyacrylic acid derivatives, polymethacrylic acid derivatives, and polyamino acids. Examples of polyacrylic acid derivatives in the hydrophilic segment of the block copolymer include, but are not limited to, polyhydroxyethyl acrylate, polyacrylic acid-N,N-dimethylaminoethyl ester, polyacrylic acid-N,N-diethylaminoethyl ester , polyethylene glycol diacrylate, polyglyceryl acrylate, etc. Examples of polymethacrylic acid derivatives in the hydrophilic segment of the block copolymer include, but are not limited to, polyhydroxyethyl methacrylate, polymethacrylic acid-N,N-dimethylaminomethyl ester, polymethacrylic acid-N , N-diethylaminoethyl ester, polyethylene glycol dimethacrylate, polyglyceryl methacrylate and poly(2-methacryloyloxyethylphosphocholine), etc. Examples of polycarbonate derivatives in the hydrophobic segment of the block copolymer include, but are not limited to, poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylic acid ethyl ester), poly(5- Methyl-2-oxo-1,3-dioxane-5-carboxylic acid benzyl ester), poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylic acid propyl ester) ) and poly(5-methyl-2-oxo-1,3-dioxane-5-carboxylic acid isopropyl ester), etc. Examples of polyacrylic acid derivatives in the hydrophobic segment of the block copolymer include, but are not limited to, polymethyl acrylate, polyethyl acrylate, polypropyl acrylate, polybutyl acrylate, polybenzyl acrylate, and the like. Examples of polymethacrylic acid derivatives in the hydrophobic segment of the block copolymer include, but are not limited to, polymethyl methacrylate, polyethyl methacrylate, polypropyl methacrylate, polybutyl methacrylate, and polymethacrylate. Benzyl acrylate, etc.
在本发明的一个或多个实施方式中，所述两亲性共聚物为甲氧基聚乙二醇-聚乳酸嵌段共聚物。In one or more embodiments of the invention, the amphiphilic copolymer is a methoxy polyethylene glycol-polylactic acid block copolymer.
在本发明的一个或多个实施方式中，嵌段共聚物亲水段重均分子量为100Da-50000Da。在一个或多个实施方式中，嵌段共聚物亲水段重均分子量为1000Da-10000Da。在一个或多个实施方式中，嵌段共聚物亲水段重均分子量为2000Da-5000Da。In one or more embodiments of the present invention, the weight average molecular weight of the hydrophilic segment of the block copolymer ranges from 100 Da to 50,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophilic segment of the block copolymer ranges from 1,000 Da to 10,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophilic segment of the block copolymer ranges from 2000 Da to 5000 Da.
在本发明的一个或多个实施方式中，嵌段共聚物疏水段重均分子量为500Da-100000Da。在一个或多个实施方式中，嵌段共聚物疏水段重均分子量为2000Da-50000Da。在一个或多个实施方式中，嵌段共聚物疏水段重均分子量为5000Da-25000Da。In one or more embodiments of the present invention, the weight average molecular weight of the hydrophobic segment of the block copolymer ranges from 500 Da to 100,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophobic segment of the block copolymer ranges from 2,000 Da to 50,000 Da. In one or more embodiments, the weight average molecular weight of the hydrophobic segment of the block copolymer ranges from 5000 Da to 25000 Da.
在本发明的一个或多个实施方式中，所述两亲性共聚物为甲氧基聚(乙二醇)-b-聚(D,L-丙交酯)(mPEG-PDLLA)，其中亲水段mPEG段的重均分子量为2000Da-5000Da，疏水段PDLLA段的重均分子量为5000Da-25000Da。 In one or more embodiments of the invention, the amphiphilic copolymer is methoxypoly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA), wherein the amphiphilic copolymer is The weight average molecular weight of the water segment mPEG segment is 2000Da-5000Da, and the weight average molecular weight of the hydrophobic segment PDLLA segment is 5000Da-25000Da.
嵌段共聚物亲水段和疏水段的重均分子量影响胶束制剂的释放、体内和体外稳定性和体内组织分布。The weight average molecular weight of the hydrophilic and hydrophobic segments of the block copolymer affects the release, in vivo and in vitro stability, and in vivo tissue distribution of the micelle preparation.
在本发明的一个或多个实施方式中，嵌段共聚物的多分散指数小于2.0。In one or more embodiments of the invention, the block copolymer has a polydispersity index of less than 2.0.
在本发明的一个或多个实施方式中，所述胶束中两亲性共聚物与棘霉素类抗生素的比率范围按重量比为约0.1:1到200:1，例如是0.1:1、0.5:1、1:1、2:1、3:1、5:1、6:1、8:1、9:1、10:1、12:1、15:1、18:1、20:1、25:1、30:1、35:1、40:1、45:1、50:1、55:1、60:1、65:1、70:1、75:1、80:1、85:1、90:1、95:1、100:1、110:1、120:1、130:1、140:1、150:1、160:1、170:1、180:1、190:1或200:1，或其间的任意范围。在一个或多个实施方式中，所述胶束中两亲性共聚物与棘霉素类抗生素的比率范围按重量比为0.5:1到100:1。在一个或多个实施方式中，所述胶束中两亲性共聚物与棘霉素类抗生素的比率范围按重量比为1:1到100:1。在一个或多个实施方式中，所述胶束中两亲性共聚物与棘霉素类抗生素的比率范围按重量比为6:1到25:1。在一个或多个实施方式中，所述胶束中两亲性共聚物与棘霉素类抗生素的比率范围按重量比为8:1到20:1。在一个或多个实施方式中，所述胶束中两亲性共聚物与棘霉素类抗生素的比率按重量比为9:1。胶束中两亲性共聚物与棘霉素类抗生素的重量比与胶束制剂的稳定性和释放有关。In one or more embodiments of the present invention, the ratio of the amphiphilic copolymer to the echinomycin antibiotic in the micelle ranges from about 0.1:1 to 200:1 by weight, such as 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 5:1, 6:1, 8:1, 9:1, 10:1, 12:1, 15:1, 18:1, 20: 1. 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190: 1 or 200:1, or any range in between. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 0.5:1 to 100:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 1:1 to 100:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 6:1 to 25:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle ranges from 8:1 to 20:1 by weight. In one or more embodiments, the ratio of amphiphilic copolymer to echinomycin antibiotic in the micelle is 9:1 by weight. The weight ratio of amphiphilic copolymer to echinomycin antibiotics in micelles is related to the stability and release of micelle preparations.
在本发明的一个或多个实施方式中，所述胶束还包含pH调节剂、张力调节剂、抗氧化剂、防腐剂、缓冲剂、金属螯合剂、惰性气体中的一种或多种。在一个或多个实施方式中，所述pH调节剂选自氢氧化钠、氢氧化钾、氢氧化镁、碳酸钠、Tris、亚油酸钠、油酸钠、碳酸钾、亚油酸钾、油酸钾、乳酸中的一种或多种。在一个或多个实施方式中，所述张力调节剂选自去氧胆酸钠、氯化钠、甘油、山梨醇、木糖醇、甘露醇、葡萄糖、海藻糖、麦芽糖、蔗糖、棉子糖、乳糖、葡聚糖、聚乙二醇和丙二醇中的一种或多种。在一个或多个实施方式中，所述张力调节剂为去氧胆酸钠。在一个或多个实施方式中，所述防腐剂选自羟苯烷基酯类、苯甲酸、苯甲酸钠、山梨酸、醋酸氯乙定、苯扎溴铵中的一种或多种。在一个或多个实施方式中，所述抗氧化剂选自亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、抗坏血酸、叔丁基对羟基茴香醚、2,6-二叔丁基化羟基甲苯、维生素E、抗坏血酸棕榈酸酯中的一种或多种。在一个或多个实施方式中，所述缓冲剂选自磷酸盐缓冲液、柠檬酸盐缓冲液、Tris缓冲液、碳酸盐缓冲液、琥珀酸盐缓冲液、马来酸盐缓冲液和硼酸盐缓冲液的一种或多种。在一个或多个实施方式中，所述金属螯合剂选自乙二胺四乙酸、乙二胺四乙酸二钠和乙二胺四乙酸钙钠中的一种或多种。在一个或多个实施方式中，所述惰性气体选自氮气和氩气中的一种或两种。 In one or more embodiments of the present invention, the micelles further include one or more of a pH adjuster, a tension adjuster, an antioxidant, a preservative, a buffer, a metal chelating agent, and an inert gas. In one or more embodiments, the pH adjuster is selected from sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium carbonate, Tris, sodium linoleate, sodium oleate, potassium carbonate, potassium linoleate, One or more of potassium oleate and lactic acid. In one or more embodiments, the tonicity adjusting agent is selected from sodium deoxycholate, sodium chloride, glycerin, sorbitol, xylitol, mannitol, glucose, trehalose, maltose, sucrose, raffinose , one or more of lactose, dextran, polyethylene glycol and propylene glycol. In one or more embodiments, the tonicity adjusting agent is sodium deoxycholate. In one or more embodiments, the preservative is selected from one or more of hydroxyphenyl alkyl esters, benzoic acid, sodium benzoate, sorbic acid, chlorethidine acetate, and benzalkonium bromide. In one or more embodiments, the antioxidant is selected from the group consisting of sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, ascorbic acid, tert-butyl-p-hydroxyanisole, 2,6-di-tert-butylated hydroxyl One or more of toluene, vitamin E, and ascorbyl palmitate. In one or more embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, Tris buffer, carbonate buffer, succinate buffer, maleate buffer, and boron buffer. One or more salt buffers. In one or more embodiments, the metal chelating agent is selected from one or more of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, and calcium sodium ethylenediaminetetraacetate. In one or more embodiments, the inert gas is selected from one or both of nitrogen and argon.
在本发明的一个或多个实施方式中，胶束中棘霉素类抗生素的浓度为0.1mg/ml-20mg/ml，例如为0.2mg/ml、0.5mg/ml、1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、8mg/ml、10mg/ml、12mg/ml、15mg/ml、18mg/ml或20mg/ml。这样的浓度有利于临床使用和生产。In one or more embodiments of the present invention, the concentration of echinomycin antibiotics in the micelles is 0.1 mg/ml-20 mg/ml, such as 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml. ml, 3mg/ml, 4mg/ml, 5mg/ml, 8mg/ml, 10mg/ml, 12mg/ml, 15mg/ml, 18mg/ml or 20mg/ml. Such concentrations are beneficial for clinical use and production.
在本发明的一个或多个实施方式中，胶束的粒径为10nm-150nm。这样的粒径利于载药胶束产生渗透滞留增强效应(EPR效应)。In one or more embodiments of the invention, the particle size of the micelles is 10 nm-150 nm. Such a particle size is conducive to the enhanced penetration and retention effect (EPR effect) of drug-loaded micelles.
在本发明的一个或多个实施方式中，胶束的多分散指数(PDI)为小于0.3，例如小于0.2。PDI表征颗粒粒径的大小分布，数值越小，表明粒径大小分布越均匀。In one or more embodiments of the invention, the micelles have a polydispersity index (PDI) of less than 0.3, such as less than 0.2. PDI characterizes the size distribution of particle size. The smaller the value, the more uniform the particle size distribution.
在本发明的一个或多个实施方式中，胶束为液体或冻干粉形式。In one or more embodiments of the invention, the micelles are in liquid or lyophilized powder form.
在本发明的一个或多个实施方式中，胶束为液体形式，其含有水，例如去离子水或纯化水。In one or more embodiments of the invention, the micelles are in liquid form, containing water, such as deionized or purified water.
在本发明的一个或多个实施方式中，胶束为冻干粉形式，其不含有冻干保护剂。在一个或多个实施方式中，胶束为冻干粉形式，其含有冻干保护剂。在一个或多个实施方式中，所述冻干保护剂选自甘露醇、葡萄糖、蔗糖、麦芽糖、乳糖、甘露糖、海藻糖、甘氨酸、赖氨酸、脯氨酸、精氨酸、羟丙基-β-环糊精、右旋糖酐、聚乙二醇、聚乙烯醇中的一种或多种。In one or more embodiments of the invention, the micelles are in the form of a lyophilized powder that does not contain a lyoprotectant. In one or more embodiments, the micelles are in the form of a lyophilized powder containing a lyoprotectant. In one or more embodiments, the lyoprotectant is selected from the group consisting of mannitol, glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, lysine, proline, arginine, hydroxypropyl One or more of β-cyclodextrin, dextran, polyethylene glycol, and polyvinyl alcohol.
在本发明的一个或多个实施方式中，液体形式的胶束包含棘霉素、mPEG-PDLLA、去氧胆酸钠以及纯化水或由其组成。In one or more embodiments of the invention, the micelles in liquid form comprise or consist of echinomycin, mPEG-PDLLA, sodium deoxycholate, and purified water.
在本发明的一个或多个实施方式中，冻干粉形式的胶束包含棘霉素、mPEG-PDLLA以及去氧胆酸钠或由其组成。In one or more embodiments of the present invention, micelles in the form of lyophilized powder comprise or consist of echinomycin, mPEG-PDLLA and sodium deoxycholate.
另一方面，本发明提供了一种制备本发明胶束的方法。在本发明的一个或多个实施方式中，胶束制备方法选自直接分散法、固相分散法、有机溶剂注入法、乳化法、膜乳化法、微流控法、超声法、均质法中的一种或多种。In another aspect, the present invention provides a method for preparing micelles of the present invention. In one or more embodiments of the present invention, the micelle preparation method is selected from the group consisting of direct dispersion method, solid phase dispersion method, organic solvent injection method, emulsification method, membrane emulsification method, microfluidic method, ultrasonic method, and homogenization method. one or more of them.
在本发明的一个或多个实施方式中，胶束制备方法选自有机溶剂注入法、微流控法和乳化法。In one or more embodiments of the present invention, the micelle preparation method is selected from the group consisting of organic solvent injection method, microfluidic method and emulsification method.
在本发明的一个或多个实施方式中，胶束制备方法为有机溶剂注入法。In one or more embodiments of the present invention, the micelle preparation method is an organic solvent injection method.
在本发明的一个或多个实施方式中，有机溶剂注入法包括以下步骤：In one or more embodiments of the present invention, the organic solvent injection method includes the following steps:
(1)将棘霉素类抗生素、两亲性共聚物和有机溶剂混合均匀，得到含两亲性共聚物的棘霉素类抗生素有机溶液；(1) Mix the echinomycin antibiotics, the amphiphilic copolymer and the organic solvent evenly to obtain an echinomycin antibiotic organic solution containing the amphiphilic copolymer;
(2)将步骤(1)得到的含两亲性共聚物的棘霉素类抗生素有机溶液注入搅拌着的水 相溶液中得到含有机溶剂的载药胶束溶液；(2) Pour the organic solution of echinomycin antibiotics containing the amphiphilic copolymer obtained in step (1) into the stirring water. The drug-loaded micelle solution containing organic solvent is obtained from the phase solution;
(3)从步骤(2)中得到的载药胶束溶液去除有机溶剂，得到药物胶束；(3) Remove the organic solvent from the drug-loaded micelle solution obtained in step (2) to obtain drug micelles;
(4)对步骤(3)中获得的药物胶束进行除菌。(4) Sterilize the drug micelles obtained in step (3).
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(1)中，所述有机溶剂选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、甲醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或多种。In one or more embodiments of the present invention, in step (1) of the above organic solvent injection method, the organic solvent is selected from the group consisting of chloroform, dichloromethane, tert-butyl alcohol, isopropyl alcohol, ethyl acetate, ethanol , one or more of methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(2)中，所述水相溶液为纯化水、缓冲液、单糖溶液、二糖溶液、多糖溶液、电解质溶液、氨基酸溶液、表面活性剂溶液中的一种或多种。在一个或多个实施方式中，所述缓冲液选自磷酸盐缓冲液、醋酸盐缓冲液、Tris缓冲液、HEPES缓冲液、枸橼酸缓冲液、琥珀酸缓冲液、碳酸氢钠缓冲液等。在一个或多个实施方式中，所述单糖溶液选自果糖、葡萄糖、半乳糖、甘露醇、山梨醇、木糖醇等溶液。在一个或多个实施方式中，所述二糖溶液选自蔗糖、乳糖、海藻糖、麦芽糖等溶液。在一个或多个实施方式中，所述多糖溶液选自右旋糖酐、海藻酸、透明质酸等溶液。在一个或多个实施方式中，所述电解质溶液选自氯化钠、氯化钙、氯化钾、氯化镁、硫酸铜、葡萄糖酸铜、葡萄糖酸钙、葡萄糖酸锌、乳酸钠等溶液。在一个或多个实施方式中，所述氨基酸溶液选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸(蛋氨酸)、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天门冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸等溶液。在一个或多个实施方式中，所述表面活性剂溶液选自吐温80、吐温20、泊洛沙姆188、聚乙二醇15-羟基硬脂酸脂(HS15)等溶液。In one or more embodiments of the present invention, in step (2) of the above organic solvent injection method, the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution and surfactant solution. In one or more embodiments, the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait. In one or more embodiments, the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions. In one or more embodiments, the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions. In one or more embodiments, the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions. In one or more embodiments, the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions. In one or more embodiments, the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions . In one or more embodiments, the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, polyethylene glycol 15-hydroxystearate (HS15) and other solutions.
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(2)中，所述水相溶液为含0.1％-2％的去氧胆酸钠、1％-10％乙酸乙酯、0.5％-5％苯甲醇的水溶液。在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(2)中，所述水相溶液由0.25％去氧胆酸钠、4％乙酸乙酯、2％苯甲醇及去离子水或净化水组成。In one or more embodiments of the present invention, in step (2) of the above organic solvent injection method, the aqueous solution contains 0.1%-2% sodium deoxycholate, 1%-10% acetic acid Ethyl ester, 0.5%-5% benzyl alcohol aqueous solution. In one or more embodiments of the present invention, in step (2) of the above organic solvent injection method, the aqueous solution is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and Composed of deionized or purified water.
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(2)中，根据工艺目的，必要时可以对获得的载药胶束溶液进行一定程度的稀释。In one or more embodiments of the present invention, in step (2) of the above-mentioned organic solvent injection method, the obtained drug-loaded micelle solution can be diluted to a certain extent if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(3)中，去除有机溶剂的方式选自挥发、透析和超滤中的一种或多种。In one or more embodiments of the present invention, in step (3) of the above-mentioned organic solvent injection method, the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(3)中，根据工艺目的，必要时可以对去除有机溶剂后的胶束溶液进行浓缩。 In one or more embodiments of the present invention, in step (3) of the above-mentioned organic solvent injection method, the micelle solution after removing the organic solvent may be concentrated if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述有机溶剂注入法的步骤(4)中，通过过滤方式进行除菌。In one or more embodiments of the present invention, in step (4) of the above-mentioned organic solvent injection method, sterilization is performed by filtration.
在本发明的一个或多个实施方式中，胶束制备方法为微流控法。In one or more embodiments of the invention, the micelle preparation method is a microfluidic method.
在本发明的一个或多个实施方式中，所述微流控法包括以下步骤：In one or more embodiments of the invention, the microfluidic method includes the following steps:
(1)将棘霉素类抗生素、两亲性共聚物和有机溶剂混合均匀，得到含两亲性共聚物的棘霉素类抗生素有机溶液；(1) Mix the echinomycin antibiotics, the amphiphilic copolymer and the organic solvent evenly to obtain an echinomycin antibiotic organic solution containing the amphiphilic copolymer;
(2)将步骤(1)得到的含两亲性共聚物的棘霉素类抗生素有机溶液和水相溶液由不同的进液口同时注入微混合器中得到含有机溶剂的载药胶束溶液；(2) Inject the organic solution of echinomycin antibiotics containing the amphiphilic copolymer and the aqueous phase solution obtained in step (1) into the micromixer simultaneously from different liquid inlets to obtain a drug-loaded micellar solution containing an organic solvent. ;
(3)从步骤(2)中得到的棘霉素类抗生素载药胶束溶液去除有机溶剂，得到药物胶束；(3) Remove the organic solvent from the echinomycin antibiotic drug-loaded micelle solution obtained in step (2) to obtain drug micelles;
(4)对步骤(3)中获得的药物胶束进行除菌。(4) Sterilize the drug micelles obtained in step (3).
在本发明的一个或多个实施方式中，在上述微流控法的步骤(1)中，所述有机溶剂选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、甲醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或多种。In one or more embodiments of the present invention, in step (1) of the above-mentioned microfluidic method, the organic solvent is selected from chloroform, dichloromethane, tert-butyl alcohol, isopropyl alcohol, ethyl acetate, ethanol , one or more of methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(2)中，所述水相溶液为纯化水、缓冲液、单糖溶液、二糖溶液、多糖溶液、电解质溶液、氨基酸溶液、表面活性剂溶液、含低浓度有机溶剂的水溶液中的一种或多种。在一个或多个实施方式中，所述缓冲液选自磷酸盐缓冲液、醋酸盐缓冲液、Tris缓冲液、HEPES缓冲液、枸橼酸缓冲液、琥珀酸缓冲液、碳酸氢钠缓冲液等。在一个或多个实施方式中，所述单糖溶液选自果糖、葡萄糖、半乳糖、甘露醇、山梨醇、木糖醇等溶液。在一个或多个实施方式中，所述二糖溶液选自蔗糖、乳糖、海藻糖、麦芽糖等溶液。在一个或多个实施方式中，所述多糖溶液选自右旋糖酐、海藻酸、透明质酸等溶液。在一个或多个实施方式中，所述电解质溶液选自氯化钠、氯化钙、氯化钾、氯化镁、硫酸铜、葡萄糖酸铜、葡萄糖酸钙、葡萄糖酸锌、乳酸钠等溶液。在一个或多个实施方式中，所述氨基酸溶液选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸(蛋氨酸)、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天门冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸等溶液。在一个或多个实施方式中，所述表面活性剂溶液选自吐温80、吐温20、泊洛沙姆188、HS15等溶液。在一个或多个实施方式中，所述含低浓度有机溶剂的水溶液选自有机溶剂浓度范围为0.1％-10％的乙醇、甲醇、丙酮、四氢呋喃、二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)等水溶液。 In one or more embodiments of the present invention, in step (2) of the above-mentioned microfluidic method, the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution, surfactant solution, aqueous solution containing low concentration organic solvent. In one or more embodiments, the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait. In one or more embodiments, the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions. In one or more embodiments, the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions. In one or more embodiments, the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions. In one or more embodiments, the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions. In one or more embodiments, the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions . In one or more embodiments, the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, HS15 and other solutions. In one or more embodiments, the aqueous solution containing a low concentration of organic solvent is selected from ethanol, methanol, acetone, tetrahydrofuran, dimethylformamide (DMF), dimethyl, etc. with an organic solvent concentration ranging from 0.1% to 10%. Aqueous solutions such as methyl sulfoxide (DMSO).
在本发明的一个或多个实施方式中，在上述微流控法的步骤(2)中，通过压缩空气或泵实现各相溶液由不同的进液口同时注入微混合器。在一个或多个实施方式中，各相溶液的总流速为1ml/min-1L/min，例如为1ml/min、2ml/min、3ml/min、5ml/min、10ml/min、15ml/min、20ml/min、25ml/min、30ml/min、50ml/min、80ml/min、100ml/min、150ml/min、200ml/min、300ml/min、400ml/min、500ml/min、600ml/min、800ml/min或1L/min，或其间任意的范围。在一个或多个实施方式中，各相溶液的总流速为20ml/min至50ml/min。在一个或多个实施方式中，各相溶液的总流速为25ml/min至30ml/min。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:1-1:300，例如为1:1、1:2、1:3、1:5、1:6、1:8、1:9、1:10、1:12、1:15、1:18、1:20、1:30、1:50、1:80、1:100、1:150、1:200、1:250或1:300，或其间任意的范围。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:8-1:15。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:9-1:12。在一个或多个实施方式中，所述微混合器可以选自T型三通、x型混合器和多进口涡流混合器(MIVM)。在一个或多个实施方式中，所述微混合器为T型三通。在一个或多个实施方式中，所述微混合器的进口和出口内径分别可以为0.1mm-5mm，二者可以相同，也可以不同。在一个或多个实施方式中，所述微混合器的材质可以选自聚醚醚酮(PEEK)、不锈钢、聚四氟乙烯(PTFE)等。In one or more embodiments of the present invention, in step (2) of the above-mentioned microfluidic method, each phase solution is simultaneously injected into the micromixer from different liquid inlets through compressed air or a pump. In one or more embodiments, the total flow rate of each phase solution is 1ml/min-1L/min, such as 1ml/min, 2ml/min, 3ml/min, 5ml/min, 10ml/min, 15ml/min, 20ml/min, 25ml/min, 30ml/min, 50ml/min, 80ml/min, 100ml/min, 150ml/min, 200ml/min, 300ml/min, 400ml/min, 500ml/min, 600ml/min, 800ml/ min or 1L/min, or any range in between. In one or more embodiments, the total flow rate of each phase solution is 20 ml/min to 50 ml/min. In one or more embodiments, the total flow rate of each phase solution is 25 ml/min to 30 ml/min. In one or more embodiments, the flow rate ratio of the organic phase solution and the aqueous phase solution is 1:1-1:300, such as 1:1, 1:2, 1:3, 1:5, 1:6, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1:150, 1: 200, 1:250 or 1:300, or any range in between. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:8-1:15. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:9-1:12. In one or more embodiments, the micromixer may be selected from a T-type tee, an x-type mixer, and a multi-inlet vortex mixer (MIVM). In one or more embodiments, the micromixer is a T-shaped tee. In one or more embodiments, the inner diameters of the inlet and outlet of the micromixer can be 0.1mm-5mm respectively, and they can be the same or different. In one or more embodiments, the material of the micromixer can be selected from polyetheretherketone (PEEK), stainless steel, polytetrafluoroethylene (PTFE), etc.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(2)中，根据工艺目的，必要时可以对获得的含有机溶剂的载药胶束溶液进行一定程度的稀释。In one or more embodiments of the present invention, in step (2) of the above-mentioned microfluidic method, the obtained drug-loaded micelle solution containing an organic solvent can be diluted to a certain extent if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，通过选自挥发、透析和超滤中的一种或多种方式去除有机溶剂。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，根据工艺目的，必要时可以对去除有机溶剂后的胶束溶液进行浓缩。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, the micellar solution after removing the organic solvent can be concentrated if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(4)中，通过过滤方式进行除菌。In one or more embodiments of the present invention, in step (4) of the above-mentioned microfluidic method, sterilization is performed by filtration.
在本发明的一个或多个实施方式中，所述微流控法包括以下步骤：In one or more embodiments of the invention, the microfluidic method includes the following steps:
(1)将棘霉素类抗生素和有机溶剂混合均匀，得到棘霉素类抗生素有机溶液；(1) Mix the echinomycin antibiotics and the organic solvent evenly to obtain an echinomycin antibiotic organic solution;
(2)将两亲性共聚物和有机溶剂混合均匀，得到聚合物有机溶液；(2) Mix the amphiphilic copolymer and organic solvent evenly to obtain a polymer organic solution;
(3)将步骤(1)得到的棘霉素类抗生素有机溶液、步骤(2)得到的聚合物有机溶液和水相溶液由不同的进液口同时注入微混合器中得到含有机溶剂的载药胶束溶液；(3) Inject the echinomycin antibiotic organic solution obtained in step (1), the polymer organic solution and the aqueous phase solution obtained in step (2) simultaneously into the micromixer through different liquid inlets to obtain a carrier containing organic solvents. Pharmaceutical micelle solution;
(4)从步骤(3)中得到的载药胶束溶液去除有机溶剂，得到药物胶束； (4) Remove the organic solvent from the drug-loaded micelle solution obtained in step (3) to obtain drug micelles;
(5)对步骤(4)中获得的药物胶束进行除菌。(5) Sterilize the drug micelles obtained in step (4).
在本发明的一个或多个实施方式中，在上述微流控法的步骤(1)中，所述有机溶剂用于溶解棘霉素类抗生物，其可以选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、甲醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或多种。In one or more embodiments of the present invention, in step (1) of the above-mentioned microfluidic method, the organic solvent is used to dissolve echinomycin antibiotics, which can be selected from chloroform, dichloromethane, tert. One or more of butanol, isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(2)中，所述有机溶剂用于溶解两亲性共聚物，其可以选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、甲醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或多种。In one or more embodiments of the present invention, in step (2) of the above-mentioned microfluidic method, the organic solvent is used to dissolve the amphiphilic copolymer, which can be selected from chloroform, dichloromethane, tert-butyl One or more of alcohol, isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
在本发明的一个或多个实施方式中，步骤(1)中用于溶解棘霉素类抗生物的有机溶剂与步骤(2)中用于溶解两亲性共聚物的有机溶剂可以相同，也可以不同。In one or more embodiments of the present invention, the organic solvent used to dissolve echinomycin antibiotics in step (1) and the organic solvent used to dissolve the amphiphilic copolymer in step (2) can be the same, or Can be different.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，所述水相溶液为纯化水、缓冲液、单糖溶液、二糖溶液、多糖溶液、电解质溶液、氨基酸溶液、表面活性剂溶液、含低浓度有机溶剂的水溶液中的一种或多种。在一个或多个实施方式中，所述缓冲液选自磷酸盐缓冲液、醋酸盐缓冲液、Tris缓冲液、HEPES缓冲液、枸橼酸缓冲液、琥珀酸缓冲液、碳酸氢钠缓冲液等。在一个或多个实施方式中，所述单糖溶液选自果糖、葡萄糖、半乳糖、甘露醇、山梨醇、木糖醇等溶液。在一个或多个实施方式中，所述二糖溶液选自蔗糖、乳糖、海藻糖、麦芽糖等溶液。在一个或多个实施方式中，所述多糖溶液选自右旋糖酐、海藻酸、透明质酸等溶液。在一个或多个实施方式中，所述电解质溶液选自氯化钠、氯化钙、氯化钾、氯化镁、硫酸铜、葡萄糖酸铜、葡萄糖酸钙、葡萄糖酸锌、乳酸钠等溶液。在一个或多个实施方式中，所述氨基酸溶液选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸(蛋氨酸)、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天门冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸等溶液。在一个或多个实施方式中，所述表面活性剂溶液选自吐温80、吐温20、泊洛沙姆188、HS15等溶液。在一个或多个实施方式中，所述含低浓度有机溶剂的水溶液选自有机溶剂浓度范围为0.1％-10％的乙醇、甲醇、丙酮、四氢呋喃、DMF、DMSO等水溶液。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution, surfactant solution, aqueous solution containing low concentration organic solvent. In one or more embodiments, the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait. In one or more embodiments, the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions. In one or more embodiments, the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions. In one or more embodiments, the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions. In one or more embodiments, the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions. In one or more embodiments, the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions . In one or more embodiments, the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, HS15 and other solutions. In one or more embodiments, the aqueous solution containing a low-concentration organic solvent is selected from aqueous solutions such as ethanol, methanol, acetone, tetrahydrofuran, DMF, DMSO, etc. with an organic solvent concentration ranging from 0.1% to 10%.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，通过压缩空气或泵实现各相溶液由不同的进液口同时注入微混合器。在一个或多个实施方式中，各相溶液的总流速为1ml/min-1L/min，例如为1ml/min、2ml/min、3ml/min、5ml/min、10ml/min、 15ml/min、20ml/min、25ml/min、30ml/min、50ml/min、80ml/min、100ml/min、150ml/min、200ml/min、300ml/min、400ml/min、500ml/min、600ml/min、800ml/min或1L/min，或其间任意的范围。在一个或多个实施方式中，各相溶液的总流速为20ml/min至50ml/min。在一个或多个实施方式中，各相溶液的总流速为25ml/min至30ml/min。在一个或多个实施方式中，总有机相溶液与水相溶液的流速比为1:1-1:300，例如为1:1、1:2、1:3、1:5、1:6、1:8、1:9、1:10、1:12、1:15、1:18、1:20、1:30、1:50、1:80、1:100、1:150、1:200、1:250或1:300，或其间任意的范围。在一个或多个实施方式中，棘霉素类抗生素有机溶液与聚合物有机溶液的流速比为1:1-1:300，例如为1:1、1:2、1:3、1:5、1:6、1:8、1:9、1:10、1:12、1:15、1:18、1:20、1:30、1:50、1:80、1:100、1:150、1:200、1:250或1:300，或其间任意的范围。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:8-1:15。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:9-1:12。在一个或多个实施方式中，所述微混合器可以是x型混合器和多进口涡流混合器(MIVM)。在一个或多个实施方式中，所述微混合器的进口和出口内径分别可以为0.1mm-5mm，二者可以相同，也可以不同。在一个或多个实施方式中，所述微混合器的材质可以选自聚醚醚酮(PEEK)、不锈钢、聚四氟乙烯(PTFE)等。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, each phase solution is simultaneously injected into the micromixer from different liquid inlets through compressed air or a pump. In one or more embodiments, the total flow rate of each phase solution is 1ml/min-1L/min, such as 1ml/min, 2ml/min, 3ml/min, 5ml/min, 10ml/min, 15ml/min, 20ml/min, 25ml/min, 30ml/min, 50ml/min, 80ml/min, 100ml/min, 150ml/min, 200ml/min, 300ml/min, 400ml/min, 500ml/min, 600ml/ min, 800ml/min or 1L/min, or any range in between. In one or more embodiments, the total flow rate of each phase solution is 20 ml/min to 50 ml/min. In one or more embodiments, the total flow rate of each phase solution is 25 ml/min to 30 ml/min. In one or more embodiments, the flow rate ratio of the total organic phase solution to the aqueous phase solution is 1:1-1:300, such as 1:1, 1:2, 1:3, 1:5, 1:6 , 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1:150, 1 :200, 1:250, or 1:300, or any range in between. In one or more embodiments, the flow rate ratio of the echinomycin antibiotic organic solution and the polymer organic solution is 1:1-1:300, for example, 1:1, 1:2, 1:3, 1:5 , 1:6, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1 :150, 1:200, 1:250 or 1:300, or any range in between. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:8-1:15. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:9-1:12. In one or more embodiments, the micromixer may be an x-type mixer and a multi-inlet vortex mixer (MIVM). In one or more embodiments, the inner diameters of the inlet and outlet of the micromixer can be 0.1mm-5mm respectively, and they can be the same or different. In one or more embodiments, the material of the micromixer can be selected from polyetheretherketone (PEEK), stainless steel, polytetrafluoroethylene (PTFE), etc.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，根据工艺目的，必要时可以对获得的含有机溶剂的载药胶束溶液进行一定程度的稀释。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, the obtained drug-loaded micelle solution containing an organic solvent can be diluted to a certain extent if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(4)中，通过选自挥发、透析和超滤中的一种或多种方式去除有机溶剂。In one or more embodiments of the present invention, in step (4) of the above-mentioned microfluidic method, the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(4)中，根据工艺目的，必要时可以对去除有机溶剂后的胶束溶液进行浓缩。In one or more embodiments of the present invention, in step (4) of the above-mentioned microfluidic method, the micelle solution after removing the organic solvent can be concentrated if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(5)中，通过过滤方式进行除菌。In one or more embodiments of the present invention, in step (5) of the above-mentioned microfluidic method, sterilization is performed by filtration.
在本发明的一个或多个实施方式中，所述微流控法包括以下步骤：In one or more embodiments of the invention, the microfluidic method includes the following steps:
(1)将棘霉素类抗生素和有机溶剂混合均匀，得到棘霉素类抗生素有机溶液；(1) Mix the echinomycin antibiotics and the organic solvent evenly to obtain an echinomycin antibiotic organic solution;
(2)将两亲性共聚物和水相溶液混合均匀，得到聚合物水相溶液；(2) Mix the amphiphilic copolymer and the aqueous phase solution evenly to obtain the polymer aqueous phase solution;
(3)将步骤(1)得到的棘霉素类抗生素有机溶液和步骤(2)得到的聚合物水相溶液由不同的进液口同时注入微混合器中得到含有机溶剂的载药胶束溶液；(3) Inject the echinomycin antibiotic organic solution obtained in step (1) and the polymer aqueous phase solution obtained in step (2) simultaneously into the micromixer through different liquid inlets to obtain drug-loaded micelles containing organic solvents. solution;
(4)从步骤(3)中得到的棘霉素类抗生素载药胶束溶液去除有机溶剂，得到药物胶束； (4) Remove the organic solvent from the echinomycin antibiotic drug-loaded micelle solution obtained in step (3) to obtain drug micelles;
(5)对步骤(4)中得到的药物胶束进行除菌。(5) Sterilize the drug micelles obtained in step (4).
在本发明的一个或多个实施方式中，在上述微流控法的步骤(1)中，所述有机溶剂用于溶解棘霉素类抗生物，其可以选自氯仿、二氯甲烷、叔丁醇、异丙醇、乙酸乙酯、乙醇、甲醇、四氢呋喃、二氧六环、乙腈、丙酮、二甲基亚砜、二甲基甲酰胺、甲基吡咯烷酮中的一种或多种。In one or more embodiments of the present invention, in step (1) of the above-mentioned microfluidic method, the organic solvent is used to dissolve echinomycin antibiotics, which can be selected from chloroform, dichloromethane, tert. One or more of butanol, isopropyl alcohol, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(2)中，所述水相溶液为纯化水、缓冲液、单糖溶液、二糖溶液、多糖溶液、电解质溶液、氨基酸溶液、表面活性剂溶液、含低浓度有机溶剂的水溶液中的一种或多种。在一个或多个实施方式中，所述缓冲液选自磷酸盐缓冲液、醋酸盐缓冲液、Tris缓冲液、HEPES缓冲液、枸橼酸缓冲液、琥珀酸缓冲液、碳酸氢钠缓冲液等。在一个或多个实施方式中，所述单糖溶液选自果糖、葡萄糖、半乳糖、甘露醇、山梨醇、木糖醇等溶液。在一个或多个实施方式中，所述二糖溶液选自蔗糖、乳糖、海藻糖、麦芽糖等溶液。在一个或多个实施方式中，所述多糖溶液选自右旋糖酐、海藻酸、透明质酸等溶液。在一个或多个实施方式中，所述电解质溶液选自氯化钠、氯化钙、氯化钾、氯化镁、硫酸铜、葡萄糖酸铜、葡萄糖酸钙、葡萄糖酸锌、乳酸钠等溶液。在一个或多个实施方式中，所述氨基酸溶液选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸(蛋氨酸)、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天门冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸、组氨酸等溶液。在一个或多个实施方式中，所述表面活性剂溶液选自吐温80、吐温20、泊洛沙姆188、HS15等溶液。在一个或多个实施方式中，所述含低浓度有机溶剂的水溶液选自有机溶剂浓度范围为0.1％-10％的乙醇、甲醇、丙酮、四氢呋喃、DMF、DMSO等水溶液。In one or more embodiments of the present invention, in step (2) of the above-mentioned microfluidic method, the aqueous phase solution is purified water, buffer, monosaccharide solution, disaccharide solution, polysaccharide solution, electrolyte solution , one or more of amino acid solution, surfactant solution, aqueous solution containing low concentration organic solvent. In one or more embodiments, the buffer is selected from phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, citrate buffer, succinate buffer, sodium bicarbonate buffer wait. In one or more embodiments, the monosaccharide solution is selected from fructose, glucose, galactose, mannitol, sorbitol, xylitol and other solutions. In one or more embodiments, the disaccharide solution is selected from sucrose, lactose, trehalose, maltose and other solutions. In one or more embodiments, the polysaccharide solution is selected from dextran, alginic acid, hyaluronic acid and other solutions. In one or more embodiments, the electrolyte solution is selected from sodium chloride, calcium chloride, potassium chloride, magnesium chloride, copper sulfate, copper gluconate, calcium gluconate, zinc gluconate, sodium lactate and other solutions. In one or more embodiments, the amino acid solution is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, Serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine and other solutions . In one or more embodiments, the surfactant solution is selected from Tween 80, Tween 20, poloxamer 188, HS15 and other solutions. In one or more embodiments, the aqueous solution containing a low-concentration organic solvent is selected from aqueous solutions such as ethanol, methanol, acetone, tetrahydrofuran, DMF, DMSO, etc. with an organic solvent concentration ranging from 0.1% to 10%.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，通过压缩空气或泵实现各相溶液由不同的进液口同时注入微混合器。在一个或多个实施方式中，各相溶液的总流速为1ml/min-1L/min，例如为1ml/min、2ml/min、3ml/min、5ml/min、10ml/min、15ml/min、20ml/min、25ml/min、30ml/min、50ml/min、80ml/min、100ml/min、150ml/min、200ml/min、300ml/min、400ml/min、500ml/min、600ml/min、800ml/min或1L/min，或其间任意的范围。在一个或多个实施方式中，各相溶液的总流速为20ml/min至50ml/min。在一个或多个实施方式中，各相溶液的总流速为25ml/min至30ml/min。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:1-1:300，例如为1:1、1:2、1:3、1:5、1:6、1:8、1:9、1:10、1:12、1:15、1:18、1:20、1:30、1:50、1:80、1:100、1:150、 1:200、1:250或1:300，或其间任意的范围。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:8-1:15。在一个或多个实施方式中，有机相溶液与水相溶液的流速比为1:9-1:12。在一个或多个实施方式中，所述微混合器可以选自T型三通、x型混合器和多进口涡流混合器(MIVM)。在一个或多个实施方式中，所述微混合器为T型三通。在一个或多个实施方式中，所述微混合器的进口和出口内径分别可以为0.1mm-5mm，二者可以相同，也可以不同。在一个或多个实施方式中，所述微混合器的材质可以选自聚醚醚酮(PEEK)、不锈钢、聚四氟乙烯(PTFE)等。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, each phase solution is simultaneously injected into the micromixer from different liquid inlets through compressed air or a pump. In one or more embodiments, the total flow rate of each phase solution is 1ml/min-1L/min, such as 1ml/min, 2ml/min, 3ml/min, 5ml/min, 10ml/min, 15ml/min, 20ml/min, 25ml/min, 30ml/min, 50ml/min, 80ml/min, 100ml/min, 150ml/min, 200ml/min, 300ml/min, 400ml/min, 500ml/min, 600ml/min, 800ml/ min or 1L/min, or any range in between. In one or more embodiments, the total flow rate of each phase solution is 20 ml/min to 50 ml/min. In one or more embodiments, the total flow rate of each phase solution is 25 ml/min to 30 ml/min. In one or more embodiments, the flow rate ratio of the organic phase solution and the aqueous phase solution is 1:1-1:300, such as 1:1, 1:2, 1:3, 1:5, 1:6, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, 1:20, 1:30, 1:50, 1:80, 1:100, 1:150, 1:200, 1:250 or 1:300, or any range in between. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:8-1:15. In one or more embodiments, the flow rate ratio of the organic phase solution to the aqueous phase solution is 1:9-1:12. In one or more embodiments, the micromixer may be selected from a T-type tee, an x-type mixer, and a multi-inlet vortex mixer (MIVM). In one or more embodiments, the micromixer is a T-shaped tee. In one or more embodiments, the inner diameters of the inlet and outlet of the micromixer can be 0.1mm-5mm respectively, and they can be the same or different. In one or more embodiments, the material of the micromixer can be selected from polyetheretherketone (PEEK), stainless steel, polytetrafluoroethylene (PTFE), etc.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(3)中，根据工艺目的，必要时可以对获得的含有机溶剂的载药胶束溶液进行一定程度的稀释。In one or more embodiments of the present invention, in step (3) of the above-mentioned microfluidic method, the obtained drug-loaded micelle solution containing an organic solvent can be diluted to a certain extent if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(4)中，通过选自挥发、透析和超滤中的一种或多种方式去除有机溶剂。In one or more embodiments of the present invention, in step (4) of the above-mentioned microfluidic method, the organic solvent is removed by one or more methods selected from the group consisting of volatilization, dialysis and ultrafiltration.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(4)中，根据工艺目的，必要时可以对去除有机溶剂后的胶束溶液进行浓缩。In one or more embodiments of the present invention, in step (4) of the above-mentioned microfluidic method, the micelle solution after removing the organic solvent can be concentrated if necessary according to the purpose of the process.
在本发明的一个或多个实施方式中，在上述微流控法的步骤(5)中，通过过滤方式进行除菌。In one or more embodiments of the present invention, in step (5) of the above-mentioned microfluidic method, sterilization is performed by filtration.
在本发明的一个或多个实施方式中，胶束制备方法为乳化法。In one or more embodiments of the invention, the micelle preparation method is an emulsification method.
在本发明的一个或多个实施方式中，所述乳化法包括以下步骤：In one or more embodiments of the invention, the emulsification method includes the following steps:
(1)将棘霉素类抗生素、两亲性共聚物和有机溶剂混合均匀，得到棘霉素类抗生素有机溶液；(1) Mix the echinomycin antibiotics, the amphiphilic copolymer and the organic solvent evenly to obtain an echinomycin antibiotic organic solution;
(2)将棘霉素类抗生素有机溶液和水相溶液混合均匀，形成初乳液；(2) Mix the echinomycin antibiotic organic solution and the aqueous phase solution evenly to form colostrum;
(3)初乳液经高压均质后形成纳米乳液，将其置于冷的水性介质中骤冷，得到载药纳米胶束溶液；(3) The colostrum is homogenized under high pressure to form a nanoemulsion, which is then quenched in a cold aqueous medium to obtain a drug-loaded nanomicelle solution;
(4)从步骤(3)中得到的载药胶束溶液去除有机溶剂，并浓缩，得到载药纳米胶束悬浮液；(4) Remove the organic solvent from the drug-loaded micelle solution obtained in step (3) and concentrate to obtain a drug-loaded nanomicelle suspension;
(5)对步骤(4)中得到的纳米胶束悬浮液进行除菌。(5) Sterilize the nanomicelle suspension obtained in step (4).
在本发明的一个或多个实施方式中，在所述乳化法的步骤(1)中，所述有机溶剂选自氯仿、二氯甲烷、乙酸乙酯中的一种或多种。In one or more embodiments of the present invention, in step (1) of the emulsification method, the organic solvent is selected from one or more of chloroform, dichloromethane, and ethyl acetate.
在本发明的一个或多个实施方式中，在所述乳化法的步骤(1)中，所述棘霉素类抗生素有机溶液中固体比例为0.1％-50％(重量/体积比)，例如0.5％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、15％、18％、20％、25％、30％、35％、40％、 45％或50％，或其间任意的范围。在一个实施方式中，所述棘霉素类抗生素有机溶液中固体比例为10％(重量/体积比)。In one or more embodiments of the present invention, in step (1) of the emulsification method, the solid proportion in the echinomycin antibiotic organic solution is 0.1%-50% (weight/volume ratio), for example 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 18%, 20%, 25% ,30%,35%,40%, 45% or 50%, or any range in between. In one embodiment, the solid proportion in the echinomycin antibiotic organic solution is 10% (weight/volume ratio).
在本发明的一个或多个实施方式中，在所述乳化法的步骤(2)中，所述水相溶液为包含0.1％-2％的去氧胆酸钠、1％-10％乙酸乙酯、0.5％-5％苯甲醇的水溶液。在本发明的一个或多个实施方式中，在所述乳化法的步骤(2)中，所述水相溶液由0.25％去氧胆酸钠、4％乙酸乙酯、2％苯甲醇及去离子水或净化水组成。In one or more embodiments of the present invention, in step (2) of the emulsification method, the aqueous solution contains 0.1%-2% sodium deoxycholate, 1%-10% ethyl acetate Ester, 0.5%-5% benzyl alcohol aqueous solution. In one or more embodiments of the present invention, in step (2) of the emulsification method, the aqueous phase solution is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and deoxycholate. Composed of ionized water or purified water.
在本发明的一个或多个实施方式中，在所述乳化法的步骤(2)中，所述棘霉素类抗生素有机溶液和水相溶液以1:1至1:9(体积比)的比率进行混合。在一个具体实施方式中，所述棘霉素类抗生素有机溶液和水相溶液以1:5(体积比)的比率进行混合。In one or more embodiments of the present invention, in step (2) of the emulsification method, the echinomycin antibiotic organic solution and the aqueous phase solution are mixed in a ratio of 1:1 to 1:9 (volume ratio). ratio to mix. In a specific embodiment, the echinomycin antibiotic organic solution and the aqueous phase solution are mixed at a ratio of 1:5 (volume ratio).
在本发明的一个或多个实施方式中，在上述乳化法的步骤(2)中，通过高速剪切将棘霉素类抗生素有机溶液和水相溶液混合均匀，形成初乳液。In one or more embodiments of the present invention, in step (2) of the above-mentioned emulsification method, the echinomycin antibiotic organic solution and the aqueous phase solution are mixed evenly through high-speed shearing to form a colostrum.
在本发明的一个或多个实施方式中，在上述乳化法的步骤(3)中，冷的水性介质是指温度低于5℃的水性介质。在一个实施方式中，水性介质是去离子水或纯化水。In one or more embodiments of the present invention, in step (3) of the above-mentioned emulsification method, the cold aqueous medium refers to an aqueous medium with a temperature lower than 5°C. In one embodiment, the aqueous medium is deionized or purified water.
在本发明的一个或多个实施方式中，在所述乳化法的步骤(4)中，通过选自挥发、透析和超滤中的一种或多种方式去除有机溶剂。在一个具体实施方式中，通过超滤去除有机溶剂。In one or more embodiments of the present invention, in step (4) of the emulsification method, the organic solvent is removed by one or more means selected from volatilization, dialysis and ultrafiltration. In a specific embodiment, the organic solvent is removed by ultrafiltration.
在本发明的一个或多个实施方式中，在所述乳化法的步骤(5)中，通过过滤方式进行除菌。In one or more embodiments of the present invention, in step (5) of the emulsification method, sterilization is performed by filtration.
在本发明的一个或多个实施方式中，胶束制备方法还包括将得到的药物胶束冻干的步骤。In one or more embodiments of the present invention, the micelle preparation method further includes the step of freeze-drying the obtained drug micelles.
通过本发明的胶束制备方法得到的胶束稳定性更好，且便于放大研究和生产。The micelles obtained by the micelle preparation method of the present invention have better stability and are convenient for scale-up research and production.
在另一个方面，本发明提供了由所述棘霉素类抗生素胶束制成的药物制剂。在本发明的一个或多个实施方式中，可以将本发明所述的棘霉素类抗生素胶束制备成通过非肠道、通过吸入、腹腔内、膀胱内、肌肉内、静脉内、气管内、皮下、眼内、鞘内、透皮给药、直肠或***内给药的药物制剂。In another aspect, the present invention provides pharmaceutical formulations made from the echinomycin antibiotic micelles. In one or more embodiments of the present invention, the echinomycin antibiotic micelles of the present invention can be prepared parenterally, by inhalation, intraperitoneally, intravesically, intramuscularly, intravenously, or intratracheally. , pharmaceutical preparations for subcutaneous, intraocular, intrathecal, transdermal, rectal or vaginal administration.
在本发明的一个或多个实施方式中，可以将所述棘霉素类抗生素胶束制备成通过静脉内给药的药物制剂。In one or more embodiments of the present invention, the echinomycin antibiotic micelles can be prepared into pharmaceutical preparations for intravenous administration.
在一个或多个实施方式中，所述药物制剂可以为固体制剂、液体制剂或气体制剂。In one or more embodiments, the pharmaceutical formulation may be a solid formulation, a liquid formulation, or a gas formulation.
在一个或多个实施方式中，所述药物制剂可以为注射用液体制剂。In one or more embodiments, the pharmaceutical formulation may be an injectable liquid formulation.
在一个或多个实施方式中，所述药物制剂可以为胶束无菌冻干粉制剂，其在使用前 用合适的介质如注射用水、生理盐水、葡萄糖溶液等复溶。In one or more embodiments, the pharmaceutical preparation may be a micellar sterile freeze-dried powder preparation, which is prepared before use. Reconstitute with appropriate media such as water for injection, physiological saline, glucose solution, etc.
在一个或多个实施方式中，所述药物制剂还包含其他药物。在一个或多个实施方式中，所述其他药物包括另外的抗癌药物。In one or more embodiments, the pharmaceutical formulation further contains other drugs. In one or more embodiments, the other drugs include additional anti-cancer drugs.
在一个或多个实施方式中，所述另外的抗癌药物为化疗药物、靶向抗肿瘤药物或/和免疫治疗药物。In one or more embodiments, the additional anti-cancer drug is a chemotherapy drug, a targeted anti-tumor drug, or/and an immunotherapy drug.
在又一个方面，本发明提供了所述棘霉素类抗生素胶束在制备用于预防或治疗癌症的药物中的用途。In yet another aspect, the present invention provides the use of the echinomycin antibiotic micelles in the preparation of medicaments for preventing or treating cancer.
在另一个方面，本发明提供了如本发明所述的棘霉素类抗生素胶束或包含所述棘霉素类抗生素胶束的药物制剂，用于预防或治疗癌症。In another aspect, the present invention provides the echinomycin antibiotic micelles or pharmaceutical preparations containing the echinomycin antibiotic micelles according to the present invention for preventing or treating cancer.
在另一个方面，本发明提供了一种预防或治疗受试者的癌症的方法，该方法包括向患者施用治疗有效量的本发明所述的棘霉素类抗生素胶束或包含所述棘霉素类抗生素胶束的药物制剂。In another aspect, the present invention provides a method for preventing or treating cancer in a subject, the method comprising administering to the patient a therapeutically effective amount of an echinomycin antibiotic micelle of the present invention or a micelle containing the echinobacterium Pharmaceutical preparations of antibiotic micelles.
本文所用的术语“受试者”包括哺乳动物和非哺乳动物。哺乳动物的实例包括但不限于哺乳动物纲的任何成员：人，非人的灵长类动物(例如黑猩猩和其它猿类和猴)；家畜，例如牛、马、绵羊、山羊、猪；家养动物，例如兔、狗和猫；实验室动物，包括啮齿类动物，例如大鼠、小鼠和豚鼠等。在一些实施方式中，所述受试者为人。The term "subject" as used herein includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (eg, chimpanzees and other apes and monkeys); domestic animals, such as cattle, horses, sheep, goats, pigs; , such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. In some embodiments, the subject is a human.
本文所用的术语“治疗”包括缓解、减轻或改善疾病或病症症状，抑制疾病或病症，例如阻止疾病或病症的发展，缓解疾病或病症，使疾病或病症好转，缓解由疾病或病症导致的症状，或者中止疾病或病症的症状，预防其他症状，改善或预防导致症状的潜在代谢原因，此外，该术语包含预防的目的。该术语还包括获得治疗效果和/或预防效果。所述治疗效果是指治愈或改善所治疗的潜在疾病。此外，对与潜在疾病相关的一种或多种生理症状的治愈或改善也是治疗效果，例如尽管受试者可能仍然受到潜在疾病的影响，但观察到受试者情况改善。就预防效果而言，可向具有患特定疾病风险例如癌症的受试者施用本发明所述棘霉素类抗生素胶束或药物制剂，或者即便尚未做出疾病诊断，但向出现该疾病的一个或多个生理症状的受试者施用本发明所述棘霉素类抗生素胶束或药物制剂。As used herein, the term "treatment" includes alleviating, alleviating or ameliorating symptoms of a disease or condition, inhibiting a disease or condition, such as preventing the progression of a disease or condition, ameliorating a disease or condition, making a disease or condition better, alleviating symptoms caused by a disease or condition , or to abort symptoms of a disease or condition, prevent other symptoms, ameliorate or prevent underlying metabolic causes of symptoms, and further, the term includes preventive purposes. The term also includes obtaining therapeutic and/or prophylactic effects. The therapeutic effect refers to the cure or improvement of the underlying disease being treated. Additionally, cure or amelioration of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect, such as a subject's condition being observed to improve although the subject may still be affected by the underlying disease. In terms of preventive effect, the echinomycin antibiotic micelles or pharmaceutical formulations of the present invention can be administered to subjects who are at risk of developing a specific disease, such as cancer, or even if a diagnosis of the disease has not yet been made, to a subject who has developed the disease. Or subjects with multiple physiological symptoms are administered the echinomycin antibiotic micelles or pharmaceutical preparations of the present invention.
本文所用的术语“治疗有效量”是指服用后足以在某种程度上缓解所治疗的疾病或病症的一个或多个症状的至少一种活性物质(如本发明所述的棘霉素类抗生素胶束)的量。其结果可以为迹象、症状或病因的消减和/或缓解，或生物***的任何其他所需变化。例如，“治疗有效量”是在临床上提供显著的病症缓解效果所需的包含本发明所述的棘霉 素类抗生素胶束的组合物的量。可使用诸如剂量递增试验的技术测定适合于任意个体病例中的治疗有效量。The term "therapeutically effective amount" as used herein refers to at least one active substance (such as the echinomycin antibiotics of the present invention) that is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. micelles). The result may be a reduction and/or alleviation of signs, symptoms or causes, or any other desired change in the biological system. For example, a "therapeutically effective amount" is the amount of the Aspergillus species of the invention required to provide clinically significant symptom relief. The amount of antibiotic micelles in the composition. The therapeutically effective amount appropriate in any individual case can be determined using techniques such as dose escalation trials.
本发明的技术方案具有以下有益效果：The technical solution of the present invention has the following beneficial effects:
本发明将棘霉素类抗生素制成胶束，提高了棘霉素类抗生素的溶解度，使其更有利于临床使用，同时制备的胶束具备粒径小、粒径分布均一、载药量大和稳定性高的特点，与注射液相比降低了在血浆中的消除速度，延长了药物在体内的作用时间，改善了棘霉素类抗生素体外及体内抗肿瘤效果，同时还降低了棘霉素类抗生素的体内毒性。本发明提供的制备棘霉素类抗生素载药胶束的技术重现性好，载药量高，有利于放大进行工业化生产。The present invention makes the echinomycin antibiotics into micelles, which improves the solubility of the echinomycin antibiotics and makes them more conducive to clinical use. At the same time, the prepared micelles have the characteristics of small particle size, uniform particle size distribution, large drug loading capacity and The characteristics of high stability, compared with the injection, reduce the elimination rate in the plasma, prolong the action time of the drug in the body, improve the anti-tumor effect of echinomycin antibiotics in vitro and in vivo, and also reduce the echinomycin In vivo toxicity of antibiotics. The technology for preparing echinomycin antibiotic drug-loaded micelles provided by the invention has good reproducibility, high drug loading capacity, and is conducive to scale-up and industrial production.
附图说明Description of drawings
图1是根据实施例5制备得到的棘霉素载药胶束的水化粒径图。Figure 1 is a hydration particle size diagram of echinomycin drug-loaded micelles prepared according to Example 5.
具体实施方式Detailed ways
以下实施例是对本发明的进一步说明，并非对本发明范围的限制。下面参考实施例进一步详细阐述本发明，但是本领域技术人员应当理解，本发明并不限于这些实施例以及使用的制备方法。而且，本领域技术人员根据本发明的描述可以对本发明进行等同替换、组合、改良或修饰，但这些都将包括在本发明的范围内。The following examples further illustrate the present invention and do not limit the scope of the present invention. The present invention will be further described in detail below with reference to the examples, but those skilled in the art will understand that the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art can make equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will be included in the scope of the present invention.
测试例1胶束的粒径检测Test Example 1 Particle size detection of micelles
使用动态光散射(DLS)(仪器：马尔文纳米粒径仪，型号：NANO ZS90)采用动态光散射(DLS)检测胶束的粒径。检测模式：自动；检测待测样品折射率：1.340；分散介质：超纯水；温度25℃；黏度：0.8872cp；分散介质折射率：1.330，得到粒径图。Use dynamic light scattering (DLS) (instrument: Malvern Nanoparticle Size Analyzer, model: NANO ZS90) to detect the particle size of micelles using dynamic light scattering (DLS). Detection mode: automatic; detect the refractive index of the sample to be tested: 1.340; dispersion medium: ultrapure water; temperature 25°C; viscosity: 0.8872cp; refractive index of the dispersion medium: 1.330, and obtain the particle size diagram.
测试例2棘霉素类抗生素的含量测定Test Example 2 Content Determination of Echinomycin Antibiotics
含量测定按照下述方法进行。Content measurement was performed according to the following method.
1、色谱条件：1. Chromatographic conditions:
色谱柱：GL Sciences Inertsil ODS-3(4.6mm×250mm，5μm)Column: GL Sciences Inertsil ODS-3 (4.6mm×250mm, 5μm)
流动相：水-乙腈(40:60) Mobile phase: water-acetonitrile (40:60)
流速：1.0mL/minFlow rate: 1.0mL/min
检测波长：243nmDetection wavelength: 243nm
柱温：30℃Column temperature: 30℃
进样量：20μLInjection volume: 20μL
2、样品处理过程：2. Sample processing process:
空白溶剂：乙腈Blank solvent: acetonitrile
对照品溶液制备：精密称取棘霉素类抗生素对照品约20mg置入100ml量瓶，加入适量乙腈溶解，并稀释至刻度，摇匀，精密量取1ml置入10ml量瓶，加入乙腈稀释至刻度，摇匀。制成每1ml溶液中含有棘霉素类抗生素约0.02mg的对照品溶液。Preparation of reference substance solution: Precisely weigh about 20 mg of the echinomycin antibiotic reference substance into a 100 ml measuring flask, add an appropriate amount of acetonitrile to dissolve, and dilute to the mark, shake well, accurately measure 1 ml and place it into a 10 ml measuring flask, add acetonitrile and dilute to Scale and shake well. Prepare a reference solution containing approximately 0.02 mg of echinomycin antibiotics per 1 ml of solution.
供试品溶液制备：精密量取棘霉素类抗生素胶束溶液0.5ml置入5ml量瓶，加入乙腈并稀释至刻度，摇匀。滤过，取滤液检测(Waters ACQUITY Arc 2998PDA)，得到样品中棘霉素类抗生素含量。Preparation of test solution: Precisely measure 0.5 ml of the echinomycin antibiotic micellar solution into a 5 ml volumetric flask, add acetonitrile and dilute to the mark, shake well. Filter, take the filtrate for testing (Waters ACQUITY Arc 2998PDA), and obtain the echinomycin antibiotic content in the sample.
测试例3棘霉素类抗生素胶束载药量检测Test Example 3 Detection of micelle drug loading of echinomycin antibiotics
将过滤后的棘霉素类抗生素胶束溶液分装至西林瓶中，使用冻干机冻干，取少许冻干品称重后照测试例2方法检测冻干品中棘霉素类抗生素含量(w/w％)，即为棘霉素类抗生素胶束载药量。The filtered echinomycin antibiotic micelle solution is divided into vials and freeze-dried using a freeze-drying machine. A small amount of the freeze-dried product is weighed and the content of the echinomycin antibiotics in the freeze-dried product is measured according to the method in Test Example 2. (w/w%), which is the drug loading capacity of echinomycin antibiotic micelles.
测试例4棘霉素类抗生素胶束的药代动力学试验。Test Example 4: Pharmacokinetic test of echinomycin antibiotic micelles.
对本发明的棘霉素类抗生素胶束进行药代动力学试验。以每种性别各3只的SD大鼠为试验动物，采用交叉给药法给药，第1次给药时对雄性SD大鼠给予棘霉素注射液(其制备方法如下所述：称取棘霉素原料药7.5mg，加入Kolliphor EL和乙醇的混合溶液(1：1，v/v)约8g，溶解。取0.25ml溶液于25ml容量瓶，采用0.9％生理盐水稀释并定容，得到棘霉素注射液)，雌性SD大鼠给予棘霉素类抗生素胶束冻干制剂，将其用0.9％生理盐水复溶后，每只SD大鼠静脉给予0.1mg/kg，第1次给药后洗脱3天后进行第2次给药，第2次给药时，对雄性SD大鼠给予棘霉素类抗生素胶束，雌性SD大鼠给予棘霉素注射液，每只SD大鼠静脉给予0.1mg/kg。每次给药时，分别在给药前及给药后的0.5h、1h、1.5h、2h、2.5h、4h、8h、12h静脉取血。血液样品经离心分离出血浆后，用LC-MS/MS法测定血浆中棘霉素类抗生素的量。 A pharmacokinetic test was conducted on the echinomycin antibiotic micelles of the present invention. Three SD rats of each gender were used as experimental animals, and administration was carried out using a cross-administration method. During the first administration, male SD rats were given echinomycin injection (its preparation method is as follows: weigh For 7.5 mg of echinomycin raw material, add about 8 g of a mixed solution of Kolliphor EL and ethanol (1:1, v/v) and dissolve it. Take 0.25 ml of the solution in a 25 ml volumetric flask, dilute it with 0.9% normal saline and set it to volume to obtain Echinomycin injection), female SD rats were given micellar freeze-dried preparations of echinomycin antibiotics, which were reconstituted with 0.9% normal saline, and each SD rat was given 0.1 mg/kg intravenously. The first dose The second administration was performed 3 days after drug washout. During the second administration, male SD rats were given echinomycin antibiotic micelles, and female SD rats were given echinomycin injection. Each SD rat 0.1 mg/kg administered intravenously. During each administration, blood was collected from the vein before administration and at 0.5h, 1h, 1.5h, 2h, 2.5h, 4h, 8h, and 12h after administration. After the blood sample is centrifuged to separate the plasma, the LC-MS/MS method is used to determine the amount of echinomycin antibiotics in the plasma.
实施例1Example 1
称取15mg棘霉素和135mg甲氧基聚乙二醇-聚乳酸嵌段共聚物(济南岱罡，mPEG-PDLLA，mPEG段重均分子量2000，PDLLA段重均分子量18000)加入西林瓶中，再加入3ml丙酮溶解得到棘霉素有机相溶液。将36ml纯化水加入烧杯中，开启剪切机(IKA T25)对水相进行剪切，剪切速率为3000rpm。使用注射泵(保定雷弗，TYB01-01)将棘霉素有机相溶液注入水相中，注入速率为0.03ml/s，注入完毕后再剪切3分钟。剪切完毕后，加入纯化水稀释，使胶束溶液中丙酮含量降低至2％(v/w％)以下。使用透析袋(索莱宝，截留分子量为8000道尔顿)对稀释后的胶束溶液进行透析，每隔一定时间更换透析液，即纯化水，透析过夜后使用0.22μm聚醚砜滤膜(密理博，针头式过滤器)对胶束溶液进行过滤，如测试例1所述使用动态光散射(DLS)检测胶束水化粒径。平均粒径为102.5nm，PDI为0.133。Weigh 15 mg of echinomycin and 135 mg of methoxypolyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 2000, PDLLA segment weight average molecular weight 18000) into a vial, and then add 3 ml acetone Dissolve to obtain an organic phase solution of echinomycin. Add 36 ml of purified water into the beaker, and turn on the shearer (IKA T25) to shear the water phase at a shear rate of 3000 rpm. Use a syringe pump (Baoding Leifu, TYB01-01) to inject the echinomycin organic phase solution into the aqueous phase at an injection rate of 0.03 ml/s. After the injection is completed, shear for 3 minutes. After shearing is completed, purified water is added to dilute the solution to reduce the acetone content in the micellar solution to less than 2% (v/w%). The diluted micellar solution was dialyzed using a dialysis bag (Soleba, molecular weight cutoff of 8000 daltons). The dialysate, that is, purified water, was replaced at regular intervals. After dialysis overnight, a 0.22 μm polyethersulfone filter membrane ( Millipore, syringe filter) was used to filter the micellar solution, and dynamic light scattering (DLS) was used to detect the micelle hydrated particle size as described in Test Example 1. The average particle size is 102.5nm and PDI is 0.133.
实施例2Example 2
称取14.7mg棘霉素和136mg甲氧基聚乙二醇-聚乳酸嵌段共聚物(济南岱罡，mPEG-PDLLA，mPEG段重均分子量5000，PDLLA段重均分子量5000)加入西林瓶中，再加入3ml丙酮溶解得到棘霉素有机相溶液。将40ml纯化水加入烧杯中，开启剪切机(IKA T25)对水相进行剪切，剪切速率为3000rpm。使用注射泵(保定雷弗，TYB01-01)将棘霉素有机相溶液注入水相中，注入速率为0.03ml/s，注入完毕后再剪切3分钟。剪切完毕后，加入纯化水稀释，使胶束溶液中丙酮含量降低至2％(v/w％)以下。使用透析袋(索莱宝，截留分子量为8000道尔顿)对稀释后的胶束溶液进行透析，每隔一定时间更换透析液，即纯化水，透析过夜后使用0.22μm聚醚砜滤膜(密理博，针头式过滤器)对胶束溶液进行过滤，如测试例1所述使用动态光散射(DLS)检测胶束水化粒径。平均粒径为78.5nm，PDI为0.120。Weigh 14.7 mg of echinomycin and 136 mg of methoxypolyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 5000, PDLLA segment weight average molecular weight 5000) into the vial, and then add 3 ml Dissolve in acetone to obtain an organic phase solution of echinomycin. Add 40ml of purified water into the beaker, turn on the shearer (IKA T25) to shear the water phase, the shearing rate is 3000rpm. Use a syringe pump (Baoding Leifu, TYB01-01) to inject the echinomycin organic phase solution into the aqueous phase at an injection rate of 0.03 ml/s. After the injection is completed, shear for 3 minutes. After shearing is completed, purified water is added to dilute the solution to reduce the acetone content in the micellar solution to less than 2% (v/w%). The diluted micellar solution was dialyzed using a dialysis bag (Soleba, molecular weight cutoff of 8000 daltons). The dialysate, that is, purified water, was replaced at regular intervals. After dialysis overnight, a 0.22 μm polyethersulfone filter membrane ( Millipore, syringe filter) was used to filter the micellar solution, and dynamic light scattering (DLS) was used to detect the micelle hydrated particle size as described in Test Example 1. The average particle size is 78.5nm and PDI is 0.120.
实施例3Example 3
称取30mg棘霉素和270mg甲氧基聚乙二醇-聚乳酸嵌段共聚物(济南岱罡，mPEG-PDLLA，mPEG段重均分子量5000，PDLLA段重均分子量5000)加入西林瓶中，再加入3ml丙酮溶解得到棘霉素有机相溶液。将棘霉素有机相溶液和纯化水通过注射泵(保定雷弗，TYB01-01)分别注入T型三通(内径0.5mm，Cole Parmer，HV-06365-77)的 进液口，总流速为25ml/min，流速比为1:9，胶束溶液由T型三通的出口处流出，制备完毕后立即加入纯化水稀释，使胶束溶液中丙酮含量降低至2％(v/w％)以下。使用透析袋(索莱宝，截留分子量为8000道尔顿)对稀释后的胶束溶液进行透析，每隔一定时间更换透析液，即纯化水，透析过夜后使用0.22μm聚醚砜滤膜(密理博，针头式过滤器)对胶束溶液进行过滤，如测试例1所述使用动态光散射(DLS)检测胶束水化粒径。平均粒径为55.2nm，PDI为0.118。Weigh 30 mg of echinomycin and 270 mg of methoxy polyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 5000, PDLLA segment weight average molecular weight 5000) and add it to the vial, then add 3 ml acetone Dissolve to obtain an organic phase solution of echinomycin. The echinomycin organic phase solution and purified water were respectively injected into the T-shaped tee (inner diameter 0.5mm, Cole Parmer, HV-06365-77) through a syringe pump (Baoding Reif, TYB01-01). At the liquid inlet, the total flow rate is 25ml/min, and the flow rate ratio is 1:9. The micellar solution flows out from the outlet of the T-shaped tee. After preparation, purified water is added immediately to dilute it to reduce the acetone content in the micellar solution to 2 % (v/w%) or less. The diluted micellar solution was dialyzed using a dialysis bag (Soleba, molecular weight cutoff of 8000 daltons). The dialysate, that is, purified water, was replaced at regular intervals. After dialysis overnight, a 0.22 μm polyethersulfone filter membrane ( Millipore, syringe filter) was used to filter the micellar solution, and dynamic light scattering (DLS) was used to detect the micelle hydrated particle size as described in Test Example 1. The average particle size is 55.2nm and PDI is 0.118.
实施例4Example 4
称取7.8mg棘霉素和152mg甲氧基聚乙二醇-聚乳酸嵌段共聚物(济南岱罡，mPEG-PDLLA，mPEG段重均分子量5000，PDLLA段重均分子量15000)加入西林瓶中，再加入3ml丙酮溶解得到棘霉素有机相溶液。将棘霉素有机相溶液和纯化水通过注射泵(保定雷弗，TYB01-01)分别注入T型三通(内径0.5mm，Cole Parmer，HV-06365-77)的进液口，总流速为30ml/min，流速比为1:12，胶束溶液由T型三通的出口处流出，制备完毕后立即加入纯化水稀释，使胶束溶液中丙酮含量降低至2％(v/w％)以下。使用中空纤维超滤柱(MidiKros)对胶束溶液进行超滤，超滤6倍体积后浓缩至原药液体积的1/3。超滤完成后使用0.22μm聚醚砜滤膜(密理博，针头式过滤器)对胶束溶液进行过滤，使用动态光散射(DLS)检测胶束水化粒径。平均粒径为60.1nm，PDI为0.156。Weigh 7.8 mg of echinomycin and 152 mg of methoxypolyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 5000, PDLLA segment weight average molecular weight 15000) into the vial, and then add 3 ml Dissolve in acetone to obtain an organic phase solution of echinomycin. The echinomycin organic phase solution and purified water were respectively injected into the liquid inlet of the T-shaped tee (inner diameter 0.5mm, Cole Parmer, HV-06365-77) through a syringe pump (Baoding Leifer, TYB01-01), with a total flow rate of 30ml/min, the flow rate ratio is 1:12, the micellar solution flows out from the outlet of the T-shaped tee, and purified water is added immediately after preparation to dilute it to reduce the acetone content in the micellar solution to 2% (v/w%) the following. Use a hollow fiber ultrafiltration column (MidiKros) to ultrafiltrate the micellar solution, ultrafiltrate 6 times the volume and then concentrate to 1/3 of the original solution volume. After ultrafiltration, a 0.22 μm polyethersulfone filter membrane (Millipore, syringe filter) was used to filter the micellar solution, and dynamic light scattering (DLS) was used to detect the hydrated particle size of the micelles. The average particle size is 60.1nm and PDI is 0.156.
实施例5Example 5
称取7.6mg棘霉素和67.3mg甲氧基聚乙二醇-聚乳酸嵌段共聚物(济南岱罡，mPEG-PDLLA，mPEG段重均分子量2000，PDLLA段重均分子量18000)加入西林瓶中，再加入3ml丙酮溶解得到棘霉素有机相溶液。将棘霉素有机相溶液和纯化水通过注射泵(保定雷弗，TYB01-01)分别注入T型三通(内径0.5mm，Cole Parmer，HV-06365-77)的进液口，总流速为25ml/min，流速比为1:9，胶束溶液由T型三通的出口处流出，制备完毕后立即加入纯化水稀释，使胶束溶液中丙酮含量降低至1.5％(v/w％)以下。使用中空纤维超滤柱(MidiKros)对胶束溶液进行超滤，超滤8倍体积后浓缩至原药液体积的1/10。超滤完成后使用0.22μm聚醚砜滤膜(密理博，针头式过滤器)对胶束溶液进行过滤，如测试例1所述使用动态光散射(DLS)检测胶束水化粒径。如图1所示，平均粒径为66.6nm，PDI为0.121。如测试例2所述测定棘霉素含量为0.21mg/ml。 Weigh 7.6 mg of echinomycin and 67.3 mg of methoxypolyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 2000, PDLLA segment weight average molecular weight 18000) into the vial, and then add Dissolve 3 ml of acetone to obtain an echinomycin organic phase solution. The echinomycin organic phase solution and purified water were respectively injected into the liquid inlet of the T-shaped tee (inner diameter 0.5mm, Cole Parmer, HV-06365-77) through a syringe pump (Baoding Leifer, TYB01-01), with a total flow rate of 25ml/min, the flow rate ratio is 1:9, the micellar solution flows out from the outlet of the T-shaped tee, and purified water is added immediately after preparation to dilute it to reduce the acetone content in the micellar solution to 1.5% (v/w%) the following. Use a hollow fiber ultrafiltration column (MidiKros) to ultrafiltrate the micellar solution, ultrafiltrate 8 times the volume and then concentrate to 1/10 of the volume of the original solution. After the ultrafiltration is completed, use a 0.22 μm polyethersulfone filter membrane (Millipore, syringe filter) to filter the micellar solution, and use dynamic light scattering (DLS) to detect the micelle hydration particle size as described in Test Example 1. As shown in Figure 1, the average particle size is 66.6nm and the PDI is 0.121. The echinomycin content was measured as described in Test Example 2 and was 0.21 mg/ml.
实施例6Example 6
称取50mg棘霉素和450mg甲氧基聚乙二醇-聚乳酸嵌段共聚物(济南岱罡，mPEG-PDLLA，mPEG段重均分子量5000，PDLLA段重均分子量15000)加入西林瓶中，加入0.5ml苯甲醇和4.5ml乙酸乙酯，完全溶解，形成棘霉素有机相溶液。将棘霉素有机相溶液与水相(水相由0.25％去氧胆酸钠、4％乙酸乙酯、2％苯甲醇及去离子水共25ml组成)以约为1:5(有机相:水相)的比率混合后，经高速剪切机(德国IKA，型号：T25DS25)高速剪切，剪切速率为5000rpm，使两相混合，形成初乳液。将初乳液经微射流均质机(诺泽流体科技(上海)有限公司，型号：Nano)在5000psi压力下高压均质后形成的纳米乳液添加到约50倍体积的5℃去离子水中，骤冷。然后用使用分子量截留值(MWCO)为300的再生纤维素膜(杭州科百特过滤器材有限公司)超滤浓缩，得纳米悬浮液。将纳米悬浮液经0.22μm聚醚砜滤膜(密理博，针头式过滤器)除菌过滤后，分装至西林瓶，冷冻干燥，得棘霉素纳米胶束。棘霉素纳米胶束用0.9％的氯化钠溶液复溶后，如测试例1所述使用动态光散射(DLS)检测胶束水化粒径，平均粒径为78.42nm，PDI为0.043。如测试例4所述对本实施例制备得到的棘霉素纳米胶束冻干粉进行药代动力学试验，结果如下表所示：
Weigh 50 mg of echinomycin and 450 mg of methoxy polyethylene glycol-polylactic acid block copolymer (Jinan Daigang, mPEG-PDLLA, mPEG segment weight average molecular weight 5000, PDLLA segment weight average molecular weight 15000) into a vial, and add 0.5 ml benzene Methanol and 4.5 ml of ethyl acetate were completely dissolved to form an echinomycin organic phase solution. Mix the echinomycin organic phase solution and the aqueous phase (the aqueous phase is composed of 0.25% sodium deoxycholate, 4% ethyl acetate, 2% benzyl alcohol and deionized water, a total of 25ml) at a ratio of about 1:5 (organic phase: After mixing at the ratio of water phase), it is sheared by a high-speed shearing machine (IKA, Germany, model: T25DS25) at a shear rate of 5000 rpm to mix the two phases to form colostrum. The nanoemulsion formed after high-pressure homogenization of the colostrum by a microjet homogenizer (Nuoze Fluid Technology (Shanghai) Co., Ltd., model: Nano) at a pressure of 5000 psi was added to about 50 times the volume of 5°C deionized water, and then cold. It was then concentrated by ultrafiltration using a regenerated cellulose membrane (Hangzhou Cobetter Filtration Equipment Co., Ltd.) with a molecular weight cutoff (MWCO) of 300 to obtain a nanosuspension. The nanosuspension was sterilized and filtered through a 0.22 μm polyethersulfone filter membrane (Millipore, syringe filter), then divided into vials and freeze-dried to obtain echinomycin nanomicelles. After the echinomycin nanomicelles were reconstituted with 0.9% sodium chloride solution, dynamic light scattering (DLS) was used to detect the hydrated particle size of the micelles as described in Test Example 1. The average particle size was 78.42 nm and the PDI was 0.043. As described in Test Example 4, a pharmacokinetic test was carried out on the echinomycin nanomicelle freeze-dried powder prepared in this example, and the results are as shown in the following table:
结果表明与棘霉素注射液相比，棘霉素胶束在血浆中的消除速度明显降低，延长了药物在体内的作用时间，有利于提高药物的抗肿瘤疗效。 The results show that compared with echinomycin injection, the elimination rate of echinomycin micelles in plasma is significantly reduced, which prolongs the action time of the drug in the body and is conducive to improving the anti-tumor efficacy of the drug.

Claims (15)

  1. 一种胶束，其包含棘霉素类抗生素和一种或多种两亲性共聚物。A micelle comprising an echinomycin antibiotic and one or more amphiphilic copolymers.
  2. 根据权利要求1所述的胶束，其中所述棘霉素类抗生素选自棘霉素、放线菌白素、三骨菌素、F-43、59266、6270和echinoserine。The micelle according to claim 1, wherein the echinomycin antibiotic is selected from the group consisting of echinomycin, actinomycin, trimostatin, F-43, 59266, 6270 and echinoserine.
  3. 根据权利要求1所述的胶束，其中所述棘霉素类抗生素为棘霉素。The micelle according to claim 1, wherein the echinomycin antibiotic is echinomycin.
  4. 根据权利要求1-3中任一项所述的胶束，其中所述两亲性共聚物选自嵌段共聚物、接枝共聚物和树枝状大分子。The micelle according to any one of claims 1 to 3, wherein the amphiphilic copolymer is selected from the group consisting of block copolymers, graft copolymers and dendrimers.
  5. 根据权利要求4所述的胶束，其中所述嵌段共聚物亲水段选自聚乙二醇、甲氧基聚乙二醇、聚丙烯酸、聚甲基丙烯酸、聚丙烯酸衍生物、聚甲基丙烯酸衍生物、聚氨基酸中的一种或多种，疏水段选自聚乳酸、丙交酯乙交酯共聚物、聚己内酯、聚碳酸酯、聚碳酸衍生物、聚丁酸酯、聚丙烯酸衍生物、聚甲基丙烯酸衍生物、聚氨基酸中的一种或多种。The micelle according to claim 4, wherein the hydrophilic segment of the block copolymer is selected from polyethylene glycol, methoxypolyethylene glycol, polyacrylic acid, polymethacrylic acid, polyacrylic acid derivatives, polymethacrylic acid One or more acrylic acid derivatives and polyamino acids, the hydrophobic segment is selected from polylactic acid, lactide-glycolide copolymer, polycaprolactone, polycarbonate, polycarbonate derivatives, polybutyrate, One or more of polyacrylic acid derivatives, polymethacrylic acid derivatives, and polyamino acids.
  6. 根据权利要求5所述的胶束，其中所述嵌段共聚物亲水段重均分子量为100Da-50000Da；所述疏水段重均分子量为500Da-100000Da；其中所述嵌段共聚物多分散指数小于2.0。The micelle according to claim 5, wherein the weight average molecular weight of the hydrophilic segment of the block copolymer is 100 Da-50000 Da; the weight average molecular weight of the hydrophobic segment is 500 Da-100000 Da; wherein the polydispersity index of the block copolymer is less than 2.0.
  7. 根据权利要求1-6中任一项所述的胶束，其中所述一种或多种两亲性共聚物与棘霉素类抗生素的重量比为0.1:1到200:1。The micelle according to any one of claims 1-6, wherein the weight ratio of the one or more amphiphilic copolymers to the echinomycin antibiotic is 0.1:1 to 200:1.
  8. 根据权利要求1-7中任一项所述的胶束，其中所述胶束为冻干粉形式。The micelle according to any one of claims 1-7, wherein the micelle is in the form of a freeze-dried powder.
  9. 根据权利要求8所述的胶束，其中所述胶束含有或不含有冻干保护剂。The micelle according to claim 8, wherein the micelle contains or does not contain a lyoprotectant.
  10. 根据权利要求1-9中任一项所述的胶束，其还包含pH调节剂、张力调节剂、抗氧化剂、防腐剂、缓冲剂、金属螯合剂、惰性气体中的一种或多种。The micelle according to any one of claims 1 to 9, further comprising one or more of a pH adjuster, a tension adjuster, an antioxidant, a preservative, a buffer, a metal chelating agent, and an inert gas.
  11. 根据权利要求1-10中任一项所述的胶束，其通过选自直接分散法、固相分散法、有机溶剂注入法、乳化法、膜乳化法、微流控法、超声法、均质法中的一种或多种方法制备得到。The micelle according to any one of claims 1-10, which is selected from the group consisting of direct dispersion method, solid phase dispersion method, organic solvent injection method, emulsification method, membrane emulsification method, microfluidic method, ultrasonic method, uniform prepared by one or more methods.
  12. 权利要求1至11中任一项所述的胶束在制备用于预防或治疗癌症的药物中的用途。Use of the micelle according to any one of claims 1 to 11 in the preparation of a medicament for preventing or treating cancer.
  13. 根据权利要求12所述的用途，其中所述胶束被制备成通过非肠道、通过吸入、腹腔内、膀胱内、肌肉内、静脉内、气管内、皮下、眼内、鞘内、透皮给药、直肠或***内给药的药物。The use according to claim 12, wherein the micelles are prepared parenterally, by inhalation, intraperitoneally, intravesically, intramuscularly, intravenously, intratracheally, subcutaneously, intraocularly, intrathecally, transdermally. Medications administered, rectally or vaginally.
  14. 根据权利要求13所述的用途，其中所述药物为固体制剂、液体制剂或气体制剂。 The use according to claim 13, wherein the drug is a solid preparation, a liquid preparation or a gas preparation.
  15. 根据权利要求12至14中任一项所述的用途，其中所述药物还包含另外的抗癌药物。 The use according to any one of claims 12 to 14, wherein the medicament further comprises an additional anti-cancer drug.
PCT/CN2023/111047 2022-08-05 2023-08-03 Echinomycin antibiotic micelle, preparation method therefor, and use WO2024002385A1 (en)

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