WO2023036276A1 - 多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途 - Google Patents

多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途 Download PDF

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WO2023036276A1
WO2023036276A1 PCT/CN2022/117973 CN2022117973W WO2023036276A1 WO 2023036276 A1 WO2023036276 A1 WO 2023036276A1 CN 2022117973 W CN2022117973 W CN 2022117973W WO 2023036276 A1 WO2023036276 A1 WO 2023036276A1
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glycol monomethyl
monomethyl ether
polyethylene glycol
product according
docetaxel
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PCT/CN2022/117973
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English (en)
French (fr)
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唐任宏
杨文清
薛黎婷
秦晓康
诸舜伟
邢辉
唐建兴
李坚
郭磊磊
丁雨寅
郝元斌
史瑞文
卞祥
刘永东
李臻
任晋生
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先声药业有限公司
江苏先声药业有限公司
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Priority to CN202280053114.5A priority Critical patent/CN117915908A/zh
Publication of WO2023036276A1 publication Critical patent/WO2023036276A1/zh

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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the application relates to the field of medicine, in particular to the use of docetaxel polymer micelles in the preparation of drugs for preventing or treating malignant pleural effusion.
  • Docetaxel also known as docetaxel, has a molecular formula of C 43 H 53 NO 14 and a molecular weight of 807.88. It is a paclitaxel antineoplastic drug, which can bind to free tubulin and promote tubulin assembly At the same time, it can inhibit the depolymerization of microtubules, resulting in the production of microtubules that lose their normal functions and the fixation of microtubules, thereby inhibiting cell mitosis and exerting anti-tumor effects.
  • docetaxel has disadvantages such as poor water solubility, short half-life and high toxicity, and is usually administered clinically by intravenous infusion.
  • Nanopolymer micelle is a drug-loading system developed in recent years for insoluble drugs. It has a core-shell structure, in which the core is the hydrophobic part and the shell is the hydrophilic part. Polymer micelles can encapsulate poorly soluble drugs in the core part to solubilize poorly soluble drugs.
  • Recombinant human endostatin is modified by modifying the nucleotide coding sequence of human endostatin so that its N-terminal has 9 additional amino acid sequences. ⁇ 192 ⁇ , ⁇ :(M)GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK ⁇
  • the currently commercially available recombinant human endostatin is common injection, which is used in combination with NP chemotherapy regimen for the treatment of patients with stage III/IV non-small cell lung cancer who have been newly or retreated.
  • the purpose of this application is to provide the use of docetaxel polymer micelles in the preparation of medicines for preventing or treating malignant pleural effusion.
  • the application provides the use of docetaxel polymer micelles in the preparation of medicines for preventing or treating malignant pleural effusion.
  • the present application provides a method for preventing or treating malignant pleural effusion in mammals, including administering docetaxel polymer micelles to mammals in need of the prevention or treatment, preferably humans.
  • the application provides the use of docetaxel polymer micelles in the prevention or treatment of malignant pleural ascites.
  • the present application provides docetaxel polymer micelles for preventing or treating malignant pleural effusion.
  • the dose of docetaxel polymer micelles is 5-200 mg/m 2 .
  • the docetaxel polymeric micelles are administered once a week.
  • the present application provides the use of docetaxel polymer micelles and recombinant human vascular endostatin in combination in the preparation of a medicament for preventing or treating malignant pleural effusion.
  • the present application provides a method for preventing or treating malignant pleural effusion in mammals, comprising administering docetaxel polymer micelles and recombinant human vascular endostatin to mammals in need of the treatment, preferably humans.
  • the present application provides the combination of docetaxel polymer micelles and recombinant human endostatin in preventing or treating malignant pleural effusion.
  • the present application provides docetaxel polymer micelles and recombinant human endostatin for preventing or treating malignant pleural effusion.
  • the dose of docetaxel polymer micelles is 5-200 mg/m 2
  • the dose of recombinant human endostatin is 5-200 mg/m 2 .
  • the dose of docetaxel polymer micelles is 5-200 mg/m2, and the dose of recombinant human endostatin is 5-400 mg/time.
  • the present application provides a product, which contains (a) docetaxel polymer micelles, and (b) recombinant human vascular endostatin, used as a combination preparation simultaneously, separately or sequentially in the prevention or treatment of malignant pleural ascites use consistently.
  • the application provides the use of the above product in the preparation of medicines for preventing or treating malignant pleural effusion.
  • the application provides the use of the above product for preventing or treating malignant pleural effusion.
  • malignant ascites is selected from malignant ascites.
  • the recombinant human endostatin is an injection.
  • the administration route of the docetaxel polymer micelle described in the present application is thoracic administration or intraperitoneal administration.
  • the route of administration of the recombinant human endostatin described in this application is either thoracic administration or intraperitoneal administration.
  • the administration frequency of the docetaxel polymer micelles is once a week, and the administration frequency of the recombinant human endostatin is once a day.
  • other active ingredients are optionally included, and the other active ingredients include but are not limited to other drugs for treating malignant hydrothorax and ascites.
  • the "combination” mentioned in this application is a way of administration, which refers to the administration of at least one dose of docetaxel polymer micelles and at least one dose of recombinant human endostatin within a certain period of time. Said time period may be within a dosing cycle.
  • Docetaxel polymer micelles and recombinant human endostatin can be administered simultaneously or sequentially. Included within this term are treatments in which the docetaxel polymeric micelles and recombinant human endostatin are administered by the same route of administration or by different routes of administration.
  • the administration mode of the combination described in the present application is selected from simultaneous administration, independent configuration and co-administration or independent configuration and sequential administration.
  • the docetaxel polymer micelle comprises docetaxel and polyethylene glycol monomethyl ether-polylactic acid block copolymer.
  • the molecular weight of the polyethylene glycol monomethyl ether-polylactic acid block copolymer is 2000-7000, and the polydispersity coefficient PD ⁇ 1.5.
  • the molecular weight of the polyethylene glycol monomethyl ether-polylactic acid block copolymer is 3000-5000, and the polydispersity coefficient PD ⁇ 1.5.
  • the polyethylene glycol monomethyl ether-polylactic acid block copolymer is a block copolymer formed by polymerization of D, L-lactide and polyethylene glycol monomethyl ether, polyethylene glycol monomethyl ether
  • the mass ratio of alcohol monomethyl ether to D,L-lactide is 1:0.6-1.6, preferably 1:0.9-1.3, more preferably 1:1.05-1.25, more preferably 1:1.1-1.2, and even more preferably 1 :1.1.
  • the polyethylene glycol monomethyl ether has a molecular weight of 1000-20000, preferably 1000-5000, more preferably 1800-2200, even more preferably 2000.
  • the polyethylene glycol monomethyl ether-polylactic acid block copolymer is prepared by the following method: (a) adding polyethylene glycol monomethyl ether to the reactor, heating and melting; (b ) Add D,L-lactide, add a metal catalyst, and then heat to 125-150°C to react; (c) Cool down after the reaction, add the first organic solvent to dissolve, add the second organic solvent, filter, and dry the filter cake in vacuum , to obtain polyethylene glycol monomethyl ether-polylactic acid block copolymer.
  • the polyethylene glycol monomethyl ether-polylactic acid block copolymer is prepared by the following method: (a) adding polyethylene glycol monomethyl ether to the reactor, heating and melting under vacuum, Inert gas replacement; (b) Add D,L-lactide, inert gas replacement, add metal catalyst, ensure negative pressure or inert gas protection in the reactor, and then heat to 125-150°C for reaction; (c) The reaction is over After cooling down, add the first organic solvent to dissolve, add the second organic solvent, filter, and vacuum-dry the filter cake to obtain polyethylene glycol monomethyl ether-polylactic acid block copolymer.
  • the polyethylene glycol monomethyl ether in step (a) is heated to 60-130° C. under vacuum.
  • the metal catalyst of step (b) is stannous isooctanoate.
  • the mass of stannous isooctanoate accounts for 0.05-0.5 wt%, preferably 0.15-0.3 wt%, of the total mass of D,L-lactide and polyethylene glycol monomethyl ether.
  • reaction time of step (b) is 1-20 h, preferably 3-10 h, more preferably 4-6 h.
  • the first organic solvent described in step (c) is one of acetonitrile, acetone, dichloromethane, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, short-chain fatty alcohol or ethyl acetate one or more, preferably one or more of dichloromethane, chloroform, methyl alcohol, ethanol, isopropanol, more preferably dichloromethane; the consumption of the first organic solvent accounts for the quality of polyethylene glycol monomethyl ether 0.1-100 times the volume (L/kg), preferably 0.2-50 times the volume (L/kg), more preferably 0.5-20 times the volume (L/kg).
  • the second organic solvent described in step (c) is one or more of diethyl ether, isopropyl ether, methyl tert-butyl ether, n-heptane, cyclohexane, petroleum ether, preferably One or more of diethyl ether, isopropyl ether or methyl tert-butyl ether, more preferably diethyl ether; the amount of the second organic solvent accounts for 1 to 100 times the volume (L/kg) of the total mass of polyethylene glycol monomethyl ether , preferably 10 to 50 times the volume (L/kg), more preferably 15 to 20 times the volume (L/kg).
  • step (b) after the reaction in step (b), it also includes the steps of adding a third organic solvent and activated carbon, stirring, filtering, and concentrating the filtrate under reduced pressure.
  • the third organic solvent is dichloromethane, chloroform, ethyl acetate, isopropyl acetate, butyl acetate, butanone, toluene, acetone, acetonitrile, dimethylformamide, di One or more of methyl sulfoxide, tetrahydrofuran, 1,4-dioxane, and short-chain fatty alcohols, preferably one or more of methylene chloride, chloroform, methanol, ethanol, and isopropanol , more preferably dichloromethane.
  • the amount of the third organic solvent accounts for 0.1 to 100 times the volume (L/kg) of polyethylene glycol monomethyl ether, preferably 0.2 to 50 times the volume (L/kg), more preferably 0.5 to 100 times the volume (L/kg). 20 times the volume (L/kg).
  • the amount of activated carbon accounts for 1-500wt% of the total mass of D,L-lactide and polyethylene glycol monomethyl ether, preferably 5-200wt%, more preferably 5-75wt%, even more preferably 20-50wt%.
  • the mass ratio of docetaxel to polyethylene glycol monomethyl ether-polylactic acid block copolymer is 0.01-0.15:1, preferably 0.02-0.12:1, more preferably 0.03-0.10: 1, more preferably 0.04:0.96.
  • the docetaxel polymeric micelles further comprise a stabilizer.
  • the stabilizer is one or more of citric acid, hydrochloric acid, sorbic acid, lactic acid, tartaric acid, malic acid, phosphoric acid, acetic acid, adipic acid, fumaric acid, preferably citric acid One or more of acid, tartaric acid, fumaric acid, more preferably citric acid.
  • the mass ratio of docetaxel to the stabilizer is 1:0.00125-0.25, preferably 1:0.005-0.25, more preferably 1:0.0125-0.125.
  • the docetaxel polymer micelles comprise the following components:
  • the docetaxel polymer micelles are prepared by the following method:
  • step (2) The micelle solution prepared in step (1) is sterilized by filtration and freeze-dried to obtain a lyophilized preparation of docetaxel polymer micelles.
  • step (1) is to dissolve the above-mentioned polyethylene glycol monomethyl ether-polylactic acid block copolymer, stabilizer and docetaxel in an organic solvent, and remove the organic solvent by rotary evaporation to obtain a gel-like
  • the drug film is then added to the drug film to dissolve and disperse the drug film to obtain a micellar solution.
  • the organic solvent described in step (1) is selected from any of acetonitrile, acetone, dichloromethane, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, short-chain fatty alcohols, and ethyl acetate.
  • the amount of the organic solvent described in step (1) is 0.5-12 ml of organic solvent per gram of polyethylene glycol monomethyl ether polylactic acid block copolymer, preferably 3-12 ml of organic solvent.
  • the amount of water added to the drug film is 2 to 40 ml of water per gram of polyethylene glycol monomethyl ether polylactic acid block copolymer, preferably per gram of polyethylene glycol monomethyl ether polylactic acid block copolymer.
  • the conditions for removing the organic solvent by rotary evaporation are as follows: a rotation speed of 10-150 rpm, a temperature of 20-80° C., and a time of 1-4 hours.
  • This application uses docetaxel polymer micelles alone or in combination with recombinant human vascular endostatin for the treatment of malignant pleural effusion, which can significantly prolong the survival period, improve the therapeutic effect of pleural effusion, increase drug plasma exposure, and reduce the concentration of VEGF. secretion.
  • VEGF refers to vascular endothelial growth factor (vascular endothelial growth factor), which plays an important role in the pathophysiological mechanism of ascites formation, can significantly increase vascular permeability, is one of the important factors in the formation of ascites, and malignant ascites The VEGF value increased significantly, and the higher the VEGF value, the worse the prognosis of the patient.
  • Malignant pleural effusion refers to malignant pleural effusion and/or malignant ascites
  • malignant pleural effusion can also be called malignant pleural effusion
  • malignant ascites can also be called malignant ascites.
  • Docetaxel polymer micelles refers to "docetaxel polymer micelles for injection”.
  • Docetaxel injection refers to injections of common dosage forms of docetaxel, such as Taxotere.
  • Polyethylene glycol monomethyl ether can be abbreviated as MPEG or mPEG.
  • Polyethylene glycol monomethyl ether-polylactic acid block copolymer can be abbreviated as MPEG-PDLLA.
  • body surface area (m 2 ) K ⁇ (W 2/3 /10000)
  • W is the general body weight (g) of the animal, and the mouse is calculated according to 20g; K is a constant, and the K value of the mouse is 9.1, the K value of the rat is 9.1, the K value of the guinea pig is 9.8, the K value of the rabbit is 10.1, and the K value of the cat is 9.1.
  • the K value is 9.8, the dog K value is 11.2, the monkey K value is 11.8, and the human K value is 10.6.
  • 5 mg/kg x 0.02 kg/0.0067 m 2 15 mg/m 2
  • the mouse dose of 5 mg/kg can be converted to 15 mg/m 2 .
  • Fig. 1 is the 1 H NMR spectrum of polyethylene glycol monomethyl ether-polylactic acid block copolymer.
  • Fig. 2 is a transmission electron micrograph of the freeze-dried preparation of docetaxel polymer micelles.
  • Figure 3 is the effect of docetaxel polymer micelles on the amount of ascites in H22 ascites tumor model mice.
  • Figure 4 shows the effect of docetaxel polymer micelles on the body weight of H22 ascites tumor model mice.
  • Figure 5 shows the effect of docetaxel polymer micelles on the survival period of H22 ascites tumor model mice.
  • Figure 6 shows the effect of docetaxel polymer micelles on the VEGF content in plasma of H22 ascites tumor model mice.
  • Figure 7 shows the effect of docetaxel polymer micelles on the amount of ascites in ES-2 ascites tumor model mice.
  • Figure 8 shows the effect of docetaxel polymer micelles on the survival period of ES-2 ascites tumor model mice.
  • Figure 9 is the mean plasma concentration-time curves of docetaxel in SD rats and Beagle dogs after administration of a single intravenous dose of HT001 or DTX.
  • the examples of this application refer to the following methods to investigate the molecular weight and distribution of polyethylene glycol monomethyl ether-polylactic acid block copolymers, and the preparation examples of this application refer to the following methods to investigate the encapsulation efficiency, related substances, particle size and span of the preparations :
  • L is the integrated area of the composite peak at 5.1ppm, representing the methine group of polylactide
  • G is the integrated area of the composite peak at 3.6ppm, representing the methylene group of polyoxyethylene
  • m is the degree of polymerization of oxyethylene in the copolymer structural formula
  • n is the degree of polymerization of D,L-lactide in the copolymer structure formula.
  • GPC was determined according to high-performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Four General Rules ⁇ 0512>).
  • Determination method Take an appropriate amount of this product, add tetrahydrofuran to prepare a 1% solution, absorb 20 ⁇ L, inject it into a liquid chromatograph, use polystyrene standard substance as a standard sample, and use GPC software to process the data.
  • Reference substance solution (containing about 0.2mg/ml docetaxel): Accurately weigh 10mg of docetaxel reference substance in a 50ml measuring bottle, add diluent to dissolve and dilute to the mark, shake well, and use it as a reference substance for content determination solution.
  • Test product stock solution (containing docetaxel 1mg/ml): take 5 bottles of this product (each bottle contains 20mg docetaxel), add 20ml of 0.9% sodium chloride injection respectively, vortex and mix for 10 minutes, As the stock solution of the test article.
  • Solution before centrifugation Take 4ml of the stock solution of the test product, centrifuge at 10000G for 10 minutes, accurately measure 2.0ml of the supernatant, place it in a 10ml measuring bottle, dilute to the mark with diluent, shake well, and use it as the solution after centrifugation.
  • HPLC chromatographic conditions are: ODS is used as filler, 0.043mol/L ammonium acetate aqueous solution-acetonitrile (45:55) is used as mobile phase, and the detection wavelength is 230nm.
  • Injection volume 20 ⁇ L.
  • Test solution Take 1 bottle of docetaxel polymer micelle freeze-dried preparation, add appropriate amount of diluent to dissolve and transfer to a 50ml measuring bottle, wash the vial with appropriate amount of diluent no less than 3 times and transfer all to the measuring bottle In the bottle, add diluent to dilute to the mark, shake well;
  • Control solution Accurately measure 1ml of the test product, put it in a 100ml measuring bottle, add diluent to the mark, and shake well.
  • Excipient solution Accurately weigh an appropriate amount of polyethylene glycol monomethyl ether-polylactic acid block copolymer (MPEG-PDLLA), add diluent to dissolve and dilute to make a solution containing about 9.6mg per 1ml.
  • MPEG-PDLLA polyethylene glycol monomethyl ether-polylactic acid block copolymer
  • Sensitivity solution Take an appropriate amount of docetaxel reference substance, weigh it accurately, add diluent to dissolve and dilute to make a solution containing about 0.2 ⁇ g per 1 ml.
  • System suitability requirements In the system suitability solution chromatogram, the separation degree of docetaxel and impurities B, C and D should meet the requirements. In the sensitivity solution chromatogram, the signal-to-noise ratio of the docetaxel peak height should be greater than 10.
  • Judgment criteria if there are impurity peaks in the chromatogram of the test solution, deduct the excipient peaks, and calculate the impurity content by the main component self-comparison method multiplied by the correction factor (1.0). In the chromatogram of the test solution, peaks that are less than 0.05 times the area of the main peak of the control solution are ignored.
  • Particle size and span detection method Take docetaxel polymer micelles freeze-dried preparation, add 20mL of 0.9% sodium chloride injection to prepare a solution, follow the guidelines for microparticle preparations (Chinese Pharmacopoeia 2020 Edition Four General Rules ⁇ 9014>) and particle size and particle size distribution determination method (Chinese Pharmacopoeia 2020 Edition Four General Rules ⁇ 0982> third method) for determination.
  • Embodiment 1 the preparation of polyethylene glycol monomethyl ether-polylactic acid block copolymer
  • Embodiment 2 the preparation of docetaxel polymer micelle lyophilized preparation
  • Preparation process (1) Weigh the polyethylene glycol monomethyl ether-polylactic acid block copolymer prepared in Example 1 of the recipe amount, add it to 50% of the recipe amount of methanol and ultrasonically dissolve it until it is completely dissolved to obtain a methanol solution.
  • Embodiment 3-5 the preparation of docetaxel polymer micelle lyophilized preparation
  • Docetaxel polymer micelles were prepared according to the following dosages, and related substances (total impurities) were investigated.
  • the product was dissolved in dichloromethane equivalent to 0.5 times the weight of the reactant, and immediately added with cold anhydrous diethyl ether equivalent to 10 times the weight of the reactant to precipitate , after stirring for 30min, let stand and filter.
  • the filter cake was refined twice according to the above operation process, and the product was vacuum-dried to obtain a polyethylene glycol monomethyl ether-polylactic acid block copolymer.
  • Example 3 Example 4
  • Example 5 Related substances (total miscellaneous) 0.43% 0.28% 0.26% .
  • Embodiment 6-7 the preparation of docetaxel polymer micelle
  • docetaxel polymer micelles according to the preparation method in embodiment 2, prepare docetaxel according to the following dosage After the polymer micelles are fully hydrated, the temperature of the micellar solution is cooled, and then diluted with 0.9% sodium chloride injection to contain about 1 mg/mL of docetaxel, and the properties and pH value are investigated.
  • Embodiment 8-9 preparation of docetaxel polymer micelles
  • docetaxel polymer micelles according to the preparation method in embodiment 2, prepare docetaxel according to the following dosage After the polymer micelles are fully hydrated, the temperature of the micellar solution is cooled, and then diluted with 0.9% sodium chloride injection to contain about 1 mg/mL of docetaxel, and the properties and pH value are investigated.
  • Embodiment 10-11 preparation of docetaxel polymer micelles lyophilized preparation
  • docetaxel polymer micelles according to the preparation method in embodiment 2, prepare docetaxel according to the following dosage After the polymer micelles are fully hydrated, the temperature of the micellar solution is cooled, and then diluted with 0.9% sodium chloride injection to contain about 1 mg/mL of docetaxel, and the properties and pH value are investigated.
  • Embodiment 12 animal experiments
  • mice Female, 6-8 weeks old, weighing 18-22 grams, 53 in total. Provided by Shanghai Lingchang Biotechnology Co., Ltd., SPF grade. Animal supplier license number: SCXK (Shanghai) 2018-0003, experimental animal certificate number: 20180003016706.
  • Mouse liver cancer H22 cells (Nanjing Kebai CBP60230) were cultured in suspension in vitro.
  • the culture conditions were RPMI Medium 1640 medium plus 10% fetal bovine serum, 1% P/S, 37°C, 5% CO 2 incubator culture. Perform routine passaging once or twice a week. When the number reaches the requirement and the cells are in the logarithmic growth phase, the cells are collected, counted, and inoculated.
  • H22 cells Collect the H22 cells in the cell culture medium, resuspend them with PBS and adjust the cell concentration to 1 ⁇ 107 cells/mL, take 3 BALB/c mice, and inject 1 mL of the cell suspension intraperitoneally into each mouse. About 7 days after the cell injection, the abdomen of the mouse was obviously bulged, and the peritoneal ascites was extracted from the mouse, and the H22 cells were collected and washed with PBS. The H22 cells were resuspended in PBS and the concentration was adjusted to 2.5 ⁇ 10 6 cells/mL, and 0.2 mL was injected intraperitoneally into each mouse. cell suspension. Three days after cell inoculation, the mice were randomly divided into 5 experimental groups according to body weight, with 10 mice in each group, and the day of grouping was Day 0.
  • the administration volume is 10ml/kg
  • group G5 the dosage of docetaxel polymer micelles (HT001) is 20mg/kg, the frequency of administration is once a week, and the administration is 3 times in total. The dosage of the drug is 48mg/kg, and the frequency of administration is once a day, with a total of 21 administrations. All were given by intraperitoneal injection.
  • Routine inspections include observation of tumor growth and the impact of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, body weight changes (weighing three times a week), appearance signs or other abnormal situation. Animal deaths and side effects within groups were recorded based on the number of animals in each group.
  • Body weight detection Inoculation, grouping (that is, before the first administration), three times a week during the administration period, and weighing the animal body weight before euthanasia, using an electronic balance to weigh, with g as the unit, and retaining one significant figure after the decimal point.
  • the weight of ascites was measured by the differential weight method, with g as the unit, and 4 significant figures after the decimal point were reserved.
  • Use a cell counter to measure the cell concentration in the ascites with 10 6 cells/mL as the unit, and keep 1 significant figure after the decimal point.
  • Plasma collection method the whole blood is collected with an anticoagulant tube, and the supernatant is taken after centrifugation for accompanying PK/PD detection, and the sample is stored at -80°C until testing.
  • mice The remaining mice continued to be administered, and the survival period was observed. At the end of the experiment, data on the body weight and survival period of the mice were provided.
  • Figure 3 shows the effect of docetaxel polymer micelles on the amount of ascites in H22 ascites tumor model mice, administration of docetaxel polymer micelles, docetaxel polymer micelles combined with recombinant human endostatin can inhibit The amount of ascites in the model mice increased, or even disappeared completely.
  • Figure 4 shows the effect of docetaxel polymer micelles on the body weight of H22 ascites tumor model mice, after administration of docetaxel polymer micelles, docetaxel polymer micelles combined with recombinant human endostatin The weight gain is gentle and has the effect of inhibiting ascites.
  • group 3 (HT001 20mg/kg) vs. group 2 (docetaxel injection 20mg/kg) P ⁇ 0.05, there is a significant difference.
  • Figure 5 shows the effect of docetaxel polymer micelles on the survival period of H22 ascites tumor model mice.
  • Administration of docetaxel polymer micelles, docetaxel polymer micelles combined with recombinant human endostatin can significantly Prolong the survival time of mice.
  • docetaxel polymer micelle single drug significantly prolongs the survival period of ascites mice, and more than half of the mice survived by the end of the experiment.
  • VEGF content was detected with a commercially available VEGF kit (Mouse VEGF Quantikine TM ELISA Kit, R&D, Catalog Number: MMV00).
  • washing buffer add 13 mL of washing buffer concentrate to 247 mL of deionized water or distilled water to prepare 260 mL of washing buffer.
  • step 4 Repeat the cleaning in step 4.
  • reaction stop solution 100 ⁇ L of reaction stop solution to each well. Shake for 5 minutes on a microplate shaker to ensure thorough mixing.
  • Mobile phase A 0.1% formic acid
  • Mobile phase B acetonitrile (0.1% formic acid).
  • Electrospray ion source temperature 5500; sheath gas pressure (GS1), heating gas pressure (GS2) and curtain gas pressure (CUR) are 55, 55 and 35 respectively; ion source spray voltage (IS); positive ion scan mode and multiple Reaction Monitoring Mode (MRM).
  • GS1 sheath gas pressure
  • GS2 heating gas pressure
  • CUR curtain gas pressure
  • IS ion source spray voltage
  • MRM multiple Reaction Monitoring Mode
  • Docetaxel stock solution Accurately weigh an appropriate amount of docetaxel, add dimethyl sulfoxide to dissolve the sample, dilute with acetonitrile until the concentration of docetaxel is 1 mg/ml, and shake well to obtain (two The volume ratio of methyl sulfoxide to acetonitrile is 1:9).
  • Standard curve solution use docetaxel stock solution, and use 50% acetonitrile as diluent to prepare different concentration standard curve working solutions (300ug/mL, 60ug/mL, 45ug/mL, 30ug/mL, 15ug/mL , 6ug/mL, 3ug/mL, 0.6ug/mL, 0.3ug/mL, 0.15ug/mL, 0.06ug/mL).
  • docetaxel polymer micelles can significantly increase drug plasma exposure and reduce VEGF secretion.
  • BALB/c nude mice female, 6-8 weeks old, weighing 18-22 grams. There are 96 in total. Provided by Shanghai Lingchang Biotechnology Co., Ltd., SPF grade. Animal supplier license number: SCXK (Shanghai) 2018-0003, laboratory animal certificate number: 20180003016704.
  • Human ovarian cancer ES-2 cells (Nanjing Kebai CBP60293) were cultured in a monolayer in vitro, and the culture conditions were RPMI Medium 1640 medium plus 10% fetal bovine serum, 1% P/S, 37°C, 5% CO 2 incubator culture . Passage with routine digestion with 0.25% trypsin-EDTA once or twice a week. When the number reaches the requirement and the cells are in the logarithmic growth phase, the cells are collected, counted, and inoculated.
  • mice Collect the ES-2 cells in the cell culture medium, resuspend them with PBS and adjust the cell concentration to 2 ⁇ 10 7 cells/mL, and inject 0.5 mL of the cell suspension intraperitoneally into each mouse. Seven days after cell inoculation, the mice were randomly divided into 6 experimental groups according to body weight, with 16 mice in each group, and the administration started on Day 0 on the day of grouping.
  • the administration volume is 10ml/kg
  • Routine inspections include observation of tumor growth and the impact of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, body weight changes (weighing three times a week), appearance signs or other abnormal situation. Animal deaths and side effects within groups were recorded based on the number of animals in each group.
  • mice were administered. The remaining mice continued to be administered, and the survival period was observed. At the end of the experiment, the survival data of the mice were provided.
  • Figure 7 shows the effect of docetaxel polymer micelles on the amount of ascites in ES-2 ascites tumor model mice, given docetaxel polymer micelles, docetaxel polymer micelles combined with recombinant human endostatin It can inhibit the increase of ascites in model mice.
  • Docetaxel polymer micelles single drug combined with docetaxel polymer micelles and recombinant human endostatin can reduce the amount of ascites and the number of cells in ascites in ES-2 model mice.
  • Figure 8 shows the effect of docetaxel polymer micelles on the survival of ES-2 ascites tumor model mice, given docetaxel polymer micelles, docetaxel polymer micelles combined with recombinant human endostatin Can significantly prolong the survival time of mice.
  • Docetaxel polymer micelles single drug combined with docetaxel polymer micelles and recombinant human endostatin can prolong the survival of ascites mice.
  • mice Female, 6-8 weeks old, weighing 18-22 grams, 83 in total. Provided by Shanghai Lingchang Biotechnology Co., Ltd., SPF grade. Animal supplier license number: SCXK (Shanghai) 2018-0003, experimental animal certificate number: 20180003016705.
  • Mouse liver cancer H22 cells (Nanjing Kebai CBP60230) were cultured in suspension in vitro.
  • the culture conditions were RPMI Medium 1640 medium plus 10% fetal bovine serum, 1% P/S, 37°C, 5% CO 2 incubator culture. Perform routine passaging once or twice a week. When the number reaches the requirement and the cells are in the logarithmic growth phase, the cells are collected, counted, and inoculated.
  • H22 cells Collect the H22 cells in the cell culture medium, resuspend them with PBS and adjust the cell concentration to 1 ⁇ 107 cells/mL, take 3 BALB/c mice, and inject 1 mL of the cell suspension intraperitoneally into each mouse. About 7 days after the cell injection, the abdomen of the mouse was obviously bulged, and the peritoneal ascites was extracted from the mouse, and the H22 cells were collected and washed with PBS. The H22 cells were resuspended in PBS and the concentration was adjusted to 2.5 ⁇ 10 6 cells/mL, and 0.2 mL was injected intraperitoneally into each mouse. cell suspension. Three days after cell inoculation, the mice were randomly divided into 5 experimental groups according to body weight, with 16 mice in each group, and the day of grouping was Day 0.
  • the administration volume is 10ml/kg
  • Routine inspections include observation of tumor growth and the impact of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake, body weight changes (weighing three times a week), appearance signs or other abnormal situation. Animal deaths and side effects within groups were recorded based on the number of animals in each group.
  • the animals were counted for ascites volume and ascites cells.
  • the volume of ascites was measured with a syringe, and the unit of mL was to keep one effective figure after the decimal point.
  • the weight of ascites was measured by the differential weight method, with g as the unit, and 4 significant figures after the decimal point were reserved. Use a cell counter to measure the cell concentration in the ascites, with 10 6 cells/mL as the unit, and keep 1 significant figure after the decimal point.
  • mice were administered, and the survival period was observed. At the end of the experiment, provide the survival data.
  • Docetaxel polymer micelles single drug, docetaxel polymer micelles and recombinant human endostatin can reduce the amount of ascites and the number of cells in ascites in H22 model mice.
  • Sprague-Dawley (SD) rats 8 weeks old, weighing 215-364 g before the start of dosing. A total of 24, half male and half male. Bought from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SPF grade.
  • Beagle dogs male/female
  • weighing 7.73-8.37kg about 6-8 months old, 12 in total, half male and half male, purchased from Mars Biotechnology Co., Ltd., ordinary grade.
  • Docetaxel injection ( hereinafter referred to as DTX) was purchased from Sanofi-Aventis GmbH (Germany). The docetaxel injection was prepared as the mother solution according to the instructions, and diluted with normal saline to the corresponding concentration of the working solution. HT001 group were all diluted with normal saline.
  • SD rats were intravenously injected with a single dose of 2.5, 5, 10 mg/kg HT001 or 5 mg/kg DTX (6 rats in each group, the administration concentrations were 0.25, 0.5, 1, 0.5 mg/mL, respectively).
  • the administration concentrations were 0.25, 0.5, 1, 0.5 mg/mL, respectively.
  • 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 24 hours after administration about 0.5 mL of whole blood was collected from the jugular vein of the animal, and the blood samples were collected and put into EP tubes containing heparin sodium for anticoagulation. Blood samples were centrifuged at 3000 revolutions per minute (rpm) for 10 minutes to obtain plasma and analyzed by LC-MS/MS (Waters Corp., Manchester, UK).
  • Beagle dogs were intravenously injected with a single dose of 1 mg/kg of DTX or HT001 (6 dogs in each group, the administration concentration was 0.2 mg/mL). Blood was collected at 0.25, 0.5, 0.55, 1, 2, 4, 6, 8, and 24 hours after administration, and about 1 mL of blood was collected from the subcutaneous vein of the forelimb or hind limb at each time point. In EP tube. Blood samples were centrifuged at 3000 rpm for 10 minutes to obtain plasma and analyzed by LC-MS/MS.
  • Observation content observe the animal's death or near-death condition, mental state, behavioral activity, eating situation, feces properties, etc. by the side of the cage.
  • LCSOLUTION system working software (Version 1.25) outputs the original spectrum and calculates the concentration of the determination sample.
  • the mean plasma concentration-time profiles of docetaxel following intravenous administration of a single dose of HT001 or DTX in SD rats are shown in Figure 9A.
  • the PK parameters of SD rats are listed in the table below.
  • both Cmax and AUClast values increased with the increase of dose, and the proportion of increased exposure was higher than that of increased dose.
  • the AUClast value of docetaxel in rats after DTX administration was significantly lower than that of the HT001 treatment group.
  • HT001 showed similar PK and tissue distribution profiles to DTX in non-rodent hosts.
  • allergic reactions such as skin flushing, eyelid swelling and redness, and shortness of breath were observed in all dogs in the DTX group, but this phenomenon was not observed in the same dose of HT001 group.

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Abstract

公开了多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途。将多西他赛聚合物胶束用于治疗恶性胸腹水，能够显著延长生存期，增加胸腹水治疗效果，提高药物血浆暴露量，减少VEGF的分泌。

Description

多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途
本申请要求2021年09月10日向中国国家知识产权局提交的，专利申请号为202111063703.5，发明名称为“多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途”以及2022年08月03日向中国国家知识产权局提交的，专利申请号为202210928962.8，发明名称为“多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途”的中国专利申请的优先权。上述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本申请涉及医药领域，具体而言，涉及多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途。
背景技术
多西他赛(docetaxel，DTX)亦称为多西紫杉醇，分子式C 43H 53NO 14，分子量为807.88，为紫杉醇类抗肿瘤药，其可与游离的微管蛋白结合，促进微管蛋白装配成稳定的微管，同时抑制其解聚，导致丧失了正常功能的微管束的产生和微管的固定，从而抑制细胞的有丝分裂，发挥抗肿瘤作用。但是，多西他赛具有水溶性差、半衰期短及毒性大等缺点，临床上通常是经静脉输注给药。纳米聚合物胶束是近年来发展起来的针对难溶性药物的载药系统，具有核-壳状结构，其中核为疏水性部分，壳为亲水性部分。聚合物胶束可以将难溶性药物包裹于核部分达到对难溶性药物的增溶。
重组人血管内皮抑制素是通过修饰人血管内皮抑制素的核苷酸编码序列使其N末端带有9个附加氨基酸序列。所生产的重组人血管内皮抑制素由192个氨基酸构成，其氨基酸序列为：(M)GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK。目前市售的重组人血管内皮抑制素为普通注射液，联合NP化疗方案用于治疗初治或复治的III/IV期非小细胞肺癌患者。
恶性肿瘤患者常常伴有恶性胸腹水并发症，严重影响患者的治疗、生活质量和生存时间。其中30-40％的病例是顽固性难治患者，临床上没有标准治疗方案，预后较差。目前临床治疗恶性胸腹水的主要手段是反复胸腹腔穿刺、置引流管、利尿和全身化疗等，但是效果并不能满足临床需求，而且会引起如壁层和脏层粘连包裹，全身性感染等不良反应。近年来，由于胸腹壁-血浆屏障存在延缓药物进入血浆，减少全身不良反应，增加药物与胸腹水中肿瘤细胞接触时间，提高抑制胸腹水的作用，腔内化疗成为治疗恶性胸腹水的手段，但也仍然存在生存时间短、预后较差、生活质量低等问题。
发明内容
本申请的目的是提供多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途。
本申请提供多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途。
本申请提供预防或治疗哺乳动物恶性胸腹水的方法，包括对需要该预防或治疗的哺乳动 物，优选人类，给予多西他赛聚合物胶束。
本申请提供多西他赛聚合物胶束在预防或者治疗恶性胸腹水中的用途。
本申请提供预防或者治疗恶性胸腹水的多西他赛聚合物胶束。
在一些实施方案中，多西他赛聚合物胶束的剂量是5～200mg/m 2。
在一些实施方案中，所述多西他赛聚合物胶束的给药频次是一周一次。
本申请提供多西他赛聚合物胶束和重组人血管内皮抑制素联合在制备预防或治疗恶性胸腹水的药物中的用途。
本申请提供预防或治疗哺乳动物恶性胸腹水的方法，包括对需要该治疗的哺乳动物，优选人类，给予多西他赛聚合物胶束和重组人血管内皮抑制素。
本申请提供多西他赛聚合物胶束和重组人血管内皮抑制素联合在预防或者治疗恶性胸腹水中的用途。
本申请提供预防或者治疗恶性胸腹水的多西他赛聚合物胶束和重组人血管内皮抑制素。
在一些实施方案中，多西他赛聚合物胶束的剂量是5～200mg/m 2，所述重组人血管内皮抑制素的剂量是5～200mg/m 2。
在一些实施方案中，多西他赛聚合物胶束的剂量是5～200mg/m2，所述重组人血管内皮抑制素的剂量是5～400mg/次。
本申请提供一种产品，所述产品含有(a)多西他赛聚合物胶束，和(b)重组人血管内皮抑制素，在预防或者治疗恶性胸腹水中作为组合制剂同时、分别或序贯使用。
本申请提供上述产品在制备预防或者治疗恶性胸腹水的药物中的用途。
本申请提供上述产品预防或治疗恶性胸腹水的用途。
在一些实施方案中，恶性胸腹水选自恶性腹水。
在一些实施方案中，所述重组人血管内皮抑制素是注射液。
在一些实施方案中，本申请所述多西他赛聚合物胶束的给药途径为胸腔给药或腹腔给药。
在一些实施方案中，本申请所述重组人血管内皮抑制素的给药途径为或胸腔给药或腹腔给药。
在一些实施方案中，所述多西他赛聚合物胶束的给药频次是一周一次，所述重组人血管内皮抑制素的给药频次是一日一次。
本申请所述的方案中，任选的还包含其它活性成分，所述其它活性成分包括但不限于其他治疗恶性胸腹水的药物等。
本申请所述的“联合”是一种给药方式，是指一定时间期限内给与至少一种剂量的多西他赛聚合物胶束和至少一种剂量的重组人血管内皮抑制素。所述的时间期限可以是一个给药周期内。可以同时或依次给予多西他赛聚合物胶束和重组人血管内皮抑制素。这种期限内包括这样的治疗，其中通过相同给药途径或不同给药途径给予多西他赛聚合物胶束和重组人血管内皮抑制素。本申请所述联合的给药方式选自同时给药、独立地配置并共给药或独立地配置并相继给药。
在一些实施方案中，所述多西他赛聚合物胶束包含多西他赛和聚乙二醇单甲醚-聚乳酸嵌段共聚物。
在一些实施方案中，所述聚乙二醇单甲醚-聚乳酸嵌段共聚物的分子量是2000～7000，且多分散系数PD≤1.5。优选地，所述聚乙二醇单甲醚-聚乳酸嵌段共聚物的分子量是3000～5000，且多分散系数PD≤1.5。
在一些实施方案中，所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物为D,L-丙交酯与聚乙二醇单甲醚聚合形成的嵌段共聚物，聚乙二醇单甲醚与D,L-丙交酯的投料质量比为1:0.6～1.6，优选1：0.9～1.3，更优选1:1.05～1.25，进一步优选1:1.1～1.2，更进一步优选1:1.1。
在一些实施方案中，所述聚乙二醇单甲醚的分子量为1000～20000，优选1000～5000，更优选1800～2200，进一步优选2000。
在一些实施方案中，所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物通过如下方法制备得到：(a)向反应器中加入聚乙二醇单甲醚，加热熔融；(b)加入D,L-丙交酯，加入金属催化剂，然后加热至125～150℃反应；(c)反应结束后降温，加入第一有机溶剂溶解，加入第二有机溶剂，过滤，滤饼真空干燥，得到聚乙二醇单甲醚-聚乳酸嵌段共聚物。
在一些实施方案中，所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物通过如下方法制备得到：(a)向反应器中加入聚乙二醇单甲醚，真空下加热熔融，惰性气体置换；(b)加入D,L-丙交酯，惰性气体置换，加入金属催化剂，保证反应器中为负压或惰性气体保护，然后加热至125～150℃反应；(c)反应结束后降温，加入第一有机溶剂溶解，加入第二有机溶剂，过滤，滤饼真空干燥，得到聚乙二醇单甲醚-聚乳酸嵌段共聚物。
在一些实施方案中，步骤(a)所述聚乙二醇单甲醚真空下加热至60～130℃。
在一些实施方案中，步骤(b)金属催化剂为异辛酸亚锡。
在一些实施方案中，异辛酸亚锡的质量占D,L-丙交酯和聚乙二醇单甲醚总质量的0.05～0.5wt％，优选0.15～0.3wt％。
在一些实施方案中，步骤(b)反应时间是1～20h，优选3～10h，更优选4～6h。
在一些实施方案中，步骤(c)所述的第一有机溶剂为乙腈、丙酮、二氯甲烷、二甲基甲酰胺、二甲亚砜、四氢呋喃、短链脂肪醇或乙酸乙酯中的一种或几种，优选二氯甲烷、三氯甲烷、甲醇、乙醇、异丙醇中的一种或几种，更优选二氯甲烷；第一有机溶剂的用量占聚乙二醇单甲醚质量0.1～100倍体积(L/kg)，优选0.2～50倍体积(L/kg)，更优选0.5～20倍体积(L/kg)。
在一些实施方案中，步骤(c)所述的第二有机溶剂为乙醚、异丙醚、甲基叔丁基醚、正庚烷、环己烷、石油醚中的一种或几种，优选乙醚、异丙醚或甲基叔丁基醚中的一种或几种，更优选乙醚；第二有机溶剂的用量占聚乙二醇单甲醚总质量1～100倍体积(L/kg)，优选10～50倍体积(L/kg)，更优选15～20倍体积(L/kg)。
在一些实施方案中，步骤(b)反应结束后还包括加入第三有机溶剂和活性炭，搅拌，过滤，滤液减压浓缩的步骤。
在一些实施方案中，所述第三有机溶剂为二氯甲烷、三氯甲烷、乙酸乙酯、乙酸异丙酯、 乙酸丁酯、丁酮、甲苯、丙酮、乙腈、二甲基甲酰胺、二甲亚砜、四氢呋喃、1,4-二氧六环、短链脂肪醇中的一种或几种，优选二氯甲烷、三氯甲烷、甲醇、乙醇、异丙醇中的一种或几种，更优选为二氯甲烷。
在一些实施方案中，所述第三有机溶剂的用量占聚乙二醇单甲醚质量0.1～100倍体积(L/kg)，优选0.2～50倍体积(L/kg)，更优选0.5～20倍体积(L/kg)。
在一些实施方案中，所述活性炭的用量占D,L-丙交酯和聚乙二醇单甲醚总质量的1～500wt％，优选5～200wt％，更优选5～75wt％，进一步优选20～50wt％。
在一些实施方案中，所述多西他赛与聚乙二醇单甲醚-聚乳酸嵌段共聚物的质量比为0.01～0.15:1，优选0.02～0.12:1，更优选0.03～0.10:1，进一步优选0.04:0.96。
在一些实施方案中，所述多西他赛聚合物胶束还包含稳定剂。
在一些实施方案中，所述稳定剂是枸橼酸、盐酸、山梨酸、乳酸、酒石酸、苹果酸、磷酸、醋酸、己二酸、富马酸中的一种或几种，优选是枸橼酸、酒石酸、富马酸中的一种或几种，更优选是枸橼酸。
在一些实施方案中，所述多西他赛与稳定剂的质量比是1：0.00125～0.25，优选是1：0.005～0.25，更优选是1:0.0125～0.125。
在一些实施方案中，所述多西他赛聚合物胶束包含以下配比的组分：
Figure PCTCN2022117973-appb-000001
在一些实施方案中，所述多西他赛聚合物胶束通过以下方法制备而成：
(1)将上述聚乙二醇单甲醚-聚乳酸嵌段共聚物和多西他赛溶于有机溶剂，旋转蒸发除去有机溶剂，得到凝胶状药膜，然后向药膜中加入水溶解分散所述药膜，制得胶束溶液；
(2)将步骤(1)制得的胶束溶液经过滤除菌、冷冻干燥后得到多西他赛聚合物胶束冻干制剂。
在一些实施方案中，步骤(1)是将上述聚乙二醇单甲醚-聚乳酸嵌段共聚物、稳定剂和多西他赛溶于有机溶剂，旋转蒸发除去有机溶剂，得到凝胶状药膜，然后向药膜中加入水溶解分散所述药膜，制得胶束溶液。
在一些实施方案中，步骤(1)中所述的有机溶剂选自乙腈、丙酮、二氯甲烷、二甲基甲酰胺、二甲亚砜、四氢呋喃、短链脂肪醇、乙酸乙酯中的任意一种或几种，优选甲醇、乙醇、异丙醇、乙腈、乙酸乙酯中的任意一种或几种，更优选甲醇。
在一些实施方案中，步骤(1)中所述的有机溶剂的用量为每克聚乙二醇单甲醚聚乳酸嵌段共聚物中加入0.5～12ml有机溶剂，优选加入3～12ml有机溶剂。
在一些实施方案中，步骤(1)中，向药膜中加入水的用量为每克聚乙二醇单甲醚聚乳酸嵌段共聚物加入2～40ml水，优选地为每克聚乙二醇单甲醚聚乳酸嵌段共聚物加入10～20ml水，更优选地为每克聚乙二醇单甲醚聚乳酸嵌段共聚物加入10～15ml水。
在一些实施方案中，步骤(2)中，旋转蒸发去除有机溶剂的条件为：旋转速度10～150rpm，温度20～80℃，时间1～4h。
本申请将多西他赛聚合物胶束单用或与重组人血管内皮抑制素联合用于治疗恶性胸腹水，能够显著延长生存期，提高胸腹水治疗效果，提高药物血浆暴露量，减少VEGF的分泌。
术语定义和说明
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
“任选”或“任选地”意味着随后所描述的事件可以但不必发生，该说明包括该事件发生或不发生的场合。
“VEGF”是指血管内皮生长因子(vascular endothelial growth factor)，其在腹水形成的病理生理机制中起重要作用，可使血管通透性明显增加，是腹水形成重要因素之一，且恶性腹水中VEGF值明显升高，VEGF值越高，患者预后越差。
恶性胸腹水是指恶性胸水和/或恶性腹水，恶性胸水也可以称为恶性胸腔积液，恶性腹水也可以称为恶性腹腔积液。
“多西他赛聚合物胶束”是指“注射用多西他赛聚合物胶束”。
“多西他赛注射液”均是指多西他赛普通剂型的注射液，例如泰索帝。
聚乙二醇单甲醚可简写为MPEG或者mPEG。
聚乙二醇单甲醚-聚乳酸嵌段共聚物可简写为MPEG-PDLLA。
剂量单位由mg/kg转换成mg/m 2的方法，按照以下步骤：
(1)由体重推算体表面积：体表面积(m 2)＝K×(W 2/3/10000)
式中W为动物的一般体重(g)，小鼠按照20g计算；K为常数，小鼠K值是9.1、大鼠K值是9.1、豚鼠K值是9.8、家兔K值是10.1、猫K值是9.8、犬K值是11.2、猴K值是11.8、人K值是10.6。
(2)剂量(mg/kg)×W(kg)/体表面积＝mg/m 2
例如，小鼠剂量是5mg/kg，体表面积是9.1×(20 2/3/10000)＝0.0067m 2，然后5mg/kg×0.02kg/0.0067m 2＝15mg/m 2。因此小鼠剂量5mg/kg可以转换成15mg/m 2。
附图说明
图1为聚乙二醇单甲醚-聚乳酸嵌段共聚物的 1HNMR图谱。
图2为多西他赛聚合物胶束冻干制剂透射电镜图。
图3为多西他赛聚合物胶束对H22腹水瘤模型小鼠腹水量的影响。
图4为多西他赛聚合物胶束对H22腹水瘤模型小鼠体重的影响。
图5为多西他赛聚合物胶束对H22腹水瘤模型小鼠生存期的影响。
图6为多西他赛聚合物胶束对H22腹水瘤模型小鼠血浆中VEGF含量的影响。
图7为多西他赛聚合物胶束对ES-2腹水瘤模型小鼠腹水量的影响。
图8为多西他赛聚合物胶束对ES-2腹水瘤模型小鼠生存期的影响。
图9为在SD大鼠、Beagle犬中通过静脉注射单剂量HT001或DTX给药后多西紫杉醇的平均血浆浓度-时间曲线。
具体实施方式
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本申请的精神和范围下可以对本申请技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
本申请实施例参考以下方法考察聚乙二醇单甲醚-聚乳酸嵌段共聚物的分子量和分布，本申请制剂实施例参考以下方法考察制剂的包封率、有关物质、粒径和跨距：
(1)分子量及分布检测方法
1HNMR 取1％四甲基硅烷的氘代氯仿10％～20％(g/mL)本品溶液0.5～1.0mL，从0ppm扫描到10ppm，以直接比较法定量，具体方法为：3.6ppm处为聚乳酸中CH的峰，5.1ppm处为聚乙二醇单甲醚中CH2的峰，峰面积与氢的数目存在以下关系：聚乙二醇单甲醚分子量2000，L/G＝2m/4n，m＝2000/44＝45.5，则可求出共聚物的组成及分子量。
Figure PCTCN2022117973-appb-000002
共聚物的分子量＝(1+PDLLA/MPEG的重量比)×2000
式中：L为5.1ppm处复合峰的积分面积，代表聚丙交酯的次甲基；
G为3.6ppm处复合峰的积分面积，代表聚氧乙烯的亚甲基；
m为共聚物结构式中氧乙烯的聚合度；
n为共聚物结构式中D,L-丙交酯的聚合度。
GPC 照高效液相色谱法(中国药典2020年版四部通则＜0512＞)测定。
色谱条件 采用凝胶色谱柱；示差折光检测；四氢呋喃为流动相；流速1.0mL/min，柱温40℃。
测定法 取本品适量，加四氢呋喃制备成1％的溶液，吸取20μL，注入液相色谱仪，用聚苯乙烯标准物质作为标样，数据采用GPC软件处理。
(2)包封率检测方法
取多西他赛聚合物胶束冻干制剂，用生理盐水溶解至浓度1mg/ml后放置在室温条件下(25±2℃)，检测包封率。
对照品溶液(含多西他赛约0.2mg/ml)：精密称取10mg多西他赛对照品于50ml量瓶中，加稀释剂溶解并稀释至刻度，摇匀，作为含量测定的对照品溶液。
供试品储备溶液(含多西他赛1mg/ml)：取本品5瓶(每瓶含多西他赛20mg)，分别 加0.9％氯化钠注射液20ml，涡旋混匀10分钟，作为供试品储备溶液。
离心前溶液：取4ml供试品储备溶液，10000G离心力离心10分钟，精密量取上清液2.0ml，置于10ml量瓶中，用稀释剂稀释至刻度，摇匀，作为离心后溶液。
HPLC色谱条件为：以ODS为填充剂，0.043mol/L醋酸铵水溶液-乙腈(45:55)为流动相，检测波长为230nm。
(3)有关物质检测方法：
色谱条件：
流动相A：水
流动相B：乙腈
按下表梯度洗脱：
Figure PCTCN2022117973-appb-000003
稀释剂：乙腈-水-冰醋酸(100：100：0.1)
色谱柱：十八烷基硅烷键合硅胶为填充剂(4.6×250mm,5μm)
柱温：35℃；
检测波长：230nm；
流速：1.0mL/min；
进样体积：20μL。
供试品溶液：取多西他赛聚合物胶束冻干制剂1瓶，加适量稀释剂溶解并转移至50ml量瓶中，用适量稀释剂清洗西林瓶不少于3次并全部转移至量瓶中，加稀释剂稀释至刻度，摇匀；
对照溶液：精密量取供试品1ml，置100ml量瓶中，加稀释剂至刻度，摇匀。
辅料溶液：精密称取聚乙二醇单甲醚-聚乳酸嵌段共聚物(MPEG-PDLLA)适量，加稀释剂溶解并稀释制成每1ml中约含9.6mg的溶液。
灵敏度溶液：取多西他赛对照品适量，精密称定，加稀释剂溶解并稀释制成每1ml中约含0.2μg的溶液。
系统适用性溶液：取多西他赛鉴别用对照品适量，精密称定，加稀释剂溶解并稀释制成每1ml中约含0.4mg的溶液。
系统适用性要求：系统适用性溶液色谱图中，多西他赛峰与杂质B、C、D的分离度应符合要求。灵敏度溶液色谱图中，多西他赛峰高的信噪比应大于10。
分析过程：照高效液相色谱法(中国药典2020年版四部通则＜0512＞)测定。精密量取供试品溶液与对照品溶液，分别注入液相色谱仪，记录色谱图。
计算公式：
Figure PCTCN2022117973-appb-000004
总杂质(％)＝∑％单个杂质
其中：
A SPL 供试品溶液中杂质峰面积
A STD 对照溶液中主峰峰面积
f    校正因子
判断标准：供试品溶液色谱图中如有杂质峰，扣除辅料峰外，按乘以校正因子(1.0)的主成分自身对照法计算杂质含量。供试品溶液色谱图中小于对照溶液主峰面积0.05倍的峰忽略不计。
Figure PCTCN2022117973-appb-000005
(4)粒径与跨距检测方法：取多西他赛聚合物胶束冻干制剂，加0.9％氯化钠注射液20mL配制成溶液，照微粒制剂指导原则(中国药典2020年版四部通则＜9014＞)和粒度和粒度分布测定法(中国药典2020版四部通则＜0982＞第三法)进行测定。
实施例1、聚乙二醇单甲醚-聚乳酸嵌段共聚物的制备
氮气保护下向反应釜中加入4.50kg聚乙二醇单甲醚(2000)，充氮气、抽真空，加热至120℃，待全部熔融，继续加热并保持真空，干燥2h后用氮气置换，在氮气保护下加入4.95kg D,L-丙交酯，充氮气、抽真空，使反应釜中保持真空；搅拌均匀后氮气置换。升温至140℃，氮气保护下，加入18.000g异辛酸亚锡，氮气置换，氮气保护加热至140±5℃反应5h。反应结束后，产物降温至40℃，加入36L二氯甲烷和4.50kg活性炭，搅拌脱色1h，过滤，滤液减压浓缩至干。
加入4.5L二氯甲烷，搅拌下加入90L冷无水乙醚，搅拌30min后静置，过滤，滤饼按上述操作过程再精制两次，真空干燥得到聚乙二醇单甲醚-聚乳酸嵌段共聚物6.204kg，收率为65.65％。得到的共聚物用核磁共振进行表征，结果如图1所示。分子量为3647，GPC分析结果：多分散系数PD值＝1.2。
实施例2、多西他赛聚合物胶束冻干制剂的制备
配方：
Figure PCTCN2022117973-appb-000006
制备工艺：(1)称取处方量的实施例1制备得到的聚乙二醇单甲醚-聚乳酸嵌段共聚物，加入至处方量50％的甲醇中超声直至完全溶解，得到甲醇溶液。
(2)将处方量的枸橼酸加入上述甲醇溶液中溶解，溶解完成后加入处方量的多西他赛和剩余甲醇，继续溶解，然后过滤，滤液在50-60℃、80～120rpm条件下旋蒸至少1h，得到多西他赛聚合物凝胶膜。
(3)向装有多西他赛聚合物药膜的圆底旋蒸瓶中加入50-60℃左右注射用水15kg，置于回旋振荡器中水化，待完全水化，将圆底烧瓶置于冷水中冷却，得多西他赛聚合物胶束溶液。除菌过滤，西林瓶灌装，冻干，得到多西他赛聚合物胶束冻干制剂。每瓶含多西他赛20mg。
取少量多西他赛聚合物胶束冻干制剂，复溶后通过透射电镜进行检测。结果如图2所示，显示样品近似圆球型，胶束已形成。
取少量多西他赛聚合物胶束冻干制剂，检测粒径与跨距，粒径22nm，跨距0.73。
取少量多西他赛聚合物胶束冻干制剂检测检测性状、pH值、复溶后的包封率。结果如下表所示：
检测项目 检测结果
性状 淡蓝色微乳光溶液
pH值 3.7
包封率 99.9％
。
实施例3-5、多西他赛聚合物胶束冻干制剂的制备
按照以下用量制备多西他赛聚合物胶束，考察有关物质(总杂)。
Figure PCTCN2022117973-appb-000007
(1)聚乙二醇单甲醚-聚乳酸嵌段共聚物的制备
将聚乙二醇单甲醚(2000)加入到反应瓶中，充氮气、抽真空，使反应瓶中保持真空，加热至100℃～120℃，待全部熔融，继续加热2h并保持真空；氮气置换后加入D,L-丙交酯，充氮气、抽真空，使反应品中保持真空；氮气置换后按反应瓶中物料重量计，加入0.18％wt的异辛酸亚锡，氮气置换，氮气保护，加热至140℃反应5小时，待反应物冷却至室温后，产物用相当于反应物重量0.5倍体积的二氯甲烷溶解，搅拌下立即加入相当于反应物重量10倍体积冷无水乙醚沉淀，搅拌30min后，静置，过滤。滤饼按上述操作过程再精制两次，产物真空干燥，得到聚乙二醇单甲醚-聚乳酸嵌段共聚物。
(2)多西他赛聚合物胶束冻干制剂按照实施例2中的制备方法，按照上表中的用量制备得到。
取少量实施例3-5中制备的多西他赛聚合物胶束冻干制剂，测定有关物质(总杂)。结果如下表：
考察项目 实施例3 实施例4 实施例5
有关物质(总杂) 0.43％ 0.28％ 0.26％
。
实施例6-7、多西他赛聚合物胶束的制备
聚乙二醇单甲醚-聚乳酸嵌段共聚物按照实施例3步骤(1)的方法、多西他赛聚合物胶束按照实施例2中的制备方法，按照以下用量制备多西他赛聚合物胶束，在水化完全后将胶束溶液降温后，用0.9％氯化钠注射液稀释到约含多西他赛1mg/mL，考察性状和pH值。
Figure PCTCN2022117973-appb-000008
结果如下表所示：
考察条件 实施例6 实施例7
性状 淡蓝色澄明微乳光液体 淡蓝色澄明微乳光液体
pH值 3.81 3.79
实施例8-9、多西他赛聚合物胶束的制备
聚乙二醇单甲醚-聚乳酸嵌段共聚物按照实施例3步骤(1)的方法、多西他赛聚合物胶束按照实施例2中的制备方法，按照以下用量制备多西他赛聚合物胶束，在水化完全后将胶束溶液降温后，用0.9％氯化钠注射液稀释到约含多西他赛1mg/mL，考察性状、pH值。
Figure PCTCN2022117973-appb-000009
Figure PCTCN2022117973-appb-000010
结果如下表所示：
考察项目 实施例8 实施例9
性状 淡蓝色澄明微乳光液体 淡蓝色澄明微乳光液体
pH值 3.88 3.85
实施例10-11、多西他赛聚合物胶束冻干制剂的制备
聚乙二醇单甲醚-聚乳酸嵌段共聚物按照实施例3步骤(1)的方法、多西他赛聚合物胶束按照实施例2中的制备方法，按照以下用量制备多西他赛聚合物胶束，在水化完全后将胶束溶液降温后，用0.9％氯化钠注射液稀释到约含多西他赛1mg/mL，考察性状、pH值。
Figure PCTCN2022117973-appb-000011
结果如下表所示：
考察项目 实施例10 实施例11
性状 淡蓝色澄明微乳光液体 淡蓝色澄明微乳光液体
pH值 3.81 3.80
实施例12、动物实验
1、实验动物
BALB/c小鼠，雌性，6-8周龄，体重18-22克，共53只。由上海灵畅生物科技有限公司提供，SPF级。动物供应商许可证号：SCXK(沪)2018-0003，实验动物合格证号：20180003016706。
2、受试药物
Figure PCTCN2022117973-appb-000012
3、实验方法
3.1细胞培养
鼠肝癌H22细胞(南京科佰CBP60230)体外悬浮培养，培养条件为RPMI Medium 1640培养基中加10％胎牛血清，1％P/S，37℃，5％CO 2孵箱培养。一周一到两次进行常规传代。数量到达要求以及细胞在对数生长期时，收取细胞，计数，接种。
3.2肿瘤细胞接种与分组
收集细胞培养液中H22细胞，用PBS重悬并调整细胞浓度至1×10 7个/mL，取3只BALB/c小鼠，每只小鼠腹腔注射1mL细胞悬液。细胞注射7天左右小鼠腹部明显隆起，抽取小鼠腹腔腹水，收集H22细胞并用PBS洗一遍，PBS重悬H22细胞并调整浓度至2.5×10 6个/mL，每只小鼠腹腔注射0.2mL细胞悬液。细胞接种3天后，将小鼠按体重随机分组到5个实验组中，每组10只，分组当天为Day 0开始给药。
3.3分组给药方案
1)分组
给药前称重动物，根据动物体重随机分组。
2)给药
给药方案见下表：
动物实验分组和给药方案
Figure PCTCN2022117973-appb-000013
注：1.给药体积均为10ml/kg；
2.IP:腹腔注射；QD:一天一次；QW:一周一次。例如，G5组：多西他赛聚合物胶束(HT001)的给药剂量是20mg/kg，给药频次是一周一次，共给药3次，
Figure PCTCN2022117973-appb-000014
的给药剂量是48mg/kg，给药频次是一天一次，共给药21次。均是腹腔注射给药。
观察
每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量，体重变化(每周称量三次体重)，外观体征或其它不正常情况。基于各组动物数量记录组内动物死亡数和副作用。
3.4检测指标
体重检测：接种、分组(即首次给药前)、给药期间每周3次、安乐死前称取动物体重，使用电子天平称量，以g为单位，保留小数点后一位有效数字。
采用差重法对腹水重量进行计量，以g为单位，保留小数点后4位有效数字。用细胞计数仪对腹水中细胞浓度进行测量，以10 6个/mL为单位，保留小数点后1位有效数字。
4.结束实验
1)每组3只小鼠分别于第二次给药前(第8天0h)和给药后(2/4/8/12/24h)采血，末次采血后安乐死，无菌注射器腹腔抽取收集腹水并测量腹水重量。实验结束，提供小鼠体重、腹水量和腹水细胞数量数据，收集血浆做伴随PK/PD实验。
血浆收集方法：全血用抗凝管收集，离心后取上清用于伴随PK/PD检测，样品-80℃保存待测。
2)剩余小鼠继续给药，观察生存期。实验结束，提供小鼠体重、生存期数据。
5.实验结果
1)腹水情况
Figure PCTCN2022117973-appb-000015
注：a.实验终点时小鼠腹腔内无腹水。
b.利用one-way anova分析方法用GraphPad Prism 9.0对动物腹水量和腹水细胞数进行统计学分析，在所有分析中P<0.05认为有显著性差异(*:P<0.05；**:P<0.01；***:P<0.001；****:P<0.0001；ns:不显著)。
图3为多西他赛聚合物胶束对H22腹水瘤模型小鼠腹水量的影响，给予多西他赛聚合物胶束、多西他赛聚合物胶束联合重组人血管内皮抑制素可以抑制模型小鼠腹水量增加，甚至完全消退腹水。
2)体重
图4为多西他赛聚合物胶束对H22腹水瘤模型小鼠体重的影响，给予多西他赛聚合物胶束、多西他赛聚合物胶束联合重组人血管内皮抑制素后小鼠体重上升平缓，有抑制腹水作用。
3)生存期
Figure PCTCN2022117973-appb-000016
Figure PCTCN2022117973-appb-000017
注：a.至实验终点，小鼠存活比例大于50％，所有小鼠状态良好。
b.利用Log-rank(Mantel-Cox)test分析方法用GraphPad Prism 9.0对动物生存期进行统计学分析，在所有分析中P<0.05认为有显著性差异(*:P<0.05；**:P<0.01；***:P<0.001；****:P<0.0001；ns:不显著)。
另外，组3(HT001 20mg/kg)vs.组2(多西他赛注射液20mg/kg)P<0.05，有显著性差异。
图5为多西他赛聚合物胶束对H22腹水瘤模型小鼠生存期的影响，给予多西他赛聚合物胶束、多西他赛聚合物胶束联合重组人血管内皮抑制素可以显著延长小鼠生存时间。
结果说明：
多西他赛聚合物胶束单药与多西他赛注射液相比，显著延长腹水小鼠生存期，至实验结束有半数以上小鼠存活。
多西他赛聚合物胶束和重组人血管内皮抑制素联合组与多西他赛注射液和重组人血管内皮抑制素联合组相比，延长腹水小鼠生存期，至实验结束只有一只小鼠未存活。
4)PK/PD
1.伴随PD实验(VEGF含量检测)
用市售的VEGF试剂盒(Mouse VEGF Quantikine TM ELISA Kit，R&D，Catalog Number：MMV00)进行VEGF含量检测。
1.1溶液制备
1)使用5mg小鼠VEGF标准品和5mL RD5T稀释液配制500pg/mL小鼠VEGF标准品储备溶液。
2)标准品的制备：使用储备溶液等比稀释配置标准品(浓度分别为250pg/mL、125pg/mL、62.5pg/mL、31.3pg/mL、15.6pg/mL、7.8pg/mL)，在等比稀释之前，先让储备溶液静置至少5分钟。未稀释的小鼠VEGF标准品储备溶液(500pg/mL)作为高标准，稀释液RD5T作为零标准(0pg/mL)。
3)对照品的制备：用1.0mL去离子水或蒸馏水与小鼠VEGF对照品混合配制对照品，在振荡器上充分混匀，测定未稀释的对照品。
4)底物溶液的制备：将Color Reagent A和Color Reagent B按照1:1比例混合，在振荡器上混匀半分钟，避光放置。底物溶液需在使用前静置15分钟。
5)洗涤缓冲液的制备：向247mL去离子水或蒸馏水中添加13mL洗涤缓冲液浓缩液，制备260mL洗涤缓冲液。
1.2检测
1)在96孔板中，在相应的孔加入50μL的检测稀释液RD1N。
2)向每个孔中添加50μL标准品、对照品和使用RD5T稀释液稀释后的样品溶液。在微 孔板振荡器上震荡2分钟，使其充分混合。用提供的胶带盖住，在室温下孵育2小时。
3)对每孔进行抽吸和冲洗，重复该过程四次，共冲洗五次。在每个孔中注入洗涤缓冲液(400μL)。在第五次清洗后，通过抽吸或倾倒的方式清除剩余的洗涤缓冲液。
4)向每个孔中添加100μL小鼠VEGF结合物。用新的胶带盖住，在室温下孵育2小时。
5)重复步骤4)中的清洗。
6)向每个孔中添加100μL底物溶液。在室温下孵育30分钟，避光。
7)向每个孔中添加100μL反应终止液。在微孔板振荡器上震荡5分钟，确保充分混合。
8)使用PE EnSight酶标仪上测量450nm处或附近的吸光度。建立标准曲线，计算每个样本品的VEGF含量。
2.伴随PK实验
2.1色谱条件
色谱柱：Waters HPLC HSS T3 1.8μm(2.1mm*5mm)。
流动相A：0.1％甲酸；流动相B：乙腈(0.1％甲酸)。
洗脱程序见下表。
洗脱程序
时间/min 流动相A 流动相B
0 55 45
0.1 55 45
0.6 5 95
1 5 95
1.01 55 45
1.5 55 45
柱温：40℃；流速：0.6ml/min；进样量：10μl。
2.2.质谱条件
电喷雾离子源温度：5500；鞘气压力(GS1)、加热气压力(GS2)以及气帘气压力(CUR)分别为55、55及35；离子源喷雾电压(IS)；正离子扫描模式和多重反应监测模式(MRM)。
2.3溶液配制
(1)内标溶液(Docetaxel浓度为50ng/ml)：取Docetaxel(多西他赛标准品)适量，以乙腈为溶剂，配制含Docetaxel浓度为50ng/ml的溶液。
(2)多西他赛储备液：精密称取多西他赛适量，加二甲基亚砜使样品溶解，用乙腈稀释至含多西他赛浓度为1mg/ml，摇匀即得(二甲基亚砜与乙腈的体积比为1：9)。
(3)标准曲线溶液：使用多西他赛储备液，以50％乙腈为稀释液进行不同浓度标准曲线工作溶液配制(300ug/mL、60ug/mL、45ug/mL、30ug/mL、15ug/mL、6ug/mL、3ug/mL、0.6ug/mL、0.3ug/mL、0.15ug/mL、0.06ug/mL)。然后取空白血浆47.5μL，分别加入各浓度的标准曲线工作溶液2.5μL，涡旋混匀，即得入血溶液(浓度3000ng/mL、2250ng/mL、1500ng/mL、750ng/mL、300ng/mL、150ng/mL、30ng/mL、15ng/mL、7.5ng/mL、3ng/mL)。取入血溶液20μL至2mL圆底96孔板中，加入100μL内标(Docetaxel浓度为50ng/ml)，涡旋混匀3min，4℃/4000rpm离心15min，取上清20μL至2mL圆底96孔板中，加入超纯水80μL，涡旋混匀，取10μL溶液进样分析。
(2)样品前处理：取20μL血浆至2mL圆底96孔板中，加入100μL内标(Docetaxel浓度为50ng/ml)，涡旋混匀3min，4℃/4000rpm离心15min，取上清20μL至2mL圆底96孔板中，加入超纯水80μL，涡旋混匀，取10μL溶液进样分析。
3.实验结果
PD结果见图6多西他赛聚合物胶束对H22腹水瘤模型小鼠血浆中VEGF含量的影响，给予多西他赛聚合物胶束、多西他赛聚合物胶束联合重组人血管内皮抑制素可以显著抑制VEGF分泌，起到抑制腹水的作用。
PK结果见下表：
Figure PCTCN2022117973-appb-000018
结果说明：
多西他赛聚合物胶束与多西他赛注射液相比，显著提高药物血浆暴露量，并减少VEGF的分泌。
实施例13
1、实验动物
BALB/c nude小鼠，雌性，6-8周龄，体重18-22克。共96只。由上海灵畅生物科技有限公司提供，SPF级。动物供应商许可证号：SCXK(沪)2018-0003，实验动物合格证号：20180003016704。
2、受试药物
Figure PCTCN2022117973-appb-000019
3、实验方法
3.1细胞培养
人卵巢癌ES-2细胞(南京科佰CBP60293)体外单层培养，培养条件为RPMI Medium 1640培养基中加10％胎牛血清，1％P/S，37℃，5％CO 2孵箱培养。一周一到两次用0.25％胰酶-EDTA进行常规消化处理传代。数量到达要求以及细胞在对数生长期时，收取细胞，计数，接种。
3.2肿瘤细胞接种与分组
收集细胞培养液中ES-2细胞，用PBS重悬并调整细胞浓度至2×10 7个/mL，每只小鼠腹腔注射0.5mL细胞悬液。细胞接种7天后，将小鼠按体重随机分组到6个实验组中，每组 16只，分组当天为Day 0开始给药。
3.3分组给药方案
1)分组
给药前称重动物，根据动物体重随机分组。
2)给药
给药方案见下表：
动物实验分组和给药方案
Figure PCTCN2022117973-appb-000020
注：1.给药体积均为10ml/kg；
2.IP:腹腔注射；QD:一天一次；QW:一周一次。
3)观察
每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量，体重变化(每周称量三次体重)，外观体征或其它不正常情况。基于各组动物数量记录组内动物死亡数和副作用。
3.4检测指标
Day8每组提前结束6只动物做腹水量和腹水细胞计数，腹水体积用注射器进行计量，以mL为单位，保留小数点后一位有效数字。采用差重法对腹水重量进行计量，以g为单位，保留小数点后4位有效数字。用细胞计数仪对腹水中细胞浓度进行测量，以10 6个/mL为单位，保留小数点后1位有效数字。
4.结束实验
1)Day8每组取6只动物安乐死，收集腹水。
2)剩余小鼠继续给药，观察生存期。实验结束，提供小鼠生存期数据。
5.实验结果
1)腹水情况
Figure PCTCN2022117973-appb-000021
Figure PCTCN2022117973-appb-000022
注：a.利用one-way anova分析方法用GraphPad Prism 9.0对数据进行统计学分析，在所有分析中P<0.05认为有显著性差异。(*:P<0.05；**:P<0.01；***:P<0.001；****:P<0.0001；ns:不显著)。
图7为多西他赛聚合物胶束对ES-2腹水瘤模型小鼠腹水量的影响，给予多西他赛聚合物胶束、多西他赛聚合物胶束联合重组人血管内皮抑制素可以抑制模型小鼠腹水量增加。
结果说明：
多西他赛聚合物胶束单药与多西他赛聚合物胶束和重组人血管内皮抑制素联合可以减少ES-2模型小鼠腹水量和腹水中细胞数。
2)生存期
Figure PCTCN2022117973-appb-000023
注：a.利用Log-rank(Mantel-Cox)test分析方法用GraphPad Prism 9.0对动物生存期进行统计学分析，在所有分析中P<0.05认为有显著性差异(*:P<0.05；**:P<0.01；***:P<0.001；****:P<0.0001；ns:不显著)。
图8为多西他赛聚合物胶束对ES-2腹水瘤模型小鼠生存期的影响，给予多西他赛聚合物胶束、多西他赛聚合物胶束联合重组人血管内皮抑制素可以显著延长小鼠生存时间。
结果说明：
多西他赛聚合物胶束单药与多西他赛聚合物胶束和重组人血管内皮抑制素联合可以延长腹水小鼠生存期。
实施例14
1、实验动物
BALB/c小鼠，雌性，6-8周龄，体重18-22克，共83只。由上海灵畅生物科技有限公司提供，SPF级。动物供应商许可证号：SCXK(沪)2018-0003，实验动物合格证号：20180003016705。
2、受试药物
Figure PCTCN2022117973-appb-000024
3、实验方法
3.1细胞培养
鼠肝癌H22细胞(南京科佰CBP60230)体外悬浮培养，培养条件为RPMI Medium 1640培养基中加10％胎牛血清，1％P/S，37℃，5％CO 2孵箱培养。一周一到两次进行常规传代。数量到达要求以及细胞在对数生长期时，收取细胞，计数，接种。
3.2肿瘤细胞接种与分组
收集细胞培养液中H22细胞，用PBS重悬并调整细胞浓度至1×10 7个/mL，取3只BALB/c小鼠，每只小鼠腹腔注射1mL细胞悬液。细胞注射7天左右小鼠腹部明显隆起，抽取小鼠腹腔腹水，收集H22细胞并用PBS洗一遍，PBS重悬H22细胞并调整浓度至2.5×10 6个/mL，每只小鼠腹腔注射0.2mL细胞悬液。细胞接种3天后，将小鼠按体重随机分组到5个实验组中，每组16只，分组当天为Day 0开始给药。
3.3分组给药方案
1)分组
给药前称重动物，根据动物体重随机分组。
2)给药
给药方案见下表：
动物实验分组和给药方案
Figure PCTCN2022117973-appb-000025
注：1.给药体积均为10ml/kg；
2.IP:腹腔注射；QD:一天一次；QW:一周一次。
3)观察
每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量，体重变化(每周称量三次体重)，外观体征或 其它不正常情况。基于各组动物数量记录组内动物死亡数和副作用。
3.4检测指标
给药一周后动物做腹水量和腹水细胞计数，腹水体积用注射器进行计量，以mL为单位，保留小数点后一位有效数字。采用差重法对腹水重量进行计量，以g为单位，保留小数点后4位有效数字。用细胞计数仪对腹水中细胞浓度进行测量，以10 6个/mL为单位，保留小数点后1位有效数字。
4.结束实验
1)Day8每组取6只动物安乐死，收集腹水。
2)剩余小鼠继续给药，观察生存期。实验结束，提供生存期数据。
5.实验结果
1)腹水情况
Figure PCTCN2022117973-appb-000026
注：a.实验终点时小鼠腹腔内无腹水。
b.利用one-way anova分析方法用GraphPad Prism 9.0对动物腹水量和腹水细胞数进行统计学分析，在所有分析中P<0.05认为有显著性差异(*:P<0.05；**:P<0.01；***:P<0.001；****:P<0.0001；ns：不显著)。
结果说明：
多西他赛聚合物胶束单药、多西他赛聚合物胶束和重组人血管内皮抑制素联合可以减少H22模型小鼠腹水量和腹水中细胞数。
2)生存期
Figure PCTCN2022117973-appb-000027
Figure PCTCN2022117973-appb-000028
注：a.至实验终点，小鼠存活比例大于50％，所有小鼠状态良好。
b.利用Log-rank(Mantel-Cox)test分析方法用GraphPad Prism 9.0对动物生存期进行统计学分析，在所有分析中p<0.05认为有显著性差异(*:P<0.05；**:P<0.01；***:P<0.001；****:P<0.0001；ns:不显著)。
实施例15大鼠和犬的体内药代动力学(PK)试验
Sprague-Dawley(SD)大鼠，8周龄，给药开始前体重215-364g。共24只，雌雄各半。购自北京维通利华实验动物科技有限公司，SPF级。
Beagle犬(雄性/雌性)，体重7.73-8.37kg，约6-8月龄，共12只，雌雄各半，购自玛氏生物科技有限公司，普通级。
多西他赛注射液(
Figure PCTCN2022117973-appb-000029
下称DTX)购自Sanofi-Aventis Deutschland GmbH(德国)。多西他赛注射液按说明书配置为母液，用生理盐水稀释至相应工作液浓度。HT001组都用生理盐水稀释。
SD大鼠静脉注射单剂量2.5、5、10mg/kg HT001或5mg/kg DTX(每组6只大鼠，给药浓度分别为0.25、0.5、1、0.5mg/mL)。在给药后0.083、0.25、0.5、1、2、3、4、6、8和24小时从动物颈静脉区全血约0.5mL，血样采集后放入含有肝素钠抗凝的EP管中。将血样以每分钟3000转(rpm)的速度离心10分钟以获得血浆并通过LC-MS/MS(Waters Corp.,Manchester,UK)进行分析。
Beagle犬静脉注射单剂量1mg/kg的DTX或HT001(每组6只，给药浓度均为0.2mg/mL)。在给药后0.25、0.5、0.55、1、2、4、6、8和24小时采血，每个时间点从前肢或后肢皮下静脉取血约1mL，血样采集后放入含有肝素钠抗凝的EP管中。将血样以3000rpm离心10分钟以获得血浆并通过LC-MS/MS进行分析。
一般临床观察：
观察时间：每天上午和下午各1次，直至D3。
观察内容：笼旁观察动物死亡或濒死情况、精神状态、行为活动、进食情况、粪便性状等。
数据采集及统计分析：
LCSOLUTION系统工作软件(Version 1.25)输出原始图谱并计算测定样品的浓度。使用Waters公司的Unifi软件输出原始图谱、浓度、准确度等数据。采用WinNonlin(V6.2)软件非房室模型方法(NCA)对动物个体进行AUC、Tmax、Cmax等主要代谢动力学参数的计算。
实验结果：
在SD大鼠中通过静脉注射单剂量HT001或DTX给药后多西紫杉醇的平均血浆浓度-时间曲线如图9A所示。SD大鼠的PK参数列于下表。HT001在2.5-10mg/kg剂量范围内，Cmax和AUClast值均随剂量增加而增加，且增加暴露的比例高于增加剂量。在5mg/kg剂量下，DTX给药后大鼠体内多西他赛的AUClast值显著低于HT001治疗组。
在Beagle犬中通过静脉注射单剂量HT001或DTX给药后多西紫杉醇的平均血浆浓度-时间曲线如图9B所示。HT001在非啮齿动物宿主中显示出与DTX相似的PK和组织分布曲线。另外，在给药期间，DTX组的全部犬都观察到了皮肤潮红、眼睑肿胀和发红、呼吸急促等过敏反应，但在同剂量HT001组中没有观察到这种现象。
HT001和DTX在SD大鼠体内的药代动力学(PK)参数
Figure PCTCN2022117973-appb-000030
注：利用student’s t test分析方法，计算HT001(5mg/kg)组vs.DTX组AUC last(h*ng/mL)的P值，P<0.05，有显著性差异。

Claims (35)

  1. 多西他赛聚合物胶束在制备预防或治疗恶性胸腹水的药物中的用途。
  2. 根据权利要求1所述的用途，其中，多西他赛聚合物胶束的人体剂量范围是5～200mg/m 2。
  3. 多西他赛聚合物胶束和重组人血管内皮抑制素联合在制备预防或治疗恶性胸腹水的药物中的用途。
  4. 根据权利要求3所述的用途，其中，多西他赛聚合物胶束的人体剂量范围是5～200mg/m 2，所述重组人血管内皮抑制素的人体剂量范围是5～200mg/m 2。
  5. 一种产品，所述产品含有(a)多西他赛聚合物胶束，和(b)重组人血管内皮抑制素，在预防或者治疗恶性胸腹水中作为组合制剂同时、分别或序贯使用。
  6. 权利要求5所述产品在制备预防或者治疗恶性胸腹水的药物中的用途。
  7. 根据权利要求1～4、6任一项所述的用途，其中，多西他赛聚合物胶束的给药途径为胸腔给药或腹腔给药。
  8. 根据权利要求3～4、6任一项所述的用途，其中，重组人血管内皮抑制素的给药途径为胸腔给药或腹腔给药。
  9. 根据权利要求1～8任一项所述的用途或产品，其中，恶性胸腹水选自恶性腹水。
  10. 根据权利要求1～8任一项所述的用途或产品，其中，所述重组人血管内皮抑制素是注射液。
  11. 根据权利要求1～8任一项所述的用途或产品，其中，所述多西他赛聚合物胶束包含多西他赛和聚乙二醇单甲醚-聚乳酸嵌段共聚物。
  12. 根据权利要求11所述的用途或产品，其中，所述聚乙二醇单甲醚-聚乳酸嵌段共聚物的分子量是2000～7000，且多分散系数PD≤1.5，优选地，所述聚乙二醇单甲醚-聚乳酸嵌段共聚物的分子量是3000～5000，且多分散系数PD≤1.5。
  13. 根据权利要求11所述的用途或产品，其中，所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物为D,L-丙交酯与聚乙二醇单甲醚聚合形成的嵌段共聚物，聚乙二醇单甲醚与D,L-丙交酯的投料质量比为1:0.6～1.6，优选1：0.9～1.3，更优选1:1.05～1.25，更进一步优选1:1.1～1.2，更进一步优选1:1.1。
  14. 根据权利要求13所述的用途或产品，其中，所述聚乙二醇单甲醚的分子量为1000～20000，优选1000～5000，更优选1800～2200，进一步优选2000。
  15. 根据权利要求11～14任一项所述的用途或产品，其中，所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物通过如下方法制备得到：(a)向反应器中加入聚乙二醇单甲醚，加热熔融；(b)加入D,L-丙交酯，加入金属催化剂，然后加热至125～150℃反应；(c)反应结束后降温，加入第一有机溶剂溶解，加入第二有机溶剂，过滤，滤饼真空干燥，得到聚乙二醇单甲醚-聚乳酸嵌段共聚物。
  16. 根据权利要求15所述的用途或产品，其中，所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物通过如下方法制备得到：(a)向反应器中加入聚乙二醇单甲醚，真空下加热熔融，惰性气体置换；(b)加入D,L-丙交酯，惰性气体置换，加入金属催化剂，保证反应器中为负压或惰性气体保护，然后加热至125～150℃反应；(c)反应结束后降温，加入第一有机溶剂溶解，加 入第二有机溶剂，过滤，滤饼真空干燥，得到聚乙二醇单甲醚-聚乳酸嵌段共聚物。
  17. 根据权利要求15或16所述的用途或产品，其中，步骤(a)所述聚乙二醇单甲醚真空下加热至60～130℃。
  18. 根据权利要求15或16所述的用途或产品，其中，步骤(b)金属催化剂为异辛酸亚锡。
  19. 根据权利要求15或16所述的用途或产品，其中，异辛酸亚锡的质量占D,L-丙交酯和聚乙二醇单甲醚总质量的0.05～0.5wt％，优选0.15～0.3wt％。
  20. 根据权利要求15或16所述的用途或产品，其中，步骤(b)反应时间是1～20h，优选3～10h，更优选4～6h。
  21. 根据权利要求15或16所述的用途或产品，其中，步骤(c)所述的第一有机溶剂为乙腈、丙酮、二氯甲烷、二甲基甲酰胺、二甲亚砜、四氢呋喃、短链脂肪醇或乙酸乙酯中的一种或几种，优选二氯甲烷、三氯甲烷、甲醇、乙醇、异丙醇中的一种或几种，更优选二氯甲烷；第一有机溶剂的用量占聚乙二醇单甲醚质量0.1～100倍体积(L/kg)，优选0.2～50倍体积(L/kg)，更优选0.5～20倍体积(L/kg)。
  22. 根据权利要求15或16所述的用途或产品，其中，步骤(c)所述的第二有机溶剂为乙醚、异丙醚、甲基叔丁基醚、正庚烷、环己烷、石油醚中的一种或几种，优选乙醚、异丙醚或甲基叔丁基醚中的一种或几种，更优选乙醚；第二有机溶剂的用量占聚乙二醇单甲醚总质量1～100倍体积(L/kg)，优选10～50倍体积(L/kg)，更优选15～20倍体积(L/kg)。
  23. 根据权利要求15或16所述的用途或产品，其中，步骤(b)反应结束后还包括加入第三有机溶剂和活性炭，搅拌，过滤，滤液减压浓缩的步骤。
  24. 根据权利要求23所述的用途或产品，其中，所述第三有机溶剂为二氯甲烷、三氯甲烷、乙酸乙酯、乙酸异丙酯、乙酸丁酯、丁酮、甲苯、丙酮、乙腈、二甲基甲酰胺、二甲亚砜、四氢呋喃、1,4-二氧六环、短链脂肪醇中的一种或几种，优选二氯甲烷、三氯甲烷、甲醇、乙醇、异丙醇中的一种或几种，更优选为二氯甲烷，所述第三有机溶剂的用量占聚乙二醇单甲醚质量0.1～100倍体积(L/kg)，优选0.2～50倍体积(L/kg)，更优选0.5～20倍体积(L/kg)。
  25. 根据权利要求23所述的用途或产品，其中，所述活性炭的用量占D,L-丙交酯和聚乙二醇单甲醚总质量的1～500wt％，优选5～200wt％，更优选5～75wt％，进一步优选20～50wt％。
  26. 根据权利要求11所述的用途或产品，其中，所述多西他赛与聚乙二醇单甲醚-聚乳酸嵌段共聚物的质量比为0.01～0.15:1，优选0.02～0.12:1，更优选0.03～0.10:1，进一步优选0.04:0.96。
  27. 根据权利要求11所述的用途或产品，其中，所述多西他赛聚合物胶束还包含稳定剂。
  28. 根据权利要求27所述的用途或产品，其中，所述稳定剂是枸橼酸、盐酸、山梨酸、乳酸、酒石酸、苹果酸、磷酸、醋酸、己二酸、富马酸中的一种或几种，优选是枸橼酸、酒石酸、富马酸中的一种或几种，更优选是枸橼酸。
  29. 根据权利要求27所述的用途或产品，其中，所述多西他赛与稳定剂的质量比是1：0.00125～0.25，优选是1：0.005～0.25，更优选是1:0.0125～0.125。
  30. 根据权利要求11所述的用途或产品，其中，所述多西他赛聚合物胶束包含以下配比的组分：
    Figure PCTCN2022117973-appb-100001
  31. 根据权利要求11～30任一项所述的用途或产品，其中，所述多西他赛聚合物胶束通过以下方法制备而成：
    (1)将权利要求11～25任一项所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物和多西他赛溶于有机溶剂，旋转蒸发除去有机溶剂，得到凝胶状药膜，然后向药膜中加入水溶解分散所述药膜，制得胶束溶液；
    (2)将步骤(1)制得的胶束溶液经过滤除菌、冷冻干燥后得到多西他赛聚合物胶束冻干制剂。
  32. 根据权利要求31所述的用途或产品，其中，步骤(1)是将权利要求11～25任一项所述的聚乙二醇单甲醚-聚乳酸嵌段共聚物、稳定剂和多西他赛溶于有机溶剂，旋转蒸发除去有机溶剂，得到凝胶状药膜，然后向药膜中加入水溶解分散所述药膜，制得胶束溶液。
  33. 根据权利要求31或32所述的用途或产品，其中，步骤(1)中所述的有机溶剂选自乙腈、丙酮、二氯甲烷、二甲基甲酰胺、二甲亚砜、四氢呋喃、短链脂肪醇、乙酸乙酯中的任意一种或几种，优选甲醇、乙醇、异丙醇、乙腈、乙酸乙酯中的任意一种或几种，更优选甲醇，步骤(1)中所述的有机溶剂的用量为每克聚乙二醇单甲醚聚乳酸嵌段共聚物中加入0.5～12ml有机溶剂，优选加入3～12ml有机溶剂。
  34. 根据权利要求31或32所述的用途或产品，其中，步骤(1)中，向药膜中加入水的用量为每克聚乙二醇单甲醚聚乳酸嵌段共聚物加入2～40ml水，优选地为每克聚乙二醇单甲醚聚乳酸嵌段共聚物加入10～20ml水，更优选地为每克聚乙二醇单甲醚聚乳酸嵌段共聚物加入10～15ml水。
  35. 根据权利要求31或32所述的用途或产品，其中，步骤(2)中，旋转蒸发去除有机溶剂的条件为：旋转速度10～150rpm，温度20～80℃，时间1～4h。
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