WO2024078529A1 - 一种普那布林胶束组合物及其制备方法 - Google Patents

一种普那布林胶束组合物及其制备方法 Download PDF

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WO2024078529A1
WO2024078529A1 PCT/CN2023/124000 CN2023124000W WO2024078529A1 WO 2024078529 A1 WO2024078529 A1 WO 2024078529A1 CN 2023124000 W CN2023124000 W CN 2023124000W WO 2024078529 A1 WO2024078529 A1 WO 2024078529A1
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plinabulin
composition
injection
propylene glycol
polyethylene glycol
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PCT/CN2023/124000
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English (en)
French (fr)
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黄岚
何成江
张�浩
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大连万春布林医药有限公司
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Publication of WO2024078529A1 publication Critical patent/WO2024078529A1/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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • 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
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to the field of pharmaceutical preparations, and in particular to a Plinabulin micelle composition and a preparation method thereof.
  • Plinabulin ((3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione
  • phenylahistin trimethylammonium chloride
  • Plinabulin is structurally different from colchicine and its combretastatin-like analogs (e.g., combretastatin phosphate) and binds at or near the colchicine binding site on tubulin monomers.
  • combretastatin-like analogs e.g., combretastatin phosphate
  • Previous studies have shown that Plinabulin at low concentrations induces vascular endothelial cell tubulin depolymerization and monolayer permeability compared to colchicine, and indicates that Plinabulin induces apoptosis in Jurkat leukemia cells.
  • Plinabulin has shown good pharmacokinetics, pharmacodynamics and safety characteristics as a single agent in patients with advanced malignancies (lung cancer, prostate cancer and colon cancer).
  • there is still a need to provide an injection formulation of Plinabulin with appropriate physicochemical properties.
  • the present invention aims to provide a novel Plinabulin micelle and a preparation method thereof.
  • the Plinabulin micelle has good stability.
  • the obtained micelle can not only improve the water solubility of Plinabulin, but also delay drug release and prolong biological half-life, and has good clinical application value.
  • the first aspect of the present invention provides a method for preparing a Plinabulin micelle composition, wherein the method comprises the steps of:
  • step 1) mixing the clarified mixed solution in step 1) with Plinabulin at 35° C.-65° C., and stirring to prepare a Plinabulin micelle composition.
  • the weight ratio of 15-hydroxystearate polyethylene glycol to propylene glycol is 1:5-5:1, preferably, 1:3-3:1, and more preferably 2:3.
  • the weight ratio of 15-hydroxystearate polyethylene glycol to propylene glycol is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1.
  • the micelle composition is essentially composed of Plinabulin, 15-hydroxystearate polyethylene glycol and propylene glycol.
  • step s1) the temperature is 40°C-60°C.
  • the temperature is 40°C-60°C.
  • step s1) is to melt 15-hydroxystearate polyethylene glycol at 50°C ⁇ 5°C, then add propylene glycol, maintain the temperature at 40°C ⁇ 5°C, and stir (for example, 5 min-2 h, preferably 20-40 min, more preferably 30 min) to prepare a clear mixed solution.
  • step s1) is to melt 15-hydroxystearate polyethylene glycol at 35°C-65°C, then add propylene glycol, maintain the temperature at 35°C-65°C, and stir (for example, 5 min-2 h, preferably 10-40 min, more preferably 20-30 min) to prepare a clear mixed solution.
  • step s1) is to melt 15-hydroxystearate polyethylene glycol at 35°C-65°C, add it to propylene glycol, maintain the temperature at 35°C-65°C, and stir (for example, 5 min-2 h, preferably 10-40 min, more preferably 20-30 min) to prepare a clear mixed solution.
  • the clarified mixed solution in step s2) is added in batches, preferably in 2-5 batches.
  • the concentration of Plinabulin is 0.02 mg/ml-4 mg/ml, based on the total mass of the Plinabulin micelle composition.
  • Plinabulin is Plinabulin anhydrate or Plinabulin monohydrate.
  • Plinabulin is Plinabulin anhydrate (crystalline form III) or Plinabulin monohydrate (crystalline form I).
  • step (s2) the Plinabulin used is Plinabulin monohydrate.
  • Plinabulin is Plinabulin monohydrate (crystalline form I).
  • the Plinabulin anhydrate is the anhydrous form of Plinabulin in CN113735834B.
  • the Plinabulin monohydrate is the Plinabulin monohydrate in CN113735834B.
  • the particle size D50 of the micelles in the obtained Plinabulin micelle composition is 5-100 nm.
  • a Plinabulin micelle composition comprising Plinabulin and a clear mixed solution of 15-hydroxystearate polyethylene glycol and propylene glycol, wherein the Plinabulin micelle composition is a yellow clear transparent solution, and the micelle particle size ranges from 10 to 100 nm.
  • the micelle particle size of the Plinabulin micelle composition is in the range of 80-110 nm, preferably Select 90-110nm, for example 100-110nm.
  • the micelle D50 of the Plinabulin micelle composition is 80-110 nm, preferably 90-110 nm, for example 100-105 nm.
  • the micelle D90 of the Plinabulin micelle composition is 100-170 nm, preferably 110-160 nm, such as 120-150 nm.
  • the concentration of Plinabulin in the Plinabulin micelle composition, is 1 mg/ml-10 mg/ml. Based on the total mass of the Plinabulin micelle composition, the concentration of Plinabulin is preferably 2 mg/ml-5 mg/ml, and the concentration of Plinabulin is more preferably 3 mg/ml-4 mg/ml.
  • the Plinabulin in the Plinabulin micelle composition, is Plinabulin monohydrate.
  • the weight ratio of 15-hydroxystearate polyethylene glycol to propylene glycol is 1:5-5:1, preferably, 1:3-3:1, and more preferably 2:3.
  • the Plinabulin micelle composition is prepared by the method described in the first aspect.
  • the Plinabulin micelle composition is sterile.
  • the third aspect of the present invention provides a liquid Plinabulin composition for injection, comprising:
  • the volume ratio of propylene glycol to D5W in the composition is about 6:50 to about 6:500.
  • the Plinabulin in the liquid Plinabulin composition for injection, is Plinabulin monohydrate.
  • Plinabulin is encapsulated in micelles, preferably, at least 90% of Plinabulin is encapsulated in micelles.
  • At least 95% of Plinabulin is encapsulated in micelles, preferably at least 97%, and more preferably at least 99%.
  • the volume ratio of propylene glycol to D5W in the composition is about 6:100 to about 6:400, preferably about 6:150 to about 6:250, and more preferably about 6:200.
  • the particle size D50 of the micelles in the obtained Plinabulin composition for injection is 5-50 nm, preferably 10-30 nm.
  • the concentration of Plinabulin is about 0.08 mg/ml to about 0.4 mg/ml.
  • the volume ratio of 15-hydroxystearate polyethylene glycol ester and D5W in the liquid injection Plinabulin preparation is about 4:50 to about 4:500, preferably about 4:100 to about 4:500, more preferably about 4:100 to about 4:400, for example about 4:100 to about 4:300, or about 4:150 to about 4:250.
  • liquid Plinabulin composition for injection 15-hydroxystearic acid polyethylene glycol
  • the weight ratio of the alcohol ester to propylene glycol is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1.
  • the ratio of propylene glycol to 15-hydroxystearate polyethylene glycol is about 60:40 (wt:wt).
  • the total amount of impurities contained in the liquid injection Plinabulin composition is less than 0.5%. In another preferred embodiment, the total amount of impurities contained in the liquid injection Plinabulin composition is less than 0.1%.
  • liquid Plinabulin composition for injection can be stably stored for about 8 hours to about 12 hours.
  • liquid Plinabulin composition for injection can be stably stored at a temperature of 10°C to about 37°C, preferably room temperature.
  • At least a portion of Plinabulin is encapsulated in micelles, preferably, at least 90% of Plinabulin is encapsulated in micelles.
  • greater than about 90% of Plinabulin in the liquid injectable Plinabulin composition is encapsulated in micelles.
  • greater than about 93% of Plinabulin in the liquid injection Plinabulin composition is encapsulated in micelles, preferably, greater than about 94% of Plinabulin is encapsulated in micelles, preferably, greater than about 95% of Plinabulin is encapsulated in micelles, preferably, greater than about 96% of Plinabulin is encapsulated in micelles, preferably, greater than about 97% of Plinabulin is encapsulated in micelles, preferably, greater than about 99% of Plinabulin is encapsulated in micelles.
  • liquid Plinabulin composition for injection is prepared by the method described in the fourth aspect of the present invention.
  • the present invention provides a method for preparing a liquid injection Plinabulin composition, wherein the method comprises:
  • the initial liquid formulation was diluted in D5W at a dilution ratio of about 1:5 to about 1:50.
  • the initial liquid preparation is the Plinabulin micellar composition described in the second aspect of the present invention.
  • the dilution ratio is about 1:10 to about 1:50, preferably, the dilution ratio is about 1:13 to about 1:30, and more preferably, the dilution ratio is about 1:20.
  • the initial liquid formulation comprises Plinabulin at a concentration of about 1 mg/ml to about 6 mg/ml, preferably comprises Plinabulin at a concentration of about 3 mg/ml to about 5 mg/ml, and more preferably comprises Plinabulin at a concentration of about 4 mg/ml.
  • the composition is stirred, preferably for at least 5 minutes.
  • the concentration of Plinabulin is about 0.08 mg/ml to about 0.4 mg/ml.
  • the ratio of propylene glycol to 15-hydroxystearate polyethylene glycol in the liquid injection composition is about 60:40 (wt:wt).
  • the initial liquid preparation is the Plinabulin micellar composition as described above.
  • the particle size of the micelles in the liquid Plinabulin composition for injection ranges from 90 to 200 nm, preferably from 100 to 150 nm, and more preferably from 100 to 110 nm.
  • the fifth aspect of the present invention provides a use of the Plinabulin micelle composition as described in the second aspect or the Plinabulin composition for liquid injection as described in the third aspect in the preparation of a drug for preventing and/or treating anti-tumor.
  • the tumor is selected from: lung cancer (such as small cell lung cancer, non-small cell lung cancer), prostate cancer, colon cancer, brain tumor (such as glioblastoma, glioblastoma multiforme, giant cell glioblastoma, metastatic brain tumor), head and neck cancer, gastric cancer, pancreatic cancer, breast cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, myeloma, lymphoma or leukemia.
  • lung cancer such as small cell lung cancer, non-small cell lung cancer
  • prostate cancer colon cancer
  • brain tumor such as glioblastoma, glioblastoma multiforme, giant cell glioblastoma, metastatic brain tumor
  • head and neck cancer gastric cancer, pancreatic cancer, breast cancer, kidney cancer, bladder cancer, ovarian cancer, cervical cancer, melanoma, myeloma, lymphoma or leukemia.
  • FIG1 shows a diagram of the Tyndall effect of a micellar composition of Plinabulin.
  • FIG2 shows a graph of the Tyndall effect of a liquid Plinabulin composition for injection.
  • the Plinabulin micelle composition can improve the water solubility of Plinabulin and has excellent electrochemical stability, dilution stability and storage stability.
  • the present invention uses Plinabulin monohydrate and a specific ratio of propylene glycol and 15-hydroxystearate polyethylene glycol to unexpectedly prepare a Plinabulin micelle composition, in which Plinabulin is encapsulated in micelles.
  • the micelle composition has excellent stability, electrochemical stability, dilution stability and storage stability.
  • Plinabulin micelle composition On the basis of obtaining the Plinabulin micelle composition, a D5W solution was added for dilution to obtain a Plinabulin composition for liquid injection. After dilution, the Plinabulin micelle composition still exists, and Plinabulin is encapsulated therein.
  • the diluted injection has excellent drip stability and storage stability, and the encapsulation rate of Plinabulin is high and the impurity content is low, so it can be used as an injection preparation of Plinabulin.
  • the present invention discloses Plinabulin micellar encapsulation compositions and methods for preparing and using Plinabulin compositions.
  • Plinabulin can be readily prepared according to the methods and steps detailed in U.S. Patent Nos. 7,064,201 and 7,919,497, the entire contents of which are incorporated herein by reference.
  • Plinabulin as used herein includes crystalline forms or amorphous forms, such as Plinabulin monohydrate, Plinabulin anhydrate, Plinabulin solvate, preferably monohydrate.
  • injectable micellar preparation of Plinabulin In the present invention, "injectable micellar preparation of Plinabulin”, “liquid injectable Plinabulin composition”, “liquid injectable preparation”, “liquid injectable Plinabulin preparation” and “liquid injectable Plinabulin composition” have the same meaning and can be used interchangeably.
  • agent is used herein to refer to a compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material.
  • cancer refers to cells that exhibit relatively autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation.
  • cells of interest for detection or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and non-metastatic cells. Detection of cancer cells is of particular interest.
  • subject refers to a human or non-human mammal, such as a dog, cat, mouse, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as any other vertebrate or invertebrate.
  • mammal is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, primates, including great apes (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs or the like.
  • primates including great apes (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs or the like.
  • the term "effective amount” or “therapeutically effective amount” refers to an amount of a therapeutic agent that is effective in alleviating one or more symptoms of a disease or condition to some extent, or reducing the likelihood of developing the condition, and may include therapeutic agents. Cure disease or illness.
  • the term “treat” refers to administering a compound or pharmaceutical composition to a subject for preventive and/or therapeutic purposes.
  • prolactic treatment refers to treating a subject who does not yet show symptoms of a disease or condition, but is susceptible or otherwise at risk for a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.
  • therapeutic treatment refers to treating a subject who already has a disease or condition.
  • Plinabulin is provided in the form of a concentrated liquid formulation, which can then be diluted to prepare a diluted liquid injection formulation.
  • the concentrated liquid formulation includes one or more solvents, which may include 15-hydroxystearate polyethylene glycol ester and/or propylene glycol.
  • the one or more solvents include at least 30% (by weight) of 15-hydroxystearate polyethylene glycol ester, at least 35% (by weight) of 15-hydroxystearate polyethylene glycol ester, at least 40% (by weight) of 15-hydroxystearate polyethylene glycol ester, at least 45% (by weight) of 15-hydroxystearate polyethylene glycol ester, or include and/or span the range of the above values.
  • the one or more solvents include at least 50% (by weight) of propylene glycol, at least 55% (by weight) of propylene glycol, at least 50% (by weight) of propylene glycol, at least 55% (by weight) of propylene glycol, at least 60% (by weight) of propylene glycol, or include and/or span the range of the above values.
  • the one or more solvents include 40% (weight) 15-hydroxystearate polyethylene glycol and 60% (weight) propylene glycol. In some embodiments, the ratio of propylene glycol to 15-hydroxystearate polyethylene glycol in the liquid injection formulation is about 60:40 (weight ratio).
  • the concentrated liquid formulation is a micellar composition.
  • Plinabulin is encapsulated in micelles.
  • the ingredients of the micellar composition include 15-hydroxystearate polyethylene glycol ester, propylene glycol and Plinabulin, wherein Plinabulin is encapsulated in micelles.
  • the Plinabulin in the micellar composition is Plinabulin monohydrate.
  • the resulting micellar composition has poor solubility.
  • the concentration of Plinabulin in the concentrated liquid formulation may be between 1 mg/ml and 10 mg/ml, between 2 mg/ml and 6 mg/ml, or about 4 mg/ml.
  • the concentrated liquid preparation can be diluted with a diluent to form a diluted liquid injection preparation.
  • the diluent is water.
  • the diluent is saline.
  • the diluent is a glucose aqueous solution.
  • the concentration of glucose can be 1% to 20%, 2% to 10%, or about 5% (i.e., D5W).
  • the diluted liquid injection formulation includes 15-hydroxystearate polyethylene glycol, propylene glycol, Propylene glycol and D5W (i.e., glucose and water).
  • the volume ratio of propylene glycol and D5W in the liquid injection plinabulin formulation is about 6:50-about 6:500, about 6:50-about 6:450, about 6:50-about 6:400, about 6:50-about 6:350, about 6:50-about 3:17:300, about 6:50-about 6:250, about 6:50-about 6:200, about 6:50-about 6:150, about 6:50-about 6:100, about 6:100-about 6:500, about 6:100-6:400, about 6:150-about 6:250, or about 6:200.
  • the volume ratio of propylene glycol and D5W in the liquid injection plinabulin formulation is about 6:70. In some embodiments, the volume ratio of propylene glycol to D5W in the liquid Plinabulin injection formulation is about 6: 140. In some embodiments, the volume ratio of propylene glycol to D5W in the liquid Plinabulin injection formulation is about 6: 400.
  • the volume ratio of polyethylene glycol 15-hydroxystearate and D5W in the liquid Plinabulin injection formulation is in the range of about 4:50 to about 4:500, about 4:100 to about 4:500, about 4:100 to about 4:400, about 4:100 to about 4:300, about 4:150 to about 4:250, or about 4:200.
  • the Plinabulin in the liquid Plinabulin injection preparation (composition) is Plinabulin monohydrate.
  • the concentration of Plinabulin in the liquid Plinabulin injection formulation is about 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.11 mg/ml, 0.12 mg/ml, 0.13 mg/ml, 0.14 mg/ml, 0.15 mg/ml, 0.16 mg/ml, 0.17 mg/ml, 0.18 mg/ml, 0.19 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, or a range including and/or spanning the above values.
  • the concentration of Plinabulin in the liquid Plinabulin composition for injection is about 0.08 mg/ml to about 0.4 mg/ml. In some embodiments, the concentration of Plinabulin in the liquid Plinabulin composition for injection is about 0.1 mg/ml to about 0.3 mg/ml. In some embodiments, the concentration of Plinabulin in the liquid Plinabulin composition for injection is about 0.2 mg/ml.
  • the impurities contained in the liquid injection Plinabulin formulation (composition) are less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or include and/or span the range of the above numerical values.
  • the ether impurities contained in the liquid injection Plinabulin formulation (composition) are less than 0.5%.
  • the alcohol impurities contained in the liquid injection Plinabulin formulation (composition) are less than 0.5%.
  • the water contained in the liquid injection Plinabulin formulation (composition) is less than 0.5%.
  • the liquid injectable Plinabulin formulation (composition) can be stably stored for about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, or a range including and/or spanning the above values.
  • the liquid Plinabulin injection formulation (composition) can be stably stored at 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, or a range including and/or spanning the above values.
  • part of the Plin when the concentrated liquid formulation is diluted with a diluent (such as D5W), part of the Plin is encapsulated in micelles.
  • micelles are formed by the interaction between 15-hydroxystearate polyethylene glycol ester and D5W.
  • the average encapsulation efficiency of the liquid formulation is at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or a range including and/or spanning the above values.
  • the method of preparing a liquid injection composition of Plinabulin includes providing an initial concentrated liquid formulation, which includes Plinabulin, propylene glycol, and 15-hydroxystearic acid polyethylene glycol ester (PEG15-hydroxystearates); and diluting the initial liquid formulation in D5W.
  • the dilution ratio between the initial concentrated liquid formulation and D5W is about 1:5 to about 1:50, about 1:10 to about 1:50, about 1:10 to about 1:40, about 1:15 to about 1:30, about 1:15 to about 1:25, or about 1:20.
  • the dilution ratio is about 1:10 to about 1:400. In some embodiments, the dilution ratio is about 1:10 to about 1:300. In some embodiments, the dilution ratio is about 1:10 to about 1:200. In some embodiments, the dilution ratio is from about 1:10 to about 1:100. In some embodiments, the dilution ratio is from about 1:10 to about 1:80. In some embodiments, the dilution ratio is from about 1:10 to about 1:60. In some embodiments, the dilution ratio is from about 1:10 to about 1:50. In some embodiments, the dilution ratio is from about 1:10 to about 1:40.
  • the dilution ratio is from about 1:10 to about 1:30. In some embodiments, the dilution ratio is from about 1:10 to about 1:20. In some embodiments, the dilution ratio is from about 1:20 to about 1:400. In some embodiments, the dilution ratio is from about 1:20 to about 1:300. In some embodiments, the dilution ratio is from about 1:20 to about 1:200. In some embodiments, the dilution ratio is from about 1:20 to about 1:100. In some embodiments, the dilution ratio is from about 1:20 to about 1:80. In some embodiments, the dilution ratio is from about 1:20 to about 1:60.
  • the dilution ratio is about 1:20 to about 1:50. In some embodiments, the dilution ratio is about 1:20 to about 1:40. In some embodiments, the dilution ratio is about 1:20 to about 1:30.
  • the dilution method includes introducing the initial concentrated liquid formulation into (e.g., by injection) a container containing a certain volume of D5W.
  • the container is an intravenous (IV) bag.
  • the mixture is stirred.
  • stirring methods include hand shaking, hand stirring or vortexing.
  • the mixture is stirred for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, or include and/or span the range of the above values.
  • propylene glycol is added first, followed by 15-hydroxystearate polyethylene glycol, and then plinabulin.
  • Polyethylene glycol esters are first added, followed by propylene glycol, and then Plinabulin.
  • the initial concentrated liquid formulation is mixed at room temperature.
  • propylene glycol is heated to about 25° C. and maintained at about 25° C. during the addition of polyethylene glycol 15-hydroxystearate.
  • Plinabulin is added to polyethylene glycol 15-hydroxystearate and propylene glycol and mixed at about 25° C.
  • propylene glycol is heated to about 40° C. and maintained at about 40° C. during the addition of polyethylene glycol 15-hydroxystearate.
  • Plinabulin is added to polyethylene glycol 15-hydroxystearate and propylene glycol and mixed at about 40° C.
  • polyethylene glycol 15-hydroxystearate is heated to about 25° C. and maintained at about 25° C. when propylene glycol is added.
  • Plinabulin is added to polyethylene glycol 15-hydroxystearate and propylene glycol and mixed at about 25° C.
  • polyethylene glycol 15-hydroxystearate is heated to about 40° C. and maintained at about 40° C. when propylene glycol is added.
  • Plinabulin is added to polyethylene glycol 15-hydroxystearate and propylene glycol and mixed at about 40° C.
  • polyethylene glycol 15-hydroxystearate is heated to about 60° C. and maintained at about 60° C. when propylene glycol is added.
  • Plinabulin is added to polyethylene glycol 15-hydroxystearate and propylene glycol and mixed at about 60° C.
  • Some embodiments relate to a method of preventing or reversing cancer progression in a subject.
  • the method comprises administering to a subject an injectable liquid formulation described herein.
  • Some embodiments relate to a method of inhibiting cancer progression.
  • Further uses of the injectable liquid formulations described herein include U.S. Patent Nos. 7,919,497; 10,238,650; 10,155,748; 10,076,518; and 10,596,169; and PCT Publication Nos.
  • the treatment regimen includes administering the injectable liquid formulation described herein once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks. In some embodiments, the treatment regimen includes administering the injectable liquid formulation described herein 2 times every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks. In some embodiments, the treatment regimen includes administering the injectable liquid formulation described herein once every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks.
  • the treatment regimen includes administering the injectable liquid formulation described herein twice every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks. In some embodiments, the treatment regimen includes administering the injectable liquid formulation described herein twice every 1 week in a treatment cycle of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks. In some embodiments, the treatment regimen includes administering the injectable liquid formulation described herein on the 1st day, the 8th day and the 15th day in a 21-day treatment cycle.
  • the treatment cycle can be repeated as long as the regimen is clinically tolerable.
  • the treatment cycle of the injectable liquid formulation described herein is repeated n times, wherein n is an integer in the range of 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • a new treatment cycle can occur immediately after the previous treatment cycle is completed. In some embodiments, a new treatment cycle can occur within a period of time after the previous treatment cycle is completed. In some embodiments, a new treatment cycle can occur 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks after the previous treatment cycle is completed.
  • the method comprises administering Plinabulin at a dose of about 5 mg/m 2 to 150 mg/m 2. In some embodiments, Plinabulin is administered at a dose greater than 20 mg/m 2. In some embodiments, Plinabulin is administered at a dose greater than 30 mg/m 2. In some embodiments, Plinabulin is administered at a dose greater than 40 mg/m 2 .
  • the injectable liquid formulation described herein is administered on day 1 of a 14-day dosing cycle. In some embodiments, the injectable liquid formulation described herein is administered on day 1 of a 21-day dosing cycle.
  • the injectable liquid formulations described herein are co-administered with one or more G-CSF drugs.
  • kits comprising one or more containers.
  • the container comprises plastic or glass, or a combination thereof, including but not limited to any one or more plastics or glasses used by one of skill in the art in light of the teachings herein.
  • the container is a vial.
  • the vial contains about 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg or an amount of Plinabulin including and/or spanning the range of the above values.
  • the vial contains Plinabulin, propylene glycol and 15-hydroxystearic acid polyethylene glycol ester (PEG15-hydroxystearates).
  • the vial includes about 4 mg/ml of Plinabulin, propylene glycol and 15-hydroxystearic acid polyethylene glycol ester (ratio of about 60:40 (wt:wt)). In some embodiments, the volume of the liquid formulation in the vial is about 10 ml.
  • the container is an IV bag.
  • the IV bag comprises D5W.
  • the volume of D5W in the IV bag is about 50 ml to about 500 ml, about 100 ml to about 500 ml, about 100 ml to about 400 ml, about 100 ml to about 300 ml, about 150 ml to about 250 ml, or about 200 ml.
  • the kit comprises a vial as described above and an intravenous syringe as described above.
  • the Plinabulin micelle composition described above is diluted in D5W at a dilution ratio of about 1:5 to about 1:50 (wt) to obtain a Plinabulin composition for injection, wherein a Tyndall effect diagram of a dilution ratio of 1:20 is shown in Figure 2.
  • the Plinabulin micellar composition of the present application has excellent dilution stability.
  • the Plinabulin micelle composition of the present application and especially the prepared Plinabulin composition for injection have excellent storage stability.
  • Example 1 Effects of different prescription addition orders on the dissolution rate of Plinabulin and the particle size distribution of the final product obtained.
  • the drug needs to be stirred for nearly 2 hours to dissolve, while at 40°C and 60°C, the raw material drug dissolves in about 10 minutes.
  • the average particle size of Plinabulin micelle compositions A and B was about 100 nm, and the particle size analysis of Plinabulin micelle composition C was not possible.
  • the particle size distributions of different Plinabulin micelle compositions after dilution at a ratio of 1:20 were similar, indicating that the preparation temperature had little effect on the particle size distribution after dilution.
  • Ethylene glycol was added to 15-hydroxystearate polyethylene glycol at 25°C (solid), 40°C (molten) and 60°C, and then Plinabulin was added to prepare Plinabulin micelle compositions D, E and F, which were then diluted with 5% glucose injection at a ratio of 1:20 to obtain liquid injection Plinabulin compositions (preparations).
  • the particle size was measured. The results showed that under the condition that 15-hydroxystearate polyethylene glycol was added first and then propylene glycol was added last, the dissolution rate of the drug increased with the increase of temperature. Under the condition of 25°C, the drug was still not dissolved after stirring for 5 hours; under the conditions of 40°C and 60°C, the drug was completely dissolved within 15 minutes.
  • the particle size distribution measurement results showed that the preparation temperature had almost no effect on the particle distribution of the micelle composition, and the order of adding 15-hydroxystearate polyethylene glycol and propylene glycol did not affect the particle size of the preparation.
  • the API was added, then propylene glycol was added, and then 15-hydroxystearate polyethylene glycol ester at 25°C (solid), 40°C (molten) and 60°C was added respectively to prepare Plinabulin micellar compositions G, H and I, and then diluted with 5% glucose injection at 1:20 to obtain a liquid injection Plinabulin composition (preparation), and the particle size was measured.
  • the results showed that when the temperature was not lower than 40°C, the API could be dissolved in the propylene glycol solvent alone in a short time, and after adding 15-hydroxystearate polyethylene glycol ester, the solution was still clear; but when the propylene glycol temperature was 25°C, the API was difficult to dissolve.
  • the particle size distribution measurement results showed that the preparation method of dissolving the drug in propylene glycol first had no effect on the particle size distribution.
  • the API was added, and then the propylene glycol and 15-hydroxystearate polyethylene glycol solution pre-mixed at 40°C were added to prepare the Plinabulin micelle composition J, which was then diluted with 5% glucose injection at 1:20 to obtain a liquid injection Plinabulin composition (preparation), and the particle size was measured.
  • the results showed that the average particle size of the preparation in which the API was first added and then the propylene glycol and 15-hydroxystearate polyethylene glycol mixed solution was increased from 104.61nm to 112.52nm, and D10 increased significantly, compared with the particle size result of the micelle composition of 1.1. However, there was no significant difference in the particle size of the diluted sample.
  • the particle size results show that compared with the particle size results in 1.1, the API is first dispersed in 15-hydroxystearic acid polyethylene glycol ester, and then propylene glycol is added, and the particle size of the stock solution is significantly increased.
  • the average particle size of the micelle composition increases from 104.61nm to 127.06mn, D10
  • D50 increasing from 100.08nm to 118.03nm
  • D90 increasing from 145.88nm to 192.41nm.
  • the preparation time of the micellar composition is shortened with the increase of the preparation temperature.
  • the order of 15-hydroxystearic acid polyethylene glycol ester and propylene glycol has no obvious effect on the preparation process and the particle size results of the micellar composition.
  • dispersing with propylene glycol first and then adding 15-hydroxystearic acid polyethylene glycol ester shortens the preparation time, and its particle size results are not significantly different from the production conditions; adding a mixture of 15-hydroxystearic acid polyethylene glycol ester and propylene glycol, there is no significant difference in preparation time and phenomenon, but the particle size distribution results of the micellar composition show that the average particle size, D10, D50, and D90 are all increased; first dispersing with 15-hydroxystearic acid polyethylene glycol ester and then adding propylene glycol, the preparation time is slightly increased, and the particle size distribution results of the micellar composition show that the average particle size, D10, D50, and D90 are all increased, and are greater than the sample with the addition of 15-hydroxystearic acid polyethylene glycol ester and propylene glycol mixture.
  • This example studies the micelle characteristics of the prepared injectable Plinabulin solution.
  • the encapsulation rate of the diluted Plinabulin was determined by separating the encapsulated drug and the free drug in the micelle.
  • the liquid phase assay was selected as the content determination method for this product, and the reproducibility and specificity of the method were confirmed.
  • the liquid phase conditions and the experimental procedures are shown in Table 2-1.
  • the equilibrium solubility of the drug in the solvent system of the injection preparation without a solubilizer (15-hydroxystearate polyethylene glycol) was determined to provide a reference for the determination of free drugs. Take 40 mg of Plinabulin API, add it to 6.0 g of propylene glycol, and then add it to 200 ml of D5W. The resulting solution was shaken continuously at 25°C and 100 rpm for 48 hours. The test solution was spun down by centrifugation and filtered through a 0.45 ⁇ m nylon filter. The supernatant and subsequent filtrate were injected into the HPLC system and the peak area was recorded. The results are shown in Table 2-5 below
  • the 0.45 ⁇ m nylon filter membrane has obvious adsorption of Plinabulin; after the supernatant after centrifugation is diluted with acetonitrile, the peak area of Plinabulin increases significantly, which can be judged that the supernatant still contains undissolved Plinabulin. Therefore, for Plinabulin, the test solution cannot be prepared by direct centrifugation, and a suitable filter membrane should be selected for filtration.
  • test solution was taken, filtered through a 0.45 ⁇ m glass fiber membrane, and diluted.
  • the subsequent filtrate was confirmed with acetonitrile to confirm the dissolution state of Plinabulin in the filtrate.
  • the corresponding continuous filtrate was injected into the HPLC system and the peak area was recorded. The results are shown in Table 2-6.
  • the corresponding propylene glycol-D5W injection was prepared.
  • the preparation process is as follows:
  • Diluent 1 (corresponding to a dilution ratio of 1:20). Weigh about 6g of propylene glycol, put it into a beaker, add 200ml of D5W injection solution, and stir evenly.
  • Diluent 2 (corresponding to a dilution ratio of 1:30). Weigh about 4 g of propylene glycol, put it into a beaker, add 200 ml of D5W injection, and mix well.
  • Diluent 3 (corresponding to a dilution ratio of 1:50): weigh about 2.4 g of propylene glycol, put it into a beaker, add 200 ml of D5W injection, and mix well.
  • the reference solution is prepared with absolute ethanol. Since this product is used to determine plasma protein in D5W solution, the effect of different diluents on the determination was investigated.
  • the experimental design is as follows, see Table 2-8
  • test solution was taken at different time points, filtered with a 0.45 ⁇ m glass fiber membrane, injected into a high performance liquid chromatography system, and the equilibrium solubility of Plinabulin in different proportions of solution at each time point was calculated using the external standard method. The results are shown in Table 2-10.
  • the final method for determining packaging efficiency is as follows:
  • Free test solution Take about 4 ml of the test solution of each dilution, place it in an ultrafiltration centrifuge tube with a molecular weight of 30 kd regenerated cellulose, centrifuge at 4500g, and discard the ultrafiltration centrifuge tube every 10 minutes. Add about 4 ml of the test solution to all solutions, continue centrifugation, repeat the operation 6 times (total centrifugation for 60 minutes), and take the filtrate after 60 minutes as the free test solution.
  • Stock solution of reference substance Take about 25 mg of Plinabulin reference substance, weigh accurately, put it into a 100 ml volumetric flask, add absolute ethanol to dissolve and dilute to the mark, and shake well.
  • Preparation of reference solution Accurately weigh 1 ml of the reference solution stock solution, place it in a 100 ml volumetric flask, dilute to the mark with absolute ethanol, shake well, and use it as the reference solution.
  • Sensitivity solution Accurately measure an appropriate amount of the above reference solution and dilute it with absolute ethanol to prepare a solution containing approximately 0.05 ⁇ g of Plinabulin per 1 ml.
  • the relative deviation of the peak area of Plinabulin should not exceed 2.0% when the reference solution is injected 5 times continuously; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the Plinabulin peak height should be greater than 10.
  • Determination method Accurately determine the total amount of reference solution, test solution and free test solution, inject into the liquid chromatography system, record the chromatogram, and calculate the peak area by the external standard method.
  • the calculation method is as follows:
  • a pair peak area of reference substance.
  • Cfree free concentration of the test sample ( ⁇ g/ml).
  • Afree free peak area of the test sample.
  • CTotal Total concentration of the test sample ( ⁇ g/ml).
  • ATotal Peak area of the total amount of the test sample.
  • f dilution multiple of the total amount of the test sample.
  • the encapsulation rate results are as follows:
  • Dilution level solution Accurately weigh about 10.36 g of plasma concentrate, put it into a 250 ml volumetric flask, add 200 ml of D5W injection solution, and shake up and down 30 times within 1 minute.
  • Free test solution Measure 2 ml of the above diluted solution, place it in 50 ml pH 7.4 phosphate buffer (37°C), and stir for 1 minute. Take about 4 ml of the solution (re-configure new solution every 10 minutes and replace the solution in the centrifuge tube), put it into the inner tube of the ultrafiltration centrifuge tube (regenerated cellulose molecular weight 30kd), centrifuge (speed 4500g) for 20min, 40min, 50min, 60min, 70min, 80min, and take the filtrate as the free test solution.
  • test solution Prepare the total amount of the test solution: accurately measure 2 ml of the above solution, place it in a 50 ml volumetric flask, dilute to the mark with absolute ethanol, shake well, filter, and use as the test solution.
  • the critical micelle concentration (CMC) of 15-hydroxystearic acid polyethylene glycol ester contained in the 15-hydroxystearic acid polyethylene glycol ester excipient is between 0.005% and 0.02%.
  • the critical micelle concentration of 15-hydroxystearic acid polyethylene glycol ester measured by steady-state fluorescence method is 0.0035%.
  • the concentration of 15-hydroxystearate polyethylene glycol ester gradually increases, and the encapsulation efficiency also gradually improves.
  • the final encapsulation rate of the test solution at different dilution levels is above 80%.
  • the batch samples of Plinabulin were diluted to dilution ratios of 1:10, 1:20, 1:30 and 1:50, and the diluted solutions were stored at room temperature in the dark, and samples were taken at 0h, 2h, 4h, 6h, 8h, 12h and 24h to study the stability of the micelle properties.
  • the results are as follows:
  • This product was diluted with D5W injection solution in different ratios (1:10, 1:20, 1:30, 1:50), and the total amount of the test solution was stored at room temperature in the dark for 24 hours. There was no significant change in concentration and encapsulation rate, indicating that the micelle solution was stable at room temperature for 24 hours.
  • Critical micelle concentration is used to evaluate the micelle characteristics of substances.
  • Commonly used measurement methods include surface tension method, conductivity method, fluorescent probe method, dye method, etc.
  • the fluorescent probe method has been widely used in determining the critical micelle concentration of surfactants because of its advantages such as simple operation and little interference with the research system. Therefore, it was decided to use the fluorescent probe method to determine the concentration of 15-hydroxystearate polyethylene glycol ester in the formulation system.
  • Critical microsphere concentration in 5% glucose injection and pH7.4 PBS solution. The statistics of equipment and reagents used in the study are as follows:
  • pyrene as a fluorescent reagent, place it in a 10 ml volumetric flask, dissolve it with acetone and dilute it to the mark, shake it well.
  • concentration of pyrene in the solution is 2.0 mg/ml (1.0 ⁇ 10-5 mol/ml).
  • the concentration of 15-hydroxystearate polyethylene glycol micelles in the solution is 1 ⁇ 10 -7 g/ml, 5 ⁇ 10 -7 g/ml, 1 ⁇ 10 -6 g/ml, 5 ⁇ 10 -6 g/ml, 1 ⁇ 10 -5 g/ml, 5 ⁇ 10 -5 g/ml, 1 ⁇ 10 -4 g/ml, 5 ⁇ 10 -4 g/ml, 1 ⁇ 10 -3 g/ml, 1 ⁇ 10 -2 g/ml. Measure after 24 hours at room temperature.
  • the specific measurement parameters of the fluorescence photometer are as follows:
  • the fluorescence spectra of the two groups of solutions were measured according to the above method after being placed at room temperature for 24 hours.
  • CMC is in the range of 0.005% to 0.02%.
  • the CMC of 15-hydroxystearate polyethylene glycol in water is determined to be 0.0035% as a control. There was no significant difference in CMC among the liquid systems.
  • This example describes the results of a dilution study conducted to determine the appearance, detection, impurity, and microbiological evaluation of Plinabulin (4 mg/mL) (propylene glycol/15-hydroxystearate solution) diluted in 5% dextrose (D5W) in a non-PVC IV bag (500 mL) at different time periods after dilution.
  • Plinabulin 4 mg/mL
  • D5W dextrose
  • six (6) clear vial samples of 4 mg/mL Plinabulin and six (6) turbid vial samples of 4 mg/mL Plinabulin were diluted with D5W to obtain two dilution levels: about 1:20 and about 1:200.
  • Plinabulin (4 mg/mL) vials were refrigerated turbid and then diluted by heating at 37°C for 1 hour to reverse the turbidity.
  • the diluted samples were tested initially (0 hours) at room temperature and again after being stored in the dark for 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, and 48 hours.
  • the post-dilution stability and potential microbiological risk of Plinabulin (4 mg/mL) were determined at 6 hours, 8 hours, 12 hours, 24 hours, and 48 hours after dilution with D5W in non-PVC IV bags (500 ml).
  • Filter interference studies were performed using the first batch of product at dilutions of 1:20 and 1:200. For the filter interference studies, the 1:200 dilution did not meet the acceptance criteria, so unfiltered samples were used throughout the study.
  • Results were identical for the clear vials and the vials with turbidity due to refrigeration for both the dilution study and the microbial count study. All samples met the acceptance criteria for microbial counts for both dilutions and all time points.
  • the 1:20 dilution met the stability criteria of the assay at 0, 4, 6, 8, and 12 hours.
  • the 1:20 dilution failed to meet acceptance criteria at 24 and 48 hours post dilution due to the presence of precipitate and potency ⁇ 90% of label claim.
  • the 1:200 dilution met the stability criteria of the assay at 0, 4, 6, and 8 hours post dilution.
  • the 1:200 dilution failed to meet acceptance criteria at 12, 24, and 48 hours post dilution due to the presence of precipitate and potency ⁇ 90% of label claim.
  • Possible interference from the 0.2 micron PBS filter was evaluated by filtering and analyzing a portion of the sample.
  • a portion of the filtered sample was drawn from the port with the infusion tubing and compared to a portion of the unfiltered sample drawn directly from the outlet port.
  • Three (3) samples were prepared and tested at each dilution level (1:20 and 1:200).
  • a placebo control sample was prepared at the 1:20 dilution level to determine the peak placebo value.
  • the assay and impurity values obtained from the filtered portion were compared to the values from the unfiltered portion.
  • the assay values obtained from the filtered portion were compared to the values from the unfiltered portion.
  • the results of the filter interference study are listed in Table 3-1.
  • Plinabulin Injection 4 mg/mL was diluted with D5W in a non-PVC IV bag to generate a diluted IV solution.
  • the volume of D5W solution contained in a single non-PVC IV bag (500 mL) was measured by pouring the bag into a 500 mL graduated cylinder or volumetric flask to the full, using an appropriately sized graduated cylinder with an accuracy of 1 mL or better.
  • the required volume of Plinabulin (4 mg/mL) is determined according to Formula 2.
  • a nominal concentration of 0.2 mg/mL is obtained by a dilution factor of 1:20.
  • a nominal concentration of 0.02 mg/mL is obtained by a dilution factor of 1:200.
  • C IV bag is the desired final concentration of Plinabulin in the diluted IV bag, in mg/ml (i.e., 0.2 mg/ml or 0.02 mg/ml)
  • V IV bag is the expected starting volume of D5W in the IV bag, in ml, from the manufacturer's product instructions.
  • ml Vaverage , ml + ((weight of Plinabulin injection (4mg/ml) , g)/(d of Plinabulin injection (4mg/ml) , g/ml)).
  • Vaverage refers to the average volume measured from 4 intravenous injection bags
  • unit Wt.Plinabulin Injection (4 mg/ml) is the actual weight of Plinabulin Injection (4 mg/ml) added to the IV bag, in g
  • dPlinabulin Injection (4 mg/ml) is the density of Plinabulin Injection (4 mg/ml) obtained from the execution batch record.
  • WtPlinabulin Injection (4mg/ml) refers to the actual weight added to the intravenous bag
  • dPlinabulin Injection (4mg/ml) refers to the density of Plinabulin Injection (4mg/ml) obtained from the execution batch record
  • VIntravenous Bag refers to the actual volume in the intravenous bag, determined by Formula 2.
  • the 1:20 dilution met the stability criteria for the test at 0, 4, 6, 8, and 12 hours.
  • the 1:20 dilution did not meet the acceptance criteria at 24 and 48 hours after dilution due to the presence of precipitation and potency ⁇ 90% of the label claim.
  • the 1:200 dilution met the stability criteria for the test at 0, 4, 6, and 8 hours after dilution. Stability criteria for the test.
  • the 1:200 dilution sample failed to meet the acceptance criteria at 12, 24, and 48 hours after dilution due to the presence of precipitate and potency ⁇ 90% of the label claim.

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Abstract

一种普那布林胶束组合物及其制备方法，具体地，所述普那布林胶束组合物基本上由普那布林和15-羟基硬脂酸聚乙二醇酯与丙二醇组成，并且至少有一部分普那布林被封装在胶束中，其可与葡萄糖注射液混合得到注射用普那布林胶束制剂，并用于临床治疗肿瘤疾病。普那布林胶束组合物以及胶束制剂均具有优异的电化学稳定性、稀释稳定性及存储稳定性。

Description

一种普那布林胶束组合物及其制备方法 技术领域
本发明涉及药物制剂领域，具体涉及一种普那布林胶束组合物及其制备方法。
背景技术
普那布林((3Z,6Z)-3-[(5-叔丁基-1H-咪唑-4-基)亚甲基]-6-(苯亚甲基)-2,5-哌嗪二酮)是从海洋和陆生的曲霉属菌种中发现的二酮哌嗪苯基阿夕斯丁(phenylahistin)(月卞三甲氯铵)的合成类似物，其结构如下所示：
普那布林在结构上不同于秋水仙碱及其康普瑞汀(combretastatin)样类似物(例如，康普瑞汀磷酸盐)并且在微管蛋白单体上的秋水仙碱结合位点处或其附近结合。先前的研究表明与秋水仙碱相比，在低浓度下的普那布林诱导血管内皮细胞微管蛋白解聚和单层透过性，并且表明普那布林诱导Jurkat白血病细胞内的细胞凋亡。普那布林在患有晚期恶性肿瘤(肺癌、前列腺癌和结肠癌)的患者中作为单一试剂的研究表现出良好的药代动力学、药效学和安全特性。然而，仍然需要提供具有适当物理化学性质的普利那布的注射制剂。
发明内容
本发明目的是提供一种新型的普那布林胶束及其制备方法，所述的普那布林胶束具有较好的稳定性，所得胶束既能提高普那布林的水溶性，又能延缓药物释放，延长生物半衰期，具有良好的临床应用价值。
本发明的第一方面，提供一种普那布林胶束组合物的制备方法，其中，所述方法包括步骤：
s1)35℃-65℃下，将15-羟基硬脂酸聚乙二醇酯与丙二醇混合，制备得到澄清混合液；
s2)35℃-65℃下，将步骤1)中的澄清混合液与普那布林混合，搅拌，制得普那布林胶束组合物。
在另一优选例中，15-羟基硬脂酸聚乙二醇酯与丙二醇重量比为1:5-5:1，优选地，1:3-3:1，更优选地2:3。
在另一优选例中，15-羟基硬脂酸聚乙二醇酯与丙二醇重量比为1:9、2:8、3:7、4:6、5:5、6:4、7:3、8:2、或9:1。
在另一优选例中，所述胶束组合物基本上由普那布林和15-羟基硬脂酸聚乙二醇酯与丙二醇组成。
在另一优选例中，步骤s1)中，温度为40℃-60℃。
在另一优选例中，步骤s2)中，温度为40℃-60℃。
在另一优选例中，步骤s1)为将15-羟基硬脂酸聚乙二醇酯在50℃±5℃条件下熔化后，再加入丙二醇，保持40℃±5℃条件下，搅拌(例如5min-2h，优选20-40min，更优选30min)，制备得到澄清混合液。
在另一优选例中，步骤s1)为将15-羟基硬脂酸聚乙二醇酯在35℃-65℃条件下熔化后，再加入丙二醇，保持35℃-65℃条件下，搅拌(例如5min-2h，优选10-40min，更优选20-30min)，制备得到澄清混合液。
在另一优选例中，步骤s1)为将15-羟基硬脂酸聚乙二醇酯在35℃-65℃条件下熔化后，加入到丙二醇中，保持35℃-65℃条件下，搅拌(例如5min-2h，优选10-40min，更优选20-30min)，制备得到澄清混合液。
在另一优选例中，步骤s2)中澄清混合液分批加入，优选地分为2-5个批次。
在另一优选例中，普那布林的浓度为0.02mg/ml-4mg/ml，以普那布林胶束组合物的总质量计。
在另一优选例中，普那布林为普那布林无水物或普那布林一水合物。
在另一优选例中，普那布林为普那布林无水物(晶型III)或普那布林一水合物(晶型I)。
在另一优选例中，在步骤(s2)中，所使用的普那布林为普那布林一水合物。
在另一优选例中，普那布林为普那布林一水合物(晶型I)。
在另一优选例中，所述普那布林无水物为CN113735834B中的普那布林无水形式。
在另一优选例中，所述普那布林一水合物为CN113735834B中的普那布林一水合物。
在另一优选例中，所得普那布林胶束组合物中胶束的粒径D50为5-100nm。
本发明第二方面，提供一种普那布林胶束组合物，其中，所述胶束组合物包含普那布林和15-羟基硬脂酸聚乙二醇酯与丙二醇的澄清混合液，其中，所述普那布林胶束组合物的为黄色澄清透明溶液，胶束粒径范围为10-100nm。
在另一优选例中，所述的普那布林胶束组合物的胶束粒径范围为80-110nm，优 选90-110nm，例如100-110nm。
在另一优选例中，所述的普那布林胶束组合物的胶束D50为80-110nm，优选90-110nm，例如100-105nm。
在另一优选例中，所述的普那布林胶束组合物的胶束D90为100-170nm，优选110-160nm，例如120-150nm。
在另一优选例中，在普那布林胶束组合物中，普那布林的浓度为1mg/ml-10mg/ml，以普那布林胶束组合物的总质量计，优选普那布林的浓度为2mg/ml-5mg/ml，更优选普那布林的浓度为3mg/ml-4mg/ml。
在另一优选例中，在所述普那布林胶束组合物中，所述普那布林为普那布林一水合物。
在另一优选例中，在所述普那布林胶束组合物中，15-羟基硬脂酸聚乙二醇酯与丙二醇重量比为1:5-5:1，优选地，1:3-3:1，更优选地2:3。
在另一优选例中，所述的普那布林胶束组合物是采用如第一方面所述的方法制备的。
在另一优选例中，所述的普那布林胶束组合物为无菌的。
本发明第三方面，提供一种液体注射用普那布林组合物，其包括：
普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯的D5W溶液(5％葡萄糖注射液)；
其中，所述组合物中丙二醇和D5W的体积比为约6：50至约6：500。
在另一优选例中，在所述液体注射用普那布林组合物中，所述普那布林为普那布林一水合物。
在另一优选例中，普那布林被封装在胶束中，优选地，至少有90％的普那布林被封装在胶束中。
在另一优选例中，至少有95％的普那布林被封装在胶束中，优选至少97％，更优选至少99％。
在另一优选例中，所述组合物中丙二醇和D5W的体积比为约6：100至约6：400，优选地为约6：150至约6：250，更优选地为约6：200。
在另一优选例中，所得注射用普那布林组合物中胶束的粒径D50为5-50nm，优选10-30nm。
在另一优选例中，普那布林的浓度为约0.08mg/ml至约0.4mg/ml。
在另一优选例中，液体注射用普那布林制剂中15-羟基硬脂酸聚乙二醇酯和D5W的体积比在约4:50-约4:500，优选为约4:100至约4:500，更优选为约4:100-约4:400，例如约4:100-约4:300、或约4:150-约4:250。
在另一优选例中，所述液体注射用普那布林组合物中，15-羟基硬脂酸聚乙二 醇酯与丙二醇重量比为1:9、2:8、3:7、4:6、5:5、6:4、7:3、8:2、或9:1。在另一优选例中，丙二醇与15-羟基硬脂酸聚乙二醇酯的比例约为60:40(wt:wt)。
在另一优选例中，所述的液体注射用普那布林组合物包含的杂质总量小于0.5％。在另一优选例中，所述的液体注射用普那布林组合物包含的杂质总量小于0.1％。
在另一优选例中，所述的液体注射用普那布林组合物可稳定保存约8小时至约12小时。
在另一优选例中，所述的液体注射用普那布林组合物可稳定保存的温度为10℃至约37℃，优选地为室温。
在另一优选例中，至少有一部分普那布林被封装在胶束中，优选地，至少90％的普那布林被封装在胶束中。
在另一优选例中，液体注射用普那布林组合物中大于约90％的普那布林被封装在胶束中。
在另一优选例中，液体注射用普那布林组合物中大于约93％的普那布林被封装在胶束中，优选地，大于约94％的普那布林被封装在胶束中，优选地，大于约95％的普那布林被封装在胶束中，优选地，大于约96％的普那布林被封装在胶束中，优选地，大于约97％的普那布林被封装在胶束中，优选地，大于约99％的普那布林被封装在胶束中。
在另一优选例中，所述液体注射用普那布林组合物是采用如本发明第四方面所述的方法制备的。
本发明第四方面，提供一种制备液体注射用普那布林组合物的方法，其中，所述方法包括：
提供一种初始液体制剂，其包括普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯；和
以约1:5至约1:50的稀释比在D5W中稀释所述初始液体制剂。
在另一优选例中，所述初始液体制剂为本发明第二方面所述的普那布林胶束组合物。
在另一优选例中，所述稀释比为约1:10至约1:50，优选地，所述稀释比为约1:13至约1:30，更优选地，所述稀释比为约1:20。
在另一优选例中，所述初始液体制剂包括浓度为约1mg/ml至约6mg/ml的普那布林，优选地包括浓度为约3mg/ml至约5mg/ml的普那布林，更优选地包括浓度为约4mg/ml的普那布林。
在另一优选例中，包括在D5W中稀释初始液体制剂后，搅拌所述组合物，优选地，搅拌至少5分钟。
在另一优选例中，在液体注射用普那布林组合物中，普那布林的浓度为约0.08mg/ml至约0.4mg/ml。
在另一优选例中，液体注射用普那布林组合物中丙二醇与15-羟基硬脂酸聚乙二醇酯的比例约为60:40(wt:wt)。
在另一优选例中，所述的初始液体制剂为如上所述的普那布林胶束组合物。
在另一优选例中，液体注射用普那布林组合物中胶束的粒径范围为90-200nm，优选地为100-150nm，更优选为100-110nm。
本发明第五方面.提供一种如第二方面所述的普那布林胶束组合物或如第三方面所述的液体注射用普那布林组合物在制备预防和/或治疗抗肿瘤药物中的用途。
在另一优选例中，所述肿瘤选自：肺癌(如小细胞肺癌、非小细胞肺癌)、前列腺癌、结肠癌、脑肿瘤(如胶质母细胞瘤、多形性胶质母细胞瘤、巨细胞胶质母细胞瘤、转移性脑肿瘤)、头颈癌、胃癌、胰腺癌、乳腺癌、肾癌、膀胱癌、卵巢癌、子宫颈癌、黑色素瘤、骨髓瘤、淋巴瘤或白血病。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了普那布林胶束组合物的丁达尔效应图。
图2显示了液体注射用普那布林组合物的丁达尔效应图。
具体实施方式
本发明人经过广泛而深入的研究，意外地获得一种新型的普那布林胶束组合物，其具有优异的稳定性，普那布林胶束组合物可提高普那布林的水溶性，并具有优异的电化学稳定性、稀释稳定性及存储稳定性。
具体地，本发明使用普那布林一水合物以及特定比例的丙二醇和15-羟基硬脂酸聚乙二醇酯，意外地制备了一种普那布林胶束组合物，普那布林封装在胶束中，该胶束组合物具备优异的稳定性、电化学稳定性、稀释稳定性及存储稳定性。
在得到普那布林胶束组合物的基础上，通过加入D5W溶液进行稀释，得到了液体注射用的普那布林组合物，在稀释后普那布林胶束组合物仍然存在，普那布林封装其中，该稀释的注射液具有优异的滴注稳定性、存储稳定性，且普那布林的封装率高，杂质含量低，可作为普那布林的注射制剂。
在此基础上，完成了本发明。
本发明公开了普那布林胶束封装组合物以及制备和使用普那布林组合物的方法。普那布林，(3Z,6Z)-3-[(5-叔丁基-1H-咪唑-4-基)亚甲基]-6-(苯亚甲基)-2,5-哌嗪二酮)，是天然化合物二酮哌嗪苯基阿夕斯丁的合成类似物。根据美国专利号7,064,201和7,919,497中详述的方法和步骤，可以很容易地制备普那布林，这两个专利号的全部内容通过引用并入本文。
除非另有定义，本文使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。尽管任何与本文所述相似或相当的方法和材料也可用于本发明的实践或测试，但本文描述的是首选的方法和材料。
术语
除非另有定义，本文使用的所有技术和科学术语与本公开内容所属领域的普通技术人员通常理解的含义相同。所有的专利、申请、已发表的申请和其他出版物都通过参考而全部纳入。如果这里的某个术语有多个定义，除非另有说明，否则以本节中的定义为准。
本文所用的普那布林包括晶型形式或无定形形式，例如普那布林一水合物、普那布林无水物、普那布林溶剂合物。优选为一水合物。
本发明中，“普那布林可注射胶束制剂”、“液体可注射的普那布林组合物”、“液体可注射制剂”和“液体可注射的普那布林制剂”和“液体注射用普那布林组合物”具有相同含义，可互换使用。
术语"药剂(agent)"在此用于表示一种化合物、一种化合物的混合物、一种生物大分子，或由生物材料制成的提取物。
术语"癌症(cancer)"、"肿瘤(neoplasm)"和"癌(carcinoma)"，在此可互换使用，指的是表现出相对自主生长的细胞，因此它们表现出异常的生长表型，其特点是细胞增殖的控制力明显丧失。一般来说，本申请中感兴趣的检测或治疗的细胞包括癌前(如良性)、恶性、转移前、转移性和非转移性细胞。对癌细胞的检测是特别有意义的。
本文所用的术语"受试者"是指人类或非人类哺乳动物，如狗、猫、老鼠、牛、羊、猪、山羊、非人类灵长类动物或鸟，如鸡，以及任何其他脊椎动物或无脊椎动物。
术语"哺乳动物"是在其通常的生物学意义上使用的。因此，它具体包括但不限于灵长类动物，包括类人猿(黑猩猩、猿、猴)和人类、牛、马、绵羊、山羊、猪、兔子、狗、猫、啮齿动物、大鼠、小鼠、豚鼠或类似动物。
本文所用的术语"有效量"或"治疗有效量"是指在某种程度上有效地缓解疾病或病症的一个或多个症状，或减少发病的可能性的治疗剂的量，并可包括治 愈疾病或病症。
本文所用的术语"治疗(treat)"是指为预防和/或治疗目的向受试者施用一种化合物或药物组合物。术语"预防治疗"是指治疗尚未表现出疾病或病症症状，但对某种特定疾病或病症易感或有其他风险的受试者，据此，所述治疗可降低患者发展为疾病或病症的可能性。术语"治疗性治疗"是指对已经患有某种疾病或病症的受试者进行治疗。
制剂(Formulations)
在一些实施方式中，普那布林以浓缩(concentrated)液体制剂的形式提供，然后可以通过稀释制备稀释的液体注射制剂。在一些实施方式中，浓缩的液体制剂包括一种或多种溶剂，其中可包括15-羟基硬脂酸聚乙二醇酯(polyoxyl 15-hydroxystearate)和/或丙二醇。在一些实施方式中，一种或多种溶剂包括至少30％(重量)的15-羟基硬脂酸聚乙二醇酯，至少35％(重量)的15-羟基硬脂酸聚乙二醇酯，至少40％的15-羟基硬脂酸聚乙二醇酯，至少45％(重量)的15-羟基硬脂酸聚乙二醇酯，或包括和/或跨越上述值的范围。在一些实施方式中，一种或多种溶剂包括至少50％(重量)的丙二醇、至少55％(重量)的丙二醇、至少50％(重量)的丙二醇、至少55％(重量)的丙二醇、至少60％(重量)的丙二醇，或包括和/或跨越上述值的范围。在一些实施方式中，一种或多种溶剂包括40％(重量)15-羟基硬脂酸聚乙二醇酯和60％(重量)丙二醇。在一些实施方式中，液体注射制剂中丙二醇与15-羟基硬脂酸聚乙二醇酯的比例约为60：40(重量比)。
在一些实施方式中，浓缩的液体制剂为胶束组合物。普那布林被封装在胶束中。在一些实施方式中，所述胶束组合物的成分包含15-羟基硬脂酸聚乙二醇酯、丙二醇和普那布林，其中普那布林封装于胶束中。在一些实施方式中，至少60％、至少62％、至少64％、至少66％、至少68％、至少70％、至少72％、至少74％、至少76％、至少78％、至少80％、至少82％、至少84％、至少86％、至少88％、至少90％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％，或包括和/或跨越上述数值的范围的普林布林被封装在胶束中。
在一些实施方式中，所述胶束组合物中的普那布林为普那布林一水合物。当使用普那布林无水形式时，所得到的胶束组合物溶解性差。
浓缩液体制剂中的普那布林的浓度可以在1mg/ml和10mg/ml之间，2mg/ml和6mg/ml之间，或大约4mg/ml。
在一些实施方式中，浓缩的液体制剂可以用稀释剂稀释，形成稀释的液体注射制剂。在一些实施方式中，稀释剂为水。在一些实施方式中，稀释剂为盐水。在一些实施方式中，稀释剂为葡萄糖水溶液。葡萄糖的浓度可以是1％至20％，2％至10％，或约5％(即D5W)。
在一些实施方式中，稀释的液体注射制剂包括15-羟基硬脂酸聚乙二醇酯、丙 二醇和D5W(即葡萄糖和水)。在一些实施方式中，液体注射用普利那布制剂中丙二醇和D5W的体积比在约6:50-约6:500、约6:50-约6:450、约6:50-约6:400、约6:50-约6:350、约6∶50-约6∶300、约6∶50-约6∶250、约6∶50-约6∶200、约6∶50-约6∶150、约6∶50-约6∶100、约6∶100-约6∶500、约6∶100-6∶400、约6∶150-约6∶250，或约6∶200的范围内。在一些实施方式中，液体注射用普那布林制剂中丙二醇和D5W的体积比约为6：70。在一些实施方式中，液体注射用普那布林制剂中丙二醇和D5W的体积比约为6：140。在一些实施方式中，液体注射用普那布林制剂中丙二醇和D5W的体积比约为6:400。
在一些实施方式中，液体注射用普那布林制剂中15-羟基硬脂酸聚乙二醇酯和D5W的体积比在约4:50-约4:500、约4:100至约4:500、约4:100-约4:400、约4:100-约4:300、约4:150-约4:250，或约4:200的范围内。
在一些实施方式中，液体注射用普那布林制剂(组合物)中的普那布林为普那布林一水合物。
在一些实施方式中，液体注射用普那布林制剂(组合物)中的普那布林的浓度约为0.02mg/ml、0.03mg/ml、0.04mg/ml、0.05mg/ml、0.06mg/ml、0.07mg/ml、0.08mg/ml、0.09mg/ml、0.1mg/ml、0.11mg/ml、0.12mg/ml、0.13mg/ml、0.14mg/ml、0.15mg/ml、0.16mg/ml、0.17mg/ml、0.18mg/ml、0.19mg/ml、0.2mg/ml、0.3mg/ml、0.4mg/ml、0.5mg/ml、1mg/ml、2mg/ml、3mg/ml、4mg/ml，或包括和/或跨越上述值的范围。在一些实施方式中，在液体注射用普那布林组合物中，普那布林的浓度为约0.08mg/ml至约0.4mg/ml。在一些实施方式中，在液体注射用普那布林组合物中，普那布林的浓度为约0.1mg/ml至约0.3mg/ml。在一些实施方式中，在液体注射用普那布林组合物中，普那布林的浓度约为0.2mg/ml。
在一些实施方式中，液体注射用普那布林制剂(组合物)包含的杂质小于5％、小于4％、小于3％、小于2％、小于1％、小于0.5％，或包括和/或跨越上述数值的范围。在一些实施方式中，液体注射用普那布林制剂(组合物)包含的醚杂质小于0.5％。在一些实施方式中，液体注射用普那布林制剂(组合物)包含的醇杂质少于0.5％。在一些实施方式中，液体注射用普那布林制剂(组合物)包含的水少于0.5％。
在一些实施方式中，液体注射用普那布林制剂(组合物)可稳定保存约4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、13小时、14小时、15小时、16小时、17小时、18小时、19小时、20小时、21小时、22小时、23小时、24小时、36小时、48小时，或包括和/或跨越上述值的范围。
在一些实施方式中，液体注射用普那布林制剂(组合物)可在10℃、12℃、14℃、16℃、18℃、20℃、22℃、24℃、26℃、28℃、30℃、32℃、34℃、36℃、38℃、40℃或包括和/或跨越上述数值的范围内稳定保存。
在一些实施方式中，当浓缩的液体制剂用稀释剂(如D5W)稀释时，部分普那 布林被包裹在胶束内。在一些实施方式中，胶束是由15-羟基硬脂酸聚乙二醇酯和D5W之间的相互作用形成的。在一些实施方式中，至少60％、至少62％、至少64％、至少66％、至少68％、至少70％、至少72％、至少74％、至少76％、至少78％、至少80％、至少82％、至少84％、至少86％、至少88％、至少90％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％，或包括和/或跨越上述数值的范围的可注射液体制剂(组合物)中的普林布林被封装在胶束中。
在一些实施方式中，液体制剂的平均封装效率为至少90％、至少92％、至少94％、至少96％、至少98％，或包括和/或跨越上述数值的范围。
制备方法
在某些方面，公开了制备本文所述的液体注射用制剂(组合物)的方法。在一些实施方式中，制备液体注射用普那布林组合物的方法包括提供初始浓缩液体制剂(initial concentrated liquid formulation)，其中包括普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯(PEG15-hydroxystearates)；以及将初始液体制剂在D5W中稀释。在各种实施方式中，初始浓缩液体制剂和D5W之间的稀释比例为约1:5至约1:50，约1:10至约1:50、约1:10至约1:40、约1:15至约1:30、约1:15至约1:25，或约1:20。在一些实施方式中，稀释比例为约1:10至约1:400。在一些实施方式中，稀释比例为约1:10至约1:300。在一些实施方式中，稀释比例为约1:10至约1:200。在一些实施方式中，稀释比例为约1:10至约1:100。在一些实施方式中，稀释比例为约1:10至约1:80。在一些实施方式中，稀释比为约1:10至约1:60。在一些实施方式中，稀释比例为约1:10至约1:50。在一些实施方式中，稀释比例为约1:10至约1:40。在一些实施方式中，稀释比例为约1:10至约1:30。在一些实施方式中，稀释比例为约1:10至约1:20。在一些实施方式中，稀释比例为约1:20至约1:400。在一些实施方式中，稀释比例为约1:20至约1:300。在一些实施方式中，稀释比例为约1:20至约1:200。在一些实施方式中，稀释比例为约1:20至约1:100。在一些实施方式中，稀释比例为约1:20至约1:80。在一些实施方式中，稀释比例为约1:20至约1:60。在一些实施方式中，稀释比例为约1:20至约1:50。在一些实施方式中，稀释比例为约1:20至约1:40。在一些实施方式中，稀释比例为约1:20至约1:30。
在一些实施方式中，稀释方法包括将初始浓缩液体制剂引入(例如，通过注射)含有一定体积D5W的容器中。在一些实施方式中，所述容器是一个静脉注射(IV)袋。在一些实施方式中，在将初始浓缩液体制剂引入容器后，对混合物进行搅拌。各种搅拌方法包括手摇、手搅拌或涡旋。在各种实施方式中，混合物被搅拌至少1分钟、至少2分钟、至少3分钟、至少5分钟、至少10分钟、至少20分钟、至少30分钟、至少45分钟、至少60分钟、至少90分钟，或包括和/或跨越上述值的范围。
在制备初始浓缩液体制剂的一些实施方式中，首先加入丙二醇，其次是15-羟基硬脂酸聚乙二醇酯，然后是普那布林。在一些实施方案中，首先加入15-羟基硬 脂酸聚乙二醇酯，其次是丙二醇，然后是普那布林。在一些实施方式中，初始浓缩液体制剂在室温下混合。
在制备初始浓缩液体制剂的一些实施方式中，加热丙二醇至约25℃，并在加入15-羟基硬脂酸聚乙二醇酯期间保持在约25℃。在一些实施方式中，将普那布林加入到15-羟基硬脂酸聚乙二醇酯和丙二醇中，并在约25℃下混合。
在制备初始浓缩液体制剂的一些实施方式中，加热丙二醇至约40℃，并在加入15-羟基硬脂酸聚乙二醇酯期间保持在约40℃。在一些实施方式中，将普那布林加入到15-羟基硬脂酸聚乙二醇酯和丙二醇中并在约40℃下混合。
在制备初始浓缩液体制剂的一些实施方式中，将15-羟基硬脂酸聚乙二醇酯加热至约25℃，并在加入丙二醇时保持在约25℃。在一些实施方式中，将普那布林加入到15-羟基硬脂酸聚乙二醇酯和丙二醇中，并在约25℃下混合。
在制备初始浓缩液体制剂的一些实施方式中，将15-羟基硬脂酸聚乙二醇酯加热至约40℃，并在加入丙二醇时保持在约40℃。在一些实施方式中，将普那布林加入到15-羟基硬脂酸聚乙二醇酯和丙二醇中，并在约40℃下混合。
在制备初始浓缩液体制剂的一些实施方式中，将15-羟基硬脂酸聚乙二醇酯加热至约60℃，并在加入丙二醇时保持在约60℃。在一些实施方式中，将普那布林添加到15-羟基硬脂酸聚乙二醇酯和丙二醇中，并在约60℃下混合。
用途和方法
一些实施方式涉及一种在受试者中阻止或逆转癌症进展的方法。在一些实施方式中，所述方法包括向受试者施用本文所述的可注射液体制剂。一些实施方式涉及一种抑制癌症进展的方法。本文所述的可注射液体制剂的进一步用途包括美国专利号7,919,497；10,238,650；10,155,748；10,076,518；和10,596,169；和PCT出版号WO 2016/130839；WO 2017/214052；WO 2018/144764；WO 2018/169887；WO 2019/147615；WO 2019/152530；WO 2020/037285；WO 2021/076485；和WO 2021/225908；所有这些都通过引用全部纳入本文中。
在一些实施方式中，治疗方案包括每1周、2周、3周、4周、5周、6周、7周或8周给予本文所述的可注射液体制剂一次。在一些实施方式中，治疗方案包括每1周、2周、3周、4周、5周、6周、7周或8周给予所述的可注射液体制剂2次。在一些实施方式中，治疗方案包括在1周、2周、3周、4周、5周、6周、7周或8周的治疗周期内每1周给药一次本文所述的可注射液体制剂。在一些实施方式中，治疗方案包括在1周、2周、3周、4周、5周、6周、7周或8周的治疗周期内，每1周给予所述的可注射液体制剂两次。在一些实施方式中，治疗方案包括在21天的治疗周期中的第1天、第8天和第15天给予本文所述的可注射液体制剂。
只要所述方案在临床上可以耐受，治疗周期就可以重复。在一些实施方式中，本文所述的可注射液体制剂的治疗周期重复n次，其中n为2至30范围内的整数。在 一些实施方式中，n为2、3、4、5、6、7、8、9或10。在一些实施方式中，新的治疗周期可以在前一个治疗周期完成后立即发生。在一些实施方式中，新的治疗周期可以在前一个治疗周期完成后的一段时间内发生。在一些实施方式中，新的治疗周期可以在前一个治疗周期完成后的1周、2周、3周、4周、5周、6周或7周后发生。
在一些实施方式中，所述方法包括以约5mg/m2至150mg/m2的剂量给药普那布林。在一些实施方式中，普那布林的给药剂量大于20mg/m2。在一些实施方式中，普那布林的给药剂量大于30mg/m2。在一些实施方式中，普那布林的给药剂量大于40mg/m2。
在一些实施方式中，本文所述的可注射液体制剂在14天给药周期的第1天给药。在一些实施方式中，本文所述的可注射液体制剂在21天给药周期的第1天给药。
在一些实施方式中，本文所述的可注射液体制剂与一种或多种G-CSF药物共同给药。
一些实施方式包括包含一个或多个容器的试剂盒。
在一些实施方式中，所述容器包括塑料或玻璃，或其组合，包括但不限于本领域技术人员在本文的教导下使用的任何一种或多种塑料或玻璃。
在一些实施方式中，容器是一个小瓶。在一些实施方案中，小瓶包含约1mg、2mg、5mg、10mg、20mg、30mg、40mg、50mg、60mg、70mg、80mg、90mg、100mg、110mg、120mg、130mg、140mg、150mg、160mg、170mg、180mg、190mg、200mg或包括和/或跨越上述值的范围的量的普那布林。在一些实施方式中，所述小瓶包含普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯(PEG15-hydroxystearates)。在一些实施方式中，小瓶包括约4mg/ml的普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯(比例约为60：40(wt:wt))。在一些实施方式中，小瓶中液体制剂的体积约为10ml。
在一些实施方式中，所述容器为静脉注射袋。在一些实施方式中，静脉注射袋包括D5W。在一些实施方式中，静脉注射袋中D5W的体积为约50ml至约500ml、约100ml至约500ml、约100ml至约400ml、约100ml至约300ml、约150ml至约250ml、或约200ml。
在一些实施方式中，试剂盒包括如上所述的小瓶和如上所述的静脉注射器。
普那布林胶束组合物的制备
将15-羟基硬脂酸聚乙二醇酯在50℃±5℃条件下熔化后，称取处方量的已熔化的15-羟基硬脂酸聚乙二醇酯并加入到充满氮气的配料罐中，再加入处方量的丙二醇，保持40℃±5℃条件下，搅拌30分钟。
从配料罐(DGJ-14)中取适量15-羟基硬脂酸聚乙二醇酯与丙二醇的混合液至烧杯和杜兰瓶中备用，在细胞毒性类药品称量室的隔离器内称取处方量的普那布林，将烧杯内的15-羟与丙二醇的混合液加入其中，搅拌5分钟后投入配料罐 中；用杜兰瓶装的15-羟基硬脂酸聚乙二醇酯与丙二醇的混合液分三次充分润洗烧杯后投入配料罐中。开启磁力搅拌器(转速频率40HZ)搅拌1小时后取样进行中间产品检测(样品应避光保护)，以上过程中药液温度始终保持40℃±5℃。中间产品按取样计划取样并检测：性状、水分、密度、有关物质、微生物限度、细菌内毒素、含量测定。从配制开始至除菌过滤开始，不得超过6个小时。普那布林胶束组合物的丁达尔效应图如图1所示。
注射用普那布林组合物
以约1:5至约1:50(wt)的稀释比在D5W中稀释如上所述的普那布林胶束组合物，得到注射用普那布林组合物，其中1:20稀释比的丁达尔效应图如图2所示.
本申请相对于现有技术，具有以下主要优点
1.本申请的普那布林胶束组合物具有优异的稀释稳定性。
2.本申请的普那布林胶束组合物以及尤其制备得到的注射用普那布林组合物具有优异的存储稳定性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1不同的处方加入顺序对普那布林溶解速度以及制备所得终产品的粒度分布影响。
1.不同的处方加入顺序(丙二醇/15-羟基硬脂酸聚乙二醇酯比例：6/4)
表1-1组合物组成
1.1先加丙二醇再加15-羟基硬脂酸聚乙二醇酯最后加药
分别向乙二醇中加入25℃(固态)、40℃(熔融)和60℃的15-羟基硬脂酸聚乙二醇酯，然后加入普那布林，制备得到普那布林胶束组合物A、B和C，然后分别用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：先加入丙二醇再加入15-羟基硬脂酸聚乙二醇酯最后加入药物的制备工艺条件下，随着温度的升高，原料药溶解速率加快。25℃时，需搅拌近2h药物溶解，而40℃与60℃温度下，原料药均在约10min 完全溶解。普那布林胶束组合物A和B的平均粒径约100nm，普那布林胶束组合物C无法进行粒度分析，不同普那布林胶束组合物以1:20比例稀释后的粒度分布相似，说明制备温度对稀释后的粒度分布影响较小。
1.2先加15-羟基硬脂酸聚乙二醇酯再加丙二醇最后加药
分别向25℃(固态)、40℃(熔融)和60℃的15-羟基硬脂酸聚乙二醇酯中加入乙二醇，然后加入普那布林，制备得到普那布林胶束组合物D、E和F，然后分别用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：先加15-羟基硬脂酸聚乙二醇酯再加丙二醇最后加药的条件下，药物的溶解速度随温度升高而升高，在25℃条件下，药物搅拌5h仍未溶解；40℃和60℃条件下，药物15min内完全溶解。粒度分布测定结果显示，制备温度对胶束组合物颗粒分布几乎没有影响，并且15-羟基硬脂酸聚乙二醇酯与丙二醇的加入顺序并不会对制剂粒径产生影响。
1.3先加药再加丙二醇最后加15-羟基硬脂酸聚乙二醇酯
先加入原料药，再加入丙二醇，然后分别加入25℃(固态)、40℃(熔融)和60℃的15-羟基硬脂酸聚乙二醇酯，制备得到普那布林胶束组合物G、H和I，然后分别用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：温度不低于40℃时，原料药能在较短的时间内单独溶解于丙二醇溶剂中，且加入15-羟基硬脂酸聚乙二醇酯后，溶液仍然澄清；但当丙二醇温度为25℃时，原料药很难溶解。粒度分布测定结果显示，药物先溶解于丙二醇中的配液方式对粒度分布无影响。
1.4先加药再加丙二醇与15-羟基硬脂酸聚乙二醇酯混合液
先加入原料药，再分别加入预先于40℃条件下混合的丙二醇与15-羟基硬脂酸聚乙二醇酯溶液，制备得到普那布林胶束组合物J，然后用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：先加原料药再加丙二醇与15-羟基硬脂酸聚乙二醇酯混合液的制剂，与1.1的胶束组合物粒径结果相比较，平均粒径由104.61nm增加至112.52mn，且D10显著增加。但其稀释样品的粒径并无显著差异。
1.5先加药再加15-羟基硬脂酸聚乙二醇酯最后加丙二醇
先加入原料药，再加入已经于40℃熔融的15-羟基硬脂酸聚乙二醇酯，维持温度40℃，搅拌，再加入丙二醇，制备得到普那布林胶束组合物H，然后用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：原料药在15-羟基硬脂酸聚乙二醇酯中不能溶解，加入丙二醇后，原料药的溶解时间与1.1相比有所增加。通过粒径结果可知，与1.1中粒径结果相比较，先将原料药在15-羟基硬脂酸聚乙二醇酯中分散，再加入丙二醇，原液的粒径显著增大。胶束组合物的平均粒径由104.61nm增加至127.06mn，D10 变化不明显，D50从100.08nm增加至118.03nm，D90从145.88nm增加至192.41nm。但1:20稀释样品的粒径并无显著差异。
综上，胶束组合物的制备时间随制备温度的升高而缩短。在工艺流程方面，在最后加入原料药的情况下，15-羟基硬脂酸聚乙二醇酯与丙二醇的先后顺序对制备过程以及胶束组合物的粒径结果均并无明显影响。在先加入原料药的情况下，先用丙二醇分散再加入15-羟基硬脂酸聚乙二醇酯缩短了制备时间，其粒径结果与生产条件并无显著差异；加入15-羟基硬脂酸聚乙二醇酯与丙二醇混合液，制备时间、现象并无显著差异，但胶束组合物的粒度分布结果显示，平均粒径、D10、D50、D90均有增加；先用15-羟基硬脂酸聚乙二醇酯分散再加入丙二醇，制备时间略有增加，胶束组合物的粒度分布结果显示，平均粒径、D10、D50、D90均有增加，并且大于加入15-羟基硬脂酸聚乙二醇酯与丙二醇混合液的样品。
2不同处方比例及温度
2.1丙二醇：15-羟基硬脂酸聚乙二醇酯(7:3)
表1-2组合物组成
分别向乙二醇中加入40℃(熔融)和60℃的15-羟基硬脂酸聚乙二醇酯，然后加入普那布林，制备得到普那布林胶束组合物K和L，然后分别用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：丙二醇与15-羟基硬脂酸聚乙二醇酯比例为7:3时，溶液为浑浊状态，加入原料药后虽然可以溶解，但溶液仍然为浑浊状态。原料药的溶解速率仍和温度正相关，随着温度的升高，样品溶液的澄清度也有增加。由于样品浑浊，并不是均一的体系，故难以进行粒度测定。仅60℃制备的制剂，其稀释后的样品，得到了粒径分析的结果，与1.1所制得制剂的粒径大小及分布相近。
2.2丙二醇：15-羟基硬脂酸聚乙二醇酯(3:7)
表1-3组合物组成
分别向乙二醇中加入40℃(熔融)和60℃(熔融)的15-羟基硬脂酸聚乙二醇酯，然后加入普那布林，制备得到普那布林胶束组合物M和N，然后分别用5％葡萄糖注射液以1:20稀释，得到液体注射用普那布林组合物(制剂)，进行粒度测定，结果表明：原料药的溶解速率随温度升高而加快。粒度分布结果显示，改变丙二醇与15-羟基硬脂酸聚乙二醇酯的比例为3:7时，胶束组合物的粒径显著减小。
综上，改变丙二醇与15-羟基硬脂酸聚乙二醇酯比例时，其混合溶液的性状会发生变化。当丙二醇：15-羟基硬脂酸聚乙二醇酯＝7:3时，溶液浑浊，并且显著影响了药物的溶解速率；当丙二醇：15-羟基硬脂酸聚乙二醇酯＝3:7时，制备过程并无显著差异，但其粒径结果显著减小。可能在一定范围内，随着表面活性剂浓度的增加，胶束粒径逐渐减小。
实施例2
本实施例研究了对制备的可注射用普那布林溶液的胶束特性的。通过分离胶束中封装的药物和游离的药物，测定稀释后的普那布林的封装率。
由于待测定的目标化合物是制剂中的普那布林，因此选择液相测定法作为该产品的含量测定方法，并确认了该方法的重现性和特异性。液相条件和实验的进行如表2-1所示。
表2-1液相测定法-具体参数
流速：1.2ml/min；柱温：40℃；注射器温度：20℃；检测波长：330nm；注射量：10μl
根据表2-1中的含量测定方法，制备了普那布林溶液的样品。测定了该方法 的重现性。
表2-2溶液制备过程
将10μL的普那布林溶液、对照品溶液和供试品溶液注入液相色谱仪中。计算峰面积并根据方法标准确定产品中的普那布林含量。测量结果如表2-3和表2-4所示。
表2-3检测方法确认-重现性-系统适用性
表2-4测定方法的确认-重现性-含量测定
从试验数据和色谱图可以看出，本品的测定方法具有良好的灵敏度和重现性，能够满足本品稀释制剂封装率测定的需要。
测定药物在不含增溶剂(15-羟基硬脂酸聚乙二醇酯)的注射制剂溶剂系统中的平衡溶解度，为测定游离药物提供参考。取40mg普那布林原料药，加入6.0g丙二醇中，然后加入至200ml D5W中。所得溶液在25℃和100rpm下连续摇动48h。通过离心将试验溶液旋转下来并通过0.45μm的尼龙过滤器过滤。将上清液和随后的滤液注入高效液相色谱系统，并记录峰面积，结果如下表2-5所示
表2-5溶液处理的研究-离心和尼龙过滤
从试验数据可以看出，0.45μm的尼龙滤膜对普那布林有明显的吸附现象；离心后的上清液用乙腈稀释后，普那布林的峰面积明显增大，可以判断上清液中仍含有未溶解的普那布林。因此，对于普那布林，不能用直接离心法制备试液，应筛选出合适的滤膜进行过滤。
振荡52h后取供试品溶液，用0.45μm的玻璃纤维膜过滤，并稀释。随后的滤液用乙腈确认，以确认滤液中普那布林的溶出状态。将相应的持续滤液注入高效液相色谱系统，记录峰面积，结果见表2-6。
表2-6溶液处理的研究-玻璃纤维过滤
从试验结果可以看出，用0.45μm的玻璃纤维膜直接过滤普林布林原料溶液，过滤后的峰面积乘以乙腈进行稀释。
平衡溶解度
制备了相应的丙二醇-D5W注射液。制备过程如下：
稀释剂1(对应1：20的稀释比例)。称取约6g丙二醇，放入烧杯中，加入200ml D5W注射液，搅拌均匀。
稀释剂2(对应于1:30的稀释比例)。称取约4g丙二醇，将其放入烧杯中，加入200ml D5W注射液，并混合均匀。
稀释剂3(对应1:50的稀释比例)：称取约2.4克丙二醇，放入烧杯中，加入200毫升的D5W注射液，并混合均匀。
平衡溶解度研究的方案如表2-7所示。
表2-7平衡溶解度研究方案
可接受的标准:测定每个溶液中的普林布林的浓度，直到连续两次测定之间没有明显差异，即平衡溶解度。
在本产品的含量测定方法中，参考溶液是用绝对乙醇制备的。由于本品是用于测定D5W溶液中的血浆蛋白，因此考察了不同稀释剂对测定的影响。实验设计如下，见表2-8
表2-8对照稀释溶剂的确认
将用不同稀释剂制备的对照品溶液和48小时的供试品溶液各10μl注入高效液相色谱系统，用外标法计算溶液中普那布林的溶解度。结果如表2-9所示。
表2-9不同的对照稀释溶剂-溶解度结果
结果显示，用无水乙醇溶液和稀释剂(丙二醇/D5W注射液)制备的普那布林对照品溶液在各稀释剂中的测定结果没有显著差异。因此，在随后的封装率测定方法中，用绝对乙醇制备普那布林对照品溶液。
在不同的时间点取供试品溶液，用0.45μm的玻璃纤维膜过滤，注入高效液相色谱系统，用外标法计算各时间点不同比例溶液中普那布林的平衡溶解度。结果如表2-10所示。
表2-10不同稀释等级下普那布林的平衡溶解度
稀释的注射用普那布林的封装率
基于上述研究，最终确定封装效率的方法如下：
(1:10)和(1:20)稀释试验液：取普利那布林浓缩液(4mg/ml普利那布林在丙二醇/聚氧乙烯15羟基硬脂酸酯中，60:40(重量比))，置于250ml计量瓶中，加入D5W注射液100ml(对应1:10稀释度)或200ml(对应1:20稀释度)，上下摇动30次，1分钟。
(1:30)和(1:50)稀释度试验液：取注射用普利那布林浓缩液(4mg/ml普利那布林在丙二醇/聚氧乙烯15羟基硬脂酸酯中，60:40(重量比))，置于500ml量瓶中，加入D5W注射液300ml(对应1:30稀释度)或500ml(对应1:50稀释度)，上下摇动30次，1分钟。
游离的供试品溶液。取上述各稀释度的供试品溶液约4ml，置于分子量为30kd再生纤维素的超滤离心管内管中，以4500g速度离心，每10分钟弃去超滤离心管。在所有溶液中加入约4毫升供试品溶液，继续离心，重复操作6次(共离心60分钟)，取60分钟的滤液作为自由测试溶液。
制备供试品溶液总量：取适量的各稀释供试品溶液，用绝对乙醇稀释，制备每1ml含约2.5μg普那布林的溶液，作为供试品溶液总量。
对照品溶液母液：取约25mg普那布林对照品，准确称量，放入100ml量瓶中，加绝对乙醇溶解并稀释至标线，摇匀。
对照品溶液的制备。精确称取上述对照品溶液母液1ml，置于100ml量瓶中，用绝对乙醇稀释至标线，摇匀，作为对照品溶液。
敏感度溶液。精确测量适量的上述对照品溶液，用绝对乙醇稀释，制备出每1ml中含有约0.05μg普那布林的溶液。
色谱条件：以十八烷基硅烷键合硅胶为填料(ACE C18 4.6mm×150mm，5.0μm或相当的色谱柱)；以0.01mol/L磷酸盐缓冲液(取一水磷酸二氢钠0.82g±0.01g和磷酸钠1.20g±0.01g，溶于水稀释至1000ml，摇匀)为流动相A，以乙腈为流动相B，按下表进行梯度洗脱；流速为每分钟1.2ml；柱温为40℃；进样器温度为30℃；检测波长为330nm；进样量为20μl(见表2-11)。
表2-11
系统适用性要求：对照品溶液连续进样5次，普那布林峰面积的相对偏差不超过2.0％；灵敏度溶液的色谱中，普那布林峰高的信噪比应大于10。
测定方法：精确测定对照品溶液、供试品溶液总量和游离的供试品溶液，注入液相色谱系统，记录色谱图，按外标法计算峰面积。计算方法如下：


C对：对照品浓度(μg/ml)。
A对：对照品峰面积。
C游离：供试品游离浓度(μg/ml)。
A游离：供试品游离峰面积。
C总量：供试品总量浓度(μg/ml)。
A总量：供试品总量峰面积。
f:供试品总量稀释倍数。
使用上述方法，对6批样品的各稀释级别的封装率进行了测量，测量结果如下：
表2-12每批样品的各稀释等级的封装率结果
结果显示，随着稀释比的增加，普那布林胶束的封装率下降。
搅拌方法对封装率的影响
研究了不同摇动时间和不同摇动方法对胶束特性的影响。使用004A批次的样品，浓缩的普那布林(4mg/ml，在丙二醇/15-羟基硬脂酸聚乙二醇酯中)用D5W稀释至1:10、1:20、1:30和1:50的稀释比例。用不同频率的手动摇动或不同 时间的搅拌(vortex)来制备混合物，并测定封装率。搅拌方法如下：
表2-13摇动方法研究-具体参数
封装率结果如下：
表2-14摇动方法的研究-不同稀释等级溶液的封装率结果
结果显示，采用1分钟手动摇动30次、1分钟内手动摇动90次、搅拌1分钟、搅拌3分钟四种方法制备的样品，每个样品的封装率均在95％-99％之间。在这个范围内，说明通过不同的摇动方法稀释样品对样品的封装率影响不大。因此，可以推测不同人员稀释的样品溶液在临床使用中的封装率相对稳定。
稀释后的制剂在PBS溶液中的耐稀释性研究
稀释批量样品(稀释比例为1:10、1:20、1:30和1:50)并模拟临床输液过程。稀释的供试品溶液被控制在大约30分钟内输注到5L的pH7.4磷酸盐缓冲液中。根据人体的血容量(约为体重的7％～8％，60公斤体重，血容量约为4.2L～4.8L)，本研究用5LpH7.4磷酸盐缓冲液模拟人体血容量。采样点为10分钟、20分钟和输注完成(除供试品溶液稀释比为1:50的情况外，输注完成时间约为60分钟，过 程中采样点为输注20分钟、40分钟和输注完成)。
由于稀释抗性试验中的药物浓度较低(总浓度在7μg/ml～8μg/ml范围内)，且为磷酸盐缓冲体系，因此确定截止分子量为30kd的再生纤维素滤膜在此浓度水平上具有过滤吸附作用。
(1:20)稀释级别溶液：准确称量约10.36g的血浆浓缩液，放入250ml容量瓶中，加入200ml的D5W注射液，在1分钟内上下摇动30次，即得。
游离的供试品溶液：量取上述稀释的溶液2ml，置于50ml pH7.4磷酸盐缓冲液(37℃)中，搅拌1分钟。取约4ml溶液(每10分钟重新配置新的溶液，并更换离心管中的溶液)，放入超滤离心管内管(再生纤维素分子量30kd)，离心(转速4500g)20min、40min、50min、60min、70min、80min，取滤液作为游离测试溶液。
制备供试品溶液总量：精确测量上述溶液2ml，置于50ml量瓶中，用绝对乙醇稀释至标线，摇匀，过滤，作为供试品溶液。
取游离的供试品溶液和供试品溶液总量，并将其注入液相色谱系统。外标法计算不同离心时间下滤液中普那布林的浓度，并确定滤膜的吸附情况。结果如下：
表2-15滤膜吸附研究-再生纤维素滤膜
通过对耐稀释滤膜的吸附研究，可以看出，截留分子量30kd的再生纤维素滤膜在40分钟后可以达到吸附饱和。
为模拟临床使用过程，制备了不同的稀释级别(1:10、1:20、1:30、1:50)，将稀释后的溶液约30min内输注到5L pH7.4的磷酸盐缓冲液中(注：1:50稀释级别溶液的输注时间约为1小时)，研究输注过程中封装率的变化。结果如下：
表2-16不同稀释等级溶液的封装率结果

根据输注过程中供试品溶液总量的浓度，计算出供试品的实际输注量和相应时间点的15-羟基硬脂酸聚乙二醇酯的浓度。结果如下：
表2-17不同稀释级别下的溶液输注结果分析

从结果可以看出，输注前1/3体积的封装率较低，且差异较大。15-羟基硬脂酸聚乙二醇酯赋形剂中含有的15-羟基硬脂酸聚乙二醇酯的临界胶束浓度(CMC)在0.005％至0.02％之间。作为参考，用稳态荧光法测定的15-羟基硬脂酸聚乙二醇酯的临界胶束浓度为0.0035％。结合上述输注过程和数据，可以看出，(1:10)稀释级别溶液输注10分钟后，实际输注量只有17.6％，而PBS溶液中15-羟基硬脂酸聚乙二醇酯的浓度处于临界胶束浓度水平，所以封装率较低。当其他稀释级别溶液为输注前用量的1/3时，PBS溶液中15-羟基硬脂酸聚乙二醇酯的浓度略高于临界胶束浓度和(1:10)稀释级别，封装率略高于50％～60％水平。
随着输注时间的延长，15-羟基硬脂酸聚乙二醇酯的浓度逐渐增加，包埋效率也逐渐提高。在5L pH7.4磷酸盐缓冲液中，不同稀释级别的供试品溶液的最终封装率都在80％以上。
从封装率的结果可以看出，当各稀释级别的溶液为输注前的1/3时，PBS溶液中15-羟基硬脂酸聚乙二醇酯的量达到了临界胶束浓度或略高于临界胶束浓度。包封率的结果较低。随着灌注时间的延长，稀释溶液在缓冲盐中逐渐形成胶束，封装率逐渐提高到80％的水平。
稀释制剂的胶束稳定性研究
将普那布林的批量样品稀释至1:10、1:20、1:30和1:50的稀释比，将稀释溶液在室温避光中保存，并在0h、2h、4h、6h、8h、12h和24h取样，研究胶束的性质稳定性。结果如下：
表2-18不同稀释级别的封装率结果

结果表明，将配制好的各稀释级别的供试品溶液在室温避光保存24小时，计算出0小时内供试品溶液总量的浓度为100.0％，各时间点供试品溶液总量的浓度在96.0％～103.0％范围内；封装率在95％以上，稀释溶液胶束性能稳定性好。
本品用不同比例的D5W注射液(1:10、1:20、1:30、1:50)稀释，供试品溶液总量在室温下避光保存24小时。浓度和封装率没有明显变化，说明胶束溶液在室温下24小时内是稳定的。
临界胶束浓度的测定
临界胶束浓度(CMC)用于评价物质的胶束特性，测量方法常用的有表面张力法、电导率法、荧光探针法、染料法等。其中，荧光探针法因其具有操作简单、对研究系统干扰小等优点，在测定表面活性剂的临界胶束浓度方面得到了广泛使用。因此，决定采用荧光探针法分别测定制剂体系中15-羟基硬脂酸聚乙二醇酯的浓度。5％葡萄糖注射液和pH7.4PBS溶液中的临界微球浓度。研究中使用的设备、试剂的统计如下：
芘母液
称取20mg芘作为荧光试剂，置于10ml容量瓶中，用丙酮溶解并稀释至标线，摇匀。溶液中芘的浓度为2.0mg/ml(1.0×10-5mol/ml)。
丙二醇-15-羟基硬脂酸聚乙二醇酯母液
丙二醇：15-羟基硬脂酸聚乙二醇酯＝60:40(wt:wt)，称取约7.5g丙二醇和约5g 15-羟基硬脂酸聚乙二醇酯，放入50ml量瓶中，加入丙酮溶解并稀释至标线，摇匀，浓度为100mg/ml。
样品的制备
取0.1ml芘母液，放入10个10ml容量瓶中。在丙酮挥发至干性后，加入不同量的丙二醇-15-羟基硬脂酸聚乙二醇酯母液。在丙酮挥发至干后，用D5W注射液稀释至标记值，摇匀。溶液中15-羟基硬脂酸聚乙二醇酯胶束的浓度为1×10-7g/ml、5×10-7g/ml、1×10-6g/ml、5×10-6g/ml、1×10-5g/ml、5×10-5g/ml、1×10-4g/ml、5×10-4g/ml、1×10-3g/ml、1×10-2g/ml。在室温下24小时后测量。
平行制备一系列相同浓度的溶液，用pH7.4的PBS溶液稀释至标线，并在室温下放置24小时后测量。
荧光光度计的具体测量参数如下：
表2-19荧光光度计设置参数
按照上述方法测量两组溶液在室温下放置24小时后的荧光光谱。
通过波尔兹曼公式进行曲线拟合，得到15-羟基硬脂酸聚乙二醇酯在不同溶液体系中的CMC值。
表2-20
辅料规格中，CMC在0.005％至0.02％的范围内，确定15-羟基硬脂酸聚乙二醇酯在水中的CMC为0.0035％，作为对照。15-羟基硬脂酸聚乙二醇酯在不同溶 液体系中的CMC没有明显差异。
实施例3
本实施例介绍了为确定普那布林(4mg/mL)(丙二醇/15-羟基硬脂酸聚乙二醇酯溶液)在5％葡萄糖(D5W)中稀释后在非PVC静脉输液袋(500mL)中，稀释后不同时期的外观、检测、杂质和微生物评价而进行的稀释研究结果。在本实施例中，用D5W稀释了六(6)个透明小瓶中的4mg/mL的普那布林样品和六(6)个混浊小瓶中的4mg/mL的普那布林样品，从而得到两个稀释级别：约1:20和约1:200。
将普那布林(4mg/mL)小瓶冷藏混浊，然后在37℃加热1小时稀释，以逆转混浊。稀释样品最初(0小时)在室温下测试，避光保存4小时、6小时、8小时、12小时、24小时和48小时后再次测试。在非PVC静脉输液袋(500ml)中用D5W稀释后，在6小时、8小时、12小时、24小时和48小时对普那布林(4mg/mL)的稀释后稳定性和潜在的微生物风险进行了测定。
本研究使用了两个批次的普那布林(4mg/mL)。第一批用于化学和外观分析，第二批用于微生物计数测试。
使用第一批产品对1:20和1:200的稀释液进行了过滤器干扰研究。对于过滤器干扰研究，1:200的稀释液不符合可接受标准，因此在整个研究中使用未过滤的样品。
对于稀释研究和微生物计数研究，透明小瓶和因冷藏而引起混浊的小瓶的结果是相同的。两种稀释液和所有时间点的所有样品都符合微生物计数的可接受标准。
1:20的稀释样品在0、4、6、8和12小时时符合检测的稳定性标准。1:20的稀释样品在稀释后24小时和48小时未能达到可接受标准，原因是存在沉淀物和药效<标签要求的90％。1:200的稀释样品在稀释后的0、4、6和8小时内符合检测的稳定性标准。1:200的稀释样品在稀释后12、24和48小时时，由于存在沉淀物和药效<标签要求的90％，未能达到可接受标准。
在1:20稀释级别的透明和混浊的小瓶中没有检测到≥0.10％的杂质。对于1:200的稀释级别，存在的杂质低于检测水平，没有进行定量。
过滤器干扰研究
通过过滤和分析一部分样品来评估0.2微米PBS过滤器的可能干扰。从带有输液管的端口抽取一部分过滤的样品，与直接从出口端口抽取的一部分未过滤的样品进行比较。在每个稀释级别(1:20和1:200)下制备并测试三(3)个样品。在1:20的稀释级别下准备了一个安慰剂对照样品，以确定安慰剂的峰值。对于1:20的稀释级别，将从过滤部分得到的检测值和杂质值与未过滤部分的值进行比较。对于1:200的稀释液，从过滤部分得到的测定值与未过滤部分的测定值进 行比较。过滤干扰研究的结果列于表3-1。
稀释研究方法
在本研究中，将4mg/mL的普那布林注射液用D5W稀释在非PVC静脉注射袋中，以生成稀释的静脉输液。
单个非PVC静脉输液袋中含有D5W溶液的体积(500mL)，通过将袋子倒入500mL量筒或容量瓶中测量到满量，并使用适当大小的量筒，精确度为1mL或更好。确定用于样品计算的平均体积(n＝4)。
用于测定平均体积的所有D5W非PVC N袋与用于稀释研究的所有袋子来自同一制造商/供应商和同一制造商的批次。
普那布林(4mg/mL)的所需体积是根据公式2确定的。标称浓度为0.2mg/mL，稀释倍数为1:20的获得。例如，使用有刻度的注射器从两(2)瓶普那布林(4mg/mL)中各无菌地抽取12.5mL(±0.2mL)，并通过一次注射直接注入一个覆盖有琥珀色套筒的500mL非PVC静脉注射袋中。记录注入袋中的普那布林(4mg/mL)的重量。确定所需的普那布林(4mg/mL)的体积。标称浓度为0.02mg/mL，稀释倍数为1:200的获得。例如，使用有刻度的注射器从普那布林(4mg/mL)小瓶中无菌地抽取2.5mL(±0.05mL)，并通过一次注射直接注射到覆盖有琥珀色套筒的500mL非PVC静脉袋中。记录注入袋中的普那布林4mg/mL的重量。注：存在的杂质低于检测水平，在1:200的稀释度下没有进行定量。
轻轻旋转输液袋以彻底混合输液。检查并记录非PVC输液袋中稀释后的溶液的外观。将稀释溶液存放在非PVC输液袋中，在控制室温(25℃±3℃)下避光保存共48小时。根据公式3计算出注射后的实际袋子体积，并根据公式4计算出每次稀释后的标签要求。每隔一段时间检查并记录稀释溶液的外观。样品是直接从静脉注射袋中取出的，而不是从静脉注射管中取出。化验和杂质的化学测试按本文所述进行。使用相同的静脉输液管，通过输液管直接抽取样品(不打底)，并在每隔一段时间进行记录。第一个批次用于化学测试。微生物测试使用USP<61>进行。通过出口端口抽取样品，并在每个间隔时间进行记录。稀释研究的结果列于表25。第二批产品用于微生物计数研究。
公式2
所需体积普那布林注射液(4mg/ml)＝(C静脉注射袋，mg/ml*V静脉注射袋，ml)/(4mg/ml)。其中，C静脉注射袋是稀释的静脉注射袋中所需要的普那布林的最终浓度，单位为mg/ml(即：0.2mg/ml或0.02mg/ml)，V静脉注射袋是静脉注射袋中D5W的预期起始体积，单位为ml，来自制造商的产品说明。
公式3
实际体积静脉注射袋，ml＝V平均，ml+((重量普那布林注射液(4mg/ml),g)/(d普那布林注射液 (4mg/ml),g/ml))。其中，V平均是指从4个静脉注射袋中测得的平均体积，单位 为ml。Wt.普那布林注射液(4mg/ml)为加入到静脉注射袋中的普那布林注射液(4mg/ml)的实际重量，以g为单位，d普那布林注射液(4mg/ml)是从执行批次记录中得到的普那布林注射液(4mg/ml)的密度。
公式4
标签要求静脉注射液，mg/ml＝(4mg/ml*普那布林注射液(4mg/ml),g)/((d静脉注射液(4mg/ml),g/ml*V静脉注射袋,ml)。其中，Wt普那布林注射液(4mg/ml)是指加入到静脉注射袋中的实际重量，以g为单位，d普那布林注射液(4mg/ml)是指从执行批次记录中得到的普那布林注射液(4mg/ml)的密度，V静脉注射袋是指静脉注射袋中的实际体积，由公式2确定。
结果
过滤干扰研究
稀释级别为1:20的过滤器干扰研究的检测结果符合3.0％以内的可接受标准。1:200稀释级别的检测结果不符合3.0％以内的可接受标准。由于在1:200稀释级别的检测中观察到了过滤器的干扰，所以在研究中使用了未经过滤的样品。在过滤和未过滤的样品中都没有检测到杂质(～0.10％)。使用第一批样品进行测试。
表3-1过滤干扰研究
稀释研究
稀释研究的结果列于表3-2、3-3和3-4。使用批次2进行测试。使用批号1进行微生物测试。样品是从静脉注射袋、输液管或出口端口(如适用)抽取的。
表3-2稀释研究



表3-3稀释研究续


表3-4稀释研究续


表3-5微生物举例研究
对于过滤干扰研究，1:200的稀释液不符合可接受标准，因此在整个研究中使用了未过滤的样品。
对于稀释研究和微生物计数研究，透明小瓶和通过冷藏导致的混浊小瓶的结果是一样的。两种稀释液和所有时间点的所有样品都符合微生物计数的可接受标准。
1:20的稀释样品在0、4、6、8和12小时内符合检测的稳定性标准。1:20的稀释样品在稀释后的24小时和48小时内没有达到可接受标准，原因是出现了沉淀物和药效<标签要求的90％。1:200的稀释样品在稀释后的0、4、6和8小时内符合 检测的稳定性标准。1:200的稀释样品在稀释后12、24和48小时内由于存在沉淀物和药效<标签要求的90％而未能达到可接受标准。
在1:20稀释级别的透明和混浊的小瓶中没有检测到≥0.10％的杂质。对于1:200的稀释级别，存在的杂质低于检测水平，没有进行定量。根据从透明和混浊的小瓶中获得的结果，可以得出结论，室温避光情况下，在非PVC静脉注射袋(500mL)中用D5W稀释的普那布林(4mg/mL)，在1:20稀释级别下稳定12小时，在1:200稀释级别下稳定8小时。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种普那布林胶束组合物的制备方法，其特征在于，所述方法包括步骤：
    s1)35℃-65℃下，将15-羟基硬脂酸聚乙二醇酯与丙二醇混合，制备得到澄清混合液；
    s2)35℃-65℃下，将步骤1)中的澄清混合液与普那布林混合，搅拌，制得普那布林胶束组合物。
  2. 如权利要求1所述的制备方法，其特征在于，15-羟基硬脂酸聚乙二醇酯与丙二醇重量比为1:5-5:1，优选地，1:3-3:1，更优选地2:3。
  3. 如权利要求1所述的制备方法，其特征在于，在步骤(s2)中，普那布林为普那布林一水合物。
  4. 一种普那布林胶束组合物，其特征在于，所述胶束组合物包含普那布林和15-羟基硬脂酸聚乙二醇酯与丙二醇的澄清混合液，其中，所述普那布林胶束组合物的为黄色澄清透明溶液，胶束粒径范围为10-100nm。
  5. 如权利要求4所述的普那布林胶束组合物，其特征在于，所述的普那布林胶束组合物是采用如权利要求1所述的方法制备的。
  6. 如权利要求4所述的普那布林胶束组合物，其特征在于，所述普那布林胶束组合物包含以下成分：
    丙二醇30-90wt％，优选40-70wt％，例如50wt％、55wt％、60wt％、65wt％、70wt％；
    15-羟基硬脂酸聚乙二醇酯20-60wt％，优选20-50wt％，例如25wt％、30wt％、35wt％、40wt％、45wt％；和
    普那布林1-10wt％，优选2-8wt％，例如3wt％、4wt％、5wt％、6wt％、7wt％。
  7. 如权利要求4或6所述的普那布林胶束组合物，其特征在于，15-羟基硬脂酸聚乙二醇酯与丙二醇重量比为1:5-5:1，优选地，1:3-3:1，更优选地2:3。
  8. 一种液体注射用普那布林组合物，其包括：
    普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯的D5W溶液(5％葡萄糖注射液)；
    其中，所述组合物中丙二醇和D5W的体积比为约6：50至约6：500。
  9. 如权利要求8所述的液体注射用普那布林组合物，其特征在于，所述液体注射用普那布林组合物是采用以下方法制备的，所述方法包括以下步骤：
    提供一种初始液体制剂，其包括普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯；和
    以约1:5至约1:50的稀释比在D5W中稀释所述初始液体制剂；
    其中，所述初始液体制剂为如权利要求4所述的普那布林胶束组合物。
  10. 如权利要求8所述的液体注射用普那布林组合物，其特征在于，普那布林的浓度为约0.08mg/ml至约0.4mg/ml。
  11. 如权利要求8-10中任一项所述的液体注射用普那布林组合物，其特征在于液体注射用普那布林制剂中15-羟基硬脂酸聚乙二醇酯和D5W的体积比在约4:50-约4:500，优选为约4:100至约4:500，更优选为约4:100-约4:400，
  12. 如权利要求8所述的液体注射用普那布林组合物，其特征在于，所述的液体注射用普那布林组合物包含的杂质总量小于0.5％。
  13. 如权利要求8所述的液体注射用普那布林组合物，其特征在于，组合物中大于约90％的普那布林被封装在胶束中。
  14. 一种制备液体注射用普那布林组合物的方法，其特征在于，所述方法包括：
    提供一种初始液体制剂，其包括普那布林、丙二醇和15-羟基硬脂酸聚乙二醇酯；和
    以约1:5至约1:50的稀释比在D5W中稀释所述初始液体制剂。
  15. 如权利要求14所述的制备液体注射用普那布林组合物的方法，其特征在于，所述初始液体制剂为权利要求4所述的普那布林胶束组合物。
  16. 如权利要求4所述的普那布林胶束组合物或如权利要求8所述的液体注射用普那布林组合物在制备治疗抗肿瘤药物中的用途。
  17. 如权利要求16所述的用途，其特征在于，所述肿瘤选自：肺癌(如小细胞肺癌、非小细胞肺癌)、前列腺癌、结肠癌、脑肿瘤(如胶质母细胞瘤、多形性胶质母细胞瘤、巨细胞胶质母细胞瘤、转移性脑肿瘤)、头颈癌、胃癌、胰腺癌、乳腺癌、肾癌、膀胱癌、卵巢癌、子宫颈癌、黑色素瘤、骨髓瘤、淋巴瘤或白血病。
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