Plinabulin micelle composition and method for preparing same

Abstract

A plinabulin micelle composition and a preparation method thereof are provided, specifically, the plinabulin micelle composition is essentially composed of plinabulin, polyoxyl 15-hydroxystearate, and propylene glycol, and at least a portion of the plinabulin is encapsulated in the micelles, which can be mixed with glucose injection to obtain an injectable plinabulin micelle formulation, which is used in the clinical treatment of tumor diseases. The plinabulin micelle composition and micelle formulation all have excellent electrochemical stability, dilution stability, and storage stability.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceutical formulations, and in particular to plinabulin micelle compositions and methods for preparing same. [Background technology]

[0002] Plinabulin ((3Z,6Z)-3-[(5-t-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione) is a synthetic analogue of the diketopiperazine phenylahistin (benzyltrimethylammonium chloride) found in marine and terrestrial Aspergillus species, and its structure is: [ka]

[0003] Plinabulin is structurally distinct from colchicine and its combretastatin-like analogs (e.g., combretastatin phosphate) and binds at or near the colchicine binding site on tubulin monomers. Previous studies have shown that, compared with colchicine, plinabulin at low concentrations induces tubulin depolymerization and monolayer permeability in vascular endothelial cells, and plinabulin induces apoptosis in Jurkat leukemia cells. It has been shown that plinabulin has good pharmacokinetic, pharmacodynamic and safety profiles as a single agent for patients with advanced malignancies (lung cancer, prostate cancer, and colon cancer). However, there remains a need to provide an injectable formulation of plinabulin with suitable physicochemical properties. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide novel plinabulin micelles and a method for preparing the same, which have excellent stability. The obtained micelles can not only improve the water solubility of plinabulin, but also delay the release of the drug, prolong the biological half-life, and have good clinical application value. [Means for solving the problem]

[0005] A first aspect of the present invention provides a method for preparing a plinabulin micelle composition, said method comprising: s1) Mixing polyoxyl 15-hydroxystearate and propylene glycol at 35°C to 65°C to prepare a clear mixture; s2) mixing and stirring the clear mixture obtained in step 1) with plinabulin at 35°C to 65°C to prepare a plinabulin micelle composition. In another preferred example, the weight ratio of polyoxyl 15-hydroxystearate to propylene glycol is 1:5 to 5:1, preferably 1:3 to 3:1, more preferably 2:3. In another preferred example, the weight ratio of polyoxyl 15-hydroxystearate to propylene glycol is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1.

[0006] In another preferred embodiment, the micelle composition consists essentially of plinabulin, polyoxyl 15-hydroxystearate, and propylene glycol.

[0007] In another preferred embodiment, in step s1), the temperature is 40°C to 60°C.

[0008] In another preferred embodiment, in step s2), the temperature is 40°C to 60°C.

[0009] In another preferred example, step s1) is to melt polyoxyl 15-hydroxystearate at 50°C±5°C, add propylene glycol, maintain the temperature at 40°C±5°C, and stir (for example, for 5 minutes to 2 hours, preferably 20 to 40 minutes, more preferably 30 minutes) to prepare a clear mixture.

[0010] In another preferred example, in step s1), polyoxyl 15-hydroxystearate is melted at 35°C to 65°C, and then propylene glycol is added, and the mixture is maintained at 35°C to 65°C and stirred (for example, for 5 minutes to 2 hours, preferably for 10 to 40 minutes, more preferably for 20 to 30 minutes) to prepare a clear mixture.

[0011] In another preferred example, in step s1), polyoxyl 15-hydroxystearate is melted at 35 to 65°C, and then added to propylene glycol, and the mixture is maintained at 35 to 65°C and stirred (for example, for 5 minutes to 2 hours, preferably for 10 to 40 minutes, more preferably for 20 to 30 minutes) to prepare a clear mixture. In another preferred embodiment, the clear mixture in step s2) is added in batches, preferably 2 to 5 batches.

[0012] In another preferred example, the concentration of plinabulin is 0.02 mg / ml to 4 mg / ml, calculated as the total mass of the plinabulin micelle composition. In another preferred embodiment, plinabulin is plinabulin anhydrate or plinabulin monohydrate.

[0013] In another preferred embodiment, plinabulin is plinabulin anhydrate (crystalline form III) or plinabulin monohydrate (crystalline form I).

[0014] In another preferred example, the plinabulin used in step (s2) is plinabulin monohydrate.

[0015] In another preferred example, plinabulin is plinabulin monohydrate (crystalline form I).

[0016] In another preferred embodiment, the plinabulin anhydrate is the anhydrous form of plinabulin in CN113735834B.

[0017] In another preferred example, the plinabulin monohydrate is the plinabulin monohydrate in CN113735834B.

[0018] In another preferred example, the particle size D50 of the micelles in the obtained plinabulin micelle composition is 5 to 100 nm.

[0019] A second aspect of the present invention provides a plinabulin micelle composition, wherein the micelle composition comprises plinabulin and a clear mixture of polyoxyl 15-hydroxystearate and propylene glycol, wherein the plinabulin micelle composition is a yellow, clear, transparent solution, and the particle size of the micelles ranges from 10 to 100 nm.

[0020] In another preferred example, the particle size range of the micelles in the plinabulin micelle composition is 80 to 110 nm, preferably 90 to 110 nm, for example, 100 to 110 nm.

[0021] In another preferred example, the micelle D50 of the plinabulin micelle composition is 80 to 110 nm, preferably 90 to 110 nm, for example, 100 to 105 nm.

[0022] In another preferred example, the micelle D90 of the plinabulin micelle composition is 100 to 170 nm, preferably 110 to 160 nm, for example, 120 to 150 nm.

[0023] In another preferred example, the concentration of plinabulin in the plinabulin micelle composition is 1 mg / ml to 10 mg / ml, calculated as the total mass of the plinabulin micelle composition, preferably the concentration of plinabulin is 2 mg / ml to 5 mg / ml, and more preferably the concentration of plinabulin is 3 mg / ml to 4 mg / ml.

[0024] In another preferred example, in the plinabulin micelle composition, the plinabulin is plinabulin monohydrate.

[0025] In another preferred example, in the plinabulin micelle composition, the weight ratio of polyoxyl 15-hydroxystearate to propylene glycol is 1:5 to 5:1, preferably 1:3 to 3:1, more preferably 2:3.

[0026] In another preferred embodiment, the plinabulin micelle composition is prepared by the method according to the first aspect.

[0027] In another preferred embodiment, the plinabulin micelle composition is sterilized.

[0028] A third aspect of the present invention provides a liquid injectable plinabulin composition, comprising: Contains plinabulin, propylene glycol, and polyoxyl 15-hydroxystearate in D5W (5% glucose injection) Here, the volume ratio of propylene glycol to D5W in the composition is about 6:50 to about 6:500.

[0029] In another preferred embodiment, in the liquid injectable plinabulin composition, the plinabulin is plinabulin monohydrate.

[0030] In another preferred example, plinabulin is encapsulated within micelles, preferably at least 90% of plinabulin is encapsulated within micelles.

[0031] In another preferred example, at least 95% of the plinabulin is encapsulated within the micelles, preferably at least 97%, more preferably at least 99%.

[0032] In another preferred example, 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.

[0033] In another preferred example, the particle size D50 of the micelles in the obtained injectable plinabulin composition is 5 to 50 nm, preferably 10 to 30 nm. In another preferred embodiment, the concentration of plinabulin is from about 0.08 mg / ml to about 0.4 mg / ml.

[0034] In another preferred example, the volume ratio of polyoxyl 15-hydroxystearate to D5W in the liquid injectable plinabulin formulation 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.

[0035] In another preferred embodiment, the weight ratio of polyoxyl 15-hydroxystearate to propylene glycol in the liquid injectable plinabulin composition is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9: 1. In another preferred embodiment, the ratio of propylene glycol to polyoxyl 15-hydroxystearate is about 60:40 (wt:wt).

[0036] In another preferred embodiment, the total amount of impurities contained in the liquid injectable plinabulin composition is less than 0.5%. In another preferred embodiment, the total amount of impurities contained in the liquid injectable plinabulin composition is less than 0.1%.

[0037] In another preferred embodiment, the liquid injectable plinabulin composition can be stably stored for about 8 to about 12 hours.

[0038] In another preferred embodiment, the temperature at which the liquid injectable plinabulin composition can be stably stored is 10°C to about 37°C, preferably room temperature.

[0039] In another preferred embodiment, at least a portion of the plinabulin is encapsulated within the micelles, preferably at least 90% of the plinabulin is encapsulated within the micelles.

[0040] In another preferred example, greater than about 90% of the plinabulin in the liquid injectable plinabulin composition is encapsulated within micelles.

[0041] In another preferred example, in the liquid injectable plinabulin composition, more than about 93% of the plinabulin is encapsulated within the micelles, preferably more than about 94% of the plinabulin is encapsulated within the micelles, preferably more than about 95% of the plinabulin is encapsulated within the micelles, preferably more than about 96% of the plinabulin is encapsulated within the micelles, preferably more than about 97% of the plinabulin is encapsulated within the micelles, and preferably more than about 99% of the plinabulin is encapsulated within the micelles.

[0042] In another preferred embodiment, the liquid injectable plinabulin composition is prepared by the method according to the fourth aspect of the present invention.

[0043] A fourth aspect of the present invention provides a method for preparing a liquid injectable plinabulin composition, said method comprising: providing an initial liquid formulation comprising plinabulin, propylene glycol and polyoxyl 15-hydroxystearate; and and diluting the initial liquid formulation in D5W at a dilution ratio of about 1:5 to about 1:50.

[0044] In another preferred embodiment, the initial liquid formulation is a plinabulin micelle composition according to the second aspect of the present invention.

[0045] In another preferred example, the dilution ratio is about 1:10 to about 1:50, preferably about 1:13 to about 1:30, and more preferably about 1:20.

[0046] In another preferred example, the initial liquid formulation contains plinabulin at a concentration of about 1 mg / ml to about 6 mg / ml, preferably at a concentration of about 3 mg / ml to about 5 mg / ml, and more preferably at a concentration of about 4 mg / ml.

[0047] In another preferred embodiment, after diluting the initial liquid formulation in D5W, the composition is stirred, preferably for at least 5 minutes.

[0048] In another preferred example, the concentration of plinabulin in the liquid injectable plinabulin composition is about 0.08 mg / ml to about 0.4 mg / ml.

[0049] In another preferred example, the ratio of propylene glycol to polyoxyl 15-hydroxystearate in the liquid injectable plinabulin composition is about 60:40 (wt:wt).

[0050] In another preferred embodiment, the initial liquid formulation is the plinabulin micelle composition described above.

[0051] In another preferred example, the particle size range of micelles in the liquid injectable plinabulin composition is 90 to 200 nm, preferably 100 to 150 nm, and more preferably 100 to 110 nm.

[0052] A fifth aspect of the present invention provides use of the plinabulin micelle composition according to the second aspect or the liquid injectable plinabulin composition according to the third aspect in the preparation of a medicament for the prevention and / or treatment of antitumor.

[0053] In another preferred example, the tumor is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), prostate cancer, colon cancer, brain tumor (e.g., 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. [Effects of the Invention]

[0054] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. DETAILED DESCRIPTION OF THE INVENTION

[0055] As a result of extensive and thorough research, the present inventors have unexpectedly obtained a novel plinabulin micelle composition with excellent stability, which can improve the water solubility of plinabulin and has excellent electrochemical stability, dilution stability and storage stability.

[0056] Specifically, the present invention unexpectedly prepares a plinabulin micelle composition using plinabulin monohydrate and a specific ratio of propylene glycol and polyoxyl 15-hydroxystearate, in which plinabulin is encapsulated within the micelles, and the micelle composition has excellent stability, electrochemical stability, dilution stability, and storage stability.

[0057] The obtained plinabulin micelle composition is diluted with D5W solution to obtain a liquid injectable plinabulin composition. After dilution, the plinabulin micelle composition still exists, and plinabulin is encapsulated therein. The diluted injection solution has excellent infusion stability and storage stability, a high plinabulin encapsulation rate, and a low impurity content, and can be used as an injectable formulation of plinabulin.

[0058] Based on this, the present invention was completed.

[0059] The present invention discloses plinabulin micelle-encapsulated compositions and methods for preparing and using plinabulin compositions. Plinabulin (3Z,6Z)-3-[(5-t-butyl-1H-imidazol-4-yl)methylene]-6-(benzylidene)-2,5-piperazinedione) is a synthetic analog of the naturally occurring diketopiperazine phenylahistine. 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.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although similar or equivalent methods and materials described herein can be used in the practice or testing of the present invention, the preferred methods and materials described herein are described.

[0061] <Terminology> Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are multiple definitions for a particular term herein, the definition in this specification shall prevail unless otherwise specified.

[0062] As used herein, plinabulin includes crystalline or amorphous forms, such as plinabulin monohydrate, plinabulin anhydrate, plinabulin solvate, etc. Preferably, it is the monohydrate.

[0063] In the present invention, the terms "plinabulin injectable micelle formulation," "liquid injectable plinabulin composition," "liquid injectable formulation," "liquid injectable plinabulin formulation," and "liquid injectable plinabulin composition" have the same meaning and are used interchangeably.

[0064] The term "agent" is used to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material.

[0065] The terms "cancer," "neoplasm," and "carcinoma" are used interchangeably to refer to cells exhibiting relatively autonomous growth, displaying an abnormal growth phenotype characterized by a marked loss of control over cell proliferation. Generally, cells of interest for detection or treatment in this application include precancerous (e.g., benign), malignant, premetastatic, metastatic, and non-metastatic cells. Detection of cancer cells is particularly meaningful.

[0066] As used herein, the term "subject" refers to a human or non-human mammal, such as, for example, a dog, cat, mouse, cow, sheep, pig, goat, non-human primate, or bird, such as, for example, a chicken, as well as any other vertebrate or invertebrate.

[0067] The term "mammal" is used in its ordinary biological sense, and thus specifically includes, but is not limited to, primates, including apes (chimpanzees, monkeys, and monkeys), as well as humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or similar animals.

[0068] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent that effectively relieves to some extent one or more symptoms of a disease or condition or effectively reduces the likelihood of illness, and may include treating a disease or condition.

[0069] As used herein, the term "treat" refers to the administration of a compound or pharmaceutical composition to a subject for purposes of prevention and / or treatment. The term "prophylactic treatment" refers to treatment of a subject who is not yet symptomatic of the disease or condition, but who is susceptible to or otherwise at risk for a particular disease or condition, whereby the treatment may reduce the likelihood that the patient will develop the disease or condition. The term "therapeutic treatment" refers to treating a subject already suffering from a particular disease or condition.

[0070] <Formulations> In some embodiments, plinabulin is provided in the form of a concentrated liquid formulation, which can then be diluted to prepare a diluted liquid injectable formulation. In some embodiments, the concentrated liquid formulation comprises one or more solvents, which can comprise polyoxyl 15-hydroxystearate and / or propylene glycol. In some embodiments, the one or more solvents comprise at least 30% (by weight) polyoxyl 15-hydroxystearate, at least 35% (by weight) polyoxyl 15-hydroxystearate, at least 40% (by weight) polyoxyl 15-hydroxystearate, at least 45% (by weight) polyoxyl 15-hydroxystearate, or a range affecting the above values. In some embodiments, the one or more solvents comprise at least 50% (by weight) propylene glycol, at least 55% (by weight) propylene glycol, at least 50% (by weight) propylene glycol, at least 55% (by weight) propylene glycol, at least 60% (by weight) propylene glycol, or a range encompassing any of the foregoing values. In some embodiments, the one or more solvents comprise 40% (by weight) polyoxyl 15-hydroxystearate and 60% (by weight) propylene glycol. In some embodiments, the ratio of propylene glycol to polyoxyl 15-hydroxystearate in the liquid injectable formulation is about 60:40 (by weight).

[0071] In some embodiments, the concentrated liquid formulation is a micellar composition. Plinabulin is encapsulated within the micelles. In some embodiments, the components of the micellar composition include polyoxyl 15-hydroxystearate, propylene glycol, and plinabulin, wherein the plinabulin is encapsulated within the micelles. In some embodiments, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a range including and / or corresponding to the foregoing values, of plinabulin is encapsulated within the micelles.

[0072] In some embodiments, the plinabulin in the micelle composition is plinabulin monohydrate. When an anhydrous form of plinabulin is used, the solubility of the resulting micelle composition is low.

[0073] The concentration of plinabulin in the concentrated liquid formulation can be between 1 mg / ml and 10 mg / ml, between 2 mg / ml and 6 mg / ml, or about 4 mg / ml.

[0074] In some embodiments, the concentrated liquid formulation can be diluted with a diluent to form a diluted liquid injectable formulation. In some embodiments, the diluent is water. In some embodiments, the diluent is saline. In some embodiments, the diluent is an aqueous glucose solution. The glucose concentration can be 1% to 20%, 2% to 10%, or about 5% (i.e., D5W).

[0075] In some embodiments, the diluted liquid injectable formulation comprises polyoxyl 15-hydroxystearate, propylene glycol, and D5W (i.e., glucose and water). In some embodiments, the volume ratio of propylene glycol to D5W in the liquid injectable plinabulin formulation is within the range of about 6:50 to about 6:500, about 6:50 to about 6:450, about 6:50 to about 6:400, about 6:50 to about 6:350, about 6:50 to about 6:300, about 6:50 to about 6:250, about 6:50 to about 6:200, about 6:50 to about 6:150, about 6:50 to about 6:100, about 6:100 to about 6:500, about 6:100 to about 6:400, about 6:150 to about 6:250, or about 6:200. In some embodiments, the volume ratio of propylene glycol to D5W in the liquid injectable plinabulin formulation is about 6:70. In some embodiments, the volume ratio of propylene glycol to D5W in the liquid injectable plinabulin formulation is about 6:140. In some embodiments, the volume ratio of propylene glycol to D5W in the liquid injectable plinabulin formulation is about 6:400.

[0076] In some embodiments, the volume ratio of polyoxyl 15-hydroxystearate to D5W in the liquid injectable plinabulin 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.

[0077] In some embodiments, the plinabulin in the liquid injectable plinabulin formulation (composition) is plinabulin monohydrate.

[0078] In some embodiments, the concentration of plinabulin in the liquid injectable plinabulin formulation (composition) 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 extending to the above values. In some embodiments, the concentration of plinabulin in the liquid injectable plinabulin composition is from about 0.08 mg / ml to about 0.4 mg / ml.

[0079] In some embodiments, the concentration of plinabulin in the liquid injectable plinabulin composition is about 0.1 mg / ml to about 0.3 mg / ml, hi some embodiments, the concentration of plinabulin in the liquid injectable plinabulin composition is about 0.2 mg / ml.

[0080] In some embodiments, the liquid injectable plinabulin formulation (composition) contains impurities at less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or a range including and / or ranging from the above values. In some embodiments, the liquid injectable plinabulin formulation (composition) contains impurities at less than 0.5%. In some embodiments, the liquid injectable plinabulin formulation (composition) contains impurities at less than 0.5%. In some embodiments, the liquid injectable plinabulin formulation (composition) contains less than 0.5% water.

[0081] In some embodiments, the liquid injectable plinabulin formulation (composition) can be stably stored for approximately 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 affecting the above values.

[0082] In some embodiments, the liquid injectable plinabulin 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 within a range including and / or affecting the above values.

[0083] In some embodiments, when the concentrated liquid formulation is diluted with a diluent (e.g., D5W), a portion of the plinabulin is encapsulated in micelles. In some embodiments, the micelles are formed by the interaction between polyoxyl 15-hydroxystearate and D5W. In some embodiments, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a range comprising and / or reflecting the above values, of the plinabulin in the injectable liquid formulation (composition) is encapsulated in micelles.

[0084] In some embodiments, 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 ranging from the above values.

[0085] <Modulation Method> Certain aspects disclose methods for preparing the liquid injectable formulations (compositions) described herein. In some embodiments, the method for preparing a liquid injectable plinabulin composition includes providing an initial concentrated liquid formulation, comprising plinabulin, propylene glycol, and polyoxyl 15-hydroxystearates (PEG15-hydroxystearates), and diluting the initial liquid formulation with D5W. In various embodiments, 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. In some embodiments, 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 about 1:10 to about 1:100. In some embodiments, the dilution ratio is about 1:10 to about 1:80. In some embodiments, the dilution ratio is about 1:10 to about 1:60. In some embodiments, the dilution ratio is about 1:10 to about 1:50. In some embodiments, the dilution ratio is about 1:10 to about 1:40. In some embodiments, the dilution ratio is about 1:10 to about 1:30. In some embodiments, the dilution ratio is about 1:10 to about 1:20. In some embodiments, the dilution ratio is about 1:20 to about 1:400. In some embodiments, the dilution ratio is about 1:20 to about 1:300. In some embodiments, the dilution ratio is about 1:20 to about 1:200. In some embodiments, the dilution ratio is about 1:20 to about 1:100. In some embodiments, the dilution ratio is about 1:20 to about 1:80. In some embodiments, the dilution ratio is from about 1:20 to about 1:60. In some embodiments, the dilution ratio is from about 1:20 to about 1:50. In some embodiments, the dilution ratio is from about 1:20 to about 1:40.In some embodiments, the dilution ratio is from about 1:20 to about 1:30.

[0086] In some embodiments, the dilution method involves introducing (e.g., by injection) the initial concentrated liquid formulation into a container containing a volume of D5W. In some embodiments, the container is an intravenous (IV) bag. In some embodiments, after introducing the initial concentrated liquid formulation into the container, the mixture is agitated. Various agitation methods include hand shaking, hand stirring, or vortexing. In various embodiments, the mixture is agitated 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 for a range including and / or extending beyond the above values.

[0087] In some embodiments of preparing the initial concentrated liquid formulation, propylene glycol is added first, followed by polyoxyl 15-hydroxystearate, and finally plinabulin is added. In some embodiments, polyoxyl 15-hydroxystearate is added first, followed by propylene glycol, and finally plinabulin is added. In some embodiments, the initial concentrated liquid formulation is mixed at room temperature.

[0088] In some embodiments of preparing the initial concentrated liquid formulation, propylene glycol is heated to about 25° C. and maintained at about 25° C. during the addition of polyoxyl 15-hydroxystearate. In some embodiments, plinabulin is added to the polyoxyl 15-hydroxystearate and propylene glycol and mixed at about 25° C.

[0089] In some embodiments of preparing the initial concentrated liquid formulation, propylene glycol is heated to about 40° C. and maintained at about 40° C. during the addition of polyoxyl 15-hydroxystearate. In some embodiments, plinabulin is added to the polyoxyl 15-hydroxystearate and propylene glycol and mixed at about 40° C.

[0090] In some embodiments of preparing the initial concentrated liquid formulation, polyoxyl 15-hydroxystearate is heated to about 25° C. and maintained at about 25° C. during the addition of propylene glycol. In some embodiments, plinabulin is added to polyoxyl 15-hydroxystearate and propylene glycol and mixed at about 25° C.

[0091] In some embodiments of preparing the initial concentrated liquid formulation, polyoxyl 15-hydroxystearate is heated to about 40° C. and maintained at about 40° C. during the addition of propylene glycol. In some embodiments, plinabulin is added to polyoxyl 15-hydroxystearate and propylene glycol and mixed at about 40° C.

[0092] In some embodiments of preparing the initial concentrated liquid formulation, polyoxyl 15-hydroxystearate is heated to about 60° C. and maintained at about 60° C. during the addition of propylene glycol. In some embodiments, plinabulin is added to polyoxyl 15-hydroxystearate and propylene glycol and mixed at about 60° C.

[0093] <Uses and Methods> Some embodiments relate to methods of preventing or reversing the progression of cancer in a subject. In some embodiments, the method comprises administering to the subject an injectable liquid formulation described herein. Some embodiments relate to methods of inhibiting the progression of cancer. Additional 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. WO 2016 / 130839, WO 2017 / 214052, WO 2018 / 144764, WO 2018 / 169887, WO 2019 / 147615, WO 2019 / 152530, WO 2020 / 037285, WO 2021 / 076485, and WO 2021 / 225908, all of which are incorporated herein by reference in their entireties.

[0094] In some embodiments, the treatment regimen comprises administering an injectable liquid formulation described herein once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen comprises administering the injectable liquid formulation twice every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen comprises administering an injectable liquid formulation described herein once weekly for a 1, 2, 3, 4, 5, 6, 7, or 8-week treatment cycle. In some embodiments, the treatment regimen comprises administering the injectable liquid formulation described herein twice weekly for a 1, 2, 3, 4, 5, 6, 7, or 8-week treatment cycle. In some embodiments, the treatment regimen comprises administering an injectable liquid formulation described herein on days 1, 8, and 15 of a 21-day treatment cycle.

[0095] The treatment cycle can be repeated as long as the regimen can be clinically tolerated. In some embodiments, the treatment cycle of the injectable liquid formulation described herein is repeated n times, where n is an integer ranging from 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a new treatment cycle can occur immediately after the completion of a previous treatment cycle. In some embodiments, a new treatment cycle can occur immediately after the completion of a previous treatment cycle. 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 completion of a previous treatment cycle.

[0096] In some embodiments, the method comprises administering plinabulin at a dose of about 5 mg / m to 150 mg / m. In some embodiments, the dose of plinabulin is greater than 20 mg / m. In some embodiments, the dose of plinabulin is greater than 30 mg / m. In some embodiments, the dose of plinabulin is greater than 40 mg / m.

[0097] In some embodiments, the injectable liquid formulations described herein are administered on day 1 of a 14-day administration cycle. In some embodiments, the injectable liquid formulations described herein are administered on day 1 of a 21-day administration cycle.

[0098] In some embodiments, the injectable liquid formulations described herein are administered in combination with one or more G-CSF drugs.

[0099] Some embodiments include a kit comprising one or more containers.

[0100] In some embodiments, the container comprises plastic or glass, or a combination thereof, including, but not limited to, any one or more types of plastic or glass that one skilled in the art would use given the teachings herein.

[0101] In some embodiments, the container is a vial. In some embodiments, the vial contains plinabulin in an amount of 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 a range including or equal to the above values. In some embodiments, the vial contains plinabulin, propylene glycol, and polyoxyl 15-hydroxystearate (PEG 15-hydroxystearates). In some embodiments, the vial contains about 4 mg / ml of plinabulin, propylene glycol, and polyoxyl 15-hydroxystearate (the ratio is about 60:40 (wt:wt)). In some embodiments, the volume of the liquid formulation in the vial is about 10 ml.

[0102] In some embodiments, the container is an IV bag. In some embodiments, the IV bag contains D5W. In some embodiments, 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. In some embodiments, the kit comprises the vial described above and the intravenous syringe described above.

[0103] <Preparation of Plinabulin Micelle Composition> After polyoxyl 15-hydroxystearate is melted at 50°C±5°C, the prescribed amount of molten polyoxyl 15-hydroxystearate is weighed and added to a mixing tank filled with nitrogen gas, and the prescribed amount of propylene glycol is added. The temperature is maintained at 40°C±5°C and the mixture is stirred for 30 minutes.

[0104] The appropriate amount of polyoxyl 15-hydroxystearate and propylene glycol mixture was taken from the mixing tank (DGJ-14) and placed in a beaker and Duran bottle for storage. The specified amount of plinabulin was weighed in the isolator in the cytotoxic drug weighing room. The 15-hydroxystearate and propylene glycol mixture in the beaker was added and stirred for 5 minutes before being added to the mixing tank. The beaker was thoroughly rinsed three times with the polyoxyl 15-hydroxystearate and propylene glycol mixture in the Duran bottle and then added to the mixing tank. The magnetic stirrer (rotational frequency 40Hz) was turned on and stirred for 1 hour. After that, samples were taken to check the intermediate product (the sample should be kept in the dark). During this process, the temperature of the drug solution was maintained at 40°C ± 5°C. The intermediate product was sampled according to the sampling plan and subjected to characterization, moisture content, density, related substances, microbial limits, bacterial endotoxin, and content determination. The time from blending to sterilization and filtration should not exceed 6 hours. The Tyndall phenomenon diagram of the plinabulin micelle composition is shown in FIG.

[0105] <Plinabulin composition for injection> The plinabulin micelle composition described above is diluted with D5W at a dilution ratio of about 1:5 to about 1:50 (wt) to obtain an injectable plinabulin composition, where the Tyndall diagram at a 1:20 dilution ratio is as shown in Figure 2.

[0106] Compared with the prior art, the present application has the following main advantages: 1. The plinabulin micelle composition of the present application has excellent dilution stability. 2. The plinabulin micelle composition and the particularly prepared injectable plinabulin composition of the present application have excellent storage stability.

[0107] Hereinafter, the present invention will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are usually in accordance with conventional conditions or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated by weight. [Example]

[0108] Effect of different formulation addition orders on the dissolution rate of plinabulin and particle size distribution of the final product prepared

[0109] <1. Addition order of different formulations (propylene glycol / polyoxyl 15-hydroxystearate ratio: 6 / 4)> [Table 1]

[0110] 1. Propylene glycol was added first, followed by polyoxyl 15-hydroxystearate, and finally the drug. Polyoxyl 15-hydroxystearate was added to ethylene glycol at 25°C (solid), 40°C (molten), and 60°C, respectively, followed by plinabulin. Plinabulin micelle compositions A, B, and C were prepared. Each was then diluted 1:20 with 5% glucose injection to obtain liquid injectable plinabulin compositions (preparations), and the particle size was measured. The results showed that under the preparation process conditions of first adding propylene glycol, then polyoxyl 15-hydroxystearate, and finally adding the drug, the dissolution rate of the drug substance accelerated with increasing temperature. At 25°C, approximately two hours of stirring was required for the drug to dissolve. At 40°C and 60°C, all of the drug substances were completely dissolved within approximately 10 minutes. The average particle size of plinabulin micelle compositions A and B was approximately 100 nm, and the particle size of plinabulin micelle composition C could not be analyzed. Various plinabulin micelle compositions had similar particle size distributions after dilution at a ratio of 1:20, indicating that the preparation temperature had little effect on the particle size distribution after dilution.

[0111] 1.2. Polyoxyl 15-hydroxystearate was added first, followed by propylene glycol, and finally the drug. Ethylene glycol was added to polyoxyl 15-hydroxystearate at 25°C (solid), 40°C (molten), and 60°C, followed by plinabulin, to prepare plinabulin micelle compositions D, E, and F. These were then diluted 1:20 with 5% glucose injection to obtain liquid injectable plinabulin compositions (formulations), and their particle sizes were measured. The results showed that when polyoxyl 15-hydroxystearate was added first, followed by propylene glycol, and finally the drug, the drug dissolution rate increased with increasing temperature. At 25°C, the drug remained undissolved even after 5 hours of stirring, whereas at 40°C and 60°C, the drug was completely dissolved within 15 minutes. Particle size distribution measurements indicated that the preparation temperature had little effect on the particle size distribution of the micelle compositions, and that the order of addition of polyoxyl 15-hydroxystearate and propylene glycol did not affect the particle size of the formulation.

[0112] 1.3. The drug was added first, followed by propylene glycol, and finally polyoxyl 15-hydroxystearate. Plinabulin micelle compositions G, H, and I were prepared by first adding the drug substance, followed by propylene glycol, and then adding polyoxyl 15-hydroxystearate at 25°C (solid), 40°C (molten), and 60°C, respectively. These compositions were then diluted 1:20 with 5% glucose injection to obtain liquid injectable plinabulin compositions (preparations), and their particle sizes were measured. The results showed that when the temperature was 40°C or higher, the drug substance could be dissolved in the propylene glycol solvent alone in a relatively short time, and the solution remained clear even after the addition of polyoxyl 15-hydroxystearate. However, when the propylene glycol temperature was 25°C, the drug substance was difficult to dissolve. The particle size distribution measurement results indicated that the preparation method of first dissolving the drug in propylene glycol did not affect the particle size distribution.

[0113] 1.4. The drug was added first, followed by the addition of a mixture of propylene glycol and polyoxyl 15-hydroxystearate. The drug substance was added first, followed by the addition of propylene glycol and polyoxyl 15-hydroxystearate solutions premixed at 40°C, to prepare plinabulin micelle composition J, which was then diluted 1:20 with 5% glucose injection to obtain a liquid injectable plinabulin composition (preparation), and the particle size was measured. The results showed that the drug substance was added first, followed by the addition of the propylene glycol and polyoxyl 15-hydroxystearate mixed solution, and compared to the particle size results for the micelle composition in 1.1, the mean particle size increased from 104.61 nm to 112.52 nm, and D10 significantly increased. However, there was no significant difference in the particle size of the diluted sample.

[0114] 1.5. The drug was added first, followed by polyoxyl 15-hydroxystearate, and then propylene glycol. The drug substance was first added, followed by polyoxyl 15-hydroxystearate melted at 40°C, and the temperature was maintained at 40°C while stirring. Propylene glycol was then added to prepare plinabulin micelle composition H, which was then diluted 1:20 with 5% glucose injection to obtain a liquid injectable plinabulin composition (preparation). The particle size was then measured. The results showed that the drug substance could not be dissolved in polyoxyl 15-hydroxystearate, and the dissolution time of the drug substance increased after the addition of propylene glycol compared to that of 1.1. As can be seen from the particle size results, compared to the particle size results of 1.1, the particle size of the original solution was significantly increased when the drug substance was first dispersed in polyoxyl 15-hydroxystearate, followed by the addition of propylene glycol. The mean particle size of the micelle composition increased from 104.61 nm to 127.06 nm, D10 remained unchanged, D50 increased from 100.08 nm to 118.03 nm, and D90 increased from 145.88 nm to 192.41 nm, but the particle size of the diluted sample at a 1:20 ratio was not significantly different.

[0115] In summary, the preparation time of the micelle composition was shortened with increasing preparation temperature. From the viewpoint of process flow, when the active pharmaceutical ingredient is added last, the addition order of polyoxyl 15-hydroxystearate and propylene glycol does not significantly affect the preparation process and particle size results of the micelle composition. When the drug substance was added first, dispersion with propylene glycol followed by the addition of polyoxyl 15-hydroxystearate shortened the preparation time, and the particle size results showed no significant difference from the manufacturing conditions. When a mixture of polyoxyl 15-hydroxystearate and propylene glycol was added, there was no significant difference in the preparation time or phenomenon, but the particle size distribution of the micellar composition showed an increase in the average particle size, D10, D50, and D90. When polyoxyl 15-hydroxystearate was first dispersed followed by the addition of propylene glycol, the preparation time increased slightly, and the particle size distribution of the micellar composition showed an increase in the average particle size, D10, D50, and D90, all of which were larger than those of the sample dispersed with a mixture of polyoxyl 15-hydroxystearate and propylene glycol.

[0116] <2. Different prescription ratios and temperatures>

[0117] 2.1. Propylene glycol: Polyoxyl 15-hydroxystearate (7:3) [Table 2]

[0118] Plinabulin micelle compositions K and L were prepared by adding polyoxyl 15-hydroxystearate to ethylene glycol at 40°C (molten) and 60°C, respectively, followed by the addition of plinabulin. These were then diluted with 5% glucose injection at a ratio of 1:20 to obtain liquid injectable plinabulin compositions (formulations), and particle size measurements were performed. The results showed that when the ratio of propylene glycol to polyoxyl 15-hydroxystearate was 7:3, the solution became cloudy, indicating that the drug substance could be dissolved after addition, but the solution remained cloudy. The dissolution rate of the drug substance remained positively correlated with temperature, and the transparency of the sample solution increased with increasing temperature. Because the samples were cloudy and not a homogeneous system, particle size measurement was difficult. Analysis of the particle size of the diluted sample of the formulation prepared at 60°C showed similar particle size and distribution to the formulation prepared in 1.1.

[0119] 2.2 Propylene glycol: Polyoxyl 15-hydroxystearate (3:7) [Table 3]

[0120] Polyoxyl 15-hydroxystearate was added to ethylene glycol at 40°C (molten) and 60°C (molten), respectively, followed by plinabulin. The resulting mixture was then diluted with 5% glucose injection at a ratio of 1:20 to obtain liquid injectable plinabulin compositions (preparations), and the particle size was measured. The dissolution rate of the active ingredient accelerated with increasing temperature. The particle size distribution analysis showed that the particle size of the micelle composition significantly decreased when the ratio of propylene glycol to polyoxyl 15-hydroxystearate was changed to 3:7.

[0121] In summary, the properties of the mixed solution changed when the ratio of propylene glycol to polyoxyl 15-hydroxystearate was changed. When the ratio of propylene glycol to polyoxyl 15-hydroxystearate was 7:3, the solution became cloudy and did not significantly affect the drug dissolution rate. When the ratio of propylene glycol to polyoxyl 15-hydroxystearate was 3:7, there was no significant difference in the preparation process, but the particle size decreased significantly. Within a certain range, the particle size of the micelles may gradually decrease as the surfactant concentration increases. [Example]

[0122] This example investigates the micelle properties of the prepared injectable plinabulin solution. The encapsulation rate of plinabulin after dilution was measured by separating the drug encapsulated in the micelles and the free drug.

[0123] Because the target compound to be measured was plinabulin in the formulation, a liquid-phase assay was selected as the content measurement method for the product, and the reproducibility and specificity of the method were confirmed. The liquid-phase conditions and experimental procedures are shown in Table 2-1. [Table 4]

[0124] A sample of plinabulin solution was prepared according to the content measurement method in Table 2-1. The reproducibility of the method was measured. [Table 5]

[0125] 10 μL of plinabulin solution, control solution, and test solution were injected into a liquid chromatograph. The peak areas were calculated, and the plinabulin content in the products was determined according to the method standard. The measurement results are shown in Tables 2-3 and 2-4. [Table 6]

[0126] [Table 7]

[0127] As can be seen from the test data and chromatograms, the measurement method of this product has good sensitivity and reproducibility, and can meet the needs of measuring the encapsulation rate of the diluted formulation of this product.

[0128] To provide a reference for the measurement of free drug, the equilibrium solubility of the drug in the solvent system of the injectable formulation without the solubilizing agent (polyoxyl 15-hydroxystearate) was measured. 40 mg of plinabulin drug substance was taken and added to 6.0 g of propylene glycol, and then added to 200 ml of D5W. The resulting solution was continuously shaken at 25°C and 100 rpm for 48 hours. The test solution was centrifuged and filtered through a 0.45 μm nylon filter. The supernatant and the subsequent filtrate were injected into a high-performance liquid chromatography system, and the peak areas were recorded. The results were as shown in Tables 2-5 below. [Table 8]

[0129] The test data showed that plinabulin was clearly adsorbed onto the 0.45 μm nylon filter membrane, and after the supernatant after centrifugation was diluted with acetonitrile, the peak area of ​​plinabulin increased significantly, indicating that the supernatant still contained no undissolved plinabulin. Therefore, in the case of plinabulin, the test solution could not be prepared directly by centrifugation, and a suitable filter membrane had to be screened for filtration.

[0130] After shaking for 52 hours, the test solution was taken, filtered through a 0.45 μm glass fiber membrane, and diluted. The filtrate was then checked with acetonitrile to confirm the elution status of plinabulin in the filtrate. The corresponding continuous filtrate was injected into a high-performance liquid chromatography system, and the peak areas were recorded. The results are shown in Table 2-6. [Table 9]

[0131] As can be seen from the test results, the raw solution of plinabulin was directly filtered through a 0.45 μm glass fiber membrane, and the peak area after filtration was multiplied by acetonitrile to dilute.

[0132] <Equilibrium solubility> The corresponding propylene glycol-D5W injection solution was prepared by the following process: Diluent 1 (corresponding to a dilution ratio of 1:20): Approximately 6 g of propylene glycol was weighed and placed in a beaker, and 200 ml of D5W injection solution was added and stirred uniformly. Diluent 2 (corresponding to a dilution ratio of 1:30): Approximately 4 g of propylene glycol was weighed and placed in a beaker, and 200 ml of D5W injection solution was added and mixed uniformly. Diluent 3 (corresponding to a dilution ratio of 1:50): Approximately 2.4 g of propylene glycol was weighed and placed in a beaker, and 200 mL of D5W injection solution was added and mixed uniformly.

[0133] The equilibrium solubility study design is as shown in Table 2-7. [Table 10]

[0134] In the content measurement method of this product, the reference solution was prepared using absolute ethanol. Since this product is used to measure plasma proteins in D5W solution, the effects of different diluents on the measurement were investigated. The experimental design was as follows, as shown in Table 2-8. [Table 11]

[0135] 10 μl of the control solution and the 48-hour test solution prepared with different diluents were injected into the high-performance liquid chromatography system, and the solubility of plinabulin in the solution was calculated using the external standard method. The results are shown in Table 2-9. [Table 12]

[0136] The results showed no significant differences between the measurement results of the plinabulin control solution prepared in absolute ethanol and the diluent (propylene glycol / D5W injection) in each diluent. Therefore, in the subsequent encapsulation rate measurement method, the plinabulin control solution was prepared in absolute ethanol.

[0137] The test solution was taken at different time points, filtered through a 0.45 μm glass fiber membrane, and injected into a high performance liquid chromatography system. The equilibrium solubility of plinabulin in the solution with different ratios at each time point was calculated by the external standard method. The results are shown in Table 2-10. [Table 13]

[0138] <Encapsulation rate of diluted plinabulin for injection> Based on the above studies, the final method for determining encapsulation efficiency was as follows.

[0139] (1:10) and (1:20) diluted test solutions: Plinabulin concentrate (4 mg / ml plinabulin in propylene glycol / polyoxyl 15-hydroxystearate, 60:40 (weight ratio)) was taken and placed in a 250 ml measuring bottle, and 100 ml (corresponding to a dilution of 1:10) or 200 ml (corresponding to a dilution of 1:20) of D5W injection solution was added, followed by shaking up and down 30 times for 1 minute.

[0140] (1:30) and (1:50) dilution test solution: Plinabulin concentrate for injection (4 mg / ml plinabulin in propylene glycol / polyoxyl 15-hydroxystearate, 60:40 (weight ratio)) was taken and placed in a 500 ml measuring flask, and 300 ml (corresponding to a dilution of 1:30) or 500 ml (corresponding to a dilution of 1:50) of D5W injection solution was added, followed by shaking up and down 30 times for 1 minute.

[0141] Free test product solution. Approximately 4 ml of the test product solution at each dilution level was taken and placed in the inner tube of an ultrafiltration centrifuge tube containing regenerated cellulose with a molecular weight of 30 kDa. The tube was centrifuged at 4,500 g, and the ultrafiltration centrifuge tube was discarded every 10 minutes. Approximately 4 ml of test product solution was added to each solution, and the tube was continuously centrifuged. This procedure was repeated six times (centrifugation for a total of 60 minutes), and the filtrate after 60 minutes was used as the free test solution.

[0142] Preparation of the total volume of test sample solution: An appropriate amount of each diluted test sample solution was taken and diluted with absolute ethanol to prepare a total volume of test sample solution containing approximately 2.5 μg of plinabulin per ml.

[0143] Stock solution of control solution: About 25 mg of plinabulin control was taken and accurately weighed, placed in a 100 ml measuring flask, dissolved in absolute ethanol, and diluted to the mark, and shaken uniformly.

[0144] Preparation of control solution: Accurately weigh 1 ml of the above-mentioned stock control solution, place it in a 100 ml measuring flask, dilute it to the mark with absolute ethanol, and shake it to make a control solution.

[0145] Sensitivity Solution: An appropriate amount of the above control solution was accurately measured and diluted with absolute ethanol to prepare a solution containing approximately 0.05 μg of plinabulin per ml.

[0146] Chromatography conditions: Octadecylsilane-bonded silica gel was used as the packing material (ACE C18 4.6 mm × 150 mm, 5.0 μm or equivalent chromatography column), 0.01 mol / L phosphate buffer (0.82 g ± 0.01 g of sodium dihydrogen phosphate monohydrate and 1.20 g ± 0.01 g of sodium phosphate were dissolved in water, diluted to 1000 ml, and shaken evenly) was used as mobile phase A, and acetonitrile was used as mobile phase B. Gradient elution was performed according to the following table, with a flow rate of 1.2 ml per minute, a column temperature of 40°C, a sample injector temperature of 30°C, a detection wavelength of 330 nm, and a sample size of 20 μl (shown in Table 2-11). [Table 14]

[0147] System suitability requirements: the relative deviation of the peak area of ​​plinabulin in five consecutive injections of the control solution should not exceed 2.0%, and the signal-to-noise ratio of the plinabulin peak height in the chromatography of the sensitivity solution should be greater than 10.

[0148] Measurement method: The total amount of the control solution, test solution, and the released test solution were accurately measured and injected into a liquid chromatography system, the chromatogram was recorded, and the peak area was calculated according to the external standard method. The calculation method is as follows:

number

[0149] Using the above method, the encapsulation rate of each dilution level of the six batches of samples was measured, and the measurement results were as follows: [Table 15]

[0150] The results showed that the encapsulation rate of plinabulin micelles decreased with increasing dilution ratio.

[0151] <Effect of Stirring Method on Encapsulation Rate> The effects of different shaking times and different shaking methods on micelle properties were studied. Samples of Batch 004A were used and concentrated prinabulin (4 mg / ml in propylene glycol / polyoxyl 15-hydroxy stearate) was diluted with D5W at dilution ratios of 1:10, 1:20, 1:30 and 1:50. The mixtures were prepared by manually shaking at different frequencies or by stirring (vortex) for different times, and the encapsulation rate was measured. The stirring methods were as follows.

Table 16

[0152] The results of the encapsulation rate were as follows.

Table 17

[0153] According to the results, samples prepared by four methods of manual shaking 30 times per minute, manual shaking 90 times per minute, stirring for 1 minute, and stirring for 3 minutes were used, and the encapsulation rate of each sample was 95% - 99%. Within this range, even if the samples were diluted by different shaking methods, there was little effect on the encapsulation rate of the samples. Therefore, it could be inferred that the encapsulation rate of the sample solution diluted by different personnel in clinical use was relatively stable.

[0154] <Study on the Dilution Resistance of the Formulation after Dilution in PBS Solution> Batch samples were diluted (dilution ratios of 1:10, 1:20, 1:30, and 1:50) to simulate a clinical infusion process. The diluted test product solution was infused into 5 L of pH 7.4 phosphate buffer over approximately 30 minutes. Based on the blood volume of the human body (approximately 7%-8% of body weight, 60 kg body weight, blood volume approximately 4.2 L-4.8 L), 5 L of pH 7.4 phosphate buffer was used in this study to simulate the blood volume of the human body. Sample collection points were 10 minutes, 20 minutes, and at the end of the infusion (except when the dilution ratio of the test product solution was 1:50, the infusion completion time was approximately 60 minutes, and the sample collection points during the process were 20 minutes of infusion, 40 minutes of infusion, and at the end of the infusion).

[0155] Because the drug concentration in the dilution resistance test was low (total concentration in the range of 7 μg / ml to 8 μg / ml) and the system was phosphate buffered, it was determined that a regenerated cellulose filter membrane with a cutoff molecular weight of 30 kd had filtration and adsorption effects at this concentration level.

[0156] (1:20) Dilution Level Solution: Approximately 10.36 g of plasma concentrate was accurately weighed and placed in a 250 ml measuring flask, 200 ml of D5W injection solution was added, and the mixture was shaken up and down 30 times within 1 minute to obtain the solution.

[0157] Released test solution: 2 ml of the diluted solution was weighed and added to 50 ml of pH 7.4 phosphate buffer (37°C) and stirred for 1 minute. Approximately 4 ml of the solution (a new solution was prepared every 10 minutes and the solution in the centrifuge tube was replaced) was taken and placed in the inner tube of an ultrafiltration centrifuge tube (regenerated cellulose molecular weight 30 kDa) and centrifuged (rotation speed 4500 g) for 20, 40, 50, 60, 70, and 80 minutes, and the filtrate was used as the release test solution.

[0158] Preparation of the total amount of test product solution: Accurately measure 2 ml of the above solution, put it into a 50 ml measuring flask, dilute it to the mark with absolute ethanol, shake it evenly, filter it, and use it as the test product solution.

[0159] The total amount of the released test sample solution and test sample solution was taken and injected into a liquid chromatography system.The concentration of plinabulin in the filtrate at different centrifugation times was calculated using the external standard method, and the adsorption status of the filter membrane was determined.The results were as follows: [Table 18]

[0160] Through adsorption studies of dilution-resistant filter membranes, it was found that regenerated cellulose filter membranes with a molecular weight cutoff of 30 kd could reach adsorption saturation after 40 minutes.

[0161] To simulate the clinical use process, different dilution levels (1:10, 1:20, 1:30, 1:50) were prepared and the diluted solutions were injected into 5 L of pH 7.4 phosphate buffer solution within about 30 minutes (Note: The injection time for the 1:50 dilution level solution is about 1 hour), and the change in encapsulation rate during the injection process was studied. The results were as follows: [Table 19]

[0162] Based on the concentration of the total amount of test product solution during the injection process, the actual injection amount of test product and the concentration of polyoxyl 15-hydroxystearate at the corresponding time point were calculated. The results were as follows: [Table 20]

[0163] As can be seen from the results, the encapsulation rate was low and significantly different at 1 / 3 the pre-injection volume. The critical micelle concentration (CMC) of polyoxyl 15-hydroxystearate contained in the polyoxyl 15-hydroxystearate excipient was 0.005%-0.02%. For reference, the critical micelle concentration of polyoxyl 15-hydroxystearate measured by steady-state fluorescence method was 0.0035%. Combining the above injection process and data, it can be seen that after 10 minutes of infusion of the (1:10) dilution level solution, the actual injection volume was only 17.6%, and the encapsulation rate was low because the concentration of polyoxyl 15-hydroxystearate in the PBS solution was at the critical micelle concentration level. When the other dilution level solutions were 1 / 3 of the pre-injection volume, the concentration of polyoxyl 15-hydroxystearate in the PBS solution was slightly higher than the critical micelle concentration and the (1:10) dilution level, and the encapsulation rate was slightly higher than the 50%-60% level.

[0164] As the injection time increased, the concentration of polyoxyl 15-hydroxystearate gradually increased, and the embedding efficiency also gradually improved. In 5 L of pH 7.4 phosphate buffer, the final encapsulation rates of the test product solutions at different dilution levels were all above 80%.

[0165] As can be seen from the results of the encapsulation rate, when the solution at each dilution level was 1 / 3 of the amount before injection, the amount of polyoxyl 15-hydroxystearate in the PBS solution reached or was slightly higher than the critical micelle concentration. The encapsulation rate results were low. As the perfusion time was extended, the diluted solution gradually formed micelles in the buffer salt, and the encapsulation rate gradually increased to a level of 80%.

[0166] <Micellar stability study of diluted formulations> 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 in the dark at room temperature. Samples were taken at 0 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours to study the stability of the micellar properties. The results were as follows: [Table 21]

[0167] According to the results, when the test product solutions prepared at each dilution level were stored in a dark place at room temperature for 24 hours, the concentration of the total amount of the test product solution at 0 hours was calculated to be 100.0%, the concentration of the total amount of the test product solution at each time point was in the range of 96.0% to 103.0%, the encapsulation rate was above 95%, and the stability of the micelle performance of the diluted solution was excellent.

[0168] This product was diluted with D5W injection at different ratios (1:10, 1:20, 1:30, 1:50), and the total amount of the test product solution was stored at room temperature in the dark for 24 hours. No significant changes in concentration or encapsulation rate were observed, indicating that the micelle solution was stable at room temperature for 24 hours.

[0169] <Measurement of critical micelle concentration> Critical micelle concentration (CMC) is used to evaluate the micellar properties of substances. Commonly used measurement methods include the surface tension method, conductivity method, fluorescent probe method, and dye method. The fluorescent probe method is widely used to measure the critical micelle concentration of surfactants due to its advantages of simple operation and minimal interference with the research system. Therefore, we decided to use the fluorescent probe method to measure the concentration of polyoxyl 15-hydroxystearate in a formulation system, as well as the critical microsphere concentration in 5% glucose injection and pH 7.4 PBS solution. The following devices and reagents were used in this study:

[0170] <Pyrene stock solution> 20 mg of pyrene was weighed as a fluorescent reagent, placed in a 10 ml measuring flask, dissolved in acetone, diluted to the mark, and shaken until uniform. The pyrene concentration in the solution was 2.0 mg / ml (1.0 × 10 mol / ml).

[0171] <Propylene glycol-polyoxyl 15-hydroxystearate stock solution> Propylene glycol: polyoxyl 15-hydroxystearate = 60:40 (wt:wt), about 7.5 g of propylene glycol and about 5 g of polyoxyl 15-hydroxystearate were weighed and placed in a 50 ml measuring flask, dissolved in acetone, and diluted to the mark. The mixture was shaken evenly, and the concentration was 100 mg / ml.

[0172] <Sample preparation> 0.1 ml of pyrene stock solution was taken and placed in ten 10 ml measuring flasks. After acetone was evaporated to dryness, different amounts of propylene glycol-polyoxyl 15-hydroxystearate stock solution were added. After acetone was evaporated to dryness, the mixture was diluted to the indicated values ​​with D5W injection and shaken uniformly. The concentrations of polyoxyl 15-hydroxystearate micelles in the solution were 1 x 10-7 g / ml, 5 x 10-7 g / ml, 1 x 10-6 g / ml, 5 x 10-6 g / ml, 1 x 10-5 g / ml, 5 x 10-5 g / ml, 1 x 10-4 g / ml, 5 x 10-4 g / ml, 1 x 10-3 g / ml, and 1 x 10-2 g / ml. Measurements were made after 24 hours at room temperature.

[0173] A series of solutions of the same concentration were prepared in parallel, diluted to the mark with PBS solution at pH 7.4, and measured after standing at room temperature for 24 hours.

[0174] The specific measurement parameters of the fluorometer were as follows: [Table 22]

[0175] According to the above method, the two groups of solutions were left at room temperature for 24 hours, and then the fluorescence spectra were measured.

[0176] The CMC values ​​of polyoxyl 15-hydroxystearate in different solution systems were obtained by curve fitting using the Boltzmann equation. [Table 23]

[0177] In the adjuvant specifications, the CMC was in the range of 0.005% to 0.02%, and the CMC of polyoxyl 15-hydroxystearate in water as a control was determined to be 0.0035%. There was no significant difference in the CMC of polyoxyl 15-hydroxystearate in different solution systems. [Example]

[0178] In this example, a dilution study was conducted to determine the appearance, detection, impurity, and microbiological evaluation of plinabulin (4 mg / mL) (propylene glycol / polyoxyl 15-hydroxystearate solution) in non-PVC IV bags (500 mL) after dilution with 5% dextrose (D5W) at various time points after dilution. Six (6) clear vial samples of plinabulin at 4 mg / mL and six (6) cloudy vial samples of plinabulin at 4 mg / mL were diluted with D5W to obtain two dilution levels: approximately 1:20 and approximately 1:200.

[0179] Plinabulin (4 mg / mL) vials were stored refrigerated in a turbid state, then heated to 37°C for 1 hour and diluted to resolve the turbidity. Diluted samples were initially tested at room temperature (time 0) and again after 4, 6, 8, 12, 24, and 48 hours of storage in the dark. After dilution with D5W in a non-PVC IV bag (500 mL), the stability and potential microbiological risk of plinabulin (4 mg / mL) after dilution were measured at 6, 8, 12, 24, and 48 hours.

[0180] Two batches of plinabulin (4 mg / mL) were used in this study: the first batch was used for chemical and visual analysis, and the second batch was used for microbial count testing.

[0181] Filter interference studies were performed using the first batch of product at dilutions of 1:20 and 1:200. For the filter interference study, the 1:200 dilution did not meet the acceptance criteria, so unfiltered samples were used throughout the study.

[0182] For both the dilution and microbial count studies, the results for the clear and refrigerated cloudy vials were the same. All samples at both dilutions and all time points met the microbial count acceptance criteria.

[0183] The 1:20 diluted sample met the stability criteria for detection at 0, 4, 6, 8, and 12 hours. The 1:20 diluted sample did not meet the acceptance criteria at 24 and 48 hours after dilution due to the presence of precipitate and potency < 90% of the label requirement. The 1:200 diluted sample met the stability criteria for detection at 0, 4, 6, and 8 hours after dilution. The 1:200 diluted sample did not meet the acceptance criteria at 12, 24, and 48 hours after dilution due to the presence of precipitate and potency < 90% of the label requirement.

[0184] Impurities of ≥ 0.10% were not detected in the clear and turbid vials at the 1:20 dilution level. At the 1:200 dilution level, impurities present were below the level of detection and were not quantified.

[0185] <Filter interference research> Potential interference with the 0.2 μm PBS filter was evaluated by filtering and analyzing a portion of the sample. A portion of the filtered sample was taken from one port of the injection tubing and compared with a portion of the unfiltered sample taken 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 and tested at the 1:20 dilution level to confirm the placebo peak value. For the 1:20 dilution level, the detection and impurity values ​​obtained from the filtered portion were compared with those from the unfiltered portion. For the 1:200 dilution, the measurements obtained from the filtered portion were compared with those from the unfiltered portion. The results of the filtration interference study are shown in Table 3-1.

[0186] <Dilution Study Method> In this study, 4 mg / mL plinabulin injection was diluted with D5W in a non-PVC IV bag to produce a diluted IV solution.

[0187] The volume of D5W solution (500 mL) contained in one non-PVC IV bag was measured by pouring the bag to capacity into a 500 mL graduated cylinder or volumetric flask using an appropriately sized graduated cylinder with 1 mL or better accuracy. The average volume used for sample calculations was determined (n=4).

[0188] All D5W non-PVCN bags used for the mean volume determination were from the same manufacturer / supplier and the same manufacturer batch as all bags used in the dilution studies.

[0189] The required volume of plinabulin (4 mg / mL) was determined according to Equation 2. The nominal concentration was 0.2 mg / mL, and the dilution factor was 1:20. For example, using a graduated syringe, 12.5 mL (±0.2 mL) was aseptically withdrawn from each of two (2) vials of plinabulin (4 mg / mL) and injected directly into a 500 mL non-PVC IV bag covered with an amber sleeve in a single injection. The weight of the plinabulin (4 mg / mL) injected into the bag was recorded. The required volume of plinabulin (4 mg / mL) was determined. The nominal concentration was 0.02 mg / mL, and the dilution factor was 1:200. For example, using a graduated syringe, 2.5 mL (±0.05 mL) was aseptically withdrawn from a vial of plinabulin (4 mg / mL) and injected directly into a 500 mL non-PVC intravenous bag covered with an amber sleeve. The weight of the 4 mg / mL plinabulin injected into the bag was recorded. Note: Impurities present were below the level of detection and were not quantified at the 1:200 dilution.

[0190] The infusion bag was gently swirled to thoroughly mix the infusion. The appearance of the diluted solution in the non-PVC infusion bag was inspected and recorded. The diluted solution was stored in a non-PVC infusion bag at room temperature (25°C ± 3°C) in the dark for a total of 48 hours. The actual bag volume after injection was calculated according to Equation 3, and the label requirement after each dilution was calculated according to Equation 4. The appearance of the diluted solution was inspected and recorded at regular intervals. Samples were taken directly from the IV bag, not from the intravenous infusion tubing. Clinical testing and chemical testing for impurities were performed as described herein. The same intravenous infusion tubing was used, and samples (without priming) were taken directly from the infusion tubing and recorded at regular intervals. The first batch was used for chemical testing. Microbiological testing was performed according to USP <61> A dilution study was performed using a 100% ethanol solution containing 100% ethanol. Samples were taken through the outlet port and recorded at each time interval. The results of the dilution study are shown in Table 25. The second batch was used for a microbial count study.

number

number

number

[0191] <Result>

[0192] <Filtration interference research> The detection results for the filter interference study at a dilution level of 1:20 met the acceptance criteria within 3.0%. The detection results for the 1:200 dilution level did not meet the acceptance criteria within 3.0%. Because filter interference was observed at the 1:200 dilution level, unfiltered samples were used in this study. No impurities (~0.10%) were detected in both filtered and unfiltered samples. Samples from the first batch were used for testing. [Table 24]

[0193] <Dilution Research> The results of the dilution studies are shown in Tables 3-2, 3-3, and 3-4. Batch 2 was used for testing. Microbiological testing was performed using batch number 1. Samples were taken from the IV bag, injection tubing, or exit port (if applicable). [Table 25]

[0194] [Table 26]

[0195] [Table 27]

[0196] [Table 28]

[0197] [Table 29]

[0198] [Table 30]

[0199] [Table 31]

[0200] [Table 32]

[0201] [Table 33]

[0202] For the filtration interference study, unfiltered samples were used in the study body because a 1:200 dilution did not meet the acceptance criteria.

[0203] For dilution and microbial count studies, results were similar for clear and refrigerated cloudy vials. All samples at both dilutions and all time points met the microbial count acceptance criteria.

[0204] The 1:20 diluted sample met the stability criteria for detection at 0, 4, 6, 8, and 12 hours. The 1:20 diluted sample did not meet the acceptance criteria at 24 and 48 hours after dilution due to the presence of precipitate and potency < 90% of the label requirement. The 1:200 diluted sample met the stability criteria for detection at 0, 4, 6, and 8 hours after dilution. The 1:200 diluted sample did not meet the acceptance criteria at 12, 24, and 48 hours after dilution due to the presence of precipitate and potency < 90% of the label requirement.

[0205] Impurities of ≥0.10% were not detected in the clear and turbid vials at the 1:20 dilution level. At the 1:200 dilution level, the impurities present were below the detection level and therefore not quantified. Based on the results obtained from the clear and turbid vials, it could be concluded that plinabulin (4 mg / mL) diluted with D5W in a non-PVC IV bag (500 ml) at room temperature and in the dark is stable for 12 hours at the 1:20 dilution level and for 8 hours at the 1:200 dilution level.

[0206] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application. [Brief explanation of the drawings]

[0207] [Figure 1] FIG. 1 shows a Tyndall phenomenon diagram of plinabulin micelle compositions. [Figure 2] FIG. 1 shows a diagram of the Tyndall phenomenon of a liquid injectable plinabulin composition.

Claims

1. 1. A method for preparing a plinabulin micelle composition, comprising: The method comprises: s1) mixing polyoxyl 15-hydroxystearate and propylene glycol at 35°C to 65°C to prepare a clear mixture; s2) mixing the clear mixture in step 1) with plinabulin at 35°C to 65°C and stirring to prepare a plinabulin micelle composition.

2. The preparation method according to claim 1, characterized in that the weight ratio of polyoxyl 15-hydroxystearate to propylene glycol is 1:5 to 5:1, preferably 1:3 to 3:1, more preferably 2:

3.

3. The preparation method according to claim 1, characterized in that in step (s2), plinabulin is plinabulin monohydrate.

4. 1. A plinabulin micelle composition comprising: The micelle composition comprises plinabulin and a clear mixture of polyoxyl 15-hydroxystearate and propylene glycol, wherein the plinabulin micelle composition is a yellow, clear, transparent solution, and the particle size of the micelles ranges from 10 to 100 nm.

5. The plinabulin micelle composition according to claim 4, characterized in that the plinabulin micelle composition is prepared by the method of claim 1.

6. The plinabulin micelle composition comprises: Propylene glycol 30 to 90 wt%, preferably 40 to 70 wt%, for example 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, Polyoxyl 15-hydroxystearate 20 to 60 wt%, preferably 20 to 50 wt%, for example, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, and The composition contains 1 to 10 wt%, preferably 2 to 8 wt%, for example, 3 wt%, 4 wt%, 5 wt%, 6 wt%, or 7 wt% of plinabulin. The plinabulin micelle composition according to claim 4 .

7. The plinabulin micelle composition according to claim 4 or 6, characterized in that the weight ratio of polyoxyl 15-hydroxystearate to propylene glycol is 1:5 to 5:1, preferably 1:3 to 3:1, more preferably 2:

3.

8. 1. A liquid injectable plinabulin composition comprising: It contains plinabulin, propylene glycol, and polyoxyl 15-hydroxystearate in D5W solution (5% glucose injection), Here, the liquid injectable plinabulin composition is characterized in that the volume ratio of propylene glycol to D5W in the composition is about 6:50 to about 6:

500.

9. The liquid injectable plinabulin composition is prepared by the following method, which comprises: providing an initial liquid formulation comprising plinabulin, propylene glycol and polyoxyl 15-hydroxystearate; and diluting said initial liquid formulation in D5W at a dilution ratio of about 1:5 to about 1:50; The liquid injectable plinabulin composition according to claim 8, wherein the initial liquid formulation is the plinabulin micelle composition according to claim 4.

10. 9. The liquid injectable plinabulin composition according to claim 8, wherein the concentration of plinabulin is about 0.08 mg / ml to about 0.4 mg / ml.

11. 11. A liquid injectable plinabulin composition according to any one of claims 8 to 10, wherein the volume ratio of polyoxyl 15-hydroxystearate to D5W in the liquid injectable plinabulin formulation is about 4:50 to about 4:500, preferably about 4:100 to about 4:500, and more preferably about 4:100 to about 4:

400.

12. 9. The liquid injectable plinabulin composition according to claim 8, wherein the total amount of impurities contained in the liquid injectable plinabulin composition is less than 0.5%.

13. 9. The liquid injectable plinabulin composition according to claim 8, wherein more than about 90% of the plinabulin in the composition is encapsulated within micelles.

14. 1. A method for preparing a liquid injectable plinabulin composition, comprising: The method comprises: providing an initial liquid formulation comprising plinabulin, propylene glycol and polyoxyl 15-hydroxystearate; diluting said initial liquid formulation in D5W at a dilution ratio of about 1:5 to about 1:50; A method for preparing the liquid injectable plinabulin composition, comprising:

15. The method for preparing a liquid injectable plinabulin composition according to claim 14, characterized in that the initial liquid formulation is the plinabulin micelle composition according to claim 4.

16. Use of the plinabulin micelle composition of claim 4 or the liquid injectable plinabulin composition of claim 8 in the preparation of an antitumor therapeutic drug.

17. 17. The use according to claim 16, wherein the tumor is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), prostate cancer, colon cancer, brain tumor (e.g., 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.

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