Cabazitaxel micelle and preparation method therefor

Abstract

Disclosed are a cabazitaxel micelle and a preparation method therefor. The cabazitaxel micelle of the present invention comprises cabazitaxel and a polyethylene glycol-derived phospholipid. The polyethylene glycol-derived phospholipid comprises a polyethylene glycol moiety and a phospholipid moiety, and the phospholipid moiety thereof is di(C12-C24 fatty acyl)phosphatidylethanolamine. The polydispersity index of the micelle is not greater than 0.09. The cabazitaxel micelle of the present invention has one or more of the following advantages: uniform product particle size, good stability, low impurity content, high release degree, short reconstitution time, and convenient clinical administration.

Description

A carbazide micelle and its preparation method

[0001] This application claims priority to Chinese patent application 2024109526938, filed on July 16, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to a carbamate micelle and its preparation method. Background Technology

[0003] Taxanes are a large class of natural drugs derived from plants, structurally classified as diterpenoids. Cabazitaxel and docetaxel are both taxanes, and are lipid-soluble compounds. Their poor water solubility, short half-life, and high toxicity limit their clinical application. Currently, both docetaxel and cabazitaxel injections marketed domestically and internationally are dissolved in Tween-80. Clinical application requires strict adherence to dedicated injection solvents for dilution, and the procedures are demanding. Special infusion devices are also necessary, making the process cumbersome. Furthermore, the presence of large amounts of Tween-80 in the formulations easily leads to hemolysis and allergic reactions, necessitating the administration of dexamethasone and other preventative medications. This results in inconvenient clinical use and low safety profiles (Annals of Allergy, Asthma and Immunology, 2005, 95(6):593-599; Journal of Investigational Allergology and Clinical Immunology, 2016, 26, 394-396). To date, this problem remains unresolved.

[0004] Liposomes containing taxane drugs exist in the current technology, but they also have many drawbacks. For example, the preparation process of liposomes is complex, requiring the combination of multiple lipid components (at least two lipid components), and particle size control requires special equipment and devices; they are also prone to flocculation during storage.

[0005] The encapsulation efficiency of cabazitaxel liposomes prepared by Zhang Li et al. of Jiangsu University was only about 86%, and the particle size changed significantly before and after lyophilization, increasing from 68 nm to 108 nm, which increased the instability of the liposomes after reconstitution. (Zhang Li. Preparation and pharmacokinetic study of cabazitaxel lyophilized liposomes [D]. Jiangsu: Jiangsu University, 2017.)

[0006] Adding a certain proportion of polyethylene glycol-derived phospholipids to a conventional liposome formulation can prepare long-circulating liposomes. The long PEG chains form a steric hindrance and hydrophilic protective layer on the liposome surface, preventing phagocytic cell recognition and uptake of the liposomes, increasing their circulation time in the bloodstream, and giving this type of liposome long-circulating properties. However, when the mass ratio of polyethylene glycol-derived phospholipids to phospholipids exceeds a certain value, the encapsulation efficiency and stability of the drug will significantly decrease. This is because when the content of polyethylene glycol-derived phospholipids is high, it is difficult for them to insert into the phospholipid bilayer of the liposome, and they easily form mixed micelles with phospholipids, affecting the stability of the liposomes. (Colloids and Surfaces B: Biointerfaces 155(2017):266-275.)

[0007] Polymer micelles are a drug delivery system developed in recent years for poorly soluble drugs. They have a core-shell structure, with a hydrophobic core and a hydrophilic shell. Polymer micelles can load poorly soluble drugs at the core-shell boundary to achieve solubilization. Compared with commonly used solubilizers such as Tween-80 and polyoxyethylated castor oil, polymer micelle carriers are often made of biodegradable materials, which are safer. Therefore, they have good application prospects as carrier excipients for poorly soluble drugs (Journal of Controlled Release, 2001, 73, 137-172; Pharmaceutical Research, 2007, 24(1), 1-16). Currently, several nano-formulations of taxane drugs are under research (CN103990135A; CN107625730A; CN104758256A). These nano-formulations have solved, to some extent, the problems of poor water solubility, many adverse reactions, and low bioavailability of taxane drugs. However, current taxane-based drug nanoformulations suffer from low drug loading, low encapsulation efficiency, and poor stability (CN103990135A; CN107625730A; CN104758256A; CN102293736A; CN101732234A; CN101804021A; CN101829046A). Existing nanoformulations generally have drug loading below 5% and encapsulation efficiency below 90%, leading to the introduction of large amounts of carrier materials into the formulation. Since many nanomaterials have not yet undergone safety verification in clinical trials, and there is even a risk of degradation and the generation of toxic substances in the human body, their clinical use is limited (Journal of Controlled Release, 2009, 133, 11-17; International Journal of...). Pharmaceutics, 2014, 464(1-2):178-184.); On the other hand, most nano-formulations of poorly soluble drugs suffer from poor stability, and the encapsulation rate decreases in a very short time, leading to drug leakage. This is especially true for taxanes, where when the encapsulation rate drops below 95%, insoluble microparticles appear in the drug solution, affecting the quality of the formulation and preventing its clinical application (CN103990135A, CN107625730A, CN104758256A); At the same time, many nano-formulations have very complex preparation processes before use, some even requiring heating and stirring, which brings many risks to clinical application (CN101972480A; Drug Delivery and Translational Research, 2018, 5, 1365-1379).

[0008] Polyethylene glycol-derived phospholipids, such as PEG-DSPE, offer a range of unique advantages as micellar drug delivery systems. Furthermore, PEG-DSPE polymer molecules are FDA-approved pharmaceutical excipients with good safety profiles (Journal of Controlled Release, 2013, 171, 133–142; Pharmaceutical Research, 2012, 29, 1977–1989; Journal of the National Cancer Institute, 2007, 99, 1004–1015). Our previous research has shown that certain water-soluble small molecule drugs can be successfully loaded into formed PEG-DSPE nanomicelles in aqueous solution via a simple one-step self-assembly process (Pharmaceutical Research, 2010, 27(2), 361–370). These drug-loaded micelles unload their drug payload onto the cell membrane by disassembling and inserting into it, thereby increasing membrane fluidity and accelerating intracellular drug transport (Journal of Controlled Release, 2012, 160, 637–651). Meanwhile, we recently discovered that PEG-DSPE nanomicelles have the function of natural molecular chaperones. The hydrophilic nanocages formed by their shells can accommodate the A and B chains of insulin, promote the correct folding of the two chains and prevent insulin aggregation. Based on the spatial structure of the formed nanocages, PEG-DSPE nanomicelles can accommodate protein molecules with a molecular weight of less than 20 kDa (Biomaterials, 2016, 77, 139-148). Although PEG-DSPE has excellent drug loading capacity, it exhibits certain structural selectivity for the loaded drugs. For example, doxorubicin hydrochloride, vinorelbine tartrate, and vincristine sulfate can be successfully loaded into PEG-DSPE micelles with an encapsulation rate as high as 99.9% (CN1840193A; CN101322681A; CN1739525A; CN101138545A). However, PEG-DSPE micelles do not have the ability to load all small molecule drugs. For instance, gemcitabine hydrochloride and paclitaxel cannot be successfully and stably loaded into PEG-DSPE micelles, thus failing to achieve the purpose of clinical drug use (Pharmaceutical Research, 2010, 27(2), 361-370; Philosophical Transactions of The Royal Society A, 20120309, 371; Molecular Cancer Therapeutics, 2014, 13(12), 2864-2875).Patent CN113908123A discloses a micelle containing a taxane drug, its preparation method, and its application. This patent employs a film-forming hydration method to prepare cabazitaxel micelles. This method requires dissolving cabazitaxel and the micelle material PEG-PE in an organic solvent, followed by removing the organic solvent from the drug-containing organic solution through heating and rotary evaporation. This method suffers from drawbacks such as high energy consumption, the impact of heating on drug stability, and small batch sizes produced by rotary evaporation, hindering commercial-scale production. Furthermore, the cabazitaxel micelles obtained by this method exhibit uneven particle size, poor stability, high impurity content, low release rate, long reconstitution time, and poor convenience for clinical use. Summary of the Invention

[0009] The technical problem this invention aims to solve is to overcome the shortcomings of existing methods for preparing taxane-containing micelles, such as high energy consumption, the impact of heating on drug stability, small batch sizes produced by rotary evaporation which are not conducive to commercial scale-up production, and the resulting cabazitaxel micelles exhibiting non-uniform particle size, poor stability, high impurity content, low release rate, long reconstitution time, and poor convenience for clinical use. Therefore, this invention provides a method for preparing cabazitaxel micelles, which has one or more of the following advantages: low energy consumption, no need for high temperature, mass production capability, conducive to commercial scale-up, uniform product particle size, good stability, low impurity content, high release rate, short reconstitution time, and good convenience for clinical use.

[0010] The present invention provides a micelle containing cabazitaxel, the micelle comprising cabazitaxel and polyethylene glycol-derived phospholipids;

[0011] The polyethylene glycol-derived phospholipid comprises a polyethylene glycol portion and a phospholipid portion, wherein the phospholipid portion is di(C) 12 -C 24 (Fat acyl) phosphatidylethanolamine;

[0012] The polydispersity index of the micelles is no higher than 0.09.

[0013] In certain preferred embodiments of the present invention, the materials, proportions, or properties of the carbataxel-containing micelles are defined as follows, and any materials, proportions, or properties not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "in a certain embodiment of the present invention"). Other aspects of the present invention are referred to using the same abbreviations as this invention and will not be described in detail hereafter.

[0014] In one embodiment of the present invention, the carbamate-containing micelles further include one or more of the following components: C 1-6 Alcohol solvents (e.g., ethanol), water (e.g., sterile water for injection), glucose injection (e.g., 5% glucose injection), and sodium chloride injection (e.g., 0.9% sodium chloride injection).

[0015] In one embodiment of the present invention, the phospholipid portion of the polyethylene glycol-derived phospholipid may be distearate phosphatidylethanolamine (C... 18 (abbreviated as DSPE), dipalmitoyl phosphatidylethanolamine (C 16 DPPE (abbreviated as DPPE), dimyristoyl phosphatidylethanolamine (C 14 DMPE (abbreviated as DMPE) and dioleoylphosphatidylethanolamine (C 18 One or more of the following (abbreviated as DOPE); preferably distearylphosphatidylethanolamine (DSPE).

[0016] In one embodiment of the present invention, the polyethylene glycol-derived phospholipid has a polyethylene glycol weight-average molecular weight range of 1000 to 5000, for example, a polyethylene glycol weight-average molecular weight of 2000.

[0017] In one embodiment of the present invention, the polyethylene glycol-derived phospholipid is polyethylene glycol 1000 distearylphosphatidylethanolamine (PEG1000-DSPE), polyethylene glycol 2000 distearylphosphatidylethanolamine (PEG2000-DSPE), polyethylene glycol 5000 distearylphosphatidylethanolamine (PEG5000-DSPE), polyethylene glycol 2000 dioleoylphosphatidylethanolamine (PEG2000-DOPE), polyethylene glycol 2000 dipalmitoylphosphatidylacetamide (PEG2000-DPPE), or polyethylene glycol 2000 dimyristoylphosphatidylethanolamine (PEG2000-DMPE); preferably PEG2000-DSPE.

[0018] In one aspect of the present invention, the polydispersity index of the carbamate-containing micelles is not higher than 0.08, for example, 0.08, 0.07, 0.06 or 0.04.

[0019] In one aspect of the present invention, the polydispersity index of the carbamate-containing micelles is not higher than 0.06, for example, 0.04.

[0020] In one aspect of the present invention, the Zeta potential of the carbamate-containing micelles is (-45mv)-(-60mv), preferably (-45mv)-(-50mv), for example -48.77mv.

[0021] In one embodiment of the present invention, the mass ratio of polyethylene glycol-derived phospholipid to carbamate in the carbamate is not higher than 15:1, for example (0.5-1.5):1 or (5-15):1, or 1:1, 10:1 or 11:1.

[0022] In one embodiment of the present invention, the mass ratio of polyethylene glycol-derived phospholipid to carbamate in the carbamate is not higher than 15:1, for example (0.5-1.5):1 or (9-13):1, or even 1:1, 10:1 or 11:1.

[0023] In one embodiment of the present invention, the mass ratio of polyethylene glycol-derived phospholipid to carbamate in the carbamate micelles is (8-15):1, preferably (9-13):1, for example 10:1 or 11:1.

[0024] In one aspect of the present invention, the resolution time of the carbamate-containing micelles is no more than 60 minutes, preferably no more than 45 minutes, more preferably no more than 15 minutes, for example 10 minutes, 7 minutes or 5 minutes.

[0025] In one aspect of the present invention, the encapsulation efficiency of the carbamate-containing micelles is 95%-100%, preferably 98.5%-100%, for example 99.70%, 98.90% or 99.90%.

[0026] In one aspect of the present invention, the total impurity content in the carbamate micelles is not higher than 2%, preferably not higher than 1%, more preferably not higher than 0.5%, for example 0.36% or 0.06%.

[0027] In one aspect of the present invention, the total impurity content in the carbamate-containing micelles is not higher than 0.1%, for example, 0.06%.

[0028] In one aspect of the present invention, the micelles containing cabazitaxel are stably stored at room temperature for more than 6 hours.

[0029] In one aspect of the present invention, the micelles containing cabazitaxel are stably stored at 4°C for more than 24 hours.

[0030] In one aspect of the present invention, the release rate of the carbamate-containing micelles is 75%-100%, preferably 75%-85%, for example 78.3%.

[0031] In one aspect of the present invention, the particle size of the carbamate-containing micelles is 10-20 nm, for example, 13 nm, 14 nm, 15 nm or 16 nm.

[0032] In one embodiment of the present invention, the micelles containing cabazitaxel also contain a freeze-drying protectant; the freeze-drying protectant is preferably sucrose or lactose.

[0033] In one embodiment of the present invention, the carbamate-containing micelles comprise carbamate and polyethylene glycol 2000 distearate phosphatidylethanolamine; the mass ratio of carbamate to polyethylene glycol 2000 distearate phosphatidylethanolamine is preferably 1:11; the reconstitution time of the carbamate-containing micelles is preferably not higher than 60 minutes; the encapsulation efficiency of the carbamate-containing micelles is preferably 95%-100%; the total impurity content in the carbamate-containing micelles is preferably not higher than 2%; the release rate of the carbamate-containing micelles is preferably 75%-100%; the particle size of the carbamate-containing micelles is preferably 10-20 nm; and the Zeta potential of the carbamate-containing micelles is preferably (-45 mV)-(-60 mV).

[0034] Preferably, the carbazide-containing micelles are composed of carbazide and polyethylene glycol 2000 distearate phosphatidylethanolamine.

[0035] In one embodiment of the present invention, the carbataxel-containing micelles are micelles used for treating cancer; preferably, the cancer is selected from prostate cancer, breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

[0036] The present invention also provides a method for preparing micelles containing cabazitaxel, which includes the following step A:

[0037] Micelles A are obtained by spray drying a solution of cabazitaxel and polyethylene glycol-derived phospholipids in a polar organic solvent A.

[0038] The polyethylene glycol-derived phospholipid comprises a polyethylene glycol portion and a phospholipid portion, wherein the phospholipid portion is di(C) 12 -C 24 (Fat acyl) phosphatidylethanolamine;

[0039] The polar organic solvent A is a polar organic solvent with a boiling point below 85°C and a solubility of cabazitaxel of not less than 60 mg / mL at room temperature.

[0040] In one embodiment of the present invention, the polar organic solvent A is a polar organic solvent with a solubility of not less than 60 mg / mL for the polyethylene glycol-derived phospholipid at room temperature.

[0041] In one embodiment of the present invention, the polar organic solvent A is acetone, dichloroethane, chloroform, tetrahydrofuran, carbon tetrachloride, ethyl acetate, butanone, or an alcohol solvent.

[0042] In one embodiment of the present invention, the polar organic solvent A is an alcohol solvent; preferably, the alcohol solvent is C. 1-6 Alcohol solvents, more preferably C 1-3 Alcohol solvents, such as ethanol.

[0043] In one embodiment of the present invention, the phospholipid portion of the polyethylene glycol-derived phospholipid may be distearate phosphatidylethanolamine (C... 18 (abbreviated as DSPE), dipalmitoyl phosphatidylethanolamine (C 16 DPPE (abbreviated as DPPE), dimyristoyl phosphatidylethanolamine (C 14 DMPE (abbreviated as DMPE) and dioleoylphosphatidylethanolamine (C 18 One or more of the following (abbreviated as DOPE); preferably distearylphosphatidylethanolamine (DSPE).

[0044] In one embodiment of the present invention, the polyethylene glycol-derived phospholipid has a polyethylene glycol weight-average molecular weight range of 1000 to 5000, for example, a polyethylene glycol weight-average molecular weight of 2000.

[0045] In one embodiment of the present invention, the polyethylene glycol-derived phospholipid is polyethylene glycol 1000 distearylphosphatidylethanolamine (PEG1000-DSPE), polyethylene glycol 2000 distearylphosphatidylethanolamine (PEG2000-DSPE), polyethylene glycol 5000 distearylphosphatidylethanolamine (PEG5000-DSPE), polyethylene glycol 2000 dioleoylphosphatidylethanolamine (PEG2000-DOPE), polyethylene glycol 2000 dipalmitoylphosphatidylacetamide (PEG2000-DPPE), or polyethylene glycol 2000 dimyristoylphosphatidylethanolamine (PEG2000-DMPE); preferably PEG2000-DSPE.

[0046] In one embodiment of the present invention, in step A, the mass ratio of the polyethylene glycol-derived phospholipid to the cabazitaxel is (0.5-1.5):1, for example, 1:1.

[0047] In one aspect of the present invention, the concentration of cabazitaxel in the polar organic solvent A is 3-80 mg / mL, preferably 6-60 mg / mL; more preferably 30-60 mg / mL, for example 60 mg / mL.

[0048] In one embodiment of the present invention, the temperature of the polar organic solvent A solution is 20-60°C, preferably 40°C.

[0049] In one aspect of the present invention, the inlet air temperature during spray drying is 45-65°C, preferably 50-60°C, for example 55°C.

[0050] In one aspect of the present invention, the outlet air temperature during spray drying is 30-45°C, preferably 35-45°C, for example 40°C.

[0051] In one aspect of the present invention, during the spray drying, the pump speed is 5%-15%, preferably 5-10%, for example 8%.

[0052] In one aspect of the present invention, the air velocity during spray drying is 40%-80%, preferably 45%-60%, for example 50%.

[0053] In one aspect of the present invention, during the spray drying process, the nozzle cleaning frequency is 3-5 times / min, for example 4 times / min.

[0054] In one embodiment of the present invention, the method for preparing the carbamate-containing micelles further includes step B:

[0055] Micelles A and polyethylene glycol-derived phospholipids were dissolved in water to obtain micelles B.

[0056] The water is preferably sterile water for injection;

[0057] The ratio of the mass of cabazitaxel contained in micelle A to the volume of water is 2-10 mg / mL, preferably 4-8 mg / mL, for example 6 mg / mL.

[0058] In one embodiment of the present invention, in step B, the concentration of polyethylene glycol-derived phospholipids in the water is 40-80 mg / mL, for example 60 mg / mL or 66 mg / mL.

[0059] In one embodiment of the present invention, in step B, the mass ratio of the polyethylene glycol-derived phospholipid to the carbamate A contained in the micelles is (5-15):1, for example, 10:1 or 11:1.

[0060] In one embodiment of the present invention, in step B, the mass ratio of the polyethylene glycol-derived phospholipid to the cabazitaxel is (8-15):1, preferably (9-13):1, for example 10:1 or 11:1.

[0061] In one embodiment of the present invention, step B is performed at 15-30°C, for example, 25°C.

[0062] In one embodiment of the present invention, the method for preparing the carbamate-containing micelles further includes the following step C:

[0063] Micelle B is filtered to obtain micelle C.

[0064] In one embodiment of the present invention, step C employs a polymer filter membrane for filtration;

[0065] The polymer filter membrane is preferably a polyvinyl fluoride filter membrane, a mixed cellulose filter membrane (MCE), a polyethersulfone filter membrane (PES), a nylon filter membrane, a polytetrafluoroethylene filter membrane (PTFE), or a polypropylene filter membrane (PP);

[0066] The polyvinylidene fluoride filter membrane is preferably PVDF (polyvinylidene fluoride membrane);

[0067] The pore size of the polyvinyl fluoride filter membrane is preferably 0.10-0.30 mm, for example 0.22 mm.

[0068] In one embodiment of the present invention, step C is performed at 15-30°C, for example, 25°C.

[0069] In one embodiment of the present invention, the method for preparing the carbamate-containing micelles further includes the following step D:

[0070] Remove the water from micelle C to obtain micelle D.

[0071] In one embodiment of the present invention, step D involves removing water from the micelles C by freeze drying, spray drying, or fluidized bed drying.

[0072] In one aspect of the present invention, the freeze-drying is carried out in the presence of a freeze-drying protectant; the freeze-drying protectant is preferably sucrose or lactose; the amount of the freeze-drying protectant is preferably 2%-6%, for example 4%; the aforementioned amount is the mass-volume ratio of the freeze-drying protectant to micelles C.

[0073] In one aspect of the present invention, the freeze-drying is carried out in the presence of a freeze-drying protectant, or in the absence of a freeze-drying protectant.

[0074] The freeze-drying is preferably carried out without the use of a freeze-drying protectant.

[0075] In one aspect of the present invention, the freeze-drying is performed using conventional methods in the art; preferably, the freeze-drying includes a freezing stage, a primary drying stage, and a desorption drying stage.

[0076] In one embodiment of the present invention, the method for preparing the carbamate-containing micelles further includes the following step E:

[0077] The micelles D are dissolved in solvent a to obtain micelles E;

[0078] Solvent a is preferably one or more of water (e.g., sterile water for injection), glucose injection (e.g., 5% glucose injection), and sodium chloride injection (e.g., 0.9% sodium chloride injection).

[0079] In one embodiment of the present invention, step E is performed at 15-30°C, for example, 25°C.

[0080] In one aspect of the present invention, the materials used in steps A, B, C, D, and E consist only of the materials mentioned in each step and do not contain any other materials.

[0081] In one aspect of the present invention, the method for preparing the carbamate-containing micelles includes the following steps:

[0082] Step A: The polar organic solvent A solution of cabazitaxel and polyethylene glycol-derived phospholipids is spray-dried to obtain micelles A;

[0083] Step B: Dissolve the micelles A and polyethylene glycol-derived phospholipids in water to obtain micelles B;

[0084] Step C: Filter the micelles B to obtain micelles C;

[0085] Step D: Remove the water from the micelles C to obtain micelles D;

[0086] Step E: Dissolve the micelles D in solvent a to obtain micelles E;

[0087] The conditions, operations, or materials in steps A, B, C, D, and E are as described in any of the preceding schemes;

[0088] Preferably, the method for preparing the carbamate-containing micelles comprises steps A, B, C, D, and E.

[0089] In one embodiment of the present invention, the method for preparing the cabazitaxel-containing micelles comprises the following steps: weighing cabazitaxel and PEG2000-DSPE, dissolving them in a polar organic solvent A to obtain a corresponding solution, and spray-drying to obtain micelle A; dissolving micelle A and PEG2000-DSPE in water to obtain micelle B; filtering micelle B to obtain micelle C; removing water from micelle C by freeze-drying to obtain micelle D; and dissolving micelle D in solvent a to obtain micelle E; wherein the polar organic solvent A is preferably ethanol; and solvent a is preferably water.

[0090] Conditions, procedures, or materials not specifically mentioned are as described in the previous scheme.

[0091] The present invention also provides a micelle containing cabazitaxel, which is micelle A, micelle B, micelle C, micelle D or micelle E;

[0092] The micelles A were prepared via step A.

[0093] The micelles B were prepared via steps A and B.

[0094] The micelles C were prepared via steps A, B, and C.

[0095] The micelles D are prepared by steps A, B, C and D.

[0096] The micelles E are prepared by steps A, B, C, D and E.

[0097] The conditions, operations, or materials in steps A, B, C, D, and E are as described in any of the preceding schemes.

[0098] In one aspect of the present invention, the resolution time of the micelles D is no more than 60 minutes, preferably no more than 45 minutes, more preferably no more than 15 minutes, for example 10 minutes, 7 minutes or 5 minutes.

[0099] In one embodiment of the present invention, the micelles E are a clear and transparent solution.

[0100] In one aspect of the present invention, the polydispersity coefficient of the micelles E is not higher than 0.09, preferably not higher than 0.08, for example 0.08, 0.07, 0.06 or 0.04.

[0101] In one aspect of the present invention, the polydispersity coefficient of the micelles E is not higher than 0.06, for example 0.04.

[0102] In one aspect of the present invention, the encapsulation efficiency of the micelles E is 95%-100%, preferably 98.5%-100%, for example 99.70%, 98.90% or 99.90%.

[0103] In one aspect of the present invention, the Zeta potential of micelle E or micelle C is (-45mv)-(-60mv), preferably (-45mv)-(-50mv), for example -48.77mv.

[0104] In one aspect of the present invention, the total impurity content in the micelles E is not higher than 2%, preferably not higher than 1%, more preferably not higher than 0.5%, for example 0.36% or 0.06%.

[0105] In one aspect of the present invention, the total impurity content in the micelles E is not higher than 0.1%.

[0106] In one aspect of the present invention, the micelles E are stably stored at room temperature for more than 6 hours.

[0107] In one aspect of the present invention, the micelles E are stably stored at 4°C for more than 24 hours.

[0108] In one aspect of the present invention, the release rate of the micelles E is 75%-100%, preferably 75%-85%, for example 78.3%.

[0109] In one aspect of the present invention, the particle size of the micelles E is 10-20 nm, for example 13 nm, 14 nm, 15 nm or 16 nm.

[0110] In one aspect of the present invention, micelles A, B, C, D, and E are micelles used for treating cancer.

[0111] Preferably, the cancer is selected from prostate cancer, breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

[0112] The present invention also provides a pharmaceutical composition comprising any of the following as described above: micelles containing cabazitaxel, micelle A, micelle B, micelle C, micelle D or micelle E, and pharmaceutical excipients.

[0113] The present invention also provides the use of any of the aforementioned micelles, micelle A, micelle B, micelle C, micelle D, micelle E, or the aforementioned pharmaceutical composition, wherein the use is in the preparation of a medicament for treating cancer.

[0114] In one aspect of the present invention, the cancer is selected from breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

[0115] In one aspect of the present invention, the cancer is selected from prostate cancer, breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

[0116] The present invention also provides a method for treating cancer in a subject in need, comprising administering substance X to the subject; said substance X is any of the following as described above: micelles containing cabazitaxel, micelle A, micelle B, micelle C, micelle D, micelle E, or the aforementioned pharmaceutical composition;

[0117] The cancers mentioned are selected from prostate cancer, breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

[0118] The present invention also provides a substance X for treating cancer;

[0119] The substance X is any of the substances described above: micelles containing cabazitaxel, micelle A, micelle B, micelle C, micelle D, micelle E, or the aforementioned pharmaceutical composition;

[0120] The cancers mentioned are selected from prostate cancer, breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

[0121] Unless otherwise specified, the terms used in this invention may be defined as follows:

[0122] The "micelles" described in this invention can be in solid or liquid form.

[0123] In this invention, "total impurities" refers to the sum of all impurities in the analyte that exceed the reporting limit.

[0124] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0125] The reagents and raw materials used in this invention are all commercially available.

[0126] The positive and progressive effects of the present invention are as follows: the method for preparing carbamate micelles provided by the present invention has one or more of the following advantages: low energy consumption, no need for high temperature, mass production capability, conducive to commercial scale-up, uniform product particle size, good stability, low impurity content, high release rate, short reconstitution time, and good convenience for clinical use. Attached Figure Description

[0127] Figure 1 is a transmission electron microscope image of the micelles prepared by the film-forming hydration method in Example 1.

[0128] Figure 2 is a transmission electron microscope image of the micelles prepared by spray drying in Example 1.

[0129] Figure 3 shows the appearance of the micelle formulation prepared in Example 2 after hydration when the ratio of API to phosphatidylethanolamine was 1:7.

[0130] Figure 4 shows the appearance of the micelle formulation prepared in Example 2 after hydration when the ratio of API to phosphatidylethanolamine was 1:11.

[0131] Figure 5 shows the appearance of the micelle formulation prepared in Example 2 after hydration when the ratio of API to phosphatidylethanolamine was 1:15.

[0132] Figure 6 shows the appearance of the drug solution after reconstitution of the micelle formulation prepared without the use of a lyophilization protectant in Example 3.

[0133] Figure 7 shows the appearance of the drug solution after reconstitution of the micelle formulation prepared using sucrose as a freeze-drying protectant in Example 3.

[0134] Figure 8 shows the appearance of the drug solution after reconstitution of the micelle formulation prepared using lactose as a lyophilization protectant in Example 3.

[0135] Figure 9 shows the appearance of the finished micelle formulation prepared without using a freeze-drying protectant in Example 3.

[0136] Figure 10 shows the appearance of the finished micelle formulation prepared using sucrose as a freeze-drying protectant in Example 3.

[0137] Figure 11 shows the appearance of the finished micelle formulation prepared using lactose as a freeze-drying protectant in Example 3.

[0138] Figure 12 shows the tumor growth trend of each group in Example 6;

[0139] Figure 13 shows the tumor weight inhibition rate of each group in Example 6;

[0140] Figure 14 is a graph showing the average drug time curve of carbazitaxel in tumors after intravenous injection of the test carbazitaxel micelles (i.e., LN003) and the control JEVTANA solution into tumor-bearing mice in Example 7.

[0141] Figure 15 is a graph showing the average carbamate time curve in tumor-bearing mice after intravenous injection of the test carbamate micelles (i.e., LN003) and the control JEVTANA solution.

[0142] Figure 16 is a graph showing the average drug time curve of carbazide in the liver of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0143] Figure 17 is a graph showing the average drug time curve of carbazide in the spleen of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0144] Figure 18 is a graph showing the average drug time curve of carbazide in the lungs of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0145] Figure 19 is a graph showing the average drug time curve of carbazide in the kidneys of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0146] Figure 20 is a graph showing the average drug time curve of carbazide in the brain of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0147] Figure 21 is a graph showing the average drug time curve of carbazitaxel in the stomach of tumor-bearing mice after intravenous injection of the test carbazitaxel micelles (i.e., LN003) and the control JEVTANA solution.

[0148] Figure 22 is a graph showing the average drug time curve of carbazide in the lymph nodes of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0149] Figure 23 is a graph showing the average drug-time curve of carbazide in the duodenum of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0150] Figure 24 is a graph showing the average drug time curve of carbazide in the testes of tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution.

[0151] Figure 25 is a graph showing the average blood concentration of carbazide mice in tumor-bearing mice after intravenous injection of the test carbazide micelles (i.e., LN003) and the control JEVTANA solution in Example 7.

[0152] Figure 26 is a bar chart of the average drug concentration of carbazide in tissues and plasma of tumor-bearing mice after intravenous injection of the tested carbazide micelles (i.e., LN003) in Example 7; each set of data involves data at four time points, and the data bars from left to right are the average drug concentrations at four time points: 2h, 8h, 24h and 96h.

[0153] Figure 27 is a bar chart of the average drug concentration of carbazide in tissues and plasma of tumor-bearing mice after intravenous injection of the control JEVTANA solution in Example 7; each set of data involves data at four time points, and the data bars from left to right are the average drug concentrations at the four time points of 2h, 8h, 24h and 96h respectively.

[0154] Figure 28 shows the state of micelles prepared by the film-forming hydration method after release at 37°C for 4 hours in a dialysis bag;

[0155] Figure 29 shows the state of micelles prepared by spray drying after being released in a dialysis bag at 37°C for 4 hours;

[0156] Figure 30 shows the state of micelles after dilution with different dilution media and placed at 37°C for 8 hours. Detailed Implementation

[0157] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0158] Drug-loaded micelle size analysis

[0159] The particle size distribution of drug-loaded micelles (micelle hydrate or reconstituted micelles) was determined using dynamic light scattering (DLS). Samples were diluted to 1 mg / mL with physiological saline, and 1 mL of sample was used to measure the particle size distribution and polydispersity index (PDI) of the drug-loaded micelles using a Malvern Zetasizer Nano-ZS light scattering particle size analyzer. The laser beam wavelength was set to 633 nm, and the angle between the incident and scattered beams was 90°. Each sample was measured for 20 cycles at a temperature of 25°C. The results for each formulation were the average of three batches. Polydispersity index (PDI) indicates the uniformity of particle size; a lower PDI indicates more uniform particle size.

[0160] Drug encapsulation efficiency and drug loading

[0161] Take 1 mL of drug-loaded micelle solution, filter it through a 220 nm microporous membrane, and determine the concentration of drug-loaded micelles and drug-loaded micelles in the filtrate using high performance liquid chromatography (HPLC). The chromatographic conditions are as follows:

[0162] Ultracentrifugation; High-performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512)

[0163] Instrument: Agilent / DAD or VWD detector or equivalent instrument

[0164] Column: ZORBAX Extend-C18 (150×4.6mm, 3.5μm)

[0165] Mobile phases: Mobile phase A: Water; Mobile phase B: Acetonitrile;

[0166] Time gradient:

[0167] Column temperature: 30℃

[0168] Flow rate: 1.0 ml / min

[0169] Detection wavelength: UV230nm

[0170] Injection volume: 10 μl

[0171] Diluent: Acetonitrile

[0172] Solution preparation:

[0173] Mobile phase A: Measure an appropriate amount of water, degas it by sonication, and you will get the mobile phase A.

[0174] Mobile phase B: Measure an appropriate amount of acetonitrile, degas it by ultrasonication, and you will get the mobile phase B.

[0175] Blank solution: Acetonitrile

[0176] Reference solution: Accurately weigh an appropriate amount of cabazitaxel working reference standard, dissolve it in acetonitrile, and quantitatively dilute it to prepare a solution containing approximately 36 μg per ml. Prepare two parallel solutions.

[0177] Test solution A: Take the test solution from the assay section as the reconstitution stock solution; accurately measure 1 ml of the reconstitution stock solution, place it in a 50 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0178] Test solution B: Take an appropriate amount of the reconstituted stock solution, centrifuge at 10,000 rpm for 10 minutes, accurately measure 1 ml of the supernatant, place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0179] System Applicability

[0180] Requirements: Accurately measure 10 μl each of the blank solution and the reference solution, inject them into the liquid chromatograph, and record the chromatograms. For five consecutive reference solutions (STD1), the peak area RSD should be ≤2.0%, and the retention time RSD should be ≤1.0%. The recovery rate of the main peak in the reference solution (STD2) should be between 98.0% and 102.0%.

[0181] Determination: Accurately measure 10 μl each of test solution A, test solution B and reference solution, inject them into the liquid chromatograph, and record the chromatograms.

[0182] Calculation: Calculate the content (S) of test solution A and the content (E) of test solution B based on peak area using the external standard method.

[0183] Where: AspL is the peak area of ​​each main peak in test solution A / B; Astd is the average peak area of ​​5 consecutive main peaks in the reference solution; Wstd is the sample weight of the reference in the reference solution, mg; P is the content of the reference, %; Vstd is the dilution factor of the reference solution; VspL is the dilution factor of test solution A / B; B is the specification, 60; N is the quantity, 3.

[0184] It should be no less than 98%.

[0185] Example 1:

[0186] Spray drying method: Accurately weigh 60 mg of cabazitaxel and 60 mg of PEG2000-DSPE (source: Lipoid), and stir and dissolve them in 1 mL of anhydrous ethanol at 40 °C to obtain a mixed ethanol solution of the drug and PEG2000-DSPE to form a film-forming solution.

[0187] If the inlet air temperature is too low or the pump speed is too high, the solution will not have enough time to dry completely into powder, the organic solvent will not be completely removed, the powder collection rate will be low, and if the inlet air temperature is too high, the powder will melt and adhere to the inner wall of the pipeline. If the air speed is too fast or too slow, the yield will decrease and the particle size of the powder will also be affected. Parameter 2 is selected for spray drying conditions.

[0188] The spray-dried powder of the API and PEG2000-DSPE complex was prepared by spray drying (spray drying parameters are as follows: inlet air temperature 55℃, outlet air temperature 40℃, pump speed: 8%).

[0189] Dissolve 600 mg of PEG2000-DSPE in 10 mL of water for injection to prepare a phospholipid aqueous solution. Dissolve the spray-dried powder in 10 mL of the phospholipid aqueous solution to prepare a micelle hydration solution. Filter the hydration solution twice through a 0.22 μm PVDF membrane to obtain the pre-filling solution. Fill the pre-filling solution into borosilicate glass vials and freeze-dry to obtain the formulation sample. Freeze-dry according to the following parameters.

[0190] Film-forming hydration method: Dissolve 60 mg of cabazitaxel and 60 mg of PEG2000-DSPE in ethanol, then remove the solvent by rotary evaporation of the drug-containing ethanol solution at 50°C to form a drug lipid film; then dissolve the remaining 600 mg of PEG2000-DSPE in water for injection to form an aqueous phase solution; then form a micelle aqueous solution of the drug lipid film with the aqueous phase solution; finally, freeze-dry the micelle aqueous solution to obtain the drug-containing micelle product.

[0191] Reconstitution: Dissolve the lyophilized product obtained by spray drying or film-forming hydration in 10 mL of water for injection.

[0192] Table 1 summarizes the quality results of micelles (after rehydration) obtained by film-forming hydration method and spray drying method.

[0193] Table 1 Summary of micelle quality test results prepared by film-forming hydration method and spray drying method

[0194] The reconstitution time of the lyophilized cabazitaxel micelle powder prepared by spray drying is significantly reduced, greatly increasing the convenience of clinical use. Simultaneously, the polydispersity index of the particle size distribution decreases from 0.106 to 0.06, resulting in a more uniform micelle size distribution, which is beneficial for the efficacy of the drug. The micelles prepared by spray drying are stable after 6 hours at room temperature and 24 hours at 4°C. Compared with the film-forming hydration method, the micelle stability is significantly improved.

[0195] Release test conditions (nanocage reciprocating frame method):

[0196] Dialysis bags: Regenerated cellulose dialysis bags (8000-10000)

[0197] Solution in the dialysis bag: an aqueous solution containing approximately 0.2 mg / ml of cabazitaxel.

[0198] Media Name: pH 7.4 Phosphate Buffer (containing 0.1% Tween 80)

[0199] Media volume: 50ml

[0200] Sample volume: 5ml

[0201] Sampling time points: 0.5h, 2h, 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h.

[0202] Zeta potential testing method:

[0203] Take one vial of the sample to be tested (take it out and place it at room temperature), shake the vial to completely separate the lyophilized powder from the vial wall, inject 20 ml of 5% glucose injection solution using a syringe (try to wet all the powder during the injection), shake slowly several times, let stand for 5 minutes, and release the gas in the vial with the syringe needle; let it stand until completely dissolved (shake slowly a few times during the process), shake well, and prepare a solution containing 3 mg of carbatastatin per 1 mL as the potential test solution, and determine its potential according to the instrument method below.

[0204] Measurement temperature: 25℃

[0205] Medium: Water

[0206] Reconstitution solvent: 5% glucose injection

[0207] Sample concentration (mg / mL): 3 mg / mL

[0208] (Note: (Water) refractive index: 1.33; viscosity: 0.8872; dielectric constant: 78.5)

[0209] Example 2:

[0210] Following the method in Example 1, three batches of cabazitaxel micelle formulations were prepared with a cabazitaxel:penicillin phosphatidylethanolamine (DSPE-PEG2000, CAS: 147867-65-0) ratio (mass ratio) of 1:7 (batch number 3662107271), 1:11 (batch number 3662107241), and 1:15 (batch number 3662107272). (The mass ratio of cabazitaxel to pegphosphatidylethanolamine in the solution before spray drying was 1:1; the remaining pegphosphatidylethanolamine was added during the hydration of the spray powder.) The results of screening the formulation quality based on the API to pegphosphatidylethanolamine ratio are shown in Table 2, and the appearance of the hydrated solution is shown in Figures 3-5.

[0211] Table 2. Screening Results of Raw Material and Auxiliary Material Ratios in Formulation Note: " / " indicates that it has not been freeze-dried.

[0212] The results show that when the ratio of API to phosphatidylethanolamine is 1:7, the hydrated solution is turbid, indicating that phosphatidylethanolamine cannot encapsulate the corresponding amount of cabazitaxel active ingredient, and the encapsulation rate is 98.0%, which is significantly lower than that of the micelle samples with API to excipient ratios of 1:11 and 1:15. When the API to excipient ratio is 1:11 and 1:15, the hydrated solutions are clear and transparent, and the related substances, particle size, and particle size distribution of the samples are comparable, indicating a homogeneous micelle formulation. However, this formulation is an injectable drug. Following the principle of minimizing excipient input, a ratio of API to phosphatidylethanolamine of 1:11 is selected as the optimal ratio for subsequent sample preparation to reduce formulation production costs.

[0213] Example 3:

[0214] Referring to Example 2, a hydrated solution of cabazitaxel and PEG2000-DSPE was prepared at a raw material ratio of 1:11. Cabazitaxel micelle formulations were then prepared with one without a lyophilization protectant, one containing 4% sucrose (w / v mass ratio of lyophilization protectant to micelle solution before lyophilization), and one containing 4% lactose. Specific results are shown in Table 3. Figures 6 to 11 show the appearance of the reconstituted drug solution and the finished product after lyophilization for micelle formulations prepared with different types of lyophilization protectants.

[0215] Table 3. Screening Results of Lyophilization Protectants Note: " / " represents none.

[0216] The results above show that the properties, encapsulation efficiency, particle size, and particle size distribution of the three batches of formulations after reconstitution are not significantly different. However, the total impurity content of the micelle formulation prepared with 4% lactose as a lyophilization protectant is 0.42%, which is significantly higher than that of the micelle formulations without a lyophilization protectant and those with 4% sucrose. To reduce production costs and adhere to the principle of minimizing excipient addition, subsequent formulations will not use a lyophilization protectant to reduce the use of excipients.

[0217] Example 4:

[0218] Three batches of cabazitaxel micelle hydrated solutions were prepared according to the preparation method in Example 1. The hydrated solutions were filtered twice through a 0.22 μm PVDF membrane to obtain filtrates. The composition of the small-scale confirmation batch formulation of the hydrated solutions is shown in Table 4, and the test results are shown in Table 5.

[0219] The above filtrate was filled into borosilicate glass vials for injection and freeze-dried to obtain the formulation sample.

[0220] Table 4. Composition of the small-scale confirmation batch prescription Note [1] Anhydrous ethanol is used as a solvent and removed during the spray drying process.

[0221] Table 5. Results of small-scale confirmation batch testing

[0222] The results showed that the three batches of finished products prepared using the proposed formula process met the quality standards, and there were no significant differences between batches of finished products. This indicates that the proposed formula process has good reproducibility and can be further scaled up for production.

[0223] The samples obtained from the small-scale confirmation batch were placed at 25℃±2℃ / 60%RH±10%RH for 2 weeks, 4 weeks, and 8 weeks; and at 40℃±2℃ / 75%RH±5%RH for 1 week, 2 weeks, and 4 weeks to examine the stability of the samples under each condition. The results are shown in Tables 6 to 8.

[0224] Table 6. Small-scale confirmation batch test results (batch number: 3662108191)

[0225] Table 7. Test results of the small-scale confirmation batch (batch number: 3662108192)

[0226] Table 8. Test results of the small-scale confirmation batch (batch number: 3662108193)

[0227] The above results indicate that the three batches of formulations prepared by the pilot-scale process, after being placed at high temperature (40℃±2℃ / 75%RH±5%RH) for 4 weeks and at 25℃±2℃ / 60%RH±10%RH for 8 weeks, showed little change in content, encapsulation efficiency, pH value, and related substances compared to the zero point, and all met the proposed quality standards. This suggests that the pilot-scale process is stable and can be used for further scale-up production.

[0228] Example 5: Pharmacokinetic Study of Cabazitaxel

[0229] Preparation of the drug formulation: Take one bottle of test sample (according to the specification), shake the bottle appropriately to disperse it into powder, then use a sterile syringe to draw 20 mL of 5% glucose solution. The glucose solution will be automatically injected into the bottle along the bottle wall by negative pressure. Shake the bottle gently to disperse and dissolve it completely, thus obtaining a drug formulation with a concentration of 3 mg / mL.

[0230] This experiment used six beagle dogs, half male and half female. Each beagle dog received a single intravenous infusion (15 minutes) of 0.5 mg / kg of cabazitaxel micelles prepared by spray drying and hydration as described in Example 4. Whole blood was collected from all animals before and at different time points after administration, and plasma was separated. The mean concentrations of free and total cabazitaxel in the plasma of the beagle dogs after a single intravenous infusion of cabazitaxel micelles were determined using standard methods in the art.

[0231] Table 9 Summary of Mean Plasma Free and Total Cabazitaxel Drug Concentrations (ng / mL) in Beagle Dogs after Single Intravenous Infusion of Cabazitaxel Micelles Note: Mean represents the average value, SD represents the standard deviation, CV% represents the coefficient of variation, NA represents not applicable, and <LOQ represents below the lower limit of quantification.

[0232] After the cabazitaxel micelles enter the bloodstream, the drug remains encapsulated in the micelles during blood circulation. The drug-loaded micelles directly and rapidly enter the lesion tissue cells to exert their pharmacological effects.

[0233] Example 6 Pharmacodynamic Effects of Cabazitaxel Micelles

[0234] Preparation method of the positive control cabazitaxel solution: Take out the cabazitaxel injection (manufacturer: Sanofi-Aventis US LLC; formulation specification: 60 mg: 1.5 ml; the injection contains cabazitaxel, ethanol, polysorbate 80), and let it stand at room temperature for 5 minutes. Use a syringe to aspirate the cabazitaxel injection and inject it into another cabazitaxel injection vial. Dilute the cabazitaxel injection with the solvent自带 in the injection to 10 mg / mL. Invert and mix repeatedly for at least 45 seconds, and do not shake. Let the vial stand at room temperature for 5 minutes until completely dissolved. Then dilute it with 5% glucose solution to a cabazitaxel concentration of 1.5 mg / mL and 0.75 mg / mL in the solution, and use it within 4 hours at room temperature and normal light.

[0235] Preparation method of the test cabazitaxel micelles: Under sterile conditions, take the preparation sample obtained by freeze-drying in Example 1 and stored in a middle-borosilicate glass control injection vial as the test sample. Disperse and dissolve each vial of the test sample with a certain volume of 5% glucose solution. Shake the vial to completely disperse and dissolve the freeze-dried powder. There will be some foam generated due to the negative pressure state in the vial. Inject air to balance the air pressure, and the foam will subside. Let the prepared drug solution stand at room temperature for 30 min to completely dissolve the drug. Dilute it with 5% glucose solution to a cabazitaxel concentration of 3 mg / mL, 1.5 mg / mL, and 0.75 mg / mL in the solution.

[0236] When the tumor grows to 101.05 - 167.11 mm 3 36 NPG mice (NOD-Prkdcscid Il2rgnull mice; grade: SPF grade; source: Beijing Vitalstar Biotechnology Co., Ltd.) are randomly divided into 6 groups, with 6 mice in each group, namely the solvent control (5% glucose solution), positive control cabazitaxel solution (7.5, 15 mg / kg), and test cabazitaxel micelles (7.5, 15, 30 mg / kg). Administer the drug by intravenous injection once a week for 4 consecutive weeks.

[0237] In the solvent control group, the tumors gradually grew throughout the experiment, reaching an average tumor volume of 1749.33 ± 673.47 mm at the end of the experiment (day 29, abbreviated as D29, the same abbreviation will be used below). 3 The mean RTV (Relative Tumor Volume) was 13.19 ± 4.78; the mean tumor volume in the positive control groups (7.5 mg / kg and 15 mg / kg) was 612.97 ± 234.46 mm. 3 313.74±74.53mm 3 The mean RTVs were 4.74±1.79 and 2.29±0.39, respectively. The mean tumor volume and RTV of the 15 mg / kg positive control group were significantly lower than those of the solvent control group from day 19 to day 29 (p<0.05). The tumor volume and RTV of the 7.5 mg / kg positive control group were significantly lower than those of the solvent control group at certain time points (p<0.05). At other time points, the tumor volume and RTV of the 7.5 mg / kg and 15 mg / kg positive control groups were lower than those of the solvent control group, but the differences were not statistically significant (p>0.05). The mean tumor volumes of the 7.5, 15, and 30 mg / kg cabazitaxel micelle groups were 209.14±34.20 mm, respectively. 3 114.96±51.91mm 3 99.02±37.56mm 3 The mean RTVs were 1.57±0.24, 0.86±0.37, and 0.72±0.23, respectively. The mean tumor volume in the 7.5, 15, and 30 mg / kg carbazone micelle groups was significantly lower than that in the solvent control group on days 8, 15–29, and 12–29 (p≤0.05). The mean tumor volume and RTV in the 30 mg / kg group on days 26–29 were significantly lower than those in the 7.5 mg / kg carbazone micelle group (p≤0.05). At the 15 mg / kg dose, the mean tumor volume and RTV of the test sample on days 19–29 and 15–29 were significantly lower than those in the positive control group at the same dose (p≤0.05).

[0238] The relative tumor proliferation rates (T / C%) of the positive control groups (7.5, 15 mg / kg and carbamate micelles, 7.5, 15, 30 mg / kg) at the end of the experiment (D29) were 35.90%, 17.38%, 11.92%, 6.49%, and 5.49%, respectively. Although the T / C% value of the 7.5 mg / kg positive control group decreased to below 40%, there was no statistically significant difference in RTV compared to the solvent control group. The RTVs of the other groups decreased to below 40% in the later stages of the experiment. At euthanasia on D29, the IR values ​​of the positive control groups (7.5, 15 mg / kg and carbamate micelles, 7.5, 15, 30 mg / kg) were...TV The percentages (relative tumor volume inhibition rates) were 64.10%, 82.62%, 88.08%, 93.51%, and 94.51%, respectively.

[0239] At the end of the experiment, the tumor weight of the animals was measured after euthanasia. The average tumor weights of the solvent control, positive control carbazide solution (7.5, 15 mg / kg), and carbazide micelles (7.5, 15, 30 mg / kg) groups were 0.853±0.278 g, 0.256±0.107 g, 0.127±0.034 g, 0.086±0.020 g, 0.049±0.031 g, and 0.032±0.013 g, respectively. The tumor weight in both the positive control and test sample groups was significantly lower than that in the solvent control group (p≤0.05). The tumor weight in the 15 mg / kg test sample group was significantly lower than that in the positive control group at the same dose (p≤0.05), and the tumor weight in the 30 mg / kg test sample group was significantly lower than that in the 7.5 mg / kg test sample group (p≤0.05). There was no statistically significant difference between the 7.5 mg / kg test sample group and the positive control group at the same dose (p>0.05). Infrared retardation (IR) data for each group in the solvent control, positive control, and test sample groups were also presented. TW The percentages (relative tumor weight inhibition rates) were 69.95%, 85.09%, 89.94%, 94.22%, and 96.21%, respectively.

[0240] Compared with the positive control cabazitaxel solution 7.5 mg / kg, cabazitaxel micelles 7.5 mg / kg showed significant advantages and statistically significant differences in tumor volume (TV) and total tumor volume (RTV) from day 15 to day 29. Cabazitaxel micelles 7.5 mg / kg also showed significant advantages and statistically significant differences in tumor weight.

[0241] Compared with the positive control cabazitaxel solution (15 mg / kg), cabazitaxel micelles (15 mg / kg) showed significant advantages and statistically significant differences in tumor volume (TV) and renal volume (RTV) from day 15 to day 29. Cabazitaxel micelles (15 mg / kg) also showed significant advantages and statistically significant differences in tumor weight.

[0242] Compared with the positive control cabazitaxel solution 15 mg / kg, cabazitaxel micelles 7.5 mg / kg showed significant advantages and statistically significant differences in TV and RTV from D15 to D29.

[0243] The above results indicate that, at the same dosage, cabazitaxel micelles (test sample) at 7.5 mg / kg and 15 mg / kg showed significant efficacy advantages over the positive control cabazitaxel solution (positive control). Furthermore, cabazitaxel micelles at 7.5 mg / kg also showed a significant efficacy advantage over the positive control cabazitaxel solution at 15 mg / kg. The tumor growth trend graph for each group is shown in Figure 12, and the tumor weight inhibition rate graph is shown in Figure 13.

[0244] Example 7: Cabazitaxel micelles reduce drug distribution in the brain

[0245] Preparation method of test carbatastatin micelles: Take the preparation sample obtained by freeze-drying in Example 1 and stored in a borosilicate glass vial as the test sample. Take one vial of test sample (according to specifications), shake the vial appropriately to disperse it into powder, and then use a sterile syringe to draw 20 mL of 5% glucose solution. The glucose solution is automatically injected into the vial by negative pressure along the vial wall. Shake the vial gently to disperse and dissolve it completely, thus obtaining a drug preparation with a carbatastatin concentration of 3 mg / mL in the solution.

[0246] Preparation of JEVTANA reference solution: Take one bottle of JEVTANA, slowly add all the included solvent while it adheres to the bottle wall, then gently invert the bottle for at least 45 seconds. If no visible particles are observed, the stock solution with a concentration of 10 mg / mL is obtained. Take a sufficient amount of the reference stock solution into a sterile container and dilute it to the required concentration with 5% glucose injection.

[0247] Forty-eight male Balb / c nude tumor-bearing mice were divided into two groups (n=24 per group), with four sampling points in each group. Following a single intravenous injection of 30 mg / kg of the test cabazitaxel micelles or 30 mg / kg of the control JEVTANA solution, plasma and tissue samples were collected from the animals at 2 h, 8 h, 24 h, and 96 h to assess the tissue distribution characteristics of cabazitaxel in Balb / c nude tumor-bearing mice.

[0248] Test method:

[0249] Blood sample collection

[0250] Blood collection animals: All animals

[0251] Blood collection site: Blood was collected from the heart of the animal after euthanasia at the sampling site.

[0252] Blood collection time points: 2h, 8h, 24h, and 96h after drug administration (sample number is the animal number after grouping - plasma).

[0253] Blood collection time error: ±5% after drug administration.

[0254] Blood volume collected: total blood sample / per time point

[0255] Blood collection tubes: heparin sodium anticoagulation

[0256] Sample processing and preservation: After blood samples are collected in blood collection tubes, they are inverted 5-8 times to mix well, placed on wet ice and centrifuged. Within 1 hour, the plasma is separated by centrifuging at 4000 rpm for 5 minutes (4℃). The plasma is divided into two portions (first portion and backup portion) and placed in labeled EP tubes. The aliquoted samples are stored in an ultra-low temperature freezer at -70±10℃ within 30 minutes until transport and transfer.

[0257] tissue collection

[0258] Collect animals: All animals

[0259] Collection site: Tissue collected after euthanasia of the animal.

[0260] Blood collection time points: 2h, 8h, 24h, and 96h after drug administration (sample number is the animal number after grouping - tissue name).

[0261] Tissue categories: tumors, heart, liver, spleen, lungs, kidneys, brain, stomach, lymph nodes, duodenum, testes (reproductive glands)

[0262] Collection volume: The entire tissue is collected.

[0263] Blood collection tubes: ordinary sample collection tubes

[0264] Sample processing and preservation: After tumor collection, cardiac perfusion was performed using 0.9% sodium chloride injection on ice, and then the required tissue samples were collected. After collection, residual blood or other secretions were rinsed off, and the samples were blotted dry with filter paper. After weighing, the samples were stored in labeled EP tubes and temporarily placed in dry ice. After each time point sample collection, all samples were stored in an ultra-low temperature freezer at -70±10℃ until transport and transfer.

[0265] Pharmacokinetic Bioanalysis

[0266] The concentration of cabazitaxel in plasma and tissue samples from all animals was determined by LC-MS / MS.

[0267] Data processing and statistics

[0268] Pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.3.3. The pharmacokinetic parameters were then analyzed and summarized using Phoenix WinNonlin 8.3.3 software or Excel 2013.

[0269] In tumor-bearing mice, intravenous injection of 30 mg / kg of the tested carbamate micelles or JEVTANA resulted in peak drug concentrations in tissues and plasma at 2 hours. The highest drug concentrations were observed in lymph nodes, reaching peak concentrations of 8186.67 ng / g and 9380.00 ng / g, respectively; plasma peak concentrations were 2723.33±898.90 ng / mL and 3050.00±849.99 ng / mL, respectively. Peak drug concentrations in kidney tissue were 6211.67±983.06 ng / g and 6466.67±1328.78 ng / g, respectively. In tumor tissues, both drug concentrations peaked at 2 hours, at 6391.67 ng / g and 8433.33 ng / g, respectively. Drug distribution in brain tissue: The tested carbamate micelles for injection (group 1) reached a peak concentration of 303.92 ng / g at 2 h, showing a certain decrease over time. JEVTANA (group 2) reached a peak concentration of 2612.50 ng / g at 8 h. The peak drug concentration in the brain of group 1 was 11.6% of that of group 2. The average drug concentrations are summarized in Tables 10 and 11. The average drug-time curves of carbamate in each tissue and plasma are shown in Figures 14-25. The average drug concentration bar charts are shown in Figures 26-27 (tissue concentrations are in ng / g, and plasma concentrations are in ng / mL; in Figures 26 and 27, each group of data involves data at four time points, with the data bars from left to right representing the average drug concentrations at 2 h, 8 h, 24 h, and 96 h).

[0270] The “LN003 for injection” in Figures 14-25 and the “test carbamate micelles” described in Example 7 have the same meaning.

[0271] Table 10. Mean carbazide concentrations in tissues and plasma of tumor-bearing mice after injection of the tested carbazide micelles. Note: Tissue concentration is measured in ng / g, and plasma concentration is measured in ng / mL.

[0272] Table 11. Mean carbazide concentrations in tissues and plasma of tumor-bearing mice after injection of JEVTANA. Note: Tissue concentration is measured in ng / g, and plasma concentration is measured in ng / mL.

[0273] The above results indicate that the distribution of LN003 in tumor-bearing mice was as follows: tumor > lymph node > stomach > testis > kidney > spleen > duodenum > lung > liver > heart > plasma > brain; and the tissue distribution of the control drug JEVTANA solution was as follows: tumor > lymph node > brain > testis > stomach > kidney > spleen > duodenum > lung > liver > heart > plasma.

[0274] After administration of cabazitaxel micelles, the distribution of the drug in tissues such as the heart and liver was significantly less than that after administration of the control product JEVTANA solution. The distribution in the brain tissue was the least, with the peak drug concentration being only 11.6% of that of the control product JEVTANA solution, showing a very significant difference.

[0275] The experimental results show that the cabazitaxel micelles utilize a special polymer micelle technology, which significantly reduces the amount of cabazitaxel that crosses the blood-brain barrier, and is expected to reduce neurotoxicity.

[0276] Example 8: Cabazitaxel micelle allergy and hemolysis test

[0277] 8.1 Cabazitaxel micelles: hemolysis test

[0278] In this experiment, blood was collected from New Zealand rabbits and a 2% erythrocyte suspension was prepared. 0.9% sodium chloride injection (negative control), sterile water for injection (positive control), the test formulation (prepared from the freeze-dried formulation sample in Example 1, stored in a borosilicate glass vial, and formulated with 0.9% sodium chloride injection to contain 0.5 mg / mL and 1 mg / mL of cabazitaxel), and excipient formulation (PEG2000-DSPE) were added for treatment. After mixing, the solution was placed in a 37℃ biochemical incubator, and the solution properties were observed at 15, 30, 45, 60, 120, and 180 minutes after addition to determine whether hemolysis or erythrocyte aggregation occurred. After the direct observation method is completed, the solution in the test tube is transferred to a dry centrifuge tube and centrifuged at 1500 rpm at room temperature for 10 min. The supernatant is then taken and its absorbance is measured at a wavelength of 540 nm using a spectrophotometer (zeroed with sodium chloride injection solution), and its hemolysis rate is calculated.

[0279] Visual inspection revealed no hemolysis or aggregation in the negative control tube (sodium chloride injection), while only hemolysis was observed in the positive control tube (sterile water for injection). No hemolysis or aggregation was observed in any of the test formulations (0.5 mg / mL, 1 mg / mL) or excipient formulations (i.e., PEG2000-DSPE) sample tubes. Spectrophotometric analysis showed that the hemolysis rate in all test formulations (0.5 mg / mL, 1 mg / mL) and excipient formulation sample tubes was <5%, indicating no hemolysis.

[0280] In summary, under the conditions of this experiment, no hemolysis or agglutination was observed in 2% erythrocyte suspension from New Zealand rabbits when the concentration of injectable carbamate micelles was 0.5 mg / mL and 1 mg / mL.

[0281] 8.2 Cabazitaxel micelles: Active systemic anaphylaxis test in guinea pigs

[0282] This experiment used 32 guinea pigs, half male and half female, randomly divided into 4 groups, with 4 females and 4 males in each group. The negative control group was given 5% glucose injection; the positive control group was given ovalbumin (solvent: 5% glucose injection) at a sensitizing dose of 5 mg / kg; the low-dose and high-dose groups were given carbazide micelles (using the preparation sample obtained by freeze-drying in Example 1 and stored in a borosilicate glass vial as raw material, prepared with glucose injection to the required concentration for administration), at sensitizing doses of 0.15 mg / kg and 0.3 mg / kg, respectively. Sensitization was performed via intraperitoneal injection at a volume of 1.5 mL / kg, every other day for a total of 3 times. On day 14 and day 21 after the last sensitization, twice the sensitizing volume of the test sample or control was injected intravenously to challenge the guinea pigs and observe their allergic reactions. The results are as follows:

[0283] Clinical observation: Except for the high-dose group of animals which showed mild emaciation on day 9, no abnormalities were observed in the clinical observation of the other animals.

[0284] Body weight: Compared with the negative control group, the body weight of male animals in the high-dose group and female animals in the high-dose group at the first stimulation was statistically different (P≤0.05) (P≤0.01). No obvious abnormalities were observed in the other animals.

[0285] Post-challenge symptom observation: In the positive control group, allergic symptoms began to appear 12–49 seconds after challenge, and animals died 2–5 minutes later. All allergic reactions were extremely positive, with an incidence of 100%. No allergic reaction symptoms were observed in the negative control group, low-dose group, and high-dose group after challenge.

[0286] In conclusion, under the conditions of this experiment, when the sensitization doses of carbataxel micelles were 0.15 mg / kg and 0.3 mg / kg, the results of active systemic anaphylactic reactions in guinea pigs were negative.

[0287] 8.3 Cabazitaxel micelles: Passive cutaneous anaphylaxis test in guinea pigs

[0288] Preparation method of low-dose and high-dose carbamate micelles: The preparation sample obtained by freeze-drying in Example 1 and stored in a borosilicate glass vial was used as raw material and prepared with glucose injection to the concentration required for administration.

[0289] Forty guinea pigs (8 females and 8 males) were used in this experiment for sensitization serum preparation. They were divided into four groups, with half males and half females in each group: a negative control group (glucose injection), a positive control group (ovalbumin, sensitization dose 5 mg / kg), a low-dose group (cabazitaxel micelles, sensitization dose 0.15 mg / kg), and a high-dose group (cabazitaxel micelles, sensitization dose 0.3 mg / kg). Sensitization was performed via intraperitoneal injection at a dose of 1.5 mL / kg, administered every other day for a total of three times. On day 14 after the last sensitization, blood was collected from the abdominal aorta under anesthesia to prepare serum, which was stored at -20°C for later use. Twelve female and twelve male guinea pigs were used for intradermal sensitization and were also divided into four groups: a negative control group, a low-dose group, a high-dose group, and a positive control group, with half males and half females in each group. First, the corresponding sensitizing serum was administered according to the group at dilution ratios of 1:2, 1:4, and 1:8 (the amount of serum to be added was calculated based on the dosage). After 47 hours, the stimulating drug solution (a solution of equal volume of the test substance and 1% Evans blue solution mixed with the same sensitizing dose as the corresponding group) was administered intravenously. The volume of the drug was twice that of the intraperitoneal injection sensitization volume. The guinea pigs were euthanized with CO2 26–34 minutes after the stimulating drug was administered, and the size of the blue spot on the inner side of the guinea pig's back was measured.

[0290] Clinical observation and weight: Only animals in the high-dose group showed mild emaciation and weight loss; no abnormalities were observed in the clinical observation and weight of the other animals.

[0291] Challenge results: Measurements of the reaction spots after challenge showed that all animals in the positive control group exhibited distinct blue spots, with diameters ranging from 14.61 mm to 51.55 mm, indicating a positive result. No blue spots were observed at any serum dilution point in the negative control group, the low-dose group, and the high-dose group of the test sample, indicating a negative result.

[0292] In conclusion, under the conditions of this experiment, when the doses of carbazide micelles were 0.15 mg / kg and 0.3 mg / kg, the passive cutaneous anaphylaxis in guinea pigs was negative.

[0293] In the following examples, unless otherwise specified, "cabatta micelles prepared by spray drying" refers to the cabazitaxel micelles obtained by spray drying and hydration in Example 4; "cabatta micelles prepared by film-forming hydration" refers to the cabazitaxel micelles obtained by film-forming hydration in Example 1.

[0294] Example 9

[0295] (1) Resolution stability

[0296] After dialysis at 37°C for 4 hours, a large amount of flake-like drug was found to precipitate in the dialysis bag of the cabazitaxel micelle formulation prepared by the film-forming hydration method. However, no drug was detected in the dialysate, indicating that when CTX-TW80 was released, the local drug concentration was much higher than the solubility in the dialysate, leading to drug crystallization. This method is not suitable for examining the release of the CTX-TW80 formulation. The states of micelles prepared by the two methods after release at 37°C for 4 hours in the dialysis bag are shown in Figures 28 and 29. Figure 28 shows the state of micelles prepared by the film-forming hydration method after release at 37°C for 4 hours in the dialysis bag, and Figure 29 shows the state of micelles prepared by the spray drying method after release at 37°C for 4 hours in the dialysis bag.

[0297] Comparing the states of carbazone micelles prepared by the film-forming hydration method and the carbazone micelles prepared by the spray drying method in a dialysis bag after a 4-hour dissolution test at 37°C, it was found that the stability of the spray-dried micelles was much higher than that of the film-forming hydration micelles.

[0298] (2) Dilution stability

[0299] Cabazitaxel micelles prepared by spray drying and those prepared by film-forming hydration were diluted with 5% human serum albumin (5% HSA) and deionized water, respectively, to prepare micelle sample solutions with a concentration of 1 mg / ml. The state of the micelles after being placed at 37°C for 8 hours is shown in Figure 30.

[0300] Figure 30 shows the state of micelles diluted with different dilution media after being placed at 37°C for 8 hours. From left to right, the results are: micelle sample solution prepared by film-forming hydration method diluted with 5% human serum albumin, micelle sample solution prepared by film-forming hydration method diluted with deionized water, micelle sample solution prepared by spray drying method diluted with 5% human serum albumin, and micelle sample solution prepared by spray drying method diluted with deionized water.

[0301] The results show that the micelles prepared by spray drying exhibit better stability in both deionized water and 5% HSA. After 8 hours at 37°C, the micelle solution remained clear and transparent, indicating that the drug did not crystallize out of the micelles. In contrast, the micelle formulation prepared by film-forming hydration showed drug precipitation and cloudiness in deionized water after 6 hours at 37°C; and in 5% albumin solution, significant precipitation was observed in micelles prepared by film-forming hydration after 6 hours, while micelles prepared by spray drying remained clear and transparent. This indicates that the micelles prepared by spray drying have a more stable structure and stronger dilution resistance than those prepared by film-forming hydration.

[0302] Example 10

[0303] Comparison of the bioactivity of micelles prepared by spray drying and film-forming hydration methods.

[0304] (1) Pharmacodynamic comparison

[0305] Preparation methods of test carbamate micelles using two different methods:

[0306] Spray drying method: Under aseptic conditions, the preparation sample obtained by freeze-drying in Example 1 and stored in a borosilicate glass vial for injection was used as the test sample. Each vial of test sample was dispersed and dissolved with a certain volume of 5% glucose solution. The vial was shaken to completely disperse and dissolve the freeze-dried powder. Some foam will be generated under negative pressure in the vial; the foam will subside after air is injected to balance the pressure. The prepared drug solution was placed in a room temperature environment and allowed to stand for 30 minutes to allow the drug to completely dissolve. The carbataxel concentrations in the solution were diluted with 5% glucose solution to 7.5 mg / mL, 3.75 mg / mL, and 1.875 mg / mL.

[0307] Film-forming hydration method: The freeze-dried product obtained by the film-forming hydration method in Example 1 was prepared into solutions with carbatastatin concentrations of 7.5 mg / mL and 3.75 mg / mL using the above method.

[0308] When the tumor grows to an average size of approximately 158 mm 3 At approximately 3:00 PM, 60 male BALB / c nude mice with tumors of appropriate size were randomly divided into 6 groups of 10 mice each: solvent control G1 (5% glucose, 0 mg / kg), carbazirate micelles prepared by film-forming hydration method (7.5 mg / kg, G2; 3.75 mg / kg, G3), and carbazirate micelles prepared by spray drying method (7.5 mg / kg, G4; 3.75 mg / kg, G5; 1.875 mg / kg, G6). The mice were administered intravenously once a week for 3 weeks.

[0309] Compared with the solvent control, cabazitaxel micelles prepared by the film-forming hydration method significantly inhibited the growth of PC-3 subcutaneous ectopic prostate cancer xenografts, showing a significant reduction in TV (tumor volume) and RTV (relative tumor volume) starting from D4. At D21, the RTVs of the 7.5 mg / kg and 3.75 mg / kg dose groups prepared by the film-forming hydration method were 1.88 and 4.49, respectively, with relative tumor inhibition rates of 82.00% and 57.12%, and tumor weight inhibition rates of 73.60% and 51.69%, respectively.

[0310] Compared with the solvent control, spray-dried cabazitaxel micelles significantly inhibited the growth of PC-3 subcutaneous ectopic prostate cancer xenografts, showing a significant reduction in tumor volume (TV) and renal volume (RTV) starting from day 4, and the tumor-inhibiting effect was dose-dependent. The RTVs at day 21 for the spray-dried cabazitaxel micelles at doses of 7.5 mg / kg, 3.75 mg / kg, and 1.875 mg / kg were 1.53, 3.37, and 6.26, respectively, with relative tumor inhibition rates of 85.40%, 67.82%, and 40.25%, and tumor weight inhibition rates of 81.46%, 62.92%, and 29.21%, respectively.

[0311] The above experimental results show that, at the same dose, especially at 3.75 mg / kg, the cabazitaxel micelles prepared by spray drying method exhibited better tumor inhibitory effects than those prepared by film-forming hydration method, and the difference in tumor inhibitory effects was significant.

[0312] Relative tumor inhibition rate (TGI) (%) = (1 - mean RTV of treatment group (T) / mean RTV of negative control group (C)) × 100%.

[0313] The relative tumor volume (mean) of each group at each time point is shown in Table 12 below; the relative tumor inhibition rate (%) of each group at each time point is shown in Table 13 below.

[0314] Table 12

[0315] Table 13

[0316] (2) Pharmacokinetics in rats

[0317] Sixteen SD rats were randomly divided into two groups (n=8 per group, half male and half female). One group received a single intravenous injection of 3 mg / kg of spray-dried carbazitaxel micelles, while the other group received 3 mg / kg of film-forming hydration carbazitaxel micelles. The plasma drug concentration-time changes after a single intravenous administration of spray-dried and film-forming hydration carbazitaxel micelles to the SD rats are shown in Table 14.

[0318] Table 14

[0319] After a single dose of 3 mg / kg spray-dried carbazide micelles to SD rats, the maximum plasma concentration (C0) was achieved. max The concentration was 1058.7 ng / mL; the area under the curve (AUC) was 489.29 h*ng / mL; after a single intravenous injection of 3 mg / kg of carbazide micelles prepared by the film-forming hydration method, the C... maxThe concentration was 1568.52 ng / mL, and the AUC was 590.13 h*ng / mL. Compared with cabazitaxel prepared by spray drying at 3 mg / kg, the C of cabazitaxel micelles prepared by film-forming hydration method was 1568.52 ng / mL. max The AUC values ​​were 1.48 and 1.21 times, respectively, indicating that the exposure of carbataxel micelles prepared by spray drying (C0.05) was... max The exposure (C and AUC) of the spray-dried carbazone micelles was significantly lower than that of the carbazone micelles prepared by the film-forming hydration method (p<0.05). These results indicate that the exposure (C and AUC) of the spray-dried carbazone micelles was significantly lower than that of the carbazone micelles prepared by the film-forming hydration method. max The AUC of the spray-dried micelles was significantly lower than that of the carbamate micelles prepared by the film-forming hydration method (p<0.05). According to the pharmacodynamic comparison in (1), at the same dosage, the micelles prepared by the spray-drying method had better therapeutic effects. This indicates that even though the AUC of the micelles prepared by the spray-drying method is lower, they still have better therapeutic effects due to their better targeting, while maintaining the safety advantage brought by the lower AUC. Moreover, due to the lower AUC and better safety, the carbamate micelles prepared by the spray-drying method are expected to increase clinical efficacy by increasing the dosage while ensuring safety.

[0320] (3) Acute toxicity test

[0321] SD rats single intravenous injection recovery 21-day toxicity test

[0322] Thirty SD rats, half male and half female, were randomly divided into three groups: a solvent control group (5% glucose injection), a spray-dried 15 mg / kg group, and a film-forming hydration group (15 mg / kg). Five rats were administered per (sex group) (meaning each sex and each group; e.g., 5 rats per (sex group) represents 5 males and 5 females in each dosage group), with a dosage of 5 mL / kg. A single tail vein injection was administered, followed by a 21-day recovery period after drug withdrawal.

[0323] The cabazitaxel micelles prepared by spray drying at 15 mg / kg showed a decrease in white blood cell count (WBC), lymphocyte count (LYMPH), neutrophil count (NEUT), monocyte count (MONO), eosinophil count (EOS), large unstained cell count (LUC), platelet count (PLT), and reticulocyte count (RETIC) on day 3, while the mean platelet volume (MPV) increased. Specifically, the decrease in NEUT was -30.0% in male SD rats and -19.5% in female SD rats. The above-mentioned changes in indicators were also observed in the cabazitaxel micelle group prepared by the 15 mg / kg film-forming hydration method on day 3. The decrease in #NEUT in male and female SD rats was -49.2% and -41.4%, respectively, both higher than that of the same dose of spray-dried cabazitaxel micelles. Furthermore, a decrease in #NEUT was still observed in female SD rats on day 22 after administration of 15 mg / kg film-forming hydration method cabazitaxel micelles. At the same dose, the spray-dried cabazitaxel micelles showed better effects on neutropenia reduction and recovery than the film-forming hydration method, suggesting that spray-dried cabazitaxel micelles for injection may improve the effect on neutropenia and have better safety profiles in clinical use.

[0324] The maximum tolerated dose (MTD) of cabazitaxel micelles prepared by spray drying was 15 mg / kg. The main toxic effects were thinning hair, decreased body weight and average food intake, and changes in hematology and prostate. At the same dose, changes in body weight, average food intake, and hematology were also observed in the 15 mg / kg cabazitaxel micelle group prepared by film-forming hydration, with the degree being essentially the same as in the 15 mg / kg group prepared by spray drying. Furthermore, the cabazitaxel micelle group prepared by film-forming hydration also showed symptoms such as ruffled hair, perianal soiling / loose stools, scalp swelling, hair loss, and histopathological changes in the epididymis, nictitating membrane, bladder, and testes, while the cabazitaxel micelles prepared by spray drying did not exhibit these symptoms.

[0325] Therefore, at the same dosage, the safety of cabazitaxel micelles prepared by spray drying is superior to that prepared by film-forming hydration.

[0326] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

A micelle containing cabazitaxel, characterized in that, The micelles comprise cabazitaxel and polyethylene glycol derivatized phospholipid; The polyethylene glycol derivatized phospholipid includes a polyethylene glycol moiety and a phospholipid moiety, which phospholipid moiety is a di(C 12 -C 24 fatty acyl)phosphatidylethanolamine; The polydispersity index of the micelles is not higher than 0.

09. The cabazitaxel-containing micelle as claimed in claim 1, characterized in that, It meets one or more of the following conditions: (1) the cabazitaxel-containing micelle further comprises one or more of the following components: C 1-6 alcohol solvent, water, glucose injection, and sodium chloride injection; The C 1-6 The alcohol solvent is preferably ethanol; the water is preferably sterile water for injection; the glucose injection is preferably 5% glucose injection; and the sodium chloride injection is preferably 0.9% sodium chloride injection. (2) The phospholipid part of the polyethylene glycol derivatized phospholipid is one or more of distearoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl ethanolamine and dioleoyl phosphatidyl ethanolamine; preferably distearoyl phosphatidyl ethanolamine; (3) The polyethylene glycol derivatized phospholipid has a polyethylene glycol weight average molecular weight in the range of 1000-5000, for example a polyethylene glycol weight average molecular weight of 2000; (4) The polyethylene glycol derivatized phospholipid is polyethylene glycol 1000 distearoyl phosphatidyl ethanolamine, polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine, polyethylene glycol 5000 distearoyl phosphatidyl ethanolamine, polyethylene glycol 2000 dioleoyl phosphatidyl ethanolamine, polyethylene glycol 2000 dipalmitoyl phosphatidyl ethanolamine or polyethylene glycol 2000 dimyristoyl phosphatidyl ethanolamine; preferably polyethylene glycol 2000 distearoyl phosphatidyl ethanolamine; (5) The polydispersity index of the cabazitaxel-containing micelles is not higher than 0.08, for example 0.08, 0.07, 0.06 or 0.04; (6) The mass ratio of the polyethylene glycol derivatized phospholipid to the cabazitaxel in the cabazitaxel-containing micelles is not higher than 15:1, for example (0.5-1.5):1 or (5-15):1, for example 1:1, 10:1 or 11:1; Preferably, the mass ratio of the polyethylene glycol derivatized phospholipid to the cabazitaxel in the cabazitaxel-containing micelles is not higher than 15:1, for example (0.5-1.5):1 or (9-13):1, for example 1:1, 10:1 or 11:1; (7) The reconstitution time of the cabazitaxel-containing micelles is not higher than 60 minutes, preferably not higher than 45 minutes, more preferably not higher than 15 minutes, for example 10 minutes, 7 minutes or 5 minutes; (8) The encapsulation efficiency of the cabazitaxel-containing micelles is 95%-100%, preferably 98.5%-100%, for example 99.70%, 98.90% or 99.90%; (9) The total impurity content of the cabazitaxel-containing micelles is not higher than 2%, preferably not higher than 1%, more preferably not higher than 0.5%, for example 0.36% or 0.06%; (10) The total impurity content of the cabazitaxel-containing micelles is not higher than 0.1%, for example 0.06%; (11) The cabazitaxel-containing micelles are stable at room temperature for more than 6 hours; (12) The cabazitaxel-containing micelles are stable at 4°C for more than 24 hours; (13) The release degree of the cabazitaxel-containing micelles is 75%-100%, preferably 75%-85%, for example 78.3%; (14) The particle size of the cabazitaxel-containing micelles is 10-20 nm, for example 13 nm, 14 nm, 15 nm or 16 nm; (15) the Zeta potential of the Cabazitaxil-containing micelles is (-45mv)-(-60mv), preferably (-45mv)-(-50mv), for example -48.77mv; and (16) the Cabazitaxil-containing micelles comprise Cabazitaxil and polyethylene glycol 2000 distearoylphosphatidyl ethanolamine; the mass ratio of the Cabazitaxil to the polyethylene glycol 2000 distearoylphosphatidyl ethanolamine is preferably 1:11; the reconstitution time of the Cabazitaxil-containing micelles is preferably no more than 60 minutes; the entrapment efficiency of the Cabazitaxil-containing micelles is preferably 95%-100%; the content of total impurities in the Cabazitaxil-containing micelles is preferably no more than 2%; the release degree of the Cabazitaxil-containing micelles is preferably 75%-100%; the particle size of the Cabazitaxil-containing micelles is preferably 10-20nm; the Zeta potential of the Cabazitaxil-containing micelles is preferably (-45mv)-(-60mv); Preferably, the Cabazitaxil-containing micelles consist of Cabazitaxil and polyethylene glycol 2000 distearoylphosphatidyl ethanolamine. A process for the preparation of a cabazitaxel-containing micelle, characterized in that, It comprises the following step A: The Cabazitaxil and the polar organic solvent A solution of polyethylene glycol derivatized phospholipid are spray dried to obtain micelles A, which can be used directly; The polyethylene glycol derivatized phospholipid includes a polyethylene glycol moiety and a phospholipid moiety, which phospholipid moiety is a di(C 12 -C 24 fatty acyl)phosphatidylethanolamine; The polar organic solvent A is a polar organic solvent with a boiling point lower than 85℃ and a solubility of Cabazitaxil at room temperature of no less than 60mg / mL. The production method as claimed in claim 3, characterized in that It meets one or more of the following conditions: (1) the polar organic solvent A is a polar organic solvent with a solubility of the polyethylene glycol derivatized phospholipid at room temperature of no less than 60mg / mL; (2) the polar organic solvent A is acetone, dichloroethane, chloroform, tetrahydrofuran, carbon tetrachloride, ethyl acetate, butanone or an alcohol solvent; (3) the polar organic solvent A is an alcohol solvent; the alcohol solvent is preferably C 1-6 an alcohol solvent, more preferably C 1-3 an alcohol solvent, for example ethanol; (4) the phospholipid part in the polyethylene glycol derivatized phospholipid is one or more of distearoylphosphatidyl ethanolamine, dipalmitoylphosphatidyl ethanolamine, dimyristoylphosphatidyl ethanolamine and dioleoylphosphatidyl ethanolamine; preferably distearoylphosphatidyl ethanolamine; (5) the polyethylene glycol derivatized phospholipid has a weight average molecular weight range of 1000-5000, for example a weight average molecular weight of 2000; (6) the polyethylene glycol derivatized phospholipid is polyethylene glycol 1000 distearoylphosphatidyl ethanolamine, polyethylene glycol 2000 distearoylphosphatidyl ethanolamine, polyethylene glycol 5000 distearoylphosphatidyl ethanolamine, polyethylene glycol 2000 dioleoylphosphatidyl ethanolamine, polyethylene glycol 2000 dipalmitoylphosphatidyl ethanolamine or polyethylene glycol 2000 dimyristoylphosphatidyl ethanolamine; preferably polyethylene glycol 2000 distearoylphosphatidyl ethanolamine; (7) in step A, the mass ratio of the polyethylene glycol derivatized phospholipid to the Cabazitaxil is (0.5-1.5):1, for example 1:1; (8) in the polar organic solvent A, the concentration of the Cabazitaxil is 3-80mg / mL, preferably 6-60mg / mL; further preferably 30-60mg / mL, for example 60mg / mL; (9) the temperature of the polar organic solvent A solution is 20-60°C, preferably 40°C; (10) in the spray drying, the inlet air temperature is 45-65°C, preferably 50-60°C, for example 55°C; (11) in the spray drying, the outlet air temperature is 30-45°C, preferably 35-45°C, for example 40°C; (12) in the spray drying, the pump speed is 5%-15%, preferably 5-10%, for example 8%; (13) in the spray drying, the air speed is 40%-80%, preferably 45%-60%, for example 50%; (14) in the spray drying, the nozzle cleaning frequency is 3-5 times / min, for example 4 times / min. The production method as claimed in claim 3, characterized in that It further comprises the following step B: dissolving the micelle A and the polyethylene glycol derivatized phospholipid in water to obtain micelle B; the water is preferably sterile water for injection; the ratio of the mass of cabazitaxel contained in the micelle A to the volume of the water is 2-10 mg / mL, preferably 4-8 mg / mL, for example 6 mg / mL; Preferably, in step B, the concentration of the polyethylene glycol derivatized phospholipid in the water is 40-80 mg / mL, for example 60 mg / mL or 66 mg / mL; Preferably, in step B, the mass ratio of the polyethylene glycol derivatized phospholipid to the cabazitaxel-containing micelle A is (5-15):1, preferably (8-15):1, more preferably (9-13):1, for example 10:1 or 11:1; Preferably, step B is carried out at 15-30°C, for example 25°C. The method for preparing the cabazitaxel-containing micelle further comprises the following step C: The production method as claimed in claim 5, characterized in that filtering the micelle B to obtain micelle C; Preferably, step C meets one or more of the following conditions: (1) step C is carried out by using a high molecular filter membrane; the high molecular filter membrane is preferably a polyvinyl fluoride filter membrane, a mixed cellulose filter membrane, a polyether sulfone filter membrane, a nylon filter membrane, a polytetrafluoroethylene filter membrane or a polypropylene filter membrane; the polyvinyl fluoride filter membrane is preferably a polyvinylidene fluoride membrane; the pore size of the polyvinyl fluoride filter membrane is preferably 0.10-0.30 mm, for example 0.22 mm; (2) step C is carried out at 15-30°C, for example 25°C. The method for preparing the cabazitaxel-containing micelle further comprises the following step D: The production method as claimed in claim 6, characterized in that removing the water in the micelle C to obtain micelle D; Preferably, step D removes the water in the micelle C by using freeze drying, spray drying or fluidized drying; Further preferably, the freeze drying is carried out in the presence of a freeze drying protective agent, or is carried out without using a freeze drying protective agent; the freeze drying protective agent is preferably sucrose or lactose; the amount of the freeze drying protective agent is preferably 2%-6%, for example 4%; Further preferably, the freeze drying is carried out by using a conventional method in the art; the freeze drying preferably comprises a freezing stage, a primary drying stage and a secondary drying stage. The method for preparing the cabazitaxel-containing micelle further comprises the following step E: The production method as claimed in claim 7, characterized in that dissolving the micelle D in solvent a to obtain micelle E; Preferably, step E meets one or more of the following conditions: ​ (1) the solvent a is preferably one or more of water, glucose injection and sodium chloride injection; the water is preferably sterile water for injection; the glucose injection is preferably 5% glucose injection; the sodium chloride injection is preferably 0.9% sodium chloride injection; (2) the step E is performed at 15-30℃, for example 25℃. The production method as claimed in claim 3, characterized in that The preparation method of the cabazitaxel-containing micelles comprises the following steps: Step A: spray-drying a solution of cabazitaxel and PEG2000-DSPE in a polar organic solvent A to obtain micelles A; Step B: dissolving the micelles A and PEG2000-DSPE in water to obtain micelles B; Step C: filtering the micelles B to obtain micelles C; Step D: removing water in the micelles C to obtain micelles D; Step E: dissolving the micelles D in a solvent a to obtain micelles E; wherein the conditions, operations or materials in steps A, B, C, D and E are as described in any one of claims 3-8; Preferably, the preparation method of the cabazitaxel-containing micelles consists of steps A, B, C, D and E; Further preferably, the preparation method of the cabazitaxel-containing micelles is performed by the following steps: weighing cabazitaxel and PEG2000-DSPE, dissolving them in a polar organic solvent A to obtain a corresponding solution, spray-drying to obtain micelles A; dissolving the micelles A and PEG2000-DSPE in water to obtain micelles B; filtering the micelles B to obtain micelles C; removing water in the micelles C by freeze-drying to obtain micelles D; dissolving the micelles D in a solvent a to obtain micelles E; the polar organic solvent A is preferably ethanol; the solvent a is preferably water. A cabazitaxel-containing micelle, which is micelles A, micelles B, micelles C, micelles D or micelles E; The micelles A are obtained by step A; The micelles B are obtained by steps A and B; The micelles C are obtained by steps A, B and C; The micelles D are obtained by steps A, B, C and D; The micelles E are obtained by steps A, B, C, D and E; wherein The conditions, operations or materials in steps A, B, C, D and E are as described in any one of claims 3-8. The cabazitaxel-containing micelle as claimed in claim 10, characterized in that, It satisfies one or more of the following conditions: (1) the reconstitution time of the micelles D is not higher than 60 minutes, preferably not higher than 45 minutes, more preferably not higher than 15 minutes, for example 10 minutes, 7 minutes or 5 minutes; (2) the micelles E are a clear and transparent solution; (3) the polydispersity coefficient of the micelles E is not higher than 0.09, preferably not higher than 0.08, for example 0.08, 0.07, 0.06 or 0.04; (4) the encapsulation efficiency of the micelles E is 95%-100%, preferably 98.5%-100%, for example 99.70%, 98.90% or 99.90%; (5) the content of total impurities in the micelles E is not higher than 2%, preferably not higher than 1%, more preferably not higher than 0.5%, for example 0.36% or 0.06%; (6) the content of total impurities in the micelles E is not higher than 0.1%; (7) the micelle E is stable for more than 6 hours at room temperature; (8) the micelle E is stable for more than 24 hours at 4°C; (9) the release of the micelle E is between 75% and 100%, preferably between 75% and 85%, for example 78.3%; (10) the particle size of the micelle E is between 10 and 20 nm, for example 13 nm, 14 nm, 15 nm or 16 nm; (11) the Zeta potential of the micelle E or micelle C is between (-45 mv) and (-60 mv), preferably between (-45 mv) and (-50 mv), for example -48.77 mv. A pharmaceutical composition comprising a cabazitaxel-containing micelle, micelle A, micelle B, micelle C, micelle D or micelle E as described in claim 1, 2, 10 or 11, and a pharmaceutically acceptable excipient. Use of a cabazitaxel-containing micelle, micelle A, micelle B, micelle C, micelle D or micelle E as described in claim 1, 2, 10 or 11, or of a pharmaceutical composition as described in claim 12, for the manufacture of a medicament for the treatment of cancer. Preferably, the cancer is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, small cell lung cancer, gastric cancer, melanoma and soft tissue sarcoma.

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