Stable docetaxel albumin nanoparticle compositions

Abstract

The present invention provides a composition containing docetaxel albumin nanoparticles, the composition comprising docetaxel and acid-modified albumin, the acid-modified albumin being obtained by modifying human serum albumin by adding acid to adjust the pH value. The composition can be prepared into an injection solution or a lyophilized powder injection. The present invention provides a composition having sufficient physical and chemical stability, preferably under acid-modified conditions, by controlling the content of sodium octanoate in human serum albumin.

Description

[Technical field]

[0001] The present invention is in the field of drug formulations, and specifically relates to compositions including docetaxel albumin nanoparticles. [Background technology]

[0002] Docetaxel is produced by semi-synthesis of a non-cytotoxic precursor (10-deacetylbaccatin III) extracted from yew needles. It is a paclitaxel analogue that acts on cellular microtubules and can promote tubulin polymerization while also promoting microtubule depolymerization, thereby arresting cells at the G2 / M phase, inhibiting mitosis, and killing tumor cells, and has stronger antitumor activity than paclitaxel.

[0003] Docetaxel has a lower water solubility and is currently marketed by Sanofi as an injectable solution under the trade name Taxotere®, usually at 75 µm 2 It is supposed to be administered by intravenous drip infusion for 1 hour once every 3 weeks. However, docetaxel injections, such as Taxotere (registered trademark), contain ethanol and Tween-80 in the formulation, and therefore have the following disadvantages: (1) It causes severe allergic reactions, requires pretreatment with dexamethasone, and has poor patient compliance. Tween-80 causes severe allergic reactions and fluid retention, and requires anti-allergy medication to be taken in advance. For example, to prevent allergic reactions and fluid retention, 16 mg of dexamethasone should be taken daily, starting the day before injection, for at least three days. There have been reports of severe allergic reactions even when dexamethasone was taken in advance. The instruction manual clearly states that "severe fluid retention was reported in 6.5% (6 / 92) of patients who received prophylactic dexamethasone for three days."

[0004] (2) Ethanol affects the central nervous system, and the infusion rate must be reduced to alleviate the symptoms of ethanol poisoning. In 2014, the FDA published a Drug Safety Information on Intravenous Administration of the Alcohol-Containing Chemotherapy Docetaxel and the Risk of Poisoning, concluding that ethanol in docetaxel injections may affect the central nervous system and that reducing the infusion rate may alleviate the symptoms of ethanol poisoning, and called for the development of an ethanol-free docetaxel formulation.

[0005] (3) Tween-80 causes allergic reactions, can only be administered at low concentrations, and has poor patient compliance. Tween-80 causes allergic reactions and can also cause hemolysis if administered at high concentrations. Therefore, it can only be diluted to low concentrations for administration, which lengthens the infusion time and results in poor patient compliance.

[0006] (4) The product has low compatibility and requires an injection device that does not contain PVC material, which is inconvenient for clinical use. The instruction manual states, "Docetaxel pre-injection solution (10 mg / mL) should be used immediately after preparation. However, its physical and chemical properties indicate that the pre-injection solution is stable for 8 hours when stored at 2-8°C or at room temperature. The injection solution (0.74 mg / mL or less) should be used within 4 hours at room temperature and infused intravenously for 1 hour," which is inconvenient for clinical use. The instruction manual also states that the use of PVC injection devices is not recommended, and that contact with PVC injection devices will cause bis-(2-ethylhexyl) phthalate in the PVC injection device to leach out, posing a safety risk.

[0007] Development of docetaxel-loaded intravenous formulation, Nanoxel-PM, has been developed to improve the many disadvantages of conventional formulations. TMUsing polymer-based delivery systems (Sa-Won Lee, Min-Hyuk Yun, Seung Wei Jeong, Journal of Controlled Release, 155 (2011) 262-271) describes Nanoxel-PM, developed by Sanyo Corporation in South Korea. TM (Already on the market in Korea, product name: Nanoxel (registered trademark) M) has been released, and the product selects polyethylene glycol-polylactide (mPEG-PDLLA) polymer as a carrier to prepare docetaxel micelles, with an average particle size of 10-50 nm and a uniform particle size distribution. Its official website has published the method of clinical use, and pretreatment is required to minimize allergic reactions, for example, 16 mg of dexamethasone is orally taken every day from the day before administration for three consecutive days (https: / / www.samyangbiopharm.com / eng / ProductIntroduce / injection03). Ethanol and Tween-80 were not used in the formulation of the product, but pretreatment is also required in clinical practice.

[0008] Human serum albumin (HSA) is a protein in human plasma, which contains 585 amino acids and has a molecular weight of 66 kD. In plasma, its concentration is 42 g / L, accounting for about 60% of the total protein in plasma. In body fluids, human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules, while maintaining normal blood pressure, osmolality, and physiological pH value.

[0009] Proteins are polymeric compounds composed of multiple amino acids, among which the amino acids are linked by peptide bonds and disulfides to form peptide chains with a certain order, which is called the primary structure; hydrogen bonds can be formed between amino groups and acyl groups in the same or different polypeptide chains, giving the main chain of the polypeptide chain a certain regular conformation, including α-helix, β-sheet, β-turn, Ω-ring, etc., which are called the secondary structure of the protein; peptide chains can further coil and fold based on the secondary structure to form a complete spatial structure, which is called the tertiary structure; and a spatial structure formed by the aggregation of multiple peptide chains through non-covalent bonds is called a quaternary structure, among which one of the peptide chains is called a subunit.

[0010] Protein denaturation refers to the change in the internal structure and properties of a protein molecule due to the influence of physical or chemical factors. In general, the change or destruction of the secondary and tertiary structures of a protein is considered to be the result of denaturation. Protein denaturation methods are mainly divided into chemical and physical methods, among which chemical methods include the addition of strong acids, strong bases, heavy metal salts, urea, acetone, etc., and physical methods include heating, ultraviolet or X-ray irradiation, ultrasonic waves, vigorous vibration or stirring, etc. Michael Dockal, Daniel C. Carter, Florian Ruker. Conformational Transitions of the Three Recombinant Domains of Human Serum Albumin Depending on pH. (J Biol Chem, 2000, 275(5):3042-3050) reports that human serum albumin undergoes conformational changes (e.g., NF and FE conversion under acidic conditions, and NB conversion under alkaline conditions) under different pH conditions, resulting in protein denaturation. In the present invention, albumin denatured by adding acid is defined as "acid-denatured albumin", and the albumin solution pH in this case is less than 5.5.

[0011] Regardless of whether it is imported or domestically produced, the process flow of human serum albumin is basically the same, which is made by purifying healthy human plasma by low-temperature ethanol protein separation method and then heating at 60°C for 10 hours to inactivate viruses. Commercial albumin usually contains sodium octanoate as a heat protectant, for example, 0.16 mmol of sodium octanoate per gram of albumin.

[0012] Human serum albumin is an endogenous human substance, has good biocompatibility, and can act as a natural carrier for hydrophobic drugs and increase the solubility of insoluble drugs. Abraxis, Inc., USA, developed injectable paclitaxel (albumin-bound) (trade name Abraxane®) using human serum albumin as an auxiliary substance and emulsification method, which was approved by the FDA in 2005 for use in the treatment of metastatic breast cancer that has failed combination chemotherapy or breast cancer that has recurred within 6 months after adjuvant chemotherapy, and was subsequently approved as a treatment for non-small cell lung cancer, pancreatic cancer, and gastric cancer (Japan).

[0013] Compared with paclitaxel injection (Taxol), Abraxane® does not contain polyoxyethylene castor oil, a solvent that can cause severe allergic reactions, so there is no need for pre-administration of anti-allergic drugs, and it can be administered quickly at a high concentration, shortening the injection time to within 30 minutes, significantly improving patient compliance. To improve safety, the dosage of paclitaxel is 175 mg / m 2 260-300 mg / m 2 The polyoxyethylene castor oil in Taxol inhibits the binding of paclitaxel to albumin, and the formulation does not contain polyoxyethylene castor oil, so that the unique "gp60-caveolin-SPARC" channel of albumin can be more fully utilized, thereby realizing the concentration of drugs at tumor sites and increasing the therapeutic effect. Currently, researchers around the world are trying to use human serum albumin to develop albumin nanoparticles of other drugs, such as docetaxel. Among them, Neil P. Desai et al. in patent US2005 / 0004002A1 disclose an emulsification method for preparing docetaxel albumin nanoparticles, the formulation of which only contains docetaxel and human serum albumin, with a particle size of 50-220 nm, and the particle size basically remains unchanged before and after freeze-drying. However, the suspension has a short stability time, both before and after freeze-drying, which cannot meet clinical needs. In Chinese Patent CN103054798A, in order to improve the stability of the suspension, soybean oil, phospholipids, cholesterol, benzoic acid, etc. were initially added to the formula as stabilizers, but all of them failed. After a large amount of testing and searching, it was finally confirmed that the stability of the suspension can be greatly improved by adding sodium citrate or a composition of sodium citrate and sodium chloride. However, due to the use of a large amount of salt, the reconstituted suspension has a high osmotic pressure and is highly irritating, which causes obvious pain when injected, and also leads to osmotic damage of the injected local tissue cells, making it inconvenient for clinical use. The invention also clearly states that the stability of the docetaxel anhydrous form is higher than that of the nanoparticle suspension prepared in the bound water form (e.g., docetaxel trihydrate or hemihydrate). Example 28 describes that when no stabilizer is included and the active ingredient is anhydrous docetaxel (i.e., docetaxel), the nanoparticle filtrate settles on the first day of standing at 4°C, and the precipitation of the hemihydrate and trihydrate occurs within a shorter time; when a stabilizer is included and the active ingredient is anhydrous docetaxel (i.e., docetaxel), the nanoparticle filtrate does not settle for two days at 4°C, and the precipitation of the hemihydrate and trihydrate occurs within a shorter time. Therefore, the patent is limited to preparing albumin nanoparticles with anhydrous docetaxel, which greatly limits the selection range of raw materials for docetaxel. The patent further discusses the pH of the formulation and finds that "an increase in pH above 6 improved the physical stability of the formulation as measured by nanoparticle size and sedimentation, while simultaneously increasing the amount of docetaxel that degraded to 7-epi-docetaxel at room temperature. Thus, both physical and chemical stability are acceptable in the pH range between 6 and 8.5, with a more preferred pH range of 6.5 to 8 and a most preferred range of pH 7.25 to 7.75." In CN103054798A, it is believed that adding sodium citrate or a composition of sodium citrate and sodium chloride is advantageous to improve the physical stability of docetaxel albumin nanoparticle preparations, but in Chinese patent CN106137969A, it is explained that adding tartaric acid, citric acid, ascorbic acid, and other organic acids with pKa of 2.5 to 4.5 to docetaxel albumin compositions cannot effectively inhibit the increase of 7-epi-docetaxel, and even increases 7-epi-docetaxel, which reduces the chemical stability of the composition and affects the safety of the preparation. However, by selectively adding amino acid substances, docetaxel albumin can be stabilized for a long time and significantly inhibit the formation of 7-epi-docetaxel. In particular, when the amino acid is arginine, the content of 7-epi-docetaxel is only 0.72% after 30 months. After redissolution of the prepared product, the suspension can be stable at room temperature for more than 8 hours. This patent is limited to the pharmaceutical composition of said docetaxel albumin nanoparticles, in which the mass ratio of amino acid to docetaxel is 0.5 or more, preferably 1 or more. WO2018 / 059304A1 also discloses a method for improving the product quality of an albumin drug composition, in which the drug composition comprises albumin and an amino acid or its salt having a relative molecular weight of at least 145-175, the amino acid being one or more selected from arginine, histidine and lysine, preferably arginine and / or histidine, more preferably arginine, the weight ratio of the amino acid or its salt to albumin is 0.1:1-10:1, and the amino acid or its salt inhibits the formation or increase in the content of albumin dimer during preparation, storage and use of the drug composition. The albumin drug composition can effectively reduce undesirable reactions caused by albumin multimers and dimers in the human body in clinical use, such as rash, urticaria, allergic reactions and possible immune reactions, further ensuring the safety of clinical drugs. In summary, to improve the product mass of docetaxel albumin, its physical stability (stability of suspension) and chemical stability (decomposition of docetaxel and increase of albumin polymer) must be considered at the same time. One type of docetaxel albumin composition disclosed in the prior art adds a large amount of chelating agent as a stabilizer to improve the physical stability of the product, but does not consider its effect on chemical stability. In addition, a large amount of chelating agent improves stability and at the same time forms a hypertonic solution, which is inconvenient for clinical use. Another type adds a large amount of amino acid to the product, which can inhibit the decomposition of docetaxel and the increase of albumin polymer, but there is no further research into the effect of physical stability, and it is only stated that after reconstitution, the suspension is stable at room temperature for more than 8 hours. Therefore, finding a method that can simultaneously ensure the physical and chemical stability of docetaxel albumin compositions is an urgent issue in the field. Summary of the Invention

[0014] The present invention provides a composition including docetaxel albumin nanoparticles, the composition comprising docetaxel and acid-modified albumin.

[0015] Alternatively, the present invention provides a composition comprising docetaxel albumin nanoparticles, the composition being prepared by docetaxel and acid-modified albumin.

[0016] Alternatively, the present invention provides a composition comprising docetaxel albumin nanoparticles, the composition being made of docetaxel and acid-modified albumin, and optionally comprising an osmolarity adjusting agent or a pH adjusting agent.

[0017] Among them, the docetaxel is preferably anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate.

[0018] The acid-modified albumin is obtained by denaturing human serum albumin after adding an acid to an appropriate pH value, preferably (1) The acid is selected from an acidic amino acid or an acidic peptide, an organic acid, and an inorganic acid. The acidic amino acid or the acidic peptide includes, but is not limited to, cysteine ​​hydrochloride, glutathione, etc., the organic acid includes, but is not limited to, citric acid, tartaric acid, etc., and the inorganic acid includes, but is not limited to, hydrochloric acid, sulfuric acid, etc. The acid is preferably cysteine ​​hydrochloride, glutathione, hydrochloric acid, more preferably cysteine ​​hydrochloride, and / or (2) the appropriate pH value is preferably 3.5 to 5.5, preferably 3.5 to 5.0, more preferably 3.8 to 4.7, and most preferably 4.0 to 4.5; and / or (3) based on anhydrous docetaxel, the mass ratio of docetaxel to human serum albumin is 1:(2.0-10.0), preferably 1:(3.0-7.0), more preferably 1:(4.0-6.0), and / or (4) The content of sodium octanoate in the human serum albumin is 0.08 mmol / g protein or less, preferably 0.03 to 0.08 mmol / g protein, more preferably 0.04 to 0.08 mmol / g protein, and most preferably 0.04 to 0.07 mmol / g protein.

[0019] In some embodiments, the docetaxel albumin nanoparticles have a particle size of about 60 to 200 nm, preferably 90 to 150 nm, and more preferably 90 to 135 nm.

[0020] The composition containing the docetaxel albumin nanoparticles according to the present invention optionally contains an osmotic pressure adjusting agent to adjust the osmotic pressure within a suitable range. The type of the osmotic pressure adjusting agent is not particularly limited, and may be selected from, for example, sodium chloride, glucose, phosphate or citrate, and is preferably sodium chloride. The type and dosage of the osmotic pressure adjusting agent can be determined by those skilled in the art according to the specific circumstances, such as the type and dosage of the diluent or reconstitution medium used in clinical practice. In some embodiments, the osmotic pressure adjusting agent is sodium chloride, and the weight ratio of sodium chloride to docetaxel is (0.75-9):1, preferably (1-7):1, preferably (1.5-4.5):1, and most preferably 2.25:1.

[0021] Optionally, the composition of the present invention includes a pH adjuster to adjust the pH value within a suitable range, and the suspension used in the preparation of the composition of the present invention and / or the reconstituted suspension used in the composition of the present invention are stable for at least 24 hours, preferably at least 30 hours, at 25°C, and / or stable for at least 7 days, preferably at least 10 days, at 2-8°C. The term "stable" as used herein means that no precipitation of nanoparticles or turbidity of the suspension occurred. The type of the pH adjuster is not particularly limited. Preferably, the pH value range is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8.

[0022] In some embodiments, the composition of the present invention is a suspension. The pH value of the suspension is 3.4 to 5.8, preferably 3.6 to 5.6, or 3.8 to 5.0, more preferably 3.9 to 4.8. The particle size of the docetaxel albumin nanoparticles in the suspension is about 60 to 200 nm, preferably 90 to 150 nm, more preferably 90 to 135 nm. The suspension contains 0 to 1.8% (w / v) sodium chloride, preferably 0.45% to 1.8% (w / v), more preferably 0.9% to 1.8% (w / v). In some embodiments, the suspension contains 2 to 10 mg / mL docetaxel, preferably 2 to 8 mg / mL. The suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours, and at 2 to 8°C for at least 7 days, preferably at least 10 days.

[0023] In another embodiment, the composition of the present invention is a lyophilized powder. The particle size of the docetaxel albumin nanoparticles is about 60-200 nm, preferably 90-150 nm, and more preferably 90-135 nm. In some embodiments, the lyophilized powder contains sodium chloride, and the weight ratio of sodium chloride:docetaxel is (0.75-9):1, preferably (1-7):1, preferably (1.5-4.5):1, and most preferably 2.25:1.

[0024] The freeze-dried powder is produced by freeze-drying a suspension containing docetaxel albumin nanoparticles. The pH value of the suspension is 3.4 to 5.8, preferably 3.6 to 5.6, or 3.8 to 5.0, more preferably 3.9 to 4.8. The particle size of the docetaxel albumin nanoparticles in the suspension is about 60 to 200 nm, preferably 90 to 150 nm, more preferably 90 to 135 nm. The suspension contains 0 to 1.8% (w / v) sodium chloride, preferably 0.45% to 1.8% (w / v), more preferably 0.9% to 1.8% (w / v). The suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours, and at 2 to 8°C for at least 7 days, preferably at least 10 days.

[0025] The freeze-dried powder is reconstituted into a suspension using a reconstitution medium. The reconstitution medium is selected from water for injection, sodium chloride solution, or glucose solution, and is preferably water for injection. The pH value of the reconstituted suspension is 3.4 to 5.8, preferably 3.6 to 5.6, or 3.8 to 5.0, and more preferably 3.9 to 4.8. The particle size of the docetaxel albumin nanoparticles in the reconstituted suspension is about 60 to 200 nm, preferably 90 to 150 nm, and more preferably 90 to 135 nm. The reconstituted suspension contains 0 to 1.8% (w / v) sodium chloride, preferably 0.45% to 1.8% (w / v), and more preferably 0.9% to 1.8% (w / v). The reconstituted isotonic suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours, and at 2 to 8°C for at least 7 days, and preferably at least 10 days.

[0026] In some embodiments, the composition comprising the docetaxel albumin nanoparticles according to the present invention is stable for at least 36 months at 25°C after lyophilization. The term "stable" as used herein includes, but is not limited to, no obvious decomposition of docetaxel, no obvious aggregation of protein nanoparticles, no obvious increase in particle size, no obvious change in docetaxel content, moisture, acidity, osmotic pressure or molarity, etc. The term "stable" may refer to one or more of the above conditions. In some embodiments, the term "stable" refers to no obvious change in the content of 7-epi-docetaxel and / or albumin polymer.

[0027] In another aspect, the present invention further provides a drug, which is prepared by the above composition comprising docetaxel albumin nanoparticles, said drug being in a clinically acceptable dosage form, preferably an injection, more preferably a liquid injection or a lyophilized powder injection.

[0028] When the injectable agent is a liquid injectable agent, the pH value of the liquid injectable agent is 3.4 to 5.8, preferably 3.6 to 5.6, or 3.8 to 5.0, more preferably 3.9 to 4.8. The particle size of the docetaxel albumin nanoparticles in the liquid injectable agent is about 60 to 200 nm, preferably 90 to 150 nm, more preferably 90 to 135 nm, and the liquid injectable agent contains 0 to 1.8% (w / v) sodium chloride, preferably 0.45% to 1.8% (w / v), more preferably 0.9% to 1.8% (w / v), more preferably 0.9% (w / v). In some embodiments, the liquid injectable agent contains 2 to 10 mg / mL docetaxel, preferably 2 to 8 mg / mL. The liquid injectable preparation is stable for at least 24 hours, preferably at least 30 hours, at 25° C., and for at least 7 days, preferably at least 10 days, at 2-8° C. In this context, "stable" means that no precipitation of nanoparticles or turbidity of the liquid injectable preparation occurs.

[0029] When the injection is a freeze-dried powder injection, the freeze-dried powder contains sodium chloride, and the weight ratio of sodium chloride:docetaxel is (0.75-9):1, preferably (1-7):1, preferably (1.5-4.5):1, and most preferably 2.25:1. The particle size of the docetaxel albumin nanoparticles is about 60-200 nm, preferably 90-150 nm, and more preferably 90-135 nm. The pH value before freeze-drying of the suspension for preparing the freeze-dried powder injection is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, and more preferably 3.9-4.8. The suspension is stable at 25°C for at least 24 hours and at 2-8°C for at least 10 days.

[0030] The freeze-dried powder injection is reconstituted using a reconstitution medium. The reconstitution medium is selected from water for injection, sodium chloride solution, or glucose solution, and is preferably water for injection. The pH value of the obtained reconstituted suspension is 3.4 to 5.8, preferably 3.6 to 5.6, or 3.8 to 5.0, and more preferably 3.9 to 4.8. The particle size of the docetaxel albumin nanoparticles in the reconstituted suspension is about 60 to 200 nm, preferably 90 to 150 nm, and more preferably 90 to 135 nm. The reconstituted suspension contains 0 to 1.8% (w / v) sodium chloride, preferably 0.45% to 1.8% (w / v), more preferably 0.9% to 1.8% (w / v), and more preferably 0.9%. In some embodiments, the reconstituted suspension contains 2 to 10 mg / mL docetaxel, and preferably 2 to 8 mg / mL. The reconstituted suspension is stable at 25° C. for at least 24 hours, preferably at least 30 hours, and at 2-8° C. for at least 7 days, preferably at least 10 days.

[0031] The present invention further provides a method for preparing said combination, comprising the steps of: (1) Dissolve docetaxel in an organic solvent, and obtain an organic phase solution after dissolution; (2) Take a human serum albumin solution and add acid to adjust the pH value to obtain an acid-denatured albumin aqueous phase solution, and take another salt solution; (3) mixing the organic phase solution, the aqueous phase solution and the salt solution to load a drug to obtain a drug-loaded solution; (4) Dialysis to obtain a dialyzed suspension; Wherein, before the step (2) of adjusting the pH value by adding an acid, the method optionally includes a step of diluting the human serum albumin solution with water for injection to obtain an albumin dilution solution.

[0032] Among these, the method optionally includes an incubation step after step (2) of adjusting the pH value by adding an acid.

[0033] Among them, after the mixing and drug loading step (3), a step of lowering the temperature is optionally included.

[0034] Among them, the method optionally includes a step of concentrating the drug-loaded solution obtained in step (3) to obtain a concentrate before the dialysis step (4).

[0035] Among them, after the dialyzing step (4), a concentrating or diluting step is optionally included to adjust the concentration of docetaxel in the dialyzed suspension.

[0036] Among them, after step (4), a step (5) of sterilizing and filtering is optionally included.

[0037] Of these, step (5) may optionally include a freeze-drying step (6).

[0038] Among them, the docetaxel in step (1) may be in any form, preferably anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate. Based on anhydrous docetaxel, the mass ratio of docetaxel to human serum albumin is 1:(2.0-10.0), preferably 1:(3.0-7.0), more preferably 1:(4.0-6.0).

[0039] Wherein, the organic solvent in step (1) is selected from solvents that can dissolve in water, such as ethanol, methanol, acetone, DMSO, etc., and preferably ethanol. Based on anhydrous docetaxel, the organic phase solution contains 45-90 mg / mL docetaxel, preferably 45-70 mg / mL.

[0040] In step (2), the content of sodium octanoate in the human serum albumin solution is 0.12 mmol / g protein or less, preferably 0.08 mmol / g protein or less, and preferably 0.045 to 0.08 mmol / g protein.

[0041] In step (2), the albumin diluent contains 6 to 25 mg / mL of albumin, preferably 10 to 20 mg / mL, more preferably 12 to 18 mg / mL, and most preferably 15 mg / mL.

[0042] Among them, in step (2), the acid is selected from an acidic amino acid or an acidic peptide, an organic acid, and an inorganic acid. The acidic amino acid or the acidic peptide includes, but is not limited to, cysteine ​​hydrochloride, glutathione, etc., the organic acid includes, but is not limited to, citric acid, tartaric acid, etc., and the inorganic acid includes, but is not limited to, hydrochloric acid, sulfuric acid, etc. The acid is preferably cysteine ​​hydrochloride, glutathione, hydrochloric acid, more preferably cysteine ​​hydrochloride. The pH value is preferably 3.5 to 5.5, preferably 3.5 to 5.0, more preferably 3.8 to 4.7, and most preferably 4.0 to 4.5.

[0043] In step (2), the incubation step refers to adding an acid to adjust the pH value, then heating to 35°C to 42°C, preferably 38°C to 42°C, and incubating for 30 minutes or more, preferably 30 minutes to 60 minutes.

[0044] Wherein, in step (2), the salt solution is selected from an aqueous solution of sodium chloride, potassium chloride, sodium sulfate or magnesium sulfate, preferably an aqueous solution of sodium chloride, and the concentration of the salt solution is 2% or more, preferably 2% to 35%, more preferably 10% to 20%.

[0045] In step (3), the drug is loaded under the conditions that the organic phase solution and the aqueous phase solution are heated to 35° C. to 42° C., preferably 38° C. to 42° C., and the three are mixed to load the drug.

[0046] Among these, the temperature reduction described in step (3) refers to reducing the temperature to a temperature lower than room temperature, preferably 0 to 20°C, and more preferably 7 to 15°C.

[0047] Of these, the concentrated solution described in step (4) contains about 4 to 10 mg / mL of docetaxel, preferably 6 to 10 mg / mL, more preferably 7 to 9 mg / mL, and most preferably 8 mg / mL.

[0048] Among them, in the step (4) of removing the excess small molecule compound by dialysis, there are no particular limitations on the type and dose of the dialysis solution and the molecular weight cutoff of the dialysis membrane, and a person skilled in the art can select them according to general technical knowledge or experience. Furthermore, for the convenience of formulation and clinical use, the dialysis solution is preferably an aqueous solution of a clinically acceptable osmotic pressure adjusting agent, such as an aqueous solution of sodium chloride, glucose, phosphate or citrate. In some embodiments, in step (4), dialysis is performed using a sodium chloride solution as the dialysis solution. The molecular weight cutoff of the dialysis membrane is 10 to 50 KDa, preferably 10 to 30 KDa, more preferably 10 KDa or 30 KDa. The volume of the dialysis solution is about 3 times or more, preferably 3 to 10 times, more preferably 3 to 6 times that of the drug-loaded solution or concentrated solution. The concentration of the sodium chloride solution is 1.8% (w / v) or less, preferably 0.45% to 1.8% (w / v), more preferably 0.9% to 1.8% (w / v).

[0049] In some embodiments, the human serum albumin solution used in step (2) needs to be adjusted in advance to have a sodium octanoate content. Those skilled in the art can select a suitable method according to their general technical knowledge or experience to adjust the sodium octanoate content in the human serum albumin solution, including but not limited to dialysis. In some embodiments, the method for adjusting the content of sodium octanoate in the human serum albumin solution is as follows: A commercially available human serum albumin solution is taken, diluted with water for injection or saline to obtain a diluted albumin solution, and the diluted albumin solution is dialyzed using water for injection or saline as a dialysis fluid to partially remove sodium octanoate and obtain a human serum albumin solution with a low content of sodium octanoate.

[0050] In some embodiments, the dilution ratio of the human serum albumin solution is 4 times or more, preferably 4 to 7 times. In the dialysis, the molecular weight cutoff of the dialysis membrane is 10 to 50 KDa, preferably 10 to 30 KDa, more preferably 10 KDa or 30 KDa. The volume of the dialysis solution can be determined by a person skilled in the art through routine testing based on the required content of sodium octanoate. Preferably, the volume of the dialysis solution is about 3 times or more, preferably 3 to 10 times, more preferably 3 to 6 times, the volume of the diluted albumin solution.

[0051] The human serum albumin solution with low sodium octanoate content contains less than 0.16 mmol / g protein of sodium octanoate, preferably less than 0.12 mmol / g protein, preferably less than 0.10 mmol / g protein, preferably less than 0.08 mmol / g protein. The human serum albumin solution with low sodium octanoate content can be directly used for preparing the docetaxel albumin nanoparticle composition according to the present invention, or can be mixed with other human serum albumin solutions with sodium octanoate content in proportion to obtain the desired content, and then used for preparing the docetaxel albumin nanoparticle composition.

[0052] In another aspect, the present invention further provides a composition comprising docetaxel albumin nanoparticles, which are prepared by the above method.

[0053] In the preparation method, the dialysis step removes excess small molecule compounds in the drug-loaded solution or concentrate. For example, in some embodiments, the acid-modified albumin is prepared in step (2) by adjusting the pH using acidic amino acids or acidic peptides, and the dialysis step essentially removes excess acidic amino acids or acidic peptides, so that the composition contains almost no free acidic amino acids or acidic peptides. The term "almost no free acidic amino acids or acidic peptides" means that the content of free acidic amino acids or acidic peptides in the composition is less than 0.25% (w / w) of docetaxel. For example, in some embodiments, the acid-modified albumin is prepared by adjusting the pH using cysteine ​​hydrochloride or glutathione, and the content of free cysteine ​​or glutathione in the composition is less than 0.25% (w / w) of docetaxel.

[0054] Furthermore, the present invention further provides a drug, which is prepared by a composition comprising the docetaxel albumin nanoparticles produced by the above method, said drug being in a clinically acceptable dosage form, preferably an injection, more preferably a liquid injection or a lyophilized powder injection.

[0055] In another aspect, the present invention further provides a method for adjusting the content of sodium octanoate in a human serum albumin solution, comprising the steps of: A commercially available human serum albumin solution is taken, diluted with water for injection or saline to obtain a diluted albumin solution, and the diluted albumin solution is dialyzed using water for injection or saline as a dialysis fluid to partially remove sodium octanoate and obtain a human serum albumin solution with a low content of sodium octanoate.

[0056] In some embodiments, the dilution ratio of the human serum albumin solution is 4 times or more, preferably 4 to 7 times. In the dialysis, the molecular weight cutoff of the dialysis membrane is 10 to 50 KDa, preferably 10 to 30 KDa, more preferably 10 KDa or 30 KDa. The volume of the dialysis solution can be determined by a person skilled in the art through routine testing based on the required content of sodium octanoate. Preferably, the volume of the dialysis solution is about 3 times or more, preferably 3 to 10 times, more preferably 3 to 6 times, the volume of the diluted albumin solution.

[0057] The human serum albumin solution having a low sodium octanoate content comprises less than 0.16 mmol sodium octanoate / g protein, preferably less than 0.12 mmol sodium octanoate / g protein, preferably less than 0.10 mmol sodium octanoate / g protein, preferably less than 0.08 mmol sodium octanoate / g protein.

[0058] The human serum albumin solution having a low content of sodium octanoate may be directly used in the preparation of the docetaxel albumin nanoparticle composition according to the present invention, or may be mixed in proportion with human serum albumin solutions having other sodium octanoate contents to obtain the desired content and then used in the preparation of the docetaxel albumin nanoparticle composition.

[0059] The docetaxel contents described in the present invention are all based on anhydrous docetaxel.

[0060] The numerical values ​​or numerical ranges described in the present invention can vary up or down within a range that can be understood by a person skilled in the art without affecting the implementation of the present invention, and the variation range is, for example, ±20%, or ±17%, or ±15%, or ±12%, or ±10%, or ±9%, or ±8%, or ±7%, or ±6%, or ±5%, or ±4%, or ±3%, or ±2%, or ±1%.

[0061] The phrase "produced by" in the present invention is an open expression and does not exclude the presence of other optional components. For example, the above-mentioned "the composition is produced by docetaxel and acid-modified albumin" should be understood as "the composition is produced by a raw material composition made of docetaxel and acid-modified albumin" or "the composition is produced by raw materials containing docetaxel and acid-modified albumin as main components."

[0062] The surprising discovery of the present invention is that sodium octanoate, a thermal stabilizer in albumin, has a great impact on the physical stability of the product. The reason is that sodium octanoate can bind to the hydrophobic site of albumin competitively with drugs, reducing the binding strength between drugs and proteins, resulting in the instability of the nanosuspension. When commercially available human serum albumin solution is directly used as an auxiliary substance (containing 0.16 mmol / g protein of sodium octanoate), precipitation of nanoparticles occurs within 10 hours, but when the content of sodium octanoate in the albumin solution is reduced to less than 0.08 mmol / g protein by using dialysis, the stability of the nanoparticles is greatly improved, and they can be stably maintained at 25°C for at least 24 hours or more, and can be stably stored at 2-8°C for at least 10 days or more.

[0063] Next, pH value is also an important factor affecting the physical stability of docetaxel albumin nanoparticles. The pH values ​​of docetaxel albumin nanoparticle suspensions prepared in the prior art are all above the isoelectric point of albumin, and in order to maintain the stability of the suspension, a large amount of organic acid or its salt needs to be added as a stabilizer. This makes the osmotic pressure of the drug higher, which will cause obvious pain and osmotic damage to the injected local cells and tissues when used in clinical practice. However, the present application uses acid-denatured albumin to prepare docetaxel albumin nanoparticles, controls the content of sodium octanoate in the albumin solution, and does not need to add other salt-based stabilizers, and the reconstituted stable isotonic suspension has less irritation to blood vessels.

[0064] In addition, CN103054798A teaches that the stability of nanoparticles prepared with anhydrous docetaxel is significantly better than that of docetaxel trihydrate and hemihydrate.However, in the technical solution of the present invention, the crystal water state of docetaxel, i.e., anhydrous, hemihydrate, and trihydrate, does not affect the stability of the composition of docetaxel albumin nanoparticles prepared.This greatly expands the selection scope of the use form of docetaxel, and has great industrial application value.

[0065] The present invention provides a physically and chemically stable docetaxel albumin composition. Accelerated and long-term stability studies of the composition provided by the present invention show that there is little change in 7-epi-docetaxel and protein polymers. Accelerated stability tests already completed show that the freeze-dried powder can be stably stored for 18 months at 30°C and 25°C, and based on existing data, it can be stably stored for at least 20 months at 30°C and at least 36 months at 25°C or lower. Observation tests of stationary stability already completed show that the composition provided by the present invention, whether it is a suspension before freeze-drying or a reconstituted suspension after freeze-drying, can be stable for at least 24 hours at room temperature and for at least 10 days under refrigerated conditions, and there is no turbidity of the suspension or sedimentation of nanoparticles, and stability studies are being conducted for longer periods of time. Compared with the reconstituted suspension of the commercially available product, which is stable for only 8 hours, the product of the present invention has greatly reduced limitations on clinical use. In addition, the docetaxel albumin composition provided by the present invention has a higher maximum tolerated dose (MTD) and lower toxicity than commercially available docetaxel injection (Taxotere). Therefore, it is expected that the docetaxel albumin composition provided by the present invention can improve the clinical use of docetaxel and improve the safety of clinical use. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

[0067] Unless otherwise specified, "stable" in the examples means that no settling of the nanoparticles or cloudiness of the suspension occurred.

[0068] Unless otherwise specified, the "reconstituted suspensions" described in the examples are isotonic suspensions.

[0069] Example 1 Preparation of a composition of docetaxel and albumin The formulation of the docetaxel albumin nanoparticle composition is as follows:

[0070] [Table 1]

[0071] (1) Weigh out 8 g of anhydrous docetaxel and dissolve it in 120 mL of ethanol. After dissolution, obtain an organic phase solution; (2) The prescribed amount of human serum albumin solution (containing 0.08 mmol / g protein of sodium octanoate) was diluted with water for injection to a solution containing 15 mg / mL of albumin, and then an appropriate amount of cysteine ​​hydrochloride was added to adjust the pH to 4.1. The solution was incubated at 42°C for 30 min to obtain an acid-denatured albumin aqueous solution, and sodium chloride was added with water for injection to prepare a salt solution with a concentration of 20%.

[0072] (3) heating the organic phase solution and the aqueous phase solution to 42°C, mixing the organic phase solution, the aqueous phase solution and the salt solution to load the drug, and leaving the obtained material in an ice-water bath to cool to 15°C to obtain a drug-loaded solution; (4) Concentrate the drug-loaded solution until it contains about 8 mg / mL of docetaxel, obtain a concentrate, use sodium chloride solution as a dialysis solution, perform 5-fold dialysis on the concentrate, obtain a suspension after dialysis with a dialysis membrane molecular weight cutoff of 30 KDa, and then add an appropriate amount of dialysis solution to adjust the concentration of the suspension until it contains 4 mg / mL of docetaxel; (5) Sterilize and filter through a 0.45 μm+0.2 μm membrane to obtain a suspension before freeze-drying. (6) The pre-lyophilized suspension obtained in step (5) was taken and freeze-dried to obtain a freeze-dried powder.

[0073] The results showed that the dialysis step reduced the content of sodium octanoate. At the end of the dialysis step, the concentration of sodium chloride in the suspension after dialysis and the initial concentration of sodium chloride in the dialysis solution are basically the same. The particle size of the nanoparticles in the suspension was detected by dynamic light scattering, and the suspension was left to stand to observe the precipitation phenomenon. The results in Table 2 showed that the content of sodium chloride in the dialysis solution had no significant effect on the particle size of docetaxel albumin nanoparticles in the suspension before freeze-drying and the reconstituted suspension. The suspension before freeze-drying and the reconstituted suspension of each formulation were left to stand for 24 hours at 25°C, and no turbidity or precipitation was observed in the solution, and even when left to stand for 10 days at 2-8°C, no turbidity or precipitation was observed in the solution. It is suggested that the suspension before freeze-drying and the reconstituted suspension of each formulation are stable for at least 24 hours at 25°C and stable for at least 10 days at 2-8°C. There was no significant change in stability before and after freeze-drying.

[0074] As can be seen from the results comparing Formulations 1-1 and 1-4, adjusting the dose ratio between docetaxel and albumin did not significantly affect the particle size of the nanoparticles or the stability of the suspension.

[0075] In addition, the inventors also referred to the Chinese Pharmacopoeia, 2020, Part 4, General Rule 0512, and detected the content of cysteine ​​in the suspension after dialysis by high performance liquid chromatography. The results showed that the suspension after dialysis contained almost no free cysteine ​​(the content was less than 0.25% (w / w) of docetaxel).

[0076] The inventors also detected the pH values ​​of the suspension before and after dialysis, and found that the pH values ​​of the suspension before and after dialysis were essentially unchanged.

[0077] [Table 2]

[0078] Example 2 Docetaxel trihydrate (8 g based on anhydrous docetaxel) was weighed and dissolved in 160 mL of absolute ethanol. After dissolution, an organic phase solution was obtained. A human serum albumin solution containing 16 g of albumin (sodium octanoate content is 0.08 mmol / g protein) was taken and diluted with water for injection to a solution containing 10 mg / mL of albumin. An appropriate amount of cysteine ​​hydrochloride was added to adjust the pH to 4.5, and incubated at 40°C for 1 hour to obtain an acid-modified albumin aqueous phase solution. Sodium chloride was prepared into a 10% salt solution with water for injection. The organic phase solution and the aqueous phase solution were heated to 40°C, and the organic phase solution, the aqueous phase solution and the salt solution were mixed to load the drug. The obtained material was placed in an ice-water bath to cool to 18°C ​​to obtain a drug-loaded solution. Approximately 10 The drug-loaded solution was concentrated until it contained docetaxel at a concentration of mg / mL to obtain a concentrate, and the concentrate was dialyzed six times using an isotonic sodium chloride (0.9%, w / v) solution as the dialysis solution to obtain a dialysis membrane molecular weight cutoff of 30 KDa. The suspension after dialysis was obtained, sterilized and filtered through a 0.45 μm+0.2 μm membrane to obtain a suspension before lyophilization, which was then lyophilized to obtain a lyophilized powder.

[0079] In the suspension before freeze-drying, the particle size of the docetaxel albumin nanoparticles was 101.3 nm, and the solution did not become turbid or precipitate even when left to stand at 25°C for 24 hours or at 2-8°C for 10 days. The freeze-dried powder was redissolved in water for injection to obtain a reconstituted suspension (isotonic suspension), and the particle size of the docetaxel albumin nanoparticles did not change significantly, being 103.5 nm. The reconstituted suspension did not become turbid or precipitate even when left to stand at 25°C for 24 hours or at 2-8°C for 10 days. It has been suggested that the suspension before freeze-drying and the reconstituted suspension are stable at 25°C for at least 24 hours and at 2-8°C for at least 10 days.

[0080] High-performance liquid chromatography detection showed that the suspension after dialysis contained almost no free cysteine ​​(content was about 0.13% (w / w) of docetaxel). The pH value of the suspension before and after dialysis was essentially unchanged.

[0081] Example 3 Docetaxel hemihydrate (based on anhydrous docetaxel, 8 g) was weighed and dissolved in 80 mL 96% ethanol. After dissolution, an organic phase solution was obtained. A human serum albumin solution (containing 50 g of albumin, with a sodium octanoate content of 0.08 mmol / g protein) was taken and diluted with water for injection to a solution containing 20 mg / mL of albumin. An appropriate amount of cysteine ​​hydrochloride was added to adjust the pH to 3.9 to obtain an acid-modified albumin aqueous phase solution. Sodium chloride was added to water for injection to prepare a 2% salt solution. The organic phase solution and the aqueous phase solution were heated to 35°C, and the organic phase solution, the aqueous phase solution and the salt solution were mixed to load the drug. The obtained material was placed in an ice-water bath to cool to 12°C to obtain a drug-loaded solution. The drug-loaded solution was then obtained. The drug-loaded solution was concentrated to contain docetaxel at a concentration of mg / mL to obtain a concentrate, and the concentrate was dialyzed 5 times using an isotonic sodium chloride (0.9%, w / v) solution as the dialysis solution to obtain a dialysis membrane molecular weight cutoff of 10 KDa. A suspension after dialysis was obtained, which was sterilized and filtered through a 0.45 μm+0.2 μm membrane to obtain a suspension before lyophilization, which was then lyophilized to obtain a lyophilized powder.

[0082] The suspension before lyophilization and the lyophilized powder were reconstituted in water for injection to obtain a reconstituted suspension (isotonic suspension). There was no significant difference in the particle size of the docetaxel albumin nanoparticles, which was 119.7 nm before lyophilization and 121.3 nm after reconstitution. Neither the suspension before lyophilization nor the reconstituted suspension showed any turbidity or precipitation even after standing at 25°C for 24 hours or at 2-8°C for 10 days. It has been suggested that the suspension before lyophilization and the reconstituted suspension are stable at 25°C for at least 24 hours and at 2-8°C for at least 10 days. Detection by high performance liquid chromatography showed that the suspension after dialysis contained almost no free cysteine ​​(content was about 0.21% (w / w) of docetaxel). The pH value of the suspension before and after dialysis was basically unchanged.

[0083] Example 4 Effect of Docetaxel Formulation on Composition Stability Anhydrous docetaxel, docetaxel hemihydrate, and docetaxel trihydrate (each 8 g based on anhydrous docetaxel) were weighed and dissolved in 120 mL of ethanol. After dissolution, an organic phase solution was obtained. A human serum albumin solution containing 36 g of albumin (sodium octanoate content is 0.08 mmol / g protein) was taken and diluted with water for injection to a solution containing 20 mg / mL of albumin. An appropriate amount of cysteine ​​hydrochloride was added to adjust the pH to 4.3, and incubated at 42°C for 60 min to obtain an acid-modified albumin aqueous phase solution. Sodium chloride was prepared into a salt solution with a concentration of 10% with water for injection. The organic phase solution and the aqueous phase solution were heated to 42°C, and the organic phase solution, the aqueous phase solution and the salt solution were mixed to load the drug. The obtained material was left in an ice-water bath to cool to 12°C to obtain a drug-loaded solution. The drug-loaded solution was then obtained. The drug-loaded solution was concentrated to contain docetaxel at a concentration of mg / mL to obtain a concentrate, and the concentrate was dialyzed 5 times using isotonic sodium chloride solution as the dialysis solution to obtain a dialysis membrane molecular weight cutoff of 30 KDa. A suspension after dialysis was obtained, which was sterilized and filtered through a 0.45 μm+0.2 μm membrane to obtain a suspension before freeze-drying, which was then freeze-dried to obtain a freeze-dried powder.

[0084] From the results in Table 3 below, it can be seen that docetaxel albumin nanoparticles were prepared by the same method using anhydrous docetaxel, docetaxel hemihydrate, and docetaxel trihydrate as raw materials, respectively, and there was no obvious difference in the particle size of the obtained nanoparticles, all of which were about 110 nm, and there was no obvious change before and after lyophilization. Moreover, there was no obvious difference in the stability of the suspension before lyophilization, the lyophilized powder, and the reconstituted suspension (containing 4 mg / mL docetaxel) redissolved in water for injection, and there was no turbidity or precipitation in the solution even when left standing at 25°C for 24 hours or at 2-8°C for 10 days. It is suggested that the suspension before lyophilization and the reconstituted suspension are stable at 25°C for at least 24 hours and at 2-8°C for at least 10 days. It can be seen that the presence or absence of crystal water in docetaxel does not affect the implementation of the present invention.

[0085] High performance liquid chromatography detection showed that the dialyzed suspension contained almost no free cysteine ​​(containing less than 0.25% (w / w) of docetaxel).

[0086] [Table 3]

[0087] Example 5 Chemical Stability Considerations The freeze-dried powder containing docetaxel albumin nanoparticles obtained by formulation 1-1 in Example 1 was left in a vial, rubber stoppers and aluminum caps were pressed, and the vial was stored for 18 months under different storage conditions. The relationship between the content of 7-epi-docetaxel in the product and the content of protein polymer and time was investigated, and the results are shown in Table 4.

[0088] [Table 4]

[0089] The results in Table 4 show that the formation of 7-epi-docetaxel is related to the storage temperature, and the higher the storage temperature, the faster the content of 7-epi-docetaxel increases. The product of the present invention can effectively control the formation of 7-epi-docetaxel. The results in Table 4 show that under the above three test conditions, the content of 7-epi-docetaxel in the product of the present invention is in a controllable range (content≦1.0%), and there is no obvious change in the content of protein polymer. Predicted according to existing data, the product of the present invention can be stably stored at 30°C for at least 20 months, and at 25°C or lower for at least 36 months.

[0090] When the API raw material is other forms of docetaxel, such as hemihydrate and trihydrate, under the above test conditions, the changes in chemical stability (content of 7-epi-docetaxel and content of protein polymer) are similar to those when anhydrous docetaxel is used as raw material.

[0091] In Chinese patent CN106137969A, arginine, proline, etc. are added as inhibitors in the formula, and the effect of arginine as an inhibitor is considered to be the best, and it can be stored at least 24 months under the condition of 2-8°C, and even up to 30 months. The data provided by this patent shows that when the product is stored under the condition of 2-8°C for 12 months, the content of 7-epi-docetaxel is 0.5-0.61%, but the content of 7-epi-docetaxel in the present invention is only about 0.41% when stored under the condition of 25°C for 12 months, and the content of 7-epi-docetaxel is only 0.14% when stored under the condition of 2-8°C for 12 months, which is enough to explain that the present invention can more effectively guarantee the chemical stability of docetaxel compared with CN106137969A.

[0092] Example 6 Effect of sodium octanoate content on composition stability 8 g of anhydrous docetaxel was weighed and dissolved in 120 mL of ethanol. After dissolution, an organic phase solution was obtained. Take human serum albumin solutions (containing 36 g of human serum albumin) containing different concentrations of sodium octanoate, dilute them with water for injection to a solution containing 15 mg / mL of albumin, add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.2, and incubate at 42°C for 30 min to obtain an acid-denatured albumin aqueous phase solution, and prepare a 15% salt solution with water for injection by adding sodium chloride. The organic phase solution and the aqueous phase solution were heated to 42°C, and the organic phase solution, the aqueous phase solution and the salt solution were mixed to load the drug. The obtained material was placed in an ice-water bath to cool to 15°C to obtain a drug-loaded solution. The drug-loaded solution was concentrated to contain docetaxel at a concentration of about 8 mg / mL to obtain a concentrate. The concentrate was dialyzed 5 times (dialysis membrane molecular weight cutoff is 30 KDa) using isotonic sodium chloride solution as the dialysis solution to obtain a suspension after dialysis. The suspension was sterilized and filtered through a 0.45 μm+0.2 μm membrane to obtain a suspension before lyophilization. The suspension was lyophilized to obtain a lyophilized powder.

[0093] The results in Table 5 show that the content of sodium octanoate in the raw material has no obvious effect on the particle size of docetaxel albumin nanoparticles. However, when docetaxel albumin nanoparticles are directly prepared using commercially available human serum albumin solution (containing 0.16 mmol of sodium octanoate / g protein) as the raw material, the solution becomes turbid after 10 hours at 25°C, and the physical stability of the suspension before freeze-drying and the reconstituted isotonic suspension is poor. Reducing the content of sodium octanoate in the human serum albumin solution is favorable for improving the physical stability of the nanoparticle suspension. When the content of sodium octanoate in the human serum albumin solution is less than 0.08 mmol / g protein, it can maintain a stable suspension state for more than 24 hours at 25°C, and the stable time is more than twice that of the sample containing 0.16 mmol of sodium octanoate / g protein. When the content of sodium octanoate in the human serum albumin solution is less than 0.08 mmol / g protein, the suspension before lyophilization and the reconstituted isotonic suspension can be stably stored at 2 to 8°C for at least 10 days or more.

[0094] [Table 5]

[0095] Example 7 Effect of pH value of aqueous phase solution on stability The effect of the pH value of the aqueous phase solution on the stability of the product was compared. The process formulation and preparation method were based on formulation 1-1 in Example 1, and the pH of the aqueous phase solution was adjusted to 7.0, 6.5, 6.0, 5.5, 5.0, 4.7, 4.1, 3.8, and 3.5 using cysteine ​​hydrochloride only in step (2). The changes in particle size and suspension stability of nanoparticles in the suspension before freeze-drying and the reconstituted suspension were observed.

[0096] The study found that when the pH of the aqueous phase solution was in the range of 3.5-5.5, the particle size of the obtained nanoparticles increased slightly as the pH increased, but freeze-drying and reconstitution had no obvious effect on the particle size. When the pH of the aqueous phase solution was in the range of 3.5-5.5, the obtained suspension before freeze-drying and the reconstituted suspension could both maintain a stable suspension state at 25°C for more than 20h and at 2-8°C for at least 7 days. Furthermore, when the pH of the aqueous phase solution was in the range of 3.8-4.7, the suspension before freeze-drying and the reconstituted suspension could maintain a stable suspension state even when left at 25°C for 30h or at 2-8°C for 10 days. Freeze-drying and reconstitution had no obvious effect on stability.

[0097] However, when the pH of the aqueous solution was 6.0 or higher, the particle size of the obtained nanoparticles increased significantly, and at pH 6.0, the particle size of the nanoparticles increased to 180 nm. Moreover, when the pH of the aqueous solution was higher than 6.0, the stability of the suspension was obviously reduced, becoming turbid within 1 h, and the nanoparticles could not be kept in a stable suspension state.

[0098] [Table 6]

[0099] Example 8 Preparation of a human serum albumin solution with low sodium octanoate content Example 8-1 A commercially available human serum albumin solution (with a sodium octanoate content of 0.16 mmol / g protein) was taken and diluted with water for injection to a solution containing 15 mg / mL of albumin to obtain a diluted albumin solution. The diluted albumin solution was dialyzed (with a membrane-encapsulated molecular weight of 30 KDa) using water for injection as the dialysis solution, with a dialysis ratio of 6 times, partially removing the sodium octanoate in the protein, and obtaining a human serum albumin solution with a low content of sodium octanoate.

[0100] The content of sodium octanoate in the obtained protein solution was detected by referring to the method for determining sodium octanoate in human serum albumin solution recorded in the Chinese Pharmacopoeia (Chinese Pharmacopoeia, 2020 Edition, Part IV, 3111). The results show that the sodium octanoate contained in the treated protein solution is approximately 0.045 mmol / g protein.

[0101] Example 8-2 A commercially available human serum albumin solution (with a sodium octanoate content of 0.16 mmol / g protein) was taken and diluted with saline to a solution containing 12 mg / mL albumin to obtain a diluted albumin solution, which was then dialyzed (with a membrane-encapsulated molecular weight of 10 KDa) using saline as the dialysis solution, with a 5-fold dialysis time, partially removing the sodium octanoate in the protein, to obtain a human serum albumin solution with a low content of sodium octanoate. From the detection, the sodium octanoate contained in the obtained human serum albumin solution with a low content of sodium octanoate was about 0.060 mmol / g protein.

[0102] Example 8-3 A commercially available human serum albumin solution (with a sodium octanoate content of 0.16 mmol / g protein) was taken and diluted with water for injection to a solution containing 20 mg / mL of albumin to obtain a diluted albumin solution, which was then dialyzed (with a membrane-encapsulated molecular weight of 30 KDa) using water for injection as the dialysis solution, with a dialysis time of 3 times, partially removing the sodium octanoate in the protein, and obtaining a human serum albumin solution with a low content of sodium octanoate. From the detection, the sodium octanoate contained in the obtained human serum albumin solution with a low content of sodium octanoate was about 0.080 mmol / g protein.

[0103] For the convenience of storage or use, the human serum albumin solutions with a low sodium octanoate content of Examples 8-1 to 8-3 above can be further concentrated.

[0104] Example 9. Effect of process flow on protein secondary structure According to the formulation and preparation method of Example 1-1, drug-loaded nanosuspension and blank suspension (no drug docetaxel was added in step (1)) were prepared, and the proportion of each secondary structure of protein in human serum albumin solution, blank suspension and drug-loaded nanosuspension was observed by circular dichroism spectrum, and the results are shown in Table 7.

[0105] The results showed that the blank suspension and drug-loaded nanosuspension prepared through the entire process flow had differences compared with human serum albumin solution in terms of protein secondary structures α-helix, β-sheet, turn and random coil, explaining that the process caused changes in the secondary structure of albumin.

[0106] The results of the study suggest that the secondary structure of acid-modified albumin still does not return to its pre-denatured state after the acid is removed by the dialysis step, and after docetaxel binding, the secondary structure of drug-loaded albumin further changes compared to non-drug-loaded acid-modified albumin.

[0107] [Table 7]

[0108] Example 10: Preparation with reference to Example 1 of CN 106137969 B (201510157393.1) patent 1.5 g of anhydrous docetaxel was weighed and dissolved in 100 mL of anhydrous ethanol, and after ultrasonic dissolution, an oil phase solution was obtained. 7.5 g of human serum albumin solution (concentration 200 mg / mL) containing albumin was taken and diluted with water for injection to a solution containing 6 mg / mL of albumin, and the aqueous phase volume was 1250 mL in total. 500 mgL of glutathione was added to the aqueous phase and incubated at 70°C for 6 min. The oil phase was uniformly dispersed in the aqueous phase by high shear at 1000 rpm to obtain a suspension.

[0109] The suspension is white and not opalescent, but is turbid and has many precipitates when observed by eye, and after standing for 10 minutes, precipitation occurs, and those skilled in the art believe that the obtained suspension has poor stability and cannot be operated according to the contents of the patent. Therefore, the inventor believes that the storage stability data of the product obtained by this research method is sufficient to prove that the stability of the product in this research is obviously superior to that of patent CN 106137969 B, when compared with the storage stability data provided in the patent.

[0110] [Table 8]

[0111] Example 11: Consideration of the effect of a protein to drug ratio of 1.5 (with simultaneous reduction of protein concentration to 5 mg / mL) Weigh out 1.6 g of anhydrous docetaxel and dissolve it in 23.7 mL of anhydrous ethanol. After dissolution, obtain an organic phase solution. Take a human serum albumin solution (sodium octanoate content is 0.08 mmol / g protein) with a concentration of 67.5 mg / mL and containing 2.4 g of albumin, dilute it with water for injection to a solution containing 5 mg / mL of albumin, and add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.0. Incubate at 40°C for 0.5h to obtain an acid-modified albumin aqueous phase solution. Prepare a salt solution with sodium chloride at a concentration of 14.4% with water for injection. Heat the organic phase solution and the aqueous phase solution to 40°C. Mix the organic phase solution, aqueous phase solution and salt solution to load the drug. Leave the obtained material in an ice water bath to cool to 18°C ​​to obtain a drug-loaded solution. Approximately 6 The drug-loaded solution was concentrated until it contained docetaxel at a concentration of mg / mL to obtain a concentrate, and the concentrate was dialyzed 5 times using an isotonic sodium chloride (0.9%, w / v) solution as the dialysis solution to obtain a dialysis membrane molecular weight cutoff of 30 KDa. A suspension after dialysis was obtained, which was sterilized and filtered through a 0.45 μm+0.2 μm membrane to obtain a suspension before lyophilization, which was then lyophilized to obtain a lyophilized powder.

[0112] In the suspension before lyophilization, the particle size of the docetaxel albumin nanoparticles was 91.38 nm, and the suspension became completely cloudy after standing for 20 hours at 25° C. The lyophilized powder was redissolved in water for injection to obtain a reconstituted suspension (isotonic suspension), and the reconstituted suspension became cloudy when visually observed.

[0113] Example 12: Consideration of the effects of different dialysis fluids Weigh out 1.8 g of anhydrous docetaxel and dissolve it in 26.7 mL of anhydrous ethanol. After dissolving, obtain an organic phase solution. Take a human serum albumin solution (sodium octanoate content is 0.08 mmol / g protein) with a concentration of 67.5 mg / mL and containing 8.1 g of albumin, dilute it with water for injection to a solution containing 15 mg / mL of albumin, and add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.0. Incubate at 40°C for 0.5h to obtain an acid-modified albumin aqueous phase solution. Prepare a salt solution with sodium chloride at a concentration of 14.4% with water for injection. Heat the organic phase solution and the aqueous phase solution to 40°C. Mix the organic phase solution, aqueous phase solution and salt solution to load the drug. Leave the obtained material in an ice water bath to cool to 18°C ​​to obtain a drug-loaded solution. Concentrate the drug-loaded solution until it contains about 6 mg / mL of docetaxel, and obtain a concentrated solution. (1) The concentrate was dialyzed 5 times using an isotonic sodium chloride (5%, w / v) solution as the dialysis fluid, with the dialysis membrane molecular weight cutoff being 30 KDa. A suspension after dialysis was obtained, which was sterilized and filtered using a 0.45 μm + 0.2 μm membrane to obtain a suspension before freeze-drying. This was then freeze-dried to obtain a freeze-dried powder. In the suspension before freeze-drying, the particle size of the docetaxel albumin nanoparticles was 102.2 nm. After standing for 16 hours at 25°C, a large amount of precipitate appeared at the bottom, and after standing for 16 hours at 2-8°C, the light transmittance decreased. The freeze-dried powder was redissolved in water for injection to obtain a reconstituted suspension (isotonic suspension). There was no obvious change in the particle size of the docetaxel albumin nanoparticles, and the particle size was 103.3 nm. The reconstituted suspension was already turbid at 25°C.

[0114] (2) The concentrate was dialyzed 5 times using isotonic PBS buffer (pH 7.31, osmotic pressure 320 mOsmol / Kg) as the dialysis fluid, with the dialysis membrane molecular weight cutoff being 30 KDa. A suspension after dialysis was obtained, which was sterilized and filtered using a 0.45 μm+0.2 μm membrane to obtain a suspension before freeze-drying. This was then freeze-dried to obtain a freeze-dried powder.

[0115] In the suspension before freeze-drying, the particle size of the docetaxel albumin nanoparticles was 92.43 nm. After standing for 16 hours at 25°C, a slight precipitate appeared at the bottom, and after standing for 24 days at 2-8°C, the light transmittance decreased. The freeze-dried powder was redissolved in water for injection to obtain a reconstituted suspension (isotonic suspension). There was no obvious change in the particle size of the docetaxel albumin nanoparticles, which was 90.91 nm. The reconstituted suspension showed precipitation for 2.5 h at 25°C.

[0116] Example 13 Comparison of maximum tolerated dose (MTD) and toxicity between injectable docetaxel (albumin-bound) (DTX~HSA) and docetaxel injection (TAXOTERE) in nude mice 1. Chemicals and test materials 1.1 Test item Docetaxel for injection (albumin-bound) (DTX-HSA) was obtained by employing formulation 1-1 in Example 1 of the present application and by the preparation method in Example 1.

[0117] 1.2 Control Docetaxel injection (brand name: Taxotere)

[0118] [Table 9]

[0119] 2. Experimental animals

[0120] [Table 10]

[0121] 3. Experimental design 3.1 Experimental principle In this study, the maximum tolerated dose (MTD) of a drug administered intravenously to mice was defined as the dose at which no animals died or experienced irreversible toxic reactions or body weight loss exceeding 15% for 3 consecutive days during the observation period, and this dose was considered to be the maximum tolerated dose for acute single administration.

[0122] 3.2 Dose setting After reconstitution, the isotonic concentration of DTX-HSA is 3.801 mg / mL. Refer to the guidelines for acceptable administration and blood collection volumes when administering or collecting blood at different loads in animals jointly issued by the European Federation of Pharmaceutical Industries and Associations and the European Centre for the Validation of Alternative Methods in 2001, and the maximum administration volume for slow intravenous injection in mice is 25 mL / kg. When administered three times within 24 h, the maximum dose can reach 285.1 mg / kg.

[0123] Preselection doses for this study: Injectable docetaxel (albumin-bound): 285.1, 228.1, 182.5, 146.0 mg / kg (gradient 1.25).

[0124] Docetaxel injection (TAXOTERE): 187.5, 150, 120, 96 mg / kg (gradient 1.25).

[0125] 4. Experimental Method 4.1 Animal Grouping and Marking Healthy mice with small body weight differences were selected and uniformly divided into 8 groups according to body weight, with 5 mice per group, which were DTX-HSA 285.1, 228.1, 182.5, 146.0 mg / kg groups and TAXOTERE 187.5, 150, 120, 96 mg / kg groups, respectively.

[0126] 4.2 Administration Administration method: Intravenous administration Dosage frequency: The dosing interval is 4 hours, and the drug is administered three times within 24 hours.

[0127] Dosage volume: 25 mL / kg Administration speed: Slow injection Dosage: Calculate dosage based on most recent weighing.

[0128] 4.3 Indicator Observation General Observation: All animals were observed daily during the study period, and the observational parameters included, but were not limited to, changes in skin, fur, eyes, ears, nose, mouth, chest, abdomen, urogenital organs, limbs, etc., as well as changes in breathing, movement, urination, defecation, and behavior. See the table below for adverse animal reactions.

[0129] Body weight: All animals are weighed once before the test, and animals of appropriate weight are selected for use in the test. Animals are weighed once at a fixed time each day.

[0130] Mortality and Moribundity: Time of death is recorded for animals that died and attention is given to increasing the frequency of observations for moribund animals to ascertain time of death during the study.

[0131] 4.4 Evaluation Index The highest dose at which no mice died, no irreversible toxic symptoms occurred, and no body weight loss of more than 15% occurred for 3 consecutive days during the observation period was considered the maximum tolerated dose (MTD) of the experimental drug.

[0132] 5. Experimental Results 5.1 Mortality No animal deaths were observed in either the DTX-HSA or TAXOTERE administration groups.

[0133] 5.2 Clinical Observations Animals in the DTX-HSA 285.1 and 228.1 mg / kg groups showed mild hindlimb tremors from D4 and D7, respectively, and the animals in the 228.1 mg / kg group recovered from hindlimb tremor symptoms on D20, and the animals in the 285.1 mg / kg group recovered from hindlimb tremor symptoms on D22. No obvious abnormalities were observed in the DTX-HSA 182.5 and 146.0 mg / kg groups.

[0134] Animals in the TAXOTERE 187.5 and 150 mg / kg groups showed mild to moderate hindlimb tremors starting on D4 and D6, respectively, while animals in the 150 mg / kg group recovered from hindlimb tremor symptoms on D19 and animals in the 187.5 mg / kg group recovered from hindlimb tremor symptoms on D24. No obvious abnormalities were observed in animals in the TAXOTERE 120 and 96 mg / kg groups.

[0135] 5.3 Weight 1 / 5 animals in the DTX-HSA 285.1 mg / kg group lost >15% of body weight on D5–D8, 1 / 5 animals lost >15% of body weight on D10, D12, and D13, and no animals in the other DTX-HSA-treated groups lost >15% of body weight. One / 5 animals in the TAXOTERE 187.5 mg / kg group lost >15% of body weight between D4 and D14; no animals in the other TAXOTERE treatment groups lost >15% of body weight.

[0136] [Table 11]

[0137] 6. Conclusion Under the present test conditions, the MTD for DTX-HSA nude mice is 228.1 mg / kg, and the MTD for TAXOTERE nude mice is 150.0 mg / kg.

Claims

1. A composition comprising docetaxel albumin nanoparticles, the composition comprising docetaxel and acid-modified albumin; The acid-modified albumin is obtained by denaturing human serum albumin after adding cysteine ​​hydrochloride to an appropriate pH value; The suitable pH value is between 3.8 and 4.7; A composition characterized in that the content of sodium octanoate in the human serum albumin is 0.08 mmol / g protein or less.

2. A composition comprising docetaxel albumin nanoparticles, the composition being prepared from docetaxel and acid-modified albumin; The acid-modified albumin is obtained by denaturing human serum albumin after adding cysteine ​​hydrochloride to an appropriate pH value; The suitable pH value is between 3.8 and 4.7; A composition characterized in that the content of sodium octanoate in the human serum albumin is 0.08 mmol / g protein or less.

3. 3. The composition of claim 1 or 2, wherein the docetaxel is anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate.

4. said suitable pH value being between 4.0 and 4.5; and / or The content of sodium octanoate in the human serum albumin is 0.03 to 0.08 mmol / g protein; and / or The composition according to any one of claims 1 to 3, characterized in that the mass ratio of docetaxel to human serum albumin is 1:(2.0-10.0) based on anhydrous docetaxel.

5. The content of sodium octanoate in the human serum albumin is 0.04 to 0.08 mmol / g protein; and / or The composition according to claim 4, characterized in that the mass ratio of docetaxel to human serum albumin is 1:(3.0-7.0) based on anhydrous docetaxel.

6. The content of sodium octanoate in the human serum albumin is 0.04 to 0.07 mmol / g protein; and / or The composition according to claim 5, characterized in that the mass ratio of docetaxel to human serum albumin is 1:(4.0-6.0) based on anhydrous docetaxel.

7. The composition according to any one of claims 1 to 6, characterized in that the particle size of the docetaxel albumin nanoparticles is 60 to 200 nm.

8. The composition according to claim 7, characterized in that the particle size of the docetaxel albumin nanoparticles is 90-135 nm.

9. The composition according to claim 1 or 2, characterized in that the composition comprises an osmolality adjusting agent.

10. 10. The composition of claim 9, wherein the osmolality adjusting agent is selected from sodium chloride, glucose, phosphate or citrate.

11. The composition according to claim 10, characterized in that the osmotic agent is sodium chloride, and the weight ratio of the sodium chloride to docetaxel is (0.75-9):

1.

12. The composition according to claim 11, characterized in that the weight ratio of sodium chloride to docetaxel is (1-7):

1.

13. The composition according to claim 12, characterized in that the weight ratio of sodium chloride to docetaxel is (1.5-4.5):

1.

14. The composition of claim 13, wherein the weight ratio of sodium chloride to docetaxel is 2.25:

1.

15. The composition according to claim 1 or 2, characterized in that the composition comprises a pH adjuster.

16. The composition according to claim 1 or 2, characterized in that the composition is a suspension, the suspension containing 2 to 10 mg / mL of docetaxel.

17. The composition of claim 16, wherein the suspension contains 2-8 mg / mL of docetaxel.

18. 18. The composition according to claim 16 or 17, characterized in that the pH value of the suspension is between 3.9 and 4.8 and the suspension contains 0 to 1.8% (w / v) sodium chloride.

19. 19. The composition of claim 18, wherein the suspension comprises 0.9-1.8% (w / v) sodium chloride.

20. A lyophilized powder produced by the composition of any one of claims 16 to 19.

21. The composition according to claim 1 or 2, characterized in that the composition is a lyophilized powder, the lyophilized powder contains sodium chloride, the weight ratio of sodium chloride to docetaxel is (0.75-9):1, the lyophilized powder is produced by lyophilizing a suspension containing docetaxel albumin nanoparticles, and the pH of the suspension is 3.9-4.

8.

22. The composition according to claim 21, characterized in that the weight ratio of sodium chloride to docetaxel is (1-7):

1.

23. The composition according to claim 22, characterized in that the weight ratio of sodium chloride to docetaxel is (1.5-4.5):

1.

24. 24. The composition of claim 23, wherein the weight ratio of sodium chloride to docetaxel is 2.25:

1.

25. A reconstituted suspension obtained by reconstituting the lyophilized powder according to claim 20 or the composition according to any one of claims 21 to 24 with a reconstitution medium, wherein the reconstitution medium is selected from water for injection, a sodium chloride solution or a glucose solution, and the pH of the reconstituted suspension is 3. 9 to 4.8 of the reconstituted suspension.

26. A drug produced by the composition of any one of claims 1 to 19 and 21 to 24, or the lyophilized powder of claim 20, or the reconstituted suspension of claim 25, wherein the drug is in a clinically acceptable dosage form.

27. The drug of claim 26, wherein the drug is an injectable drug.

28. The drug of claim 27, wherein the drug is a liquid injection or a lyophilized powder injection.

29. A method for preparing a composition comprising docetaxel albumin nanoparticles according to any one of claims 1 to 9 and 21 to 24, or a lyophilized powder according to claim 20, or a reconstituted suspension according to claim 25, comprising the steps of: (1) Docetaxel is dissolved in an organic solvent, and after dissolution, an organic phase solution is obtained; (2) Take human serum albumin solution and add acid to adjust the pH value to obtain acid-denatured albumin aqueous phase solution, and take another salt solution; (3) Mixing the organic phase solution, the aqueous phase solution and the salt solution to load the drug to obtain a drug-loaded solution; (4) A preparation method, comprising dialysis.

30. Before the step (2) of adjusting the pH value by adding an acid, the step of diluting the human serum albumin solution with water for injection to obtain an albumin dilution solution is included; After the step (2) of adjusting the pH value by adding an acid, an incubation step is included, After the mixing and drug loading step (3), a step of lowering the temperature is included, and before the dialysis step (4), a step of concentrating the drug-loaded solution obtained in step (3) to obtain a concentrate is included; After the dialysis step (4), a concentrating or diluting step is included, After step (4), a step (5) of sterilizing and filtering is included; 30. The method of claim 29, further comprising, after step (5), a step (6) of freeze-drying.

31. The preparation method according to claim 30, characterized in that in step (2), the albumin diluent is a solution containing 6 to 25 mg / mL of albumin.

32. The preparation method according to claim 31, characterized in that in step (2), the albumin diluent is a solution containing 10 to 20 mg / mL of albumin.

33. The preparation method according to claim 32, characterized in that in step (2), the albumin diluent is a solution containing 12 to 18 mg / mL of albumin.

34. The preparation method according to claim 33, characterized in that in step (2), the albumin diluent is a solution containing 15 mg / mL albumin.

35. The method according to any one of claims 29 to 34, characterized in that the organic solvent in step (1) is selected from solvents that can dissolve both water and water.

36. 36. The method according to claim 35, wherein the organic solvent in step (1) is selected from ethanol, methanol, acetone, and DMSO.

37. The method according to any one of claims 29 to 36, characterized in that the organic phase solution contains 45 to 90 mg / mL of docetaxel based on anhydrous docetaxel.

38. 38. The method of claim 37, wherein the organic phase solution contains 45-70 mg / mL of docetaxel based on anhydrous docetaxel.

39. The preparation method according to claim 30, characterized in that in step (2), the incubation comprises adding an acid to adjust the pH value, then heating to 35°C to 42°C and incubating for 30 minutes or more.

40. The preparation method according to claim 39, characterized in that in step (2), the incubation comprises adding an acid to adjust the pH value, then heating to 38°C to 42°C, and incubating for 30 min to 60 min.

41. The method according to any one of claims 29 to 40, characterized in that in step (2), the salt of the salt solution is selected from sodium chloride, potassium chloride, sodium sulfate or magnesium sulfate, and the concentration of the salt solution is 2% or more.

42. The preparation method according to claim 41, characterized in that the concentration of the salt solution is 2% to 35%.

43. The preparation method according to claim 42, characterized in that the concentration of the salt solution is 10% to 20%.

44. The method according to any one of claims 29 to 40, characterized in that in step (3), the drug loading conditions include heating the organic phase solution and the aqueous phase solution to 35°C to 42°C, and mixing the organic phase solution, the aqueous phase solution, and the salt solution to load the drug.

45. The preparation method according to claim 44, characterized in that in step (3), the drug loading conditions include heating the organic phase solution and the aqueous phase solution to 38°C to 42°C.

46. The preparation method according to claim 30, characterized in that the temperature reduction in step (3) is 0 to 20°C.

47. The method according to claim 46, characterized in that the temperature reduction in step (3) is 7 to 15°C.

48. In step (4), the dialysate is a sodium chloride solution, the concentration of which is 1.8% (w / v) or less; The molecular weight cutoff of the dialysis membrane used in the dialysis in step (4) is 10 to 50 KDa: The volume of the dialysate used for dialysis in step (4) is at least three times the volume of the drug-loaded solution or concentrate; The preparation method according to any one of claims 29 to 40, characterized in that the concentrated solution described in step (4) contains 4 to 10 mg / mL docetaxel.

49. In step (4), the concentration of the sodium chloride solution is 0.45% to 1.8%; The molecular weight cutoff of the dialysis membrane used in the dialysis in step (4) is 10 to 30 KDa: The volume of the dialysate used for dialysis in step (4) is 3 to 10 times the volume of the drug-loaded solution or concentrate; The preparation method according to claim 48, characterized in that the concentrated solution described in step (4) contains 6 to 10 mg / mL of docetaxel.

50. In step (4), the concentration of the sodium chloride solution is 0.9% to 1.8%; The molecular weight cutoff of the dialysis membrane used in the dialysis in step (4) is 10 KDa or 30 KDa: The volume of the dialysate used for dialysis in step (4) is 3 to 6 times the volume of the drug-loaded solution or concentrate; The method according to claim 49, characterized in that the concentrated solution described in step (4) contains 7 to 9 mg / mL of docetaxel.

51. The preparation method according to claim 48, characterized in that the concentrated solution described in step (4) contains 8 mg / mL docetaxel.

52. 41. The method according to any one of claims 29 to 40, characterized in that the human serum albumin solution in step (2) is obtained by preparing in the following manner: take a commercially available human serum albumin solution, dilute it with water for injection or saline to obtain a diluted albumin solution, and dialyze the diluted albumin solution with water for injection or saline as a dialysis solution to partially remove sodium octanoate and obtain a human serum albumin solution with a low content of sodium octanoate, which is directly used as the human serum albumin solution in step (2), or is mixed in proportion with other human serum albumin solutions with different sodium octanoate contents to obtain a desired content, and then used as the human serum albumin solution in step (2).

53. The dilution ratio of the human serum albumin solution is 4 times or more; The preparation method according to claim 52, characterized in that the molecular weight cutoff of the dialysis membrane used for the dialysis is 10 to 50 KDa, and the volume of the dialysis solution is three times or more the volume of the diluted albumin solution.

54. The dilution ratio of the human serum albumin solution is 4 to 7 times; The method according to claim 53, characterized in that the molecular weight cutoff of the dialysis membrane used for the dialysis is 10 to 30 KDa, and the volume of the dialysis solution is 3 to 10 times the volume of the diluted albumin solution.

55. The method according to claim 54, characterized in that the molecular weight cut-off of the dialysis membrane used for the dialysis is 10 KDa or 30 KDa, and the volume of the dialysis solution is 3 to 6 times the volume of the diluted albumin solution.

56. A composition comprising docetaxel albumin nanoparticles prepared by the method according to any one of claims 29 to 55.

Images