Combination and use of docetaxel albumin composition and immune checkpoint inhibitor

Abstract

The present application provides various applications of docetaxel albumin compositions and immune checkpoint inhibitors in treating tumors such as solid tumors, especially in treating colon cancer, liver cancer, melanoma and head and neck squamous cell carcinoma, including related pharmaceutical uses, pharmaceutical compositions, etc. The docetaxel albumin compositions of the present application do not require corticosteroid pretreatment during clinical use, so that they can be used in combination with immune checkpoint inhibitors with high efficiency, producing a synergistic anti-tumor effect.

Description

[0001] Related Applications

[0002] This application claims priority to Chinese Patent Application No. 20210577701.1 filed on May 26, 2021 and Chinese Patent Application No. 202110921678.3 filed on August 12, 2021, and the entire contents of the above applications are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present application belongs to the field of medicine, and specifically relates to the use of a docetaxel albumin composition and an immune checkpoint inhibitor in the preparation of a drug for treating tumors. Background Art

[0004] Docetaxel is semi-synthetically produced from a non-cytotoxic precursor (10-deacetylbaccatin III) extracted from the needles of Taxus chinensis. It is a paclitaxel analogue and has stronger anti-tumor activity than paclitaxel.

[0005] Docetaxel is poorly water-soluble and the currently commercially available preparation is a common injection solution, which was first developed by Sanofi and is marketed under the trade name The usual dosage is 75 mg / m2 once every 3 weeks as a 1-hour intravenous infusion. 2 However, Docetaxel injection, represented by , can easily induce severe allergic reactions due to the use of ethanol and Tween 80 in its formula, requires dexamethasone pretreatment, and has poor patient compliance. In addition, ethanol can affect the central nervous system, and the infusion rate needs to be reduced to alleviate the symptoms of poisoning.

[0006] Since human albumin is an endogenous substance in the human body, it has good biocompatibility and can be used as a natural carrier for hydrophobic drugs to increase the solubility of poorly soluble drugs. Abraxis, a US company, developed paclitaxel (albumin-bound) for injection (trade name: ) was approved by the FDA in 2005 for the treatment of metastatic breast cancer that has failed combination chemotherapy or breast cancer that has recurred within 6 months of adjuvant chemotherapy. It was subsequently approved for the treatment of non-small cell lung cancer, pancreatic cancer, and gastric cancer (Japan).

[0007] Paclitaxel injection Compared with paclitaxel, since the formula does not contain polyoxyethylene castor oil, a solvent that can cause severe allergic reactions, there is no need to pre-administer anti-allergic drugs, and high-concentration drugs can be quickly administered, shortening the infusion time to less than 30 minutes, significantly improving patient compliance. Due to the improved safety, the dosage of paclitaxel has been increased from 175 mg / m 2Can be increased to 260-300 mg / m 2 ; The polyoxyethylene castor oil in the formula will inhibit the binding of paclitaxel to albumin. The formula without polyoxyethylene castor oil can make full use of the unique "gp60-caveolin-SPARC" channel of albumin to enrich the drug in the tumor area, thereby increasing the efficacy. Currently, researchers from various countries are trying to use human albumin to develop albumin nanoparticles of other drugs, such as docetaxel.

[0008] To improve the quality of docetaxel albumin products, it is necessary to consider both its physical stability (suspension stability) and chemical stability (docetaxel degradation and albumin polymer increase). The docetaxel albumin compositions disclosed in the prior art are, in one way, to add a large amount of chelating agents as stabilizers to improve the physical stability of the product, but the effect on its chemical stability is not investigated; in addition, a large amount of chelating agents improves stability while forming a hypertonic solution, which is inconvenient for clinical use. Another way is to add a large amount of amino acids to the product, which can inhibit the degradation of docetaxel and the increase of albumin polymers, but the effect on physical stability has not been studied in depth, and it is only mentioned that the suspension can be stable at room temperature for more than 8 hours after reconstitution.

[0009] Therefore, finding a method that can simultaneously ensure the physical stability and chemical stability of docetaxel albumin compositions is an urgent problem to be solved in the art.

[0010] Currently, many clinical studies have been conducted on the combination of immune checkpoint blockade therapy and chemotherapy (CIT), including cisplatin, carboplatin, paclitaxel, docetaxel, pemetrexed, gemcitabine, irinotecan, etc. Docetaxel injection, represented by PD-1, must be pretreated with dexamethasone in clinical practice due to severe allergic reactions, which is speculated to affect its combined effect with PD-1 antibodies.

[0011] Therefore, the preparation of docetaxel formulations that do not require dexamethasone pretreatment and combined with immune checkpoint inhibitors has broad significance. Summary of the invention

[0012] The present application provides a new dosage form of docetaxel, which does not require pretreatment with corticosteroids (such as dexamethasone) during clinical use, so that it can be used in combination with immune checkpoint inhibitors efficiently. Specifically, the docetaxel albumin composition of the present application and the immune checkpoint inhibitors have significant anti-tumor efficacy and produce a synergistic anti-tumor effect.

[0013] In a first aspect, the present application provides a use of a docetaxel albumin composition and an immune checkpoint inhibitor in the preparation of a drug for treating tumors. In a specific embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0014] In a second aspect, the present application provides a use of a docetaxel albumin composition in the preparation of a drug for improving the effect of an immune checkpoint inhibitor in treating tumors. In a specific embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0015] In a third aspect, the present application provides a compound drug or drug combination product for treating tumors, comprising (1) a docetaxel albumin composition, and (2) an immune checkpoint inhibitor. In an embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0016] In a fourth aspect, the present application provides a method for treating a tumor in an individual (e.g., a patient or subject), comprising administering to the individual a therapeutically effective amount of (1) a docetaxel albumin composition and (2) an immune checkpoint inhibitor, or a compound drug or drug combination product as described in the third aspect. In a specific embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0017] In a fifth aspect, the present application provides a method for improving the efficacy of immune checkpoint inhibitors on tumors, comprising administering a therapeutically effective amount of a docetaxel albumin composition to an individual with a tumor who has been treated with an immune checkpoint inhibitor. In a specific embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

[0018] In the first to fifth aspects above, the tumor is a solid tumor. Preferably, the solid tumor is salivary gland cancer, esophageal cancer and esophagogastric junction cancer, undifferentiated thyroid cancer, ovarian cancer, head and neck cancer, colorectal cancer, liver cancer, melanoma, non-small cell lung cancer, breast cancer, gastric cancer, head and neck squamous cell carcinoma, renal cell carcinoma, bile duct cancer, bladder cancer and urinary tract tumors, cervical cancer, small cell lung cancer, pancreatic cancer, uterine tumor, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, lip cancer, maxillary sinus tumor, ethmoid sinus tumor, bone tumor. In a preferred embodiment, the solid tumor is colon cancer, liver cancer, melanoma or head and neck squamous cell carcinoma (abbreviated as head and neck squamous cell carcinoma), and in a more preferred embodiment, the solid tumor is melanoma or head and neck squamous cell carcinoma.

[0019] In the first to fifth aspects above, the docetaxel albumin composition is a docetaxel albumin nanoparticle composition, wherein the particle size of the docetaxel albumin nanoparticles is preferably about 60-200 nm, more preferably 90-150 nm, for example 90-135 nm.

[0020] In the first to fifth aspects above, the drug or the compound drug or drug combination product is formulated into a clinically acceptable preparation, preferably an injectable dosage form, including a liquid injection, an injectable powder, and an injectable tablet. In some embodiments, the docetaxel albumin composition and the immune checkpoint inhibitor are present in the same preparation, or are separately prepared.

[0021] For example, in some embodiments, the composition containing docetaxel albumin nanoparticles and the PD-1 inhibitor or PD-L1 inhibitor are present in the same formulation. In other embodiments, the composition containing docetaxel albumin nanoparticles and the PD-1 inhibitor or PD-L1 inhibitor are separately formulated.

[0022] In some embodiments, when the docetaxel albumin composition (e.g., a composition containing docetaxel albumin nanoparticles) is formulated into a liquid injection, the content of the docetaxel active ingredient is 3.3-4.3 mg / ml, preferably 4 mg / ml, calculated as anhydrous docetaxel.

[0023] In some embodiments, the PD-1 inhibitor or PD-L1 inhibitor is selected from recombinant humanized anti-PD-1 monoclonal antibody injection such as Nivolumab, recombinant anti-PD-1 fully human monoclonal antibody injection such as Pembrolizumab (PD-1 monoclonal antibody), recombinant humanized anti-PD-L1 monoclonal antibody injection such as Atezolizumab, fully human anti-PD-L1 antibody injection such as Avelumab and Durvalumab, preferably Nivolumab.

[0024] In the above first to fifth aspects, optionally, the drug or the compound drug or drug combination product further comprises other drugs for treating the tumor.

[0025] In some embodiments, the present application also provides the use of docetaxel albumin nanoparticles combined with PD-1 inhibitors or PD-L1 inhibitors in the preparation of a drug for treating tumors. The PD-1 inhibitor or PD-L1 inhibitor is selected from Nivolumab. In a preferred embodiment, the tumor is head and neck squamous cell carcinoma. Preferably, the head and neck squamous cell carcinoma is recurrent or metastatic head and neck squamous cell carcinoma.

[0026] The recurrent or metastatic squamous cell carcinoma of the head and neck described in the present application includes patients who cannot receive local radical treatment again and distant metastatic head and neck squamous cell carcinoma originating from the oral cavity, oropharynx, hypopharynx and larynx.

[0027] In some embodiments, the present application provides a drug for treating tumors comprising docetaxel albumin nanoparticles and nivolumab.

[0028] In some embodiments, the drug may further include other first-line and second-line drugs for the treatment of recurrent or metastatic head and neck squamous cell carcinoma. The drug refers to first-line and second-line drugs approved by drug regulatory authorities in China or other countries and regions (such as the United States, the European Union, Japan, South Korea, etc.) for the treatment of recurrent or metastatic head and neck squamous cell carcinoma.

[0029] In a preferred embodiment, the drug is in the form of an injection, including a liquid injection, an injection powder, an injection tablet, etc. When the docetaxel albumin nanoparticles are injection powders, the active ingredient is 80 mg / vial, and the active ingredient is 3.3-4.3 mg / ml, preferably 4 mg / ml, calculated as anhydrous docetaxel. When Nivolumab is an injection, it contains 100 mg Nivolumab / vial.

[0030] In some embodiments, the present application provides a combined regimen for treating tumors, which comprises administering a therapeutically effective amount of docetaxel albumin nanoparticles for injection and Nivolumab to a tumor patient. The administration is preferably by injection.

[0031] In some embodiments, the present application also provides a method for improving the efficacy of Nivolumab on tumors, which comprises further administering a therapeutically effective amount of docetaxel albumin nanoparticles on the basis of administering Nivolumab to tumor patients.

[0032] In a preferred embodiment, the administration of docetaxel albumin nanoparticles for injection is intravenous administration. Preferably, the administration cycle is once every 3 weeks. The dosage range is 25-200 mg / m 2 In a specific embodiment, the albumin pharmaceutical preparation is intravenously administered for 30 min to 60 min, preferably 60±10 min.

[0033] In some embodiments, the dosage of docetaxel albumin nanoparticles for injection is 50, 75, 100, 125, 150 mg / m 2 , administered once every 3 weeks (recorded as Q3W);

[0034] In some embodiments, the dosage of docetaxel albumin nanoparticles for injection is 30, 40, 50 mg / m2 , the drug is administered once a week for 3 weeks and then stopped for 1 week, with 4 weeks as one cycle (recorded as QW 3 / 4);

[0035] In some embodiments, the dosage of docetaxel albumin nanoparticles for injection is 50, 60, 75 mg / m 2 , administered once every 2 weeks (recorded as Q2W);

[0036] In some embodiments, the dosage of docetaxel albumin nanoparticles for injection is 30, 40, 50 mg / m 2 The drug was administered once a week for 2 weeks and then stopped for 1 week, with 3 weeks as a cycle (recorded as QW 2 / 3).

[0037] In some embodiments, based on Nivolumab, the administration of Nivolumab is 240 mg Q2W, or 360 mg Q3W, or 480 mg Q4W, preferably intravenous administration, and can be administered at any time before, during, or after the administration of docetaxel albumin. The administration cycle is the same as that of docetaxel albumin nanoparticles for injection.

[0038] In a preferred embodiment, nivolumab 360 mg is administered intravenously every 30 minutes, followed by docetaxel (albumin-bound) 75 mg / m 2 Nivolumab 360 mg IV every 3 weeks for 60 minutes, or nivolumab 360 mg IV every 3 weeks for 30 minutes, followed by docetaxel (albumin-bound) 100 mg / m 2 Q3W, 60min.

[0039] The dosage of docetaxel albumin nanoparticles for injection described in this application is calculated based on anhydrous docetaxel.

[0040] As used herein, the term "subject" refers to a mammal, such as a human being, but may also be other mammals, such as livestock or laboratory animals.

[0041] The term "treatment" means administering the compounds or preparations described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) inhibiting a disease or disease state, i.e., curbing its development; (ii) alleviating a disease or disease state, i.e., causing the disease or disease state to regress.

[0042] The term "therapeutically effective amount" means an amount of a compound of the present application that (i) treats a particular disease, condition or disorder, or (ii) alleviates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0043] In a sixth aspect, the present application also provides a composition containing docetaxel albumin nanoparticles, which comprises docetaxel and acid-denatured albumin or is made of docetaxel and acid-denatured albumin.

[0044] In some embodiments, the above composition further optionally comprises an osmotic pressure regulator or a pH regulator.

[0045] In some embodiments, the docetaxel is preferably anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate.

[0046] In some embodiments, the acid-denatured albumin is obtained by adding acid to human albumin and then denaturing it to an appropriate pH value. In a preferred embodiment, the acid is selected from acidic amino acids or acidic polypeptides, organic acids, and inorganic acids. The acidic amino acids or acidic polypeptides include but are not limited to cysteine ​​hydrochloride, glutathione, etc.; the organic acids include but are not limited to citric acid, tartaric acid, etc.; the inorganic acids include but are not limited to hydrochloric acid, sulfuric acid, etc. The acid is preferably cysteine ​​hydrochloride, glutathione, hydrochloric acid, and more preferably cysteine ​​hydrochloride. In a preferred embodiment, the appropriate pH value is 3.5-5.5, preferably 3.5-5.0, more preferably 3.8-4.7, and more preferably 4.0-4.5.

[0047] In some embodiments, based on anhydrous docetaxel, the mass ratio of docetaxel to human albumin is 1:(2.0-10.0), preferably 1:(3.0-7.0), and more preferably 1:(4.0-6.0).

[0048] In some embodiments, the content of sodium caprylate in the human serum albumin is no higher than 0.08 mmol / g protein, preferably 0.03-0.08 mmol / g protein, further preferably 0.04-0.08 mmol / g protein, and more preferably 0.04-0.07 mmol / g protein.

[0049] In some embodiments, the particle size of the docetaxel albumin nanoparticles is about 60-200 nm, preferably 90-150 nm, and more preferably 90-135 nm.

[0050] In an optional embodiment, the composition containing docetaxel albumin nanoparticles described in the present application comprises an osmotic pressure regulator to adjust the osmotic pressure within an appropriate range. The type of the osmotic pressure regulator is not particularly limited, for example, it can be selected from sodium chloride, glucose, phosphate or citrate, etc., preferably sodium chloride. Its type and dosage can be determined by those skilled in the art according to the specific circumstances of the type and dosage of the diluent or reconstitution medium used clinically. In some embodiments, the osmotic pressure regulator 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.

[0051] In an optional embodiment, the docetaxel albumin composition described herein (e.g., a composition containing docetaxel albumin nanoparticles) comprises a pH adjuster to adjust the pH value within an appropriate range so that the suspension for preparing the composition described herein and / or the reconstituted suspension of the composition described herein is stable at 25°C for at least 24 hours, preferably at least 30 hours; and / or stable at 2-8°C for at least 7 days, preferably at least 10 days. The "stable" described herein means that there is no precipitation of nanoparticles or turbidity of the suspension. 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.

[0052] In some embodiments, the docetaxel albumin composition described in the present application (e.g., a composition containing docetaxel albumin nanoparticles) is a suspension. Wherein, the pH value of the suspension is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8. The particle size of the docetaxel albumin nanoparticles in the suspension is about 60-200nm, preferably 90-150nm, more preferably 90-135nm. The suspension contains 0-1.8% (w / v) sodium chloride, preferably 0.45%-1.8% (w / v), more preferably 0.9%-1.8% (w / v). In some embodiments, the suspension contains docetaxel 2-10mg / ml, preferably 2-8mg / ml. The suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours; stable at 2-8°C for at least 7 days, preferably at least 10 days.

[0053] In other embodiments, the docetaxel albumin composition described herein (e.g., a composition containing docetaxel albumin nanoparticles) 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 to docetaxel is (0.75-9):1, preferably (1-7):1, preferably (1.5-4.5):1, and most preferably 2.25:1.

[0054] In some embodiments, the lyophilized powder is freeze-dried from a suspension containing docetaxel albumin nanoparticles. The pH value of the suspension is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8. The particle size of the docetaxel albumin nanoparticles in the suspension is about 60-200nm, preferably 90-150nm, more preferably 90-135nm. The suspension contains 0-1.8% (w / v) sodium chloride, preferably 0.45%-1.8% (w / v), more preferably 0.9%-1.8% (w / v). The suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours; stable at 2-8°C for at least 7 days, preferably at least 10 days.

[0055] In some embodiments, the lyophilized powder is reconstituted with a reconstitution medium. The reconstitution medium is selected from water for injection, sodium chloride solution or glucose solution, preferably water for injection. The pH value of the reconstituted suspension is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8. The particle size of docetaxel albumin nanoparticles in the reconstituted suspension is about 60-200nm, preferably 90-150nm, more preferably 90-135nm. The reconstituted suspension contains 0-1.8% (w / v) sodium chloride, preferably 0.45%-1.8% (w / v), more preferably 0.9%-1.8% (w / v). The reconstituted isotonic suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours; stable at 2-8°C for at least 7 days, preferably at least 10 days.

[0056] In some embodiments, the composition containing docetaxel albumin nanoparticles described in the present application is stable for at least 36 months at 25°C after freeze-drying. The "stable" described herein includes but is not limited to no significant degradation of docetaxel, no significant aggregation of protein nanoparticles, no significant increase in particle size, no significant change in docetaxel content, moisture, acidity, osmotic pressure or molar concentration. The "stable" can be one or more of the above-listed situations. In some embodiments, the "stable" is manifested as no significant change in the content of 7-epi-docetaxel and / or albumin polymers.

[0057] In a seventh aspect, the present application further provides a drug, which is prepared from the above-mentioned composition containing docetaxel albumin nanoparticles. The drug is in a clinically acceptable dosage form, preferably an injection, and more preferably a liquid injection or a lyophilized powder injection.

[0058] In some embodiments, when the injection is a liquid injection, the pH value of the liquid injection is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8. The particle size of docetaxel albumin nanoparticles in the liquid injection is about 60-200nm, preferably 90-150nm, more preferably 90-135nm; the liquid injection contains 0-1.8% (w / v) sodium chloride, preferably 0.45%-1.8% (w / v), more preferably 0.9%-1.8% (w / v), more preferably 0.9% (w / v). In some embodiments, the liquid injection contains docetaxel 2-10mg / ml, preferably 2-8mg / ml. The liquid injection is stable for at least 24 hours at 25°C, preferably at least 30 hours; stable for at least 7 days at 2-8°C, preferably at least 10 days. The term "stable" as used herein refers to the absence of nanoparticle sedimentation or turbidity in the liquid injection.

[0059] In some embodiments, when the injection is a lyophilized powder injection, the lyophilized powder injection 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 docetaxel albumin nanoparticles is about 60-200nm, preferably 90-150nm, and more preferably 90-135nm. The pH of the suspension for preparing the lyophilized powder injection before lyophilization 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.

[0060] In some embodiments, the lyophilized powder injection uses a reconstitution medium to reconstitute the suspension. The reconstitution medium is selected from water for injection, sodium chloride solution or glucose solution, preferably water for injection. The pH value of the reconstituted suspension obtained is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8. The particle size of docetaxel albumin nanoparticles in the reconstituted suspension is about 60-200nm, preferably 90-150nm, more preferably 90-135nm. The reconstituted suspension contains 0-1.8% (w / v) sodium chloride, preferably 0.45%-1.8% (w / v), more preferably 0.9%-1.8% (w / v), more preferably 0.9%. In some embodiments, the reconstituted suspension contains docetaxel 2-10mg / ml, preferably 2-8mg / ml. The reconstituted suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours; and is stable at 2-8°C for at least 7 days, preferably at least 10 days.

[0061] In an eighth aspect, the present application also provides a method for preparing the composition containing docetaxel albumin nanoparticles, which comprises the following steps:

[0062] (1) dissolving docetaxel in an organic solvent to obtain an organic phase solution;

[0063] (2) taking a human albumin solution, adding acid to adjust the pH value, and obtaining an acid-denatured albumin aqueous solution; and taking another salt solution;

[0064] (3) mixing the organic phase solution, the aqueous phase solution and the salt solution to carry the drug to obtain a drug-loaded solution;

[0065] (4) dialyzing to obtain a dialyzed suspension;

[0066] Wherein, before adding acid to adjust the pH value, step (2) optionally comprises the step of diluting the human albumin solution with water for injection to obtain an albumin dilution solution.

[0067] Wherein, step (2) optionally comprises an incubation step after adding acid to adjust the pH value.

[0068] Wherein, step (3) optionally comprises a cooling step after mixing the drug-loaded materials.

[0069] Wherein, step (4) optionally comprises, before dialysis, concentrating the drug-loaded solution obtained in step (3) to obtain a concentrated solution.

[0070] Wherein, after dialysis, step (4) optionally comprises a concentration or dilution step to adjust the concentration of docetaxel in the suspension after dialysis.

[0071] Wherein, step (4) optionally includes step (5) of sterilization filtration.

[0072] Wherein, step (5) optionally comprises step (6) of freeze-drying.

[0073] Wherein, the docetaxel in step (1) is in any form, preferably anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate. Based on anhydrous docetaxel, the mass ratio of docetaxel to human albumin is 1:(2.0-10.0), preferably 1:(3.0-7.0), and more preferably 1:(4.0-6.0).

[0074] Wherein, the organic solvent in step (1) is selected from solvents miscible with water, such as ethanol, methanol, acetone, DMSO, etc., preferably ethanol. Based on anhydrous docetaxel, the organic phase solution contains 45-90 mg / ml of docetaxel, preferably 45-70 mg / ml.

[0075] Wherein, the content of sodium octanoate in the human albumin solution in step (2) is not higher than 0.12 mmol / g protein, preferably not higher than 0.08 mmol / g protein, preferably 0.045-0.08 mmol / g protein.

[0076] Wherein, in step (2), the albumin diluent contains 6-25 mg / ml of albumin, preferably 10-20 mg / ml, more preferably 12-18 mg / ml, and more preferably 15 mg / ml.

[0077] Wherein, in step (2), the acid is selected from acidic amino acids or acidic polypeptides, organic acids, and inorganic acids. The acidic amino acids or acidic polypeptides include but are not limited to cysteine ​​hydrochloride, glutathione, etc.; the organic acids include but are not limited to citric acid, tartaric acid, etc.; the inorganic acids include but are not limited to hydrochloric acid, sulfuric acid, etc. The acid is preferably cysteine ​​hydrochloride, glutathione, hydrochloric acid, and more preferably cysteine ​​hydrochloride. The pH value is preferably 3.5-5.5, preferably 3.5-5.0, more preferably 3.8-4.7, and more preferably 4.0-4.5.

[0078] Wherein, in step (2), the incubation refers to heating to 35°C-42°C, preferably 38°C-42°C, after adding acid to adjust the pH value; incubating for more than 30 minutes, preferably 30 minutes-60 minutes.

[0079] The salt solution in step (2) is selected from an aqueous solution of sodium chloride, potassium chloride, sodium sulfate or magnesium sulfate, preferably an aqueous solution of sodium chloride; the concentration of the salt solution is not less than 2%, preferably 2% to 35%, more preferably 10% to 20%.

[0080] Wherein, in step (3), the drug loading condition is to heat the organic phase solution and the aqueous phase solution to 35°C-42°C, preferably 38°C-42°C, and mix the three to load the drug.

[0081] The cooling in step (3) refers to cooling to below room temperature, preferably 0-20°C, more preferably 7-15°C.

[0082] Wherein, the concentrated solution in step (4) contains about 4-10 mg / ml of docetaxel, preferably 6-10 mg / ml, more preferably 7-9 mg / ml, and more preferably 8 mg / ml.

[0083] Wherein, step (4) dialyzes to remove excess small molecule compounds, and there is no particular restriction on the type and dosage of the dialysate and the molecular weight cutoff of the dialysis membrane, and those skilled in the art can make a choice based on common technical knowledge or experience. For the convenience of further preparation and clinical use, the dialysate is preferably an aqueous solution of a clinically acceptable osmotic pressure regulator, such as an aqueous solution of sodium chloride, glucose, phosphate or citrate. In some embodiments, sodium chloride solution is used as the dialysate for dialysis in step (4). The molecular weight cutoff of the dialysis membrane is 10-50KDa, preferably 10-30KDa, more preferably 10KDa or 30KDa. The volume of the dialysate is not less than 3 times that of the drug-carrying solution or the concentrated solution, preferably 3-10 times, and more preferably 3-6 times. The concentration of the sodium chloride solution is not higher than 1.8% (w / v), preferably 0.45%-1.8% (w / v), and more preferably 0.9%-1.8% (w / v).

[0084] In some embodiments, the human albumin solution used in step (2) needs to be adjusted in advance for the content of sodium octanoate. A person skilled in the art can select an appropriate method to adjust the content of sodium octanoate in the human albumin solution based on common technical knowledge or experience, including but not limited to dialysis.

[0085] In some embodiments, the method for adjusting the content of sodium caprylate in the human albumin solution is:

[0086] A commercially available human albumin solution is taken and diluted with water for injection or physiological saline to obtain a diluted albumin solution; the diluted albumin solution is dialyzed with water for injection or physiological saline as a dialysate to partially remove sodium octanoate, thereby obtaining a human albumin solution with a low sodium octanoate content.

[0087] In some embodiments, the dilution multiple of the human albumin solution is not less than 4 times, preferably 4-7 times. For the dialysis, the molecular weight cutoff of the dialysis membrane is 10-50KDa, preferably 10-30KDa, more preferably 10KDa or 30KDa. The volume of the dialysate can be determined by a technician in the field according to the required sodium octanoate content through routine tests. Preferably, the volume of the dialysate is about 3 times or more of the volume of the diluted albumin solution, preferably 3-10 times, more preferably 3-6 times.

[0088] In some embodiments, the human albumin solution with low sodium octanoate content contains sodium octanoate less than 0.16 mmol / g protein, 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 albumin solution with low sodium octanoate content can be directly used to prepare the docetaxel albumin nanoparticle composition described in the present application, or can be mixed with other human albumin solutions with sodium octanoate content in proportion to obtain the desired content, and then used to prepare the docetaxel albumin nanoparticle composition.

[0089] In the ninth aspect, the present application also provides a composition containing docetaxel albumin nanoparticles, which is prepared by the method described in the eighth aspect.

[0090] In the preparation method, the dialysis step will remove excess small molecule compounds in the drug-carrying solution or the concentrated solution. For example, in some embodiments, step (2) uses acidic amino acids or acidic polypeptides to adjust the pH to prepare acid-denatured albumin; the dialysis step substantially removes excess acidic amino acids or acidic polypeptides; the composition contains almost no free acidic amino acids or acidic polypeptides. The "almost no free acidic amino acids or acidic polypeptides" means that the content of free acidic amino acids or acidic polypeptides in the composition is less than 0.25% (w / w) of docetaxel. For example, in some embodiments, cysteine ​​hydrochloride or glutathione is used to adjust the pH to prepare acid-denatured albumin, and the content of free cysteine ​​or glutathione in the composition is less than 0.25% (w / w) of docetaxel.

[0091] Furthermore, the present application also provides a drug, which is prepared from the composition containing docetaxel albumin nanoparticles prepared by the above method. The drug is a clinically acceptable dosage form, preferably an injection, and more preferably a liquid injection or a lyophilized powder injection.

[0092] In the tenth aspect, the present application also provides a method for regulating the content of sodium octanoate in a human albumin solution, comprising the following steps: taking a commercially available human albumin solution, diluting it with water for injection or physiological saline to obtain a diluted albumin solution; dialyzing the diluted albumin solution with water for injection or physiological saline as a dialysis fluid to partially remove the sodium octanoate, thereby obtaining a human albumin solution with a low sodium octanoate content.

[0093] In some embodiments, the dilution multiple of the human albumin solution is not less than 4 times, preferably 4-7 times. For the dialysis, the molecular weight cutoff of the dialysis membrane is 10-50KDa, preferably 10-30KDa, more preferably 10KDa or 30KDa. The volume of the dialysate can be determined by a technician in the field according to the required sodium octanoate content through routine tests. Preferably, the volume of the dialysate is about 3 times or more of the volume of the diluted albumin solution, preferably 3-10 times, more preferably 3-6 times.

[0094] In some embodiments, the human albumin solution with low sodium caprylate content contains sodium caprylate less than 0.16 mmol / g protein, preferably less than 0.12 mmol / g protein, preferably less than 0.10 mmol / g protein, preferably less than 0.08 mmol / g protein.

[0095] In some embodiments, the human albumin solution with low sodium octanoate content can be directly used to prepare the docetaxel albumin nanoparticle composition described in the present application, or can be mixed with other human albumin solutions with sodium octanoate content in proportion to obtain the desired content, and then used to prepare the docetaxel albumin nanoparticle composition. The docetaxel content described in the present application is calculated as anhydrous docetaxel.

[0096] The numerical values ​​or numerical ranges described in this application may fluctuate within the scope understood by those skilled in the art without affecting the implementation of this application, and the floating range may be, for example, ±20%, or ±17%, or ±15%, or ±12%, ±10%, or ±9%, or ±8%, or ±7%, or ±6%, or ±5%, or ±4%, or ±3%, or ±2%, or ±1%.

[0097] The inventors of the present application unexpectedly found that sodium octanoate, a heat stabilizer in albumin, has a significant effect on the physical stability of docetaxel albumin nanoparticles. The reason is that sodium octanoate can compete with the drug to bind to the hydrophobic sites of albumin, reduce the binding force between the drug and the protein, and cause the nanosuspension to be unstable. When a commercially available human albumin solution is directly used as an excipient (containing 0.16 mmol / g protein of sodium octanoate), the nanoparticles precipitate within 10 hours; however, when the sodium octanoate content in the albumin solution is reduced to less than 0.08 mmol / g protein by dialysis, the stability of the prepared nanoparticles is greatly improved, and it can remain stable for at least 24 hours at 25°C, and can be stably placed for at least 10 days at 2-8°C.

[0098] The inventors of the present application also found that pH value is also an important factor affecting the physical stability of docetaxel albumin nanoparticles. The pH value of the docetaxel albumin nanoparticle suspension prepared by the prior art is above the isoelectric point of albumin. 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, resulting in a high drug osmotic pressure, which will cause obvious pain during clinical use and cause osmotic damage to local cells and tissues of the injection site. However, the present application uses acid-denatured albumin to prepare docetaxel albumin nanoparticles, and controls the content of sodium octanoate in the albumin solution, without the need to add other salt stabilizers, to reconstruct a stable isotonic suspension with little irritation to blood vessels.

[0099] In addition, CN103054798A teaches that the stability of nanoparticles prepared by anhydrous docetaxel is significantly better than that of docetaxel trihydrate and hemihydrate. However, in the technical solution of the present application, the crystal water of docetaxel, i.e., anhydrous, hemihydrate, and trihydrate, has no effect on the stability of the prepared docetaxel albumin nanoparticle composition. This greatly expands the range of choices for the use of docetaxel and has great industrial application value.

[0100] The present application provides a physically and chemically stable docetaxel albumin composition. The accelerated and long-term stability studies of the composition provided by the present application show that 7-epi-docetaxel and protein polymers change very little. The completed accelerated stability test shows that the lyophilized powder can be stably stored at 30°C and 25°C for 18 months. According to existing data, it is predicted that it can be stably stored at 30°C for at least 20 months and at least 36 months at 25°C or below. The completed static stability observation test shows that the composition provided by the present application, whether it is a suspension before lyophilization or a reconstituted suspension after lyophilization, can be stable at room temperature for at least 24 hours and can be stable under refrigerated conditions for at least 10 days without turbidity of the suspension or precipitation of nanoparticles; stability studies for longer periods of time are in progress. Compared with the situation where the reconstituted suspension of commercially available products is only stable for 8 hours, the product of the present application greatly reduces the restrictions on clinical use.

[0101] In the present application, docetaxel is encapsulated in human albumin. Compared with common injections, the preparation does not contain Tween-80 and ethanol, and does not require pretreatment with corticosteroids (such as dexamethasone). It is not only safe and well tolerated, but also can effectively avoid the disadvantage that dexamethasone pretreatment weakens the tumor inhibition effect of docetaxel and / or PD-1 inhibitors.

[0102] The combined use of the docetaxel albumin composition and the immune checkpoint inhibitor described in the present application can significantly enhance the tumor inhibition effect and produce a synergistic effect. For example, the docetaxel albumin composition and the PD-1 inhibitor or the PD-L1 inhibitor can synergize to significantly inhibit the growth of various tumors such as colon cancer, liver cancer, melanoma or head and neck squamous cell carcinoma, especially in the treatment of melanoma or head and neck squamous cell carcinoma. It has a significant synergistic effect.

[0103] The embodiments described above represent exemplary embodiments of the present application, but the present application is not limited to the above embodiments. In addition, the various technical features in the above embodiments of the present application can be combined with each other to form one or more new technical solutions, which also fall within the scope of the present application as long as such new technical solutions are technically feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 The effect of dexamethasone (DEX) pretreatment on (TAXOTERE) inhibits the growth of mouse liver cancer Hepa1-6 transplanted tumors. Compared with the solvent control group, *P<0.05, **P<0.01; compared with the DEX / TAXOTERE group, ###P<0.001.

[0105] Figure 2A The effect of DEX on the efficacy of Opdivo (Nivolumab) in inhibiting mouse colon cancer MC38 transplanted tumors is shown. Compared with the solvent control group, *P<0.05, **P<0.01; compared with the DEX / Opdivo or TAXOTERE / DEX / Opdivo group, #P<0.05.

[0106] Figure 2B The comparison of the efficacy of Opdivo combined with DTX-HSA or DEX / TAXOTERE in inhibiting mouse colon cancer MC38 xenografts is shown. Compared with the solvent control group, **P<0.01; compared with the DEX / Opdivo or TAXOTERE / DEX / Opdivo group, ##P<0.01.

[0107] Figure 3 The results show the comparison of the efficacy of Keytruda (pembrolizumab) combined with docetaxel (albumin-bound) (DTX-HSA) or DEX / TAXOTERE in inhibiting MC38 colon cancer xenografts in mice. Compared with the solvent control group, ***P<0.001; compared with the DEX / TAXOTERE / Keytruda group, ###P<0.001.

[0108] Figure 4A The effects of DTX-HSA and DEX / TAXOTERE on the body weight of mice transplanted with Hepa1-6 liver cancer tumors are shown. Compared with the solvent control group, *P<0.05, **P<0.01; compared with the DEX / TAXOTERE group, ###P<0.001.

[0109] Figure 4B The results show the comparison of the efficacy of DTX-HSA and DEX / TAXOTERE in inhibiting mouse hepatocellular carcinoma Hepa1-6 homologous transplanted tumors. Compared with the solvent control group, *P<0.05, **P<0.01; compared with the DEX / TAXOTERE group, ###P<0.001.

[0110] Figure 5A The effect of Keytruda combined with DTX-HSA or DEX / TAXOTERE on the body weight of mice with Hepa1-6 liver cancer xenografts is shown. Compared with the solvent control group, *P<0.05; compared with the DEX / TAXOTERE / Keytruda group, #P<0.05.

[0111] Figure 5B The results show the efficacy of Keytruda combined with DTX-HSA or DEX / TAXOTERE in inhibiting Hepa1-6 transplanted tumors in mice. Compared with the solvent control group, ***P<0.001; compared with the DEX / TAXOTERE / Keytruda group, ##P<0.01.

[0112] Figure 6 The results show the comparison of the efficacy of DTX-HSA, Keytruda and their combined administration in inhibiting mouse Hepa1-6 transplanted tumors. Compared with the solvent control group, **P<0.01, ***P<0.001.

[0113] Figure 7 The results show the comparison of the efficacy of DTX-HSA, Keytruda and their combined administration regimen in inhibiting B16 / F10 transplanted tumors in mice. Compared with the solvent control group, **P<0.01, ***P<0.001. Example

[0114] The following examples are specific descriptions of the present application and should not limit the scope of the present application.

[0115] Unless otherwise specified, the term "stable" as used in the examples means that there is no precipitation of nanoparticles or turbidity of the suspension.

[0116] Unless otherwise specified, the "reconstituted suspension" described in the examples is an isotonic suspension.

[0117] Example 1 Preparation of a composition of docetaxel and albumin

[0118] The formulation of the docetaxel albumin nanoparticle composition is as follows:

[0119] Table 1 Formula of docetaxel albumin nanoparticle composition

[0120] Recipe No. 1-1 1-2 1-3 1-4 Anhydrous docetaxel 8g 8g 8g 8g Human serum albumin 36g 36g 36g 80g <![CDATA[Sodium chloride a > 18g 36g 9g 18g

[0121] Note: a is the sodium chloride content in the suspension before freeze-drying after conversion. The concentrations of sodium chloride in the corresponding dialysates (w / v) are: Formula 1-10.9%; Formula 1-21.8%; Formula 1-30.45%; Formula 1-40.9%.

[0122] (1) Weigh 8 g of anhydrous docetaxel and dissolve it in 120 ml of ethanol to obtain an organic phase solution;

[0123] (2) Take a formulated amount of human albumin solution (the content of sodium octanoate is 0.08 mmol / g protein), dilute it with water for injection to a solution containing 15 mg / ml albumin, then add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.1, incubate at 42°C for 30 minutes, and obtain an acid-denatured albumin aqueous phase solution; prepare a 20% saline solution of sodium chloride with water for injection.

[0124] (3) The organic phase solution and the aqueous phase solution were heated to 42°C, the organic phase, aqueous phase and salt solution were mixed to carry the drug, and the resulting material was placed in an ice water bath and cooled to 15°C to obtain a drug-loaded solution;

[0125] (4) concentrating the drug-loaded solution to a docetaxel concentration of about 8 mg / ml to obtain a concentrated solution; dialyzing the concentrated solution 5 times using a sodium chloride solution as a dialysate, with a dialysis membrane molecular weight cutoff of 30 KDa, to obtain a dialyzed suspension; then, adding an appropriate amount of dialysate to adjust the concentration of the suspension to 4 mg / ml of docetaxel;

[0126] (5) sterilizing and filtering through a 0.45 μm + 0.2 μm membrane to obtain a suspension before lyophilization;

[0127] (6) taking the pre-lyophilized suspension obtained in step (5), lyophilizing to obtain a lyophilized powder.

[0128] The results showed that the dialysis step would reduce the content of sodium octanoate. At the end of the dialysis step, the concentration of sodium chloride in the post-dialysis suspension was basically the same as the initial concentration of sodium chloride in the dialysate. 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 sedimentation phenomenon. The results in Table 2 showed that the content of sodium chloride in the dialysate had no significant effect on the particle size of docetaxel albumin nanoparticles in the pre-lyophilized suspension and the reconstituted suspension. The pre-lyophilized suspension and the reconstituted suspension of each formula were left to stand at 25°C for 24 hours, and no turbidity or precipitation of the solution was observed; no turbidity or precipitation of the solution was observed when the suspension was left to stand at 2-8°C for 10 days. It is suggested that the pre-lyophilized suspension and the reconstituted suspension of each formula are stable for at least 24 hours at 25°C and at least 10 days at 2-8°C. There is no significant change in stability before and after lyophilization.

[0129] Comparing the results of formulations 1-1 and 1-4, it can be seen that adjusting the dosage ratio of docetaxel and albumin has no significant effect on the particle size of the nanoparticles and the stability of the suspension.

[0130] In addition, the inventors also used high performance liquid chromatography to detect the content of cysteine ​​in the suspension after dialysis with reference to the general rule 0512 of the fourth part of the 2020 edition of the Chinese Pharmacopoeia. The results showed that the suspension after dialysis contained almost no free cysteine ​​(the content was less than 0.25% (w / w) of docetaxel).

[0131] The inventors also tested the pH value of the suspension before and after dialysis and found that the pH value of the suspension before and after dialysis remained substantially unchanged.

[0132] Table 2 Particle size and stability of docetaxel albumin nanoparticles with different formulations

[0133]

[0134] Note: a. The freeze-dried powder is reconstituted into a suspension (isotonic suspension) using water for injection. Formula 1-1 is reconstituted into an isotonic suspension containing 4 mg / ml of docetaxel; Formula 1-2 is reconstituted into an isotonic suspension containing 2 mg / ml of docetaxel; Formula 1-3 is reconstituted into an isotonic suspension containing 8 mg / ml of docetaxel; Formula 1-4 is reconstituted into an isotonic suspension containing 4 mg / ml of docetaxel.

[0135] Example 2

[0136] Weigh docetaxel trihydrate (8 g based on anhydrous docetaxel) and dissolve it in 160 ml of anhydrous ethanol to obtain an organic phase solution; take a human albumin solution containing 16 g of albumin (the content of sodium octanoate is 0.08 mmol / g protein), dilute it with water for injection to a solution containing 10 mg / ml albumin, then add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.5, incubate at 40°C for 1 hour, and obtain an acid-denatured albumin aqueous phase solution; prepare a 10% salt solution of sodium chloride with water for injection; The organic phase, aqueous phase and saline solution are heated to 40° C., the organic phase, aqueous phase and saline solution are mixed to carry the drug, and the resulting material is placed in an ice water bath and cooled to 18° C. to obtain a drug-loaded solution; the drug-loaded solution is concentrated to a docetaxel concentration of about 10 mg / ml to obtain a concentrated solution; the concentrated solution is dialyzed 6 times using an isotonic sodium chloride solution (0.9%, w / v) as a dialysate, and the molecular weight cutoff of the dialysis membrane is 30 KDa to obtain a post-dialysis suspension; sterilization filtration is performed through a 0.45 μm+0.2 μm membrane to obtain a pre-lyophilized suspension; and lyophilization is performed to obtain a lyophilized powder.

[0137] In the suspension before lyophilization, the particle size of docetaxel albumin nanoparticles was 101.3nm. After standing at 25℃ for 24 hours and at 2-8℃ for 10 days, no turbidity or precipitation of the solution was observed. The lyophilized powder was reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension). The particle size of docetaxel albumin nanoparticles did not change significantly and was 103.5nm. The reconstituted suspension was stood at 25℃ for 24 hours and at 2-8℃ for 10 days, and no turbidity or precipitation of the solution was observed. It is suggested that the suspension before lyophilization and the reconstituted suspension are stable for at least 24 hours at 25℃ and at least 10 days at 2-8℃.

[0138] High performance liquid chromatography showed that the suspension after dialysis contained almost no free cysteine ​​(the content was about 0.13% (w / w) of docetaxel). The pH value of the suspension before and after dialysis remained basically unchanged.

[0139] Example 3

[0140] Weigh docetaxel hemihydrate (8 g based on anhydrous docetaxel) and dissolve it in 80 ml of 96% ethanol to obtain an organic phase solution; take a human albumin solution containing 50 g of albumin (the content of sodium octanoate is 0.08 mmol / g protein), dilute it with water for injection to a solution containing 20 mg / ml albumin, then add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 3.9 to obtain an acid-denatured albumin aqueous phase solution; prepare a 2% saline solution of sodium chloride with water for injection; heat the organic phase solution and the aqueous phase solution to 35°C, and The solution and the salt solution are mixed to load the drug, and the obtained material is placed in an ice water bath to cool to 12° C. to obtain a drug-loaded solution; the drug-loaded solution is concentrated to a docetaxel concentration of about 7 mg / ml to obtain a concentrated solution, and the concentrated solution is dialyzed 5 times using an isotonic sodium chloride solution (0.9%, w / v) as a dialysate, and the molecular weight cutoff of the dialysis membrane is 10 KDa to obtain a post-dialysis suspension; sterilization filtration is performed through a 0.45 μm+0.2 μm membrane to obtain a pre-lyophilized suspension; and lyophilization is performed to obtain a lyophilized powder.

[0141] There was no significant difference in the particle size of docetaxel albumin nanoparticles between the suspension before lyophilization and the reconstituted suspension (isotonic suspension) obtained after reconstitution of the lyophilized powder with water for injection. The particle size was 119.7 nm before lyophilization and 121.3 nm after reconstitution. After standing at 25°C for 24 hours or at 2-8°C for 10 days, no turbidity or precipitation was observed in the suspension before lyophilization and the reconstituted suspension. It is suggested that the suspension before lyophilization and the reconstituted suspension are stable for at least 24 hours at 25°C and for at least 10 days at 2-8°C. HPLC detection showed that the suspension after dialysis contained almost no free cysteine ​​(the content was about 0.21% (w / w) of docetaxel). The pH value of the suspension before and after dialysis remained basically unchanged.

[0142] Example 4 Effect of docetaxel usage form on the stability of the composition

[0143] Anhydrous docetaxel, docetaxel hemihydrate and docetaxel trihydrate (8 g each based on anhydrous docetaxel) were weighed and dissolved in 120 ml of ethanol to obtain an organic phase solution. A human albumin solution containing 36 g of albumin (the content of sodium octanoate was 0.08 mmol / g protein) was 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 the mixture was incubated at 42°C for 60 min to obtain an acid-denatured albumin aqueous phase solution. Sodium chloride was prepared with water for injection to a concentration of 10% salt solution; 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 carry out drug loading, placing the obtained material in an ice water bath and cooling it to 12° C. to obtain a drug-loaded solution; concentrating the drug-loaded solution to contain docetaxel at a concentration of about 7 mg / ml to obtain a concentrated solution; using an isotonic sodium chloride solution as a dialysate to dialyze the concentrated solution 5 times, the molecular weight cutoff of the dialysis membrane is 30 KDa, to obtain a post-dialysis suspension; sterilizing and filtering through a 0.45 μm+0.2 μm membrane to obtain a pre-lyophilized suspension; lyophilizing to obtain a lyophilized powder.

[0144] As can be seen from the results in Table 3 below, docetaxel albumin nanoparticles were prepared using the same method using anhydrous docetaxel, docetaxel hemihydrate and docetaxel trihydrate as raw materials, respectively. There was no significant difference in the particle size of the obtained nanoparticles, all of which were about 110 nm, and there was no significant change before and after lyophilization. In addition, there was no significant difference in the stability of the suspension before lyophilization and the reconstructed suspension (containing docetaxel 4 mg / ml) after the lyophilized powder was reconstituted with water for injection. No turbidity or precipitation of the solution was observed when it was allowed to stand for 24 hours at 25°C and for 10 days at 2-8°C. It is suggested that the above-mentioned suspension before lyophilization and the reconstructed suspension can be stable for at least 24 hours at 25°C and at least 10 days at 2-8°C. It can be seen that whether docetaxel contains water of crystallization does not affect the implementation of this application.

[0145] HPLC detection showed that the suspension after dialyzation contained almost no free cysteine ​​(the content was less than 0.25% (w / w) of docetaxel).

[0146] Table 3 Effect of docetaxel usage form on the stability of the composition

[0147]

[0148] Example 5 Chemical Stability Study

[0149] The lyophilized powder containing docetaxel albumin nanoparticles obtained in formula 1-1 in Example 1 was placed in a vial, a rubber stopper and an aluminum cap were pressed on, and stored for 18 months under different storage conditions. The relationship between the percentage content of 7-epi-docetaxel and the protein polymer content in the product and time was investigated. The results are shown in Table 4.

[0150] Table 4 Stability of the product of Example 1 Formula 1-1

[0151]

[0152] Note: The acceptable limit used to define the stability of the pharmaceutical composition is "percentage content of 7-epidocetaxel ≤ 1.0%".

[0153] N / A* means the experiment has not yet reached that time point, so no data is available.

[0154] The results in Table 4 show that the generation of 7-epidocetaxel is related to the storage temperature. The higher the storage temperature, the faster the content of 7-epidocetaxel increases. The product of the present application can effectively control the generation of 7-epidocetaxel. The results in Table 4 show that under the above three experimental conditions, the content of 7-epidocetaxel in the product of the present application is within a controllable range (content ≤1.0%), and the protein polymer content does not change significantly. According to existing data predictions, the product of the present application can be stably stored at 30°C for at least 20 months and stably stored at below 25°C for at least 36 months.

[0155] When the API raw material is other forms of docetaxel, such as hemihydrate and trihydrate, under the above test conditions, the changes in its chemical stability (percentage content of 7-epi-docetaxel and protein polymer content) are similar to those when anhydrous docetaxel is used as the raw material.

[0156] Chinese patent CN106137969A adds arginine, proline, etc. as inhibitors in the formula, and believes that the use of arginine as an inhibitor has the best effect, and can be stored at 2-8°C for at least 24 months, or even up to 30 months. The data provided by the patent show that when the product is stored at 2-8°C for 12 months, the content of 7-epidocetaxel is between 0.5-0.61%; while the content of 7-epidocetaxel in the present application is only about 0.41% when stored at 25°C for 12 months, and only 0.14% when stored at 2-8°C for 12 months, which is enough to show that the present application can more effectively ensure the chemical stability of docetaxel compared with CN106137969A.

[0157] Example 6 Effect of Sodium Caprylate Content on Composition Stability

[0158] Weigh 8 g of anhydrous docetaxel and dissolve it in 120 ml of ethanol to obtain an organic phase solution;

[0159] Take human albumin solutions containing different concentrations of sodium octanoate (containing 36 grams of human albumin), dilute them with water for injection to 15 mg / ml of albumin, then add appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.2, incubate at 42°C for 30 minutes to obtain an acid-denatured albumin aqueous phase solution; prepare a 15% saline solution of sodium chloride with water for injection;

[0160] The organic phase solution and the aqueous phase solution are heated to 42°C, the organic phase solution, the aqueous phase solution and the salt solution are mixed to carry the drug, and the resulting material is placed in an ice water bath and cooled to 15°C to obtain a drug-loaded solution; the drug-loaded solution is concentrated to a docetaxel concentration of about 8 mg / ml to obtain a concentrated solution; the concentrated solution is dialyzed 5 times using an isotonic sodium chloride solution as a dialysate (dialysis membrane molecular weight cutoff 30 KDa) to obtain a post-dialysis suspension; sterile filtration is performed through a 0.45 μm+0.2 μm membrane to obtain a pre-lyophilized suspension; and lyophilization is performed to obtain a lyophilized powder.

[0161] As shown in Table 5, the content of sodium octanoate in the raw material has no significant effect on the particle size of docetaxel albumin nanoparticles. However, when commercially available human albumin solution (containing 0.16 mmol / g protein of sodium octanoate) was used as the raw material to prepare docetaxel albumin nanoparticles, turbidity appeared after being placed at 25°C for 10 hours, and the physical stability of the suspension before lyophilization and the reconstructed isotonic suspension was poor. Reducing the content of sodium octanoate in the human albumin solution helps to improve the physical stability of the nanoparticle suspension. When the content of sodium octanoate in the human albumin solution is lower than 0.08 mmol / g protein, it can maintain a stable suspension state at 25°C for more than 24 hours, and the stable time is more than twice that of the sample containing 0.16 mmol / g protein of sodium octanoate. When the content of sodium octanoate in the human albumin solution is lower than 0.08 mmol / g protein, the suspension before lyophilization and the reconstructed isotonic suspension can be stably placed at 2-8°C for at least 10 days.

[0162] Table 5 Effect of the content of sodium octanoate on the stability of the composition

[0163]

[0164] Note: a The lyophilized powder is reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension).

[0165] Example 7 Effect of pH value of aqueous solution on stability

[0166] The effect of the pH value of the aqueous solution on the stability of the product was compared. The process formula and preparation method were based on the formula 1-1 of Example 1, except that cysteine ​​hydrochloride was used in step (2) to adjust the pH of the aqueous solution to 7.0, 6.5, 6.0, 5.5, 5.0, 4.7, 4.1, 3.8, and 3.5, respectively. The changes in the nanoparticle size and the stability of the suspension were observed in the suspension before lyophilization and the reconstituted suspension.

[0167] The study found that when the pH of the aqueous solution was in the range of 3.5-5.5, the particle size of the obtained nanoparticles increased slightly with the increase of pH, but freeze-drying and reconstitution had no obvious effect on the particle size. When the pH of the aqueous solution was in the range of 3.5-5.5, the obtained pre-freeze-dried suspension and reconstituted suspension could maintain a stable suspension state at 25°C for more than 20 hours, and a stable suspension state at 2-8°C for at least 7 days. Furthermore, when the pH of the aqueous solution was in the range of 3.8-4.7, the pre-freeze-dried suspension and reconstituted suspension could still maintain a stable suspension state when left at 25°C for 30 hours and at 2-8°C for 10 days. Freeze-drying and reconstitution had no obvious effect on stability.

[0168] However, when the pH of the aqueous solution is above 6.0, the particle size of the obtained nanoparticles increases significantly, and the particle size of the nanoparticles increases to 180nm at pH 6.0. In addition, when the pH of the aqueous solution is higher than 6.0, the stability of the suspension decreases significantly, and turbidity appears within 1 hour, and the nanoparticles cannot be kept in a stable suspended state.

[0169] Table 6 Effect of aqueous protein pH on docetaxel albumin nanoparticles

[0170]

[0171] Note: a The lyophilized powder is reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension).

[0172] Example 8 Preparation of human albumin solution with low sodium octanoate content

[0173] Example 8-1

[0174] A commercially available human albumin solution (the content of sodium octanoate is 0.16 mmol / g protein) is diluted with water for injection to a solution containing 15 mg / ml albumin to obtain an albumin dilution solution; the albumin dilution solution is dialyzed with water for injection as a dialysate (the molecular weight cut-off of the membrane package is 30 KDa) at a dialysis multiple of 6 times to partially remove the sodium octanoate in the protein to obtain a human albumin solution with a low sodium octanoate content.

[0175] The content of sodium octanoate in the obtained protein solution was detected by referring to the determination method of sodium octanoate in human albumin solution included in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Part IV 3111, 2020 Edition). The results showed that the treated protein solution contained approximately 0.045 mmol / g protein of sodium octanoate.

[0176] Example 8-2

[0177] Take a commercially available human albumin solution (the content of sodium octanoate is 0.16mmol / g protein), dilute it with normal saline to a solution containing 12mg / ml albumin, and obtain an albumin dilution solution; use normal saline as a dialysate to dialyze the albumin dilution solution (the molecular weight cutoff of the membrane package is 10KDa), the dialysis multiple is 5 times, and the sodium octanoate in the protein is partially removed to obtain a human albumin solution with a low sodium octanoate content. After testing, the obtained human albumin solution with a low sodium octanoate content contains about 0.060mmol / g protein of sodium octanoate.

[0178] Example 8-3

[0179] Take a commercially available human albumin solution (the content of sodium octanoate is 0.16mmol / g protein), dilute it with water for injection to a solution containing 20mg / ml albumin, and obtain an albumin dilution solution; use water for injection as a dialysate to dialyze the albumin dilution solution (the molecular weight cut-off of the membrane package is 30KDa), the dialysis multiple is 3 times, and the sodium octanoate in the protein is partially removed to obtain a human albumin solution with a low sodium octanoate content. After testing, the obtained human albumin solution with a low sodium octanoate content contains about 0.080mmol / g protein of sodium octanoate.

[0180] For convenient storage or use, the human albumin solutions with low sodium octanoate content in the above Examples 8-1 to 8-3 can be further concentrated.

[0181] Example 9 Effect of Process on Protein Secondary Structure

[0182] According to the formulation and preparation method of Example 1-1, drug-loaded nanosuspensions and blank suspensions were prepared (docetaxel was not added in step (1)). Circular dichroism spectroscopy was used to investigate the proportion of each secondary structure of the protein in the human albumin solution, blank suspension and drug-loaded nanosuspension. The results are shown in Table 7.

[0183] The results showed that the blank suspension and drug-loaded nanosuspension prepared through the entire process had differences compared with the human albumin solution in terms of protein secondary structure α-helix, β-fold, turn and random coil, indicating that the process changed the secondary structure of albumin.

[0184] The results of the study suggest that even if the acid is removed during the dialysis step, the secondary structure of the acid-denatured albumin cannot be restored to the state before denaturation. Moreover, after binding with docetaxel, the secondary structure of the drug-loaded albumin further changes compared to the acid-denatured albumin without drug loading.

[0185] Table 7 Proportion of each secondary structure of the protein

[0186] Human albumin solution <![CDATA[Blank suspension a > <![CDATA[Drug-loaded nanosuspension a > α-helix (%) 39.5 23.9 27.9 β-fold (%) 15.1 11.7 4.4 Rotation angle (%) 16.8 26.2 30.1 Random curl (%) 28.6 38.2 37.6

[0187] Note: a: The blank suspension and drug-loaded nanosuspension described in Example 9 are both isotonic suspensions reconstituted after freeze-drying.

[0188] Example 10 was prepared with reference to Example 1 of Chinese Patent CN 106137969 B (201510157393.1)

[0189] Weigh 1.5g of anhydrous docetaxel and dissolve it in 100ml of anhydrous ethanol. After ultrasonic dissolution, obtain an oil phase solution; take a human albumin solution containing 7.5g of albumin (concentration is 200mg / ml), dilute it with water for injection to a solution containing 6mg / ml albumin, and the total volume of the aqueous phase is 1250ml. Add 500mg of L-glutathione to the aqueous phase and incubate at 70℃ for 6min. Disperse the oil phase evenly into the aqueous phase under high shear of 1000rpm to obtain a suspension.

[0190] The suspension is white without opalescence, visually turbid, with a large amount of precipitates visible, and precipitation occurs after 10 minutes of standing. The technicians believe that the obtained suspension has poor stability and cannot continue the subsequent operation according to the patent content. Therefore, the storage stability data of the product prepared by the method of the present application is compared with the storage stability data provided in the above patent, which is sufficient to prove that the stability of the product of the present application is significantly better than the stability of the product in patent CN106137969B.

[0191] Table 8

[0192]

[0193] N / A: This time point is not available in the stability study.

[0194] Example 11 Investigating the Effect of a Protein-Drug Ratio of 1.5 (while reducing the protein concentration to 5 mg / ml)

[0195] Weigh 1.6 g of anhydrous docetaxel and dissolve it in 23.7 ml of anhydrous ethanol to obtain an organic phase solution with a concentration of 67.5 mg / ml; take a human albumin solution containing 2.4 g of albumin (the content of sodium octanoate is 0.08 mmol / g protein), dilute it with water for injection to a solution containing 5 mg / ml of albumin, then add an appropriate amount of cysteine ​​hydrochloride to adjust the pH to 4.0, incubate at 40°C for 0.5 h, and obtain an acid-denatured albumin aqueous phase solution; prepare a 14.4% saline solution of sodium chloride with water for injection; The organic phase solution and the aqueous phase solution are heated to 40° C., the organic phase, the aqueous phase and the salt solution are mixed to carry the drug, and the obtained material is placed in an ice water bath to cool to 18° C. to obtain a drug-loaded solution; the drug-loaded solution is concentrated to a docetaxel concentration of about 6 mg / ml to obtain a concentrated solution; the concentrated solution is dialyzed 5 times using an isotonic sodium chloride solution (0.9%, w / v) as a dialysate, and the molecular weight cutoff of the dialysis membrane is 30 KDa to obtain a post-dialysis suspension; sterilization filtration is performed through a 0.45 μm+0.2 μm membrane to obtain a pre-lyophilized suspension; and lyophilization is performed to obtain a lyophilized powder.

[0196] In the suspension before lyophilization, the particle size of docetaxel albumin nanoparticles was 91.38 nm, and it was completely turbid when left to stand for 20 hours at 25° C. The lyophilized powder was reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension), which was visually turbid.

[0197] Example 12 Investigating the effects of different dialysates

[0198] 1.8 g of anhydrous docetaxel was weighed and dissolved in 26.7 ml of anhydrous ethanol to obtain an organic phase solution with a concentration of 67.5 mg / ml; a human albumin solution containing 8.1 g of albumin (the content of sodium octanoate was 0.08 mmol / g protein) was taken and 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 4.0, and incubated at 40°C for 0.5 h to obtain an acid-denatured albumin aqueous phase solution; sodium chloride was prepared with water for injection to a saline solution with a concentration of 14.4%; the organic phase solution and the aqueous phase solution were heated to 40°C, the organic phase, the aqueous phase and the saline solution were mixed to carry the drug, and the obtained material was placed in an ice water bath and cooled to 18°C ​​to obtain a drug-loaded solution; the drug-loaded solution was concentrated to a docetaxel concentration of about 6 mg / ml to obtain a concentrated solution; and then the following two methods were used for treatment and measurement.

[0199] (1) using an isotonic glucose solution (5%, w / v) as a dialysate to dialyze the concentrate 5 times, with a dialysis membrane having a molecular weight cutoff of 30 KDa, to obtain a post-dialysis suspension; sterilizing and filtering through a 0.45 μm + 0.2 μm membrane to obtain a pre-lyophilized suspension; and lyophilizing to obtain a lyophilized powder.

[0200] In the suspension before lyophilization, the particle size of docetaxel albumin nanoparticles was 102.2nm. When standing at 25℃ for 16 hours, there was a lot of precipitation at the bottom. When standing at 2-8℃ for 16 hours, the transmittance decreased. The lyophilized powder was reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension). The particle size of docetaxel albumin nanoparticles did not change significantly and was 103.3nm. The reconstituted suspension was turbid after 2.5h at 25℃.

[0201] (2) using isotonic PBS buffer (pH 7.31, osmotic pressure 320 mOsmol / Kg) as a dialysate to dialyze the concentrate 5 times, with a dialysis membrane molecular weight cutoff of 30 KDa, to obtain a post-dialysis suspension; sterilizing and filtering through a 0.45 μm + 0.2 μm membrane to obtain a pre-lyophilized suspension; and lyophilizing to obtain a lyophilized powder.

[0202] In the suspension before lyophilization, the particle size of docetaxel albumin nanoparticles was 92.43nm. After standing at 25℃ for 16 hours, there was a slight precipitation at the bottom. After standing at 2-8℃ for 24 hours, the transmittance decreased. The lyophilized powder was reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension). The particle size of docetaxel albumin nanoparticles did not change significantly and was 90.91nm. Precipitation was observed in the reconstituted suspension at 25℃ for 2.5h.

[0203] Example 13 Comparison of the maximum tolerated dose (MTD) and toxicity of docetaxel injection (albumin-bound) (DTX-HSA) and docetaxel injection (TAXOTERE) in nude mice

[0204] 1. Drugs and test materials

[0205] 1.1 Test sample

[0206] Docetaxel (albumin-bound) for injection (DTX-HSA) is prepared using the formulation 1-1 in Example 1 of the present application and the preparation method of Example 1.

[0207] 1.2 Comparator drugs

[0208] Docetaxel injection (trade name: Taxotere)

[0209]

[0210] 2. Experimental Animals

[0211]

[0212] 3. Experimental Design

[0213] 3.1 Experimental Principle

[0214] This study defines the maximum tolerated dose (MTD) of the drug for intravenous administration to mice as the absence of death and irreversible toxic reactions in the animals, or no weight loss exceeding 15% for three consecutive days during the observation period. This dose is considered the maximum tolerated dose of an acute single administration.

[0215] 3.2 Dosage setting

[0216] The isotonic concentration of DTX-HSA after reconstitution is 3.801 mg / mL. According to the guidelines for the permissible volume of administration and blood collection for different routes of administration or blood collection for animals jointly issued by the European Federation of Pharmaceutical Industries and Associations and the European Center for Validation of Alternative Methods in 2001, the maximum volume of slow intravenous injection for mice is 25 mL / kg. The maximum dose can be given 3 times within 24 hours, and 285.1 mg / kg.

[0217] The doses preselected for this trial are:

[0218] Docetaxel for injection (albumin-bound): 285.1, 228.1, 182.5, 146.0 mg / kg (gradient 1.25).

[0219] Docetaxel injection (TAXOTERE): 187.5, 150, 120, 96 mg / kg (gradient 1.25).

[0220] 4. Experimental Methods

[0221] 4.1 Animal grouping and labeling

[0222] Healthy mice with small weight differences were selected and divided into 8 groups according to balanced weight, with 5 mice in each group, namely DTX-HSA 285.1, 228.1, 182.5, 146.0 mg / kg groups and TAXOTERE 187.5, 150, 120, 96 mg / kg groups.

[0223] 4.2 Administration

[0224] Administration: intravenous injection

[0225] Dosage frequency: 3 times within 24 hours, with an interval of 4 hours.

[0226] Dosing volume: 25mL / kg.

[0227] Administration rate: Slow push.

[0228] Dosage: Calculate the dosage based on the most recent weighing.

[0229] 4.3 Indicator Observation

[0230] General status observation: All animals were observed every day during the test period. Observation indicators included but were not limited to skin, fur, eyes, ears, nose, mouth, chest, abdomen, urogenital area, limbs, breathing, movement, urination, defecation and behavioral changes. Animal adverse reactions are shown in the table below.

[0231] Body weight: All animals were weighed once before the experiment, and animals with appropriate weight were selected for the experiment. Animals were weighed once a day at a fixed time.

[0232] Death and dying: For dead animals, the time of death should be recorded. For dying animals, the observation frequency should be increased and the time of death should be determined during the experiment.

[0233] 4.4 Evaluation indicators

[0234] During the observation period, there was no death, irreversible toxicity symptoms, or weight loss in mice. The maximum dose exceeding 15% for 3 consecutive days was considered the maximum tolerated dose (MTD) of the experimental drug.

[0235] 5. Experimental Results

[0236] 5.1 Death

[0237] No animal death was observed in the DTX-HSA and TAXOTERE administration groups.

[0238] 5.2 Clinical Observation

[0239] The animals in the DTX-HSA 285.1 and 228.1 mg / kg groups showed slight hind limb tremor from D4 and D7, respectively. The hind limb tremor of the animals in the 228.1 mg / kg group recovered on D20, and the hind limb tremor of the animals in the 285.1 mg / kg group recovered on D22. No obvious abnormalities were observed in the animals in the DTX-HSA 182.5 and 146.0 mg / kg groups.

[0240] Animals in the TAXOTERE 187.5 and 150 mg / kg groups showed mild to moderate hind limb tremors starting from D4 and D6, respectively. The hind limb tremor symptoms of animals in the 150 mg / kg group recovered on D19, and the hind limb tremor symptoms of animals in the 187.5 mg / kg group recovered on D24. No obvious abnormalities were observed in the animals in the TAXOTERE 120 and 96 mg / kg groups.

[0241] 5.3 Weight

[0242] In the DTX-HSA 285.1 mg / kg group, 1 / 5 animals lost more than 15% body weight on D5-D8, and 1 / 5 animals lost more than 15% body weight on D10, D12, and D13. No animals in other DTX-HSA-administered groups lost more than 15% body weight.

[0243] One out of five animals in the TAXOTERE 187.5 mg / kg group lost more than 15% of their body weight from D4 to D14, while no animal in the other TAXOTERE-administered groups lost more than 15% of their body weight.

[0244] Table 9 Toxicity in mice after administration (n=5)

[0245]

[0246] MTD: During the observation period, there was no death, no irreversible toxicity symptoms, and no weight loss of mice exceeding 15% of the maximum dose for 3 consecutive days. Observation time: 24 days.

[0247] 6. Conclusion

[0248] Under the conditions of this experiment, the MTD of DTX-HSA in nude mice was 228.1 mg / kg; the MTD of TAXOTERE in nude mice was 150.0 mg / kg.

[0249] Example 14 Comparison of side effects of single administration of DTX-HSA and TAXOTERE in dogs

[0250] 36 Beagle dogs were divided into 6 groups, with 6 animals in each group (half female). The grouping and dosing methods are as follows:

[0251] Table 10 Dosage and method of administration for dogs

[0252]

[0253] Note: The solvent given to the experimental animals in group 1 was 0.9% sodium chloride injection, and the human albumin solution in the same dose as that in group 5 was given to the experimental animals in group 2. DTX-HSA was prepared according to the formula and preparation method of Example 1-1 (the same below).

[0254] Results: On the first day of administration: one male animal in group 5 (high-dose DTX-HSA group) showed reduced activity and inability to stand 1 hour after administration; all male and female animals in group 6 (TAXOTERE control group) showed reduced activity, inability to stand, reddening of ear skin / hair, salivation in 2 female animals, vomiting in 4 female animals, and loose / watery stools in 2 female animals. The above animals were given a single intramuscular injection of dexamethasone sodium phosphate injection (source: Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd., batch number: 181116) within 1 hour after administration, and the symptoms improved and the animal activities returned to normal. No animals in the other groups (groups 1-4) showed allergic reactions.

[0255] It can be seen that compared with TAXOTERE, the probability and severity of allergic reactions caused by the DTX-HSA dosage form are significantly reduced.

[0256] Example 15 Effect of dexamethasone on the efficacy of docetaxel (mouse liver cancer Hepa1-6 model)

[0257] Experimental model: C57BL / 6 mice were used to establish a mouse hepatocellular carcinoma Hepa1-6 syngeneic transplant tumor model to evaluate the effect of dexamethasone on the antitumor effect of docetaxel.

[0258] Experimental method: 50 female C57BL / 6 mice were subcutaneously inoculated with mouse liver cancer Hepa1-6 cells (4×10 6 / animal / 0.1mL), on the 6th day after inoculation (this day was recorded as D0), 32 animals with good tumor growth were selected and divided into 4 groups according to the tumor volume, with 8 animals in each group. The grouping and administration method are as follows: Groups ①, ②, and ③ were given distilled water by gavage at the same frequency as group ④. The administration volume was 10mL / kg.

[0259] Table 11 Dosage and method of administration

[0260]

[0261] The experiment ended at D15. The data were analyzed using SPSS 19.0 statistical software.

[0262] Test results: From Table 12, Figure 1 It can be seen that under the experimental conditions, compared with the solvent control group, both DTX-HSA and TAXOTERE can significantly inhibit the growth of Hepa1-6 transplanted tumors (P<0.01), and the effects are comparable. The addition of DEX greatly affected the tumor inhibition effect of TAXOTERE (DTX-HSA or TAXOTERE group compared with TAXOTERE / DEX group, P<0.001), and the tumor volume of the TAXOTERE / DEX group was even significantly higher than that of the solvent control group (D15, P<0.05), suggesting that the combined use of DEX to prevent allergic reactions and fluid retention will promote tumor growth and greatly weaken the anti-tumor effect of docetaxel. DTX-HSA does not require dexamethasone pretreatment, avoiding the adverse effects of DEX on promoting tumor growth, which is of great significance for improving the anti-tumor effect of docetaxel.

[0263] Note: Tumor volume = 1 / 2 × long diameter × short diameter 2 The long and short diameters of the tumor were measured using a vernier caliper.

[0264] Table 12 Effect of dexamethasone on the efficacy of docetaxel

[0265] Group Mean tumor volume (mean±sd) ①Solvent control group 484.6±193.1 ②DTX-HSA <![CDATA[217.9±76.2 **### ]]> ③TAXOTERE <![CDATA[233.4±84.6 **### ]]> ④TAXOTERE / DEX <![CDATA[794.6±379.4 * ]]>

[0266] Note: Compared with the solvent control group, * P < 0.05;** P<0.01; compared with TAXOTERE / DEX group, ### P<0.001

[0267] Example 16 Effect of dexamethasone on the efficacy of Opdivo and its combination with docetaxel (mouse colon cancer MC38 model)

[0268] Experimental model: A mouse colon cancer MC38 syngeneic transplant tumor model was established using Biocytogen B-hPD-1 mice to evaluate the effect of DEX on the tumor inhibition of Opdivo alone, as well as the tumor inhibition effect of DTX-HSA or DEX / TAXOTERE combined with PD-1 antibody (Opdivo).

[0269] Experimental method: 43 female humanized B-hPD-1 mice were subcutaneously inoculated with mouse colon cancer MC38 cells (2×10 6 / animal / 0.1mL), and on the 5th day after inoculation (this day was recorded as D0), 40 animals with good tumor growth were selected, and the average tumor volume was about 110mm 3 The animals were divided into 5 groups according to the tumor volume, with 8 animals in each group. The grouping and administration methods are as follows:

[0270] Groups ①② and ⑤ were gavaged with distilled water at the same frequency as group ③ was given DEX.

[0271] The administration volume of DTX-HSA was 20 mL / kg, and the others were 10 mL / kg.

[0272] Table 13 Dosage and method of administration

[0273]

[0274] The experiment was terminated at D19. The data were analyzed using SPSS 19.0 statistical software. 3 ) is the efficacy index.

[0275] Table 14 Comparison of tumor volume in each group

[0276]

[0277]

[0278] Note: Compared with the solvent control group, * P<0.05, ** P<0.01; compared with the DEX / Opdiv group, # P<0.05; compared with TAXOTERE / DEX / Opdivo group, ## P<0.01.

[0279] Test results: From Table 14, Figure 2A It can be seen that under the experimental conditions, the tumor volume of the Opdivo group was significantly reduced compared with the solvent control group (P<0.01), indicating that Opdivo 30 mg / kg alone has a significant tumor inhibitory effect; further DEX treatment was used. Although DEX / Opdivo showed a certain tumor inhibitory effect (compared with the solvent control group, P<0.05), the effect of inhibiting the growth of mouse colon cancer MC38 transplanted tumors was significantly reduced compared with Opdivo alone (P<0.05), indicating that DEX pretreatment would affect the efficacy of PD-1 antibody Opdivo.

[0280] Further combined with TAXOTERE (76 mg / kg), the tumor inhibition effect of the TAXOTERE / DEX / Opdivo group was not enhanced compared with the DEX / Opdivo group, and the tumor volume was even slightly increased. When DTX-HSA was used instead of TAXOTERE and DEX treatment was omitted, the tumor volume was significantly reduced. Compared with the TAXOTERE / DEX / Opdivo group, the inhibitory effect of DTX-HSA / Opdivo on the growth of mouse colon cancer MC38 transplanted tumors was significantly enhanced (P<0.01) (see Figure 2B ). The results of the experiment showed that when given the same dose of Opdivo and docetaxel, compared with the common docetaxel preparation, the DTX-HSA preparation does not require DEX pretreatment, can effectively avoid the disadvantage of combined use of DEX to reduce the efficacy of Opdivo, and can more effectively inhibit tumor growth.

[0281] Example 17 Effect of dexamethasone on the therapeutic efficacy of docetaxel combined with Keytruda (mouse colon cancer MC38 model)

[0282] Experimental model: A mouse colon cancer MC38 syngeneic transplant tumor model was established using Biocytogen B-hPD-1 mice to evaluate the tumor inhibitory effect of DTX-HSA or DEX / TAXOTERE combined with PD-1 antibody (Keytruda).

[0283] Experimental method: 30 female humanized B-hPD-1 mice were subcutaneously inoculated with mouse colon cancer MC38 cells (2×10 6 / animal / 0.1mL), and on the 5th day after inoculation (the day was recorded as D0), 24 animals with good tumor growth were selected, and the average tumor volume was about 68mm 3 The animals were divided into 3 groups according to the tumor volume, with 8 animals in each group. The grouping and administration methods are as follows:

[0284] Groups ① and ② were given distilled water by gavage at the same frequency as group ③, with a dosing volume of 10 mL / kg.

[0285] Table 15 Dosage and method of administration

[0286]

[0287] The experiment was terminated on D20. The data were analyzed using SPSS 19.0 statistical software. 3 ) is the efficacy index.

[0288] Table 16 Comparison of tumor volume in each group

[0289]

[0290] Note: Compared with the solvent control group, *** P<0.001; compared with TAXOTERE / DEX / Keytruda group, ### P<0.001.

[0291] Test results: Figure 3 It can be seen that under the conditions of this experiment, compared with the solvent control group, the TAXOTERE / DEX / Keytruda group did not show significant tumor inhibition efficacy, suggesting that DEX pretreatment significantly weakened the anti-tumor effects of docetaxel and Keytruda. When the ordinary docetaxel preparation (TAXOTERE) was replaced with the DTX-HSA described in this application, without the need for combined use of DEX, the DTX-HSA / Keytruda group significantly inhibited the growth of MC38 transplanted tumors, and the anti-tumor effect was significantly enhanced (compared with the solvent control group, P < 0.001; compared with the TAXOTERE / DEX / Keytruda group, P < 0.001). The experimental results show that the combination of DTX-HSA and Keytruda, omitting DEX, can effectively avoid the adverse effects of DEX pretreatment that weakens the tumor inhibition effects of docetaxel and PD-1 antibody Keytruda.

[0292] Example 18 Effect of dexamethasone on the efficacy of Keytruda, docetaxel and combination regimen (mouse liver cancer Hepa1-6 model)

[0293] Experimental model: A mouse hepatocellular carcinoma Hepa1-6 homologous transplant tumor model was established using Biocytogen B-hPD-1 mice to evaluate the effect of dexamethasone on the tumor inhibition effect of DTX-HSA and TAXOTERE combined with PD-1 antibody (Keytruda).

[0294] Experimental method: 60 female humanized B-hPD-1 mice were subcutaneously inoculated with mouse liver cancer Hepa1-6 cells (5×10 6 / / 0.1mL), on the 5th day after inoculation (the day was recorded as D0), 48 animals with good tumor growth were selected and divided into 6 groups according to the balanced tumor volume, with 8 animals in each group. The grouping and administration method are as follows: Groups ①, ②, ④, and ⑤ were given distilled water by gavage at the same frequency as group ③. The administration volume of DTX-HSA was 20mL / kg, and the others were 10mL / kg.

[0295] Table 17 Dosage and method of administration

[0296]

[0297] The experiment ended at D20. The data were analyzed using SPSS 19.0 statistical software.

[0298] Test results: Figure 4A It can be seen that the body weight of mice in the DTX-HSA group was not significantly different from that in the solvent control group, indicating that DTX-HSA has good tolerance. However, the body weight of mice in the TAXOTERE group pretreated with DEX was significantly lower than that in the solvent control group and DTX-HSA (P < 0.05), and their tolerance was worse than that in the DTX-HSA group. Figure 4B It can be seen that after TAXOTERE was pretreated with DEX, it significantly promoted the growth of Hepa1-6 tumors, and the D20 tumor volume was significantly higher than that of the solvent control group (P < 0.01). However, DTX-HSA did not require DEX pretreatment and could significantly inhibit tumor growth (P < 0.05). The experimental results showed that DTX-HSA was not only better tolerated than TAXOTERE / DEX, but also could effectively avoid the adverse effects of DEX pretreatment that reduced the anti-tumor effect of docetaxel.

[0299] Depend on Figure 5A It can be seen that the animals tolerated the combination of DTX-HSA and Keytruda well, and there was no significant difference in body weight compared with the solvent control group. TAXOTERE was given DEX pretreatment and then combined with Keytruda, and the body weight was significantly reduced (compared with the solvent control group, P < 0.05; compared with the DTX-HSA / Keytruda group, P < 0.05), and the animal tolerance was worse than the combination of DTX-HSA and Keytruda.

[0300] Depend on Figure 5B It can be seen that the combined administration of DTX-HSA and Keytruda can significantly inhibit tumor growth (P < 0.001). However, after TAXOTERE was pretreated with DEX, it had almost no anti-tumor activity after being co-administered with Keytruda (compared with the solvent control group, P > 0.05). This further indicates that DEX pretreatment will affect the anti-tumor effect of docetaxel combined with PD-1 antibody.

[0301] During the experiment, one mouse died in the DEX / TAXOTERE / Keytruda group, but no mouse died in the other groups. Figure 6 It can be seen that the use of DTX-HSA and Keytruda alone can significantly inhibit the growth of Hepa1-6 transplanted tumors. The combined administration of DTX-HSA and Keytruda, without the need for dexamethasone pretreatment, achieved a better inhibitory effect on Hepa1-6 transplanted tumors.

[0302] Table 18 Comparison of tumor weight in each group

[0303]

[0304] Note: Compared with the solvent control group, * P < 0.05; ** P<0.01; compared with the DEX / TAXOTERE group, # P<0.05, ### P<0.001.

[0305] Example 19 Synergistic efficacy of the combination of Keytruda and docetaxel (mouse melanoma B16 / F10 model)

[0306] Experimental model: A mouse melanoma B16 / F10 syngeneic transplant tumor model was established using Biocytogen B-hPD-1 mice to evaluate the anti-tumor effect of the combination of docetaxel (albumin-bound) for injection and the PD-1 antibody Keytruda.

[0307] Experimental method: 40 female humanized B-hPD-1 mice were divided into 4 groups according to their weight, with 10 mice in each group. All mice were inoculated with B16 / F10 cells (1×10 6 / / 0.1mL), the day of inoculation was recorded as D0, and the grouping and administration method were as follows:

[0308] Table 19 Dosage and method of administration

[0309]

[0310]

[0311] The experiment was terminated at D15. SPSS 19.0 statistical software was used to analyze the changes in tumor volume between multiple measurements over time using the Repeated Measure procedure. The Burgi correction formula was used: Q = E(a+b) / [E(a)+E(b)-E(a)*E(b) (where E(a+b): tumor inhibition rate after combined administration of drugs a and b, E(a): tumor inhibition rate when a is administered alone, and E(b): tumor inhibition rate when b is administered alone. Q < 0.85 indicates an antagonistic effect, 0.85 ≤ Q ≤ 1.15 indicates an additive effect, and Q > 1.15 indicates a synergistic effect) to determine the combined effect of the two drugs.

[0312] Test results: During the test, 3 mice died in the solvent control group, and no mice in the other groups died. Figure 7 As shown in Table 20, under the experimental conditions, compared with the solvent control group, the use of DTX-HSA alone can significantly inhibit the growth of B16 / F10 transplanted tumors (P < 0.001), and Keytruda also has a certain inhibitory effect on B16 / F10 transplanted tumors. The combined administration of DTX-HSA and Keytruda has a synergistic inhibitory effect on B16 / F10 transplanted tumors (P < 0.001) (Q = 0.713 / ((0.474+0.177)-0.474*0.177) = 1.26).

[0313] Table 20 Comparison of tumor weight in each group

[0314]

[0315] Note: Compared with the solvent control group, *** P<0.001.

[0316] Example 20 Clinical Study of Combination Therapy of DTX-HSA and Opdivo

[0317] 1. Experimental Design

[0318] 1.1 Overall Design

[0319] This trial is a single-arm, multicenter Phase Ib / II clinical study for subjects with recurrent or metastatic head and neck squamous cell carcinoma who have progressed on platinum-based regimens and are positive for PD-L1. It aims to evaluate the efficacy, safety and pharmacokinetic characteristics of docetaxel (albumin-bound) for injection combined with nivolumab.

[0320] 1.2 Basis for dose selection

[0321] Dosage regimen:

[0322] The dosing regimen of Nivolumab is: Nivolumab 360 mg Q3W (once every 3 weeks), iv for 30 minutes

[0323] The dosing regimen of docetaxel for injection (albumin-bound) is: 75 mg / m 2 or 100 mg / m 2 , Q3W, iv 30~60min

[0324] Basis for formulation:

[0325] Referring to the regimen of Nivolumab (360 mg, Q3W) combined with ipilimumab and two cycles of platinum-containing doublet chemotherapy for the treatment of metastatic or recurrent NSCLC without EGFR and ALK gene mutations (CheckMate-9LA), and the regimen of Nivolumab (360 mg, Q3W) combined with XELOX (oxaliplatin + capecitabine) for the treatment of advanced or metastatic gastric cancer / gastroesophageal junction adenocarcinoma, Nivolumab was administered at a dose of 360 mg Q3W in this study.

[0326] The ongoing Phase I clinical study shows that docetaxel (albumin-bound) injection 100 mg / m 2 No DLT (dose-limiting toxicity) was observed in patients with solid tumors during Q3W, and 125 mg / m 2 Q3W dose escalation phase. Combined with the clinical studies of the marketed preparation Taxotere, the MTD (maximum tolerated dose) in patients with solid tumors is 115 mg / m 2 Q3W, the commonly used clinical dosage regimen is 75-100 mg / m 2 Q3W. In summary, the phase Ib docetaxel (albumin-bound) injection in this study was selected at 75 mg / m 2 or 100mg / m 2 Q3W, iv 60min.

[0327] In summary, the combined dosing regimen of this clinical study is: Nivolumab 360 mg Q3W, iv 30 min combined with docetaxel (albumin-bound) 75 mg / m 2 Q3W, iv for 60 min, or Nivolumab 360 mg Q3W, iv for 30 min plus docetaxel (albumin-bound) 100 mg / m 2 Q3W, iv 60min. One of the dosing regimens was selected for Phase II study based on the safety and efficacy results.

[0328] 1.3 Experimental design

[0329] Phase Ib:

[0330] Explore 75 mg / m 2 and 100 mg / m 2 Safety and tolerability of two dose levels of docetaxel (albumin-bound) for injection combined with nivolumab 360 mg.

[0331] Dose exploration starts with a low dose and proceeds sequentially. Three subjects are initially enrolled in each dose group and observed for 21 days. If no DLT occurs, a higher dose level study (up to 100 mg / m 2 ); if DLT occurs in 1 of 3 subjects, 3 more subjects will be enrolled at this dose level for safety observation; if DLT occurs in less than 2 of 6 subjects, a higher dose level study (up to 100 mg / m 2 ); if DLT occurred in 2 of the 3 cases, the investigator and the sponsor would jointly discuss and decide on the follow-up study.

[0332] DLT is defined as the following toxic reactions related to the trial drug occurring within the first cycle (21 days):

[0333] (1) Non-hematological toxicity:

[0334] Toxic effects of grade 4 or above.

[0335] Grade 3 toxic effects, except for the following:

[0336] Recovered to ≤ grade 2 within 3 days after best supportive treatment;

[0337] Grade 3 liver function abnormalities, but recovered to Grade 1 or baseline within 7 days after treatment;

[0338] Simple laboratory abnormalities that are asymptomatic and do not require intervention as judged by the investigator.

[0339] (2) Hematological toxicity:

[0340] Grade 4 neutropenia lasting >5 days.

[0341] Febrile neutropenia (ANC <1.0×10 9 / L, body temperature reaches 38.3℃ once or the body temperature remains at 38℃ for more than one hour) and lasts for >3 days.

[0342] Grade 3 thrombocytopenia with grade 2 bleeding.

[0343] Grade 4 thrombocytopenia.

[0344] Grade 4 anemia.

[0345] die.

[0346] (3) Other toxic reactions that the investigator determines should warrant permanent discontinuation of the investigational drug.

[0347] Safety events occurring in subsequent dosing cycles are also one of the factors influencing the selection of combination regimen dosage.

[0348] A comprehensive analysis of the safety, tolerability, efficacy and pharmacokinetic study results of Phase Ib was conducted to determine the RP2D (Phase 2 recommended dose) and conduct a Phase II study.

[0349] Stage II:

[0350] Based on the RP2D determined in the Phase Ib study, a Phase II study of docetaxel (albumin-bound) for injection combined with nivolumab was conducted to observe the effectiveness of the combination regimen, with the primary endpoint being ORR.

[0351] Objective response rate (ORR) definition: the proportion of subjects whose best overall response in the study was complete remission (CR) or partial remission (PR) (i.e., CR+PR) as assessed by the investigator according to RECISTv1.1 (evaluation criteria for efficacy response in solid tumors based on single-pathway measurement) from the start of the trial drug to the end of the study. Subjects with the first evaluation of PR or CR need to undergo efficacy confirmation after 4 weeks.

[0352] The Phase II trial adopts Simon two-stage (Optimum) design. In the first stage, 30 subjects need to be enrolled. If the number of remissions (PR+CR) does not exceed 5, the trial will be terminated. Otherwise, the trial will be advanced to the second stage and 52 more subjects will be enrolled. If the total number of remissions in the two stages exceeds 17, the combination regimen is considered to have subsequent development value in this indication. A total of 82 subjects need to be enrolled in the two stages.

[0353] All subjects in Phase Ib and Phase II received docetaxel (albumin-bound) injection RP2D combined with nivolumab until they met the trial termination or withdrawal criteria. The trial drugs were used for a maximum of 2 years.

[0354] Tumor assessment was performed every 6 weeks (42 days ± 7 days) and confirmed by an independent imaging review center (IRC).

[0355] To evaluate the pharmacokinetic characteristics of docetaxel (albumin-bound) for injection in combination with nivolumab, all subjects in Phase Ib and Phase II were required to collect PK blood samples in cycles 1 and 3 to detect the free and total docetaxel levels.

[0356] 2. Test population:

[0357] 2.1 Inclusion Criteria

[0358] Subjects must meet all of the following criteria to be eligible for this trial:

[0359] 1. Those aged ≥ 18 years (based on the day of signing the informed consent form) and voluntarily signed the informed consent form.

[0360] 2. Patients with histologically or cytologically confirmed head and neck squamous cell carcinoma (primary tumor located in the oral cavity, oropharynx, larynx or hypopharynx), PD-L1 positive expression (tumor cells expressing PD-L1 ≥1%), and who are not suitable for local radical treatment.

[0361] 3. Failure of platinum-based therapy, defined as:

[0362] a) Recurrent or metastatic head and neck squamous cell carcinoma with disease progression during or after treatment with a platinum-containing regimen;

[0363] b) Locally advanced head and neck cancer, recurrence or metastasis within 6 months after previous multimodal treatment with platinum-containing regimen; 4. Previous or qualified tumor tissue samples for PD-L1 testing:

[0364] a) Phase Ib: PD-L1 test results from level 3 or above hospitals or central laboratories with CAP, CLIA or other equivalent certifications are acceptable, and the test results must be reviewed by the sponsor's biomarker team. Or provide fresh or archived tumor tissue within 3 years for PD-L1 immunohistochemistry (IHC) testing.

[0365] b) Phase II: Fresh or archived tumor tissue within 3 years can be provided for PD-L1 immunohistochemistry (IHC) testing.

[0366] 5. Oropharyngeal cancer subjects must provide previous HPV p16 immunohistochemistry test results, or qualified tumor tissue samples for testing HPV status.

[0367] 6. According to RECIST v1.1, at least one measurable lesion confirmed by CT or MRI (patients who have received radiotherapy before and whose disease progresses or whose tumor persists for ≥3 months after radiotherapy can be considered as measurable lesions).

[0368] 7. Subjects with ECOG performance status of 0-1.

[0369] 8. Patients with an expected survival period of ≥ 3 months.

[0370] 9. The major organ functions meet the following criteria within 7 days before treatment (no blood component transfusion, human granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), interleukin-11 and erythropoietin (EPO) and other medical supportive treatments within 2 weeks before the administration of the investigational drug):

[0371]

[0372]

[0373] 10. Women with childbearing potential must have a negative serum pregnancy test within 7 days before the first use of the investigational drug. The subjects and their spouses must agree to take adequate contraceptive measures from the signing of the informed consent form to 6 months after the last dose. During this period, women are not breastfeeding and men should avoid sperm donation.

[0374] 2.2 Exclusion criteria

[0375] Subjects who meet any of the following exclusion criteria are not eligible to participate in this study:

[0376] 1. Histologically or cytologically confirmed recurrent or metastatic nasopharyngeal carcinoma, head and neck squamous cell carcinoma of unknown primary lesion, salivary gland carcinoma, or non-squamous tissue cancer (such as mucosal melanoma).

[0377] 2. Active brain metastases and leptomeningeal metastases. Patients with brain metastases can be enrolled if MRI evidence shows no PD at least 8 weeks after treatment and within 28 days before the first use of the trial drug; patients can be enrolled if they do not need to receive systemic corticosteroid treatment (prednisone>10 mg / day or an equivalent dose of similar drugs) at least 2 weeks before the first use of the trial drug; patients with skull base lesions without clear evidence of dura mater or brain parenchyma involvement need to discuss with the medical monitor of the sponsor before considering whether they can be enrolled.

[0378] 3. A history of other malignant tumors within 5 years before the first use of the investigational drug, excluding the following situations: a. Any other invasive malignant tumor (for which the subject has received adequate treatment) that has been disease-free for more than 3 years, which the researcher assesses will not affect the evaluation of tumor efficacy; b. Locally curable cancers such as cured basal cell or squamous cell skin cancer, superficial bladder cancer, prostate cancer, cervical cancer, or breast carcinoma in situ.

[0379] 4. Subjects with known or suspected autoimmune diseases within 2 years before the first use of the investigational drug, excluding the following: a. Well-controlled type 1 diabetes; b. Hypothyroidism that only requires hormone replacement therapy and is well-controlled; c. Skin diseases that do not require systemic treatment (such as vitiligo, psoriasis, or alopecia); d. Subjects whose disease is not expected to relapse in the absence of external triggers.

[0380] 5. Patients with uncontrollable third cavity effusion (eg, pleural effusion, ascites, or pericardial effusion) who are judged by the researchers to be unsuitable for inclusion in the study.

[0381] 6. A history of severe cardiovascular disease within 6 months before the first use of the investigational drug, including but not limited to:

[0382] a). Severe cardiac rhythm or conduction abnormalities, such as ventricular arrhythmia requiring clinical intervention, third-degree atrioventricular block, etc.;

[0383] b). History of myocardial infarction, angina pectoris, angioplasty, or coronary artery bypass surgery;

[0384] c) Heart failure, New York Heart Association (NYHA) grade III or above;

[0385] d). Poorly controlled hypertension (systolic blood pressure ≥150 mmHg and / or diastolic blood pressure ≥95 mmHg despite optimal treatment).

[0386] 7. Patients with prolonged QT / QTc interval on electrocardiogram during the screening period (QTcF>480ms, Fridericia formula: QTcF=QT / RR0.33, RR=60 / heart rate) and / or left ventricular ejection fraction (LVEF) ≤50% on echocardiography (ECHO) or multi-gated acquisition (MUGA) technology during the screening period;

[0387] 8. Patients with HCV antibody (+) positive during the screening period (patients with HCV RNA negative can be included, and anti-HCV treatment other than interferon is allowed), active hepatitis B (patients with HBV DNA ≤ 500 IU / ml can be included, and anti-HBV treatment other than interferon is allowed), known HIV positive or known acquired immunodeficiency syndrome (AIDS).

[0388] 9. Patients who have undergone major organ surgery (excluding puncture biopsy, tracheotomy, and gastrostomy) within 4 weeks before the first use of the investigational drug, or who need to undergo elective surgery during the trial.

[0389] 10. Toxic reactions from previous anti-tumor treatment have not recovered to grade 1 or below (CTCAE V5.0), excluding the following situations: grade 2 neuropathy, alopecia, hypothyroidism caused by previous anti-tumor treatment (including hormone replacement therapy), and toxicity judged by the investigator to have no safety risk.

[0390] 11. Patients who have received T cell co-stimulation or drugs acting on the immune checkpoint pathway (including PD-1, PD-L1 / 2, CTLA-4 inhibitors, etc.) in the past.

[0391] 12. Patients who have received other immunotherapy before and experienced irAE (immune-related adverse reactions) of grade ≥ 3.

[0392] 13. Patients who have received taxane treatment before (patients who have received taxane-containing induction chemotherapy before and progressed after 6 months can be included in the group).

[0393] 14. Patients who have received anti-tumor treatments such as chemotherapy, radiotherapy, targeted therapy, immunotherapy, and other clinical research drugs within 4 weeks before the first use of the investigational drug, and other conditions are as follows:

[0394] Local palliative radiotherapy within 2 weeks before the first use of investigational drugs; treatment with oral fluorouracil, small molecule targeted drugs, etc. within 2 weeks before the first use of investigational drugs or within 5 half-lives of known drugs (whichever is longer); taking traditional Chinese medicine with anti-tumor indications within 2 weeks before the first use of investigational drugs.

[0395] 15. Receiving corticosteroids (prednisone >10 mg / day or an equivalent dose of similar drugs) or other immunosuppressive treatment within 2 weeks before the first use of the investigational drug, except for the following situations: a. Treatment with topical, ocular, intra-articular, intranasal and inhaled glucocorticoids; b. Short-term use of glucocorticoids for preventive treatment (e.g., prevention of contrast agent allergy).

[0396] 16. Received a live attenuated vaccine within 2 weeks before the first use of the investigational drug or planned to receive it during the study.

[0397] 17. Patients who have used strong inhibitors or inducers of CYP3A4 within 2 weeks before the first use of the investigational drug.

[0398] 18. Patients with known grade ≥ 3 hypersensitivity reaction and / or contraindications to albumin or monoclonal antibodies.

[0399] 19. Other situations that the researchers consider to be unsuitable for the subject to participate in the clinical trial, including but not limited to: the subject has concurrent serious or uncontrolled medical conditions that interfere with the interpretation of the research results and affect trial compliance, etc.

[0400] 2.3 Termination of treatment / withdrawal criteria

[0401] Subjects can continue to use the investigational drugs until they meet the criteria for discontinuation of treatment / withdrawal, or until they have completed 2 years of treatment.

[0402] Criteria for termination of treatment:

[0403] · Withdrawal of informed consent;

[0404] Pregnancy or pregnancy planning;

[0405] Poor compliance with the study intervention;

[0406] · The occurrence of clinical adverse events (AEs), laboratory abnormalities, or other medical conditions, continued participation in the study would not be in the best interest of the subject;

[0407] If the disease progresses, treatment can be continued if the following conditions are met:

[0408] √The subjects tolerate the test drugs;

[0409] √ Clinically stable as assessed by the investigator;

[0410] √ Subsequent treatment will not delay interventions for other serious complications;

[0411] Serious deviations from the protocol affect the evaluation of this study;

[0412] ·Having received the investigational drug for 2 years;

[0413] Termination by other investigators’ decision;

[0414] Exit criteria:

[0415] Withdraw the informed consent and refuse to accept follow-up;

[0416] Lost to follow-up;

[0417] ·die;

[0418] 3. Research Results

[0419] In this example, docetaxel (albumin-bound) for injection is combined with Nivolumab to study its clinical efficacy in recurrent or metastatic head and neck squamous cell carcinoma, without the need for dexamethasone pretreatment, to further improve the efficacy of immune checkpoint inhibitors in patients with recurrent or metastatic head and neck squamous cell carcinoma, increase the response rate, prolong the overall survival (OS), reduce the risk of death, and benefit patients.

[0420] The results of this study showed that the combination of the two drugs not only did not cause new safety issues, but also produced a significant synergistic effect in the treatment of head and neck squamous cell carcinoma. This shows that the combination of the docetaxel albumin composition and the immune checkpoint inhibitor of the present application can produce a synergistic effect in the treatment of tumors, significantly improving the anti-tumor effect.

Claims

1. Use of a composition containing docetaxel albumin nanoparticles in the preparation of an anti-tumor drug, characterized in that: The composition comprises docetaxel and acid-denatured albumin; the acid-denatured albumin is obtained by adding acid to human albumin and then denaturing it to an appropriate pH value, the acid is cysteine ​​hydrochloride, the appropriate pH value is 3.8-4.7, the content of sodium octanoate in the human albumin is not higher than 0.08 mmol / g protein, and the mass ratio of docetaxel to human albumin is 1:(2.0-10.0) based on anhydrous docetaxel.

2. Use of a composition containing docetaxel albumin nanoparticles in the preparation of an anti-tumor drug, characterized in that: The composition is prepared from docetaxel and acid-denatured albumin; the acid-denatured albumin is obtained by adding acid to human albumin and then denaturing it to an appropriate pH value, the acid is cysteine ​​hydrochloride, the appropriate pH value is 3.8-4.7, the content of sodium octanoate in the human albumin is not higher than 0.08 mmol / g protein, and the mass ratio of docetaxel to human albumin is 1:(2.0-10.0) based on anhydrous docetaxel.

3. The use according to claim 1 or 2, wherein the composition containing docetaxel albumin nanoparticles is combined with an immune checkpoint inhibitor for preparing the anti-tumor drug, and the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

4. Use of a docetaxel albumin composition in the preparation of a medicament for improving the efficacy of an immune checkpoint inhibitor in treating tumors, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor, wherein the composition comprises docetaxel and acid-denatured albumin; the acid-denatured albumin is obtained by denaturing human albumin after adding acid to an appropriate pH value, the acid is cysteine ​​hydrochloride, the appropriate pH value is 3.8-4.7, the content of sodium octanoate in the human albumin is not higher than 0.08 mmol / g protein, and the mass ratio of docetaxel to human albumin is 1:(2.0-10.0) based on anhydrous docetaxel.

5. Use of a docetaxel albumin composition in the preparation of a drug for improving the efficacy of immune checkpoint inhibitors in treating tumors, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor, wherein the composition is made of docetaxel and acid-denatured albumin; the acid-denatured albumin is obtained by denaturing human albumin after adding acid to an appropriate pH value, the acid is cysteine ​​hydrochloride, the appropriate pH value is 3.8-4.7, the content of sodium octanoate in the human albumin is not higher than 0.08 mmol / g protein, and the mass ratio of docetaxel to human albumin is 1:(2.0~10.0) based on anhydrous docetaxel.

6. A compound drug or drug combination product for treating tumors, comprising a docetaxel albumin composition and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor, wherein the docetaxel albumin composition comprises docetaxel and acid-denatured albumin; the acid-denatured albumin is obtained by denaturing human albumin after adding acid to an appropriate pH value, the acid is cysteine ​​hydrochloride, the appropriate pH value is 3.8-4.7, the content of sodium octanoate in the human albumin is not higher than 0.08 mmol / g protein, and the mass ratio of docetaxel to human albumin is 1:(2.0~10.0) based on anhydrous docetaxel.

7. A compound drug or drug combination product for treating tumors, comprising a docetaxel albumin composition and an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor, wherein the docetaxel albumin composition is made of docetaxel and acid-denatured albumin; the acid-denatured albumin is obtained by denaturing human albumin after adding acid to an appropriate pH value, the acid is cysteine ​​hydrochloride, the appropriate pH value is 3.8-4.7, the content of sodium octanoate in the human albumin is not higher than 0.08 mmol / g protein, and the mass ratio of docetaxel to human albumin is 1:(2.0~10.0) based on anhydrous docetaxel.

8. The use according to any one of claims 1 to 5, or the compound medicine or drug combination product according to claim 6 or 7, characterized in that: The docetaxel is anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate.

9. The use according to any one of claims 1 to 5, or the compound medicine or drug combination product according to claim 6 or 7, characterized in that: (1) The appropriate pH value is 4.0 to 4.5; and / or (2) the content of sodium octanoate in the human albumin is 0.03-0.08 mmol / g protein; and / or (3) Based on anhydrous docetaxel, the mass ratio of docetaxel to human albumin is 1:(3.0-7.0).

10. The use according to any one of claims 1 to 5, or the compound medicine or drug combination product according to claim 6 or 7, characterized in that: The composition is a suspension; the suspension contains 2-10 mg / ml of docetaxel; the pH value of the suspension is 3.9-4.8; and the suspension contains 0-1.8% (w / v) of sodium chloride.

11. The use according to any one of claims 1 to 5, or the compound medicine or drug combination product according to claim 6 or 7, characterized in that: The preparation method of the composition comprises the following steps: (1) dissolving docetaxel in an organic solvent to obtain an organic phase solution; (2) taking a human albumin solution, adding acid to adjust the pH value, and obtaining an acid-denatured albumin aqueous solution; and taking another salt solution; (3) mixing the organic phase solution, the aqueous phase solution and the salt solution to carry the drug to obtain a drug-loaded solution; (4) Dialysis; in, Calculated on the basis of anhydrous docetaxel, the mass ratio of docetaxel to human albumin is 1:(2.0-10.0); In step (2), the content of sodium octanoate in the human albumin solution is not higher than 0.08 mmol / g protein, the acid is cysteine ​​hydrochloride, and the pH value is 3.8-4.7; Step (2) optionally comprises the step of diluting the human albumin solution with water for injection to obtain an albumin dilution solution before adding acid to adjust the pH value; Step (2) optionally comprises an incubation step after adding acid to adjust the pH value; Step (3) optionally comprises a cooling step after mixing the drug-loaded mixture; Step (4) optionally comprises the step of concentrating the drug-loaded solution obtained in step (3) to obtain a concentrated solution before dialysis; In step (4), sodium chloride solution is used as the dialysate for dialysis, Step (4) optionally comprises a concentration or dilution step after dialysis; Optionally, step (5) of sterilizing and filtering is included after step (4); Optionally, step (6) of freeze-drying is included after step (5).

12. The use according to any one of claims 1 to 5, or the compound medicine or drug combination product according to claim 6 or 7, characterized in that: The particle size of the docetaxel albumin nanoparticles is 60-200 nm.

13. The use, or compound medicine or drug combination product according to claim 12, characterized in that: The particle size of the docetaxel albumin nanoparticles is 90-150 nm.

14. The use, or the compound medicine or the drug combination product according to claim 13, characterized in that: The particle size of the docetaxel albumin nanoparticles is 90-135 nm.

15. The use according to any one of claims 1 to 5, or the compound medicine or pharmaceutical combination product according to claim 6 or 7, wherein the content of sodium octanoate in the albumin is 0.045-0.08 mmol / g protein.

16. The use according to any one of claims 1 to 5, or the compound medicine or pharmaceutical combination product according to claim 6 or 7, wherein the tumor is a solid tumor.

17. The use, or compound drug or drug combination product according to claim 16, wherein the tumor is selected from salivary gland cancer, esophageal cancer and esophagogastric junction cancer, undifferentiated thyroid cancer, ovarian cancer, head and neck cancer, colorectal cancer, liver cancer, melanoma, non-small cell lung cancer, breast cancer, gastric cancer, renal cell carcinoma, bile duct cancer, bladder cancer and urinary tract tumors, small cell lung cancer, pancreatic cancer, uterine tumors, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, lip cancer, maxillary sinus tumors, ethmoid sinus tumors or bone tumors.

18. The use, or the compound medicine or the pharmaceutical combination product according to claim 17, wherein the tumor is selected from colon cancer, liver cancer, melanoma or head and neck squamous cell carcinoma.

19. The use, or the compound medicine or the pharmaceutical combination product according to claim 18, wherein the tumor is selected from melanoma or head and neck squamous cell carcinoma.

20. The use according to any one of claims 1 to 5, or the compound drug or drug combination product according to claim 6 or 7, wherein the drug or the compound drug or drug combination product is formulated into a clinically acceptable preparation; and optionally, the docetaxel albumin composition and the immune checkpoint inhibitor are present in the same preparation, or are formulated into different preparations respectively.

21. The use, compound medicine or pharmaceutical combination product according to claim 20, wherein the preparation is in the form of an injection.

22. The use, compound medicine or pharmaceutical combination product according to claim 21, wherein the preparation is a liquid injection, a powder for injection or a tablet for injection.

23. The use, compound medicine or pharmaceutical combination product according to claim 22, wherein the preparation is a liquid injection, and the content of docetaxel active ingredient is 3.3-4.3 mg / ml based on anhydrous docetaxel.

24. The use according to any one of claims 3 to 5, or the compound drug or drug combination product according to claim 6 or 7, wherein the PD-1 inhibitor or PD-L1 inhibitor is selected from recombinant humanized anti-PD-1 monoclonal antibody injection, recombinant anti-PD-1 fully human monoclonal antibody injection (PD-1 monoclonal antibody), Atezolizumab, Avelumab, Durvalumab or fully human anti-PD-L1 antibody injection.

25. The use according to any one of claims 1 to 5, or the compound medicine or drug combination product according to claim 6 or 7, wherein the medicine or the compound medicine or drug combination product further comprises other tumor therapeutic agents.

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