WO2022247846A1 - 多西他赛白蛋白组合物和免疫检查点抑制剂的组合及用途 - Google Patents

多西他赛白蛋白组合物和免疫检查点抑制剂的组合及用途 Download PDF

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WO2022247846A1
WO2022247846A1 PCT/CN2022/094859 CN2022094859W WO2022247846A1 WO 2022247846 A1 WO2022247846 A1 WO 2022247846A1 CN 2022094859 W CN2022094859 W CN 2022094859W WO 2022247846 A1 WO2022247846 A1 WO 2022247846A1
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docetaxel
albumin
drug
cancer
injection
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PCT/CN2022/094859
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French (fr)
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李春雷
张晓君
姚雪坤
赫晓林
李桂霞
祁欢欢
束云
赵媛媛
陈东健
刘佳慧
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石药集团中奇制药技术（石家庄）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the 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 medicines for treating tumors.
  • Docetaxel is produced semisynthetically from a non-cytotoxic precursor (10-deacetylbaccatin III) extracted from the needles of Taxus chinensis. It is an analogue of paclitaxel and has stronger antitumor activity than paclitaxel.
  • Docetaxel is poorly water-soluble, and the current commercially available preparation is its common injection, which was first developed by Sanofi, and its trade name is The conventional dosage is once every 3 weeks, intravenous infusion for 1 hour, and the dose is 75mg/m 2 .
  • the representative docetaxel injection due to the use of ethanol and Tween 80 in the formula, is likely to cause severe allergic reactions, requires pretreatment with dexamethasone, and the patient's compliance is poor; in addition, ethanol will affect the central nervous system, and the infusion needs to be reduced. speed to reduce poisoning symptoms.
  • human albumin is an endogenous substance in the human body and has good biocompatibility, it can be used as a natural carrier of hydrophobic drugs to increase the solubility of insoluble drugs.
  • U.S. Abraxis company uses human serum albumin as auxiliary material, adopts the paclitaxel for injection (albumin binding type) (trade name) of emulsification development ) was approved by the FDA in 2005 for the treatment of metastatic breast cancer that failed combined chemotherapy or breast cancer that recurred within 6 months of adjuvant chemotherapy, and was subsequently approved for the treatment of non-small cell lung cancer, pancreatic cancer and gastric cancer (Japan ).
  • 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 it can be administered quickly at high concentrations, shortening the infusion time to less than 30 minutes, significantly improving the Patient compliance; due to the improvement of safety, the dosage of paclitaxel can be increased from 175mg/m 2 to 260-300mg/m 2 ;
  • the polyoxyethylene castor oil in the formula will inhibit the combination of paclitaxel and albumin, and the formula does not contain polyoxyethylene castor oil, which can make full use of the unique "gp60-caveolin-SPARC" channel of albumin to enrich the drug to the tumor area , thereby increasing the curative effect.
  • researchers from various countries are trying to use human albumin to develop albumin nanoparticles for other drugs, such as docetaxel.
  • docetaxel albumin composition disclosed in the prior art, one is to add a large amount of chelating agent as a stabilizer to improve the physical stability of the product, but the impact on its chemical stability has not been investigated; in addition, a large amount of chelating agent While improving stability, a hypertonic solution is formed, which is inconvenient for clinical use.
  • Chemotherapy drugs include cisplatin, carboplatin, paclitaxel, docetaxel, Pemetrexed, gemcitabine, irinotecan, etc.
  • the representative docetaxel injection due to severe allergic reactions, must be pretreated clinically with dexamethasone, presumably this will affect the effect of its combination with PD-1 antibody.
  • This 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.
  • corticosteroids such as dexamethasone
  • the combined use of the docetaxel albumin composition of the present application and an immune checkpoint inhibitor has a significant anti-tumor effect, resulting in a synergistic anti-tumor effect.
  • the present application provides the use of a docetaxel albumin composition and an immune checkpoint inhibitor in the preparation of a drug for treating tumors.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the present application provides the use of the docetaxel albumin composition in the preparation of a drug for improving the effect of immune checkpoint inhibitors in treating tumors.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the present application provides a compound drug or drug combination product for treating tumors, which comprises (1) a docetaxel albumin composition, and (2) an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the present application provides a method for treating a tumor in an individual (such as a patient or a 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.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the present application provides a method for improving the efficacy of an immune checkpoint inhibitor on a tumor, comprising administering a therapeutically effective amount of docetaxel albumin to a tumor-bearing individual who has received immune checkpoint inhibitor therapy combination.
  • the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.
  • the tumor is a solid tumor.
  • 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, cholangiocarcinoma, bladder cancer and urinary tract tumors, cervical cancer, 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, bone tumors.
  • the solid tumor is colon cancer, liver cancer, melanoma or head and neck squamous cell carcinoma (head and neck squamous cell carcinoma for short), in a more preferred embodiment, the solid tumor is melanoma or Squamous cell carcinoma of the head and neck.
  • the docetaxel albumin composition is a docetaxel albumin nanoparticle composition, wherein the particle size of the docetaxel albumin nanoparticles is preferably about 60-200nm, more preferably 90-150nm, for example 90-135nm.
  • the drug or the compound drug or drug combination product is formulated into a clinically acceptable preparation, preferably in the form of injection, including liquid injection, powder for injection, and tablet for injection.
  • the docetaxel albumin composition and the immune checkpoint inhibitor are present in the same formulation, or are separately formulated.
  • a composition comprising docetaxel albumin nanoparticles and a PD-1 inhibitor or a PD-L1 inhibitor are present in the same formulation.
  • the composition containing docetaxel albumin nanoparticles and the PD-1 inhibitor or PD-L1 inhibitor are formulated separately.
  • the docetaxel albumin composition for example, a composition containing docetaxel albumin nanoparticles
  • the content of the active ingredient of sitaxel is 3.3-4.3 mg/ml, preferably 4 mg/ml.
  • the PD-1 inhibitor or PD-L1 inhibitor is selected from recombinant humanized anti-PD-1 monoclonal antibody injections such as Nivolumab, and recombinant anti-PD-1 fully human monoclonal antibodies such as Pembrolizumab Injection (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.
  • recombinant humanized anti-PD-1 monoclonal antibody injections such as Nivolumab
  • recombinant anti-PD-1 fully human monoclonal antibodies such as Pembrolizumab Injection (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 Ni
  • the drug or the compound drug or drug combination product further includes other drugs for treating the tumor.
  • the present application also provides the use of docetaxel albumin nanoparticles combined with PD-1 inhibitors or PD-L1 inhibitors in the preparation of drugs for treating tumors.
  • the PD-1 inhibitor or PD-L1 inhibitor is selected from Nivolumab.
  • the tumor is head and neck squamous cell carcinoma.
  • the head and neck squamous cell carcinoma is recurrent or metastatic head and neck squamous cell carcinoma.
  • the recurrent or metastatic squamous cell carcinoma of the head and neck mentioned in this application includes patients who cannot receive radical local treatment again and distantly metastatic squamous cell carcinoma of the head and neck that originates in the oral cavity, oropharynx, hypopharynx and larynx.
  • the present application provides a drug for treating tumors comprising docetaxel albumin nanoparticles and Nivolumab.
  • 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. etc.) First-line and second-line drugs approved by drug regulatory authorities for the treatment of recurrent or metastatic head and neck squamous cell carcinoma.
  • the drug is in the form of injection, including liquid injection, powder for injection, tablet for injection and the like.
  • the docetaxel albumin nanoparticles are powder for injection, based on anhydrous docetaxel
  • the active ingredient is 80 mg/bottle
  • the active ingredient is 3.3-4.3 mg/ml, preferably 4 mg/ml.
  • Nivolumab is an injection, it contains Nivolumab 100mg/bottle.
  • the present application provides a combination regimen for treating tumors, which includes administering therapeutically effective doses of docetaxel albumin nanoparticles for injection and Nivolumab to tumor patients.
  • the administration is preferably injection administration.
  • the present application also provides a method for improving the curative effect 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.
  • the administration mode of docetaxel albumin nanoparticles for injection is intravenous administration.
  • the administration period is once every 3 weeks.
  • the dosage range is 25-200mg/m 2 .
  • the infusion administration time of the albumin pharmaceutical preparation is 30 min-60 min, preferably 60 ⁇ 10 min.
  • the dosage of docetaxel albumin nanoparticles for injection is 50, 75, 100, 125, 150 mg/m 2 , once every 3 weeks (denoted as Q3W);
  • the dosage of docetaxel albumin nanoparticles for injection is 30, 40, 50 mg/m 2 , administered once a week, administered for 3 weeks, stopped for 1 week, and 4 weeks 1 cycle (denoted as QW 3/4);
  • the dosage of docetaxel albumin nanoparticles for injection is 50, 60, 75 mg/m 2 , once every 2 weeks (denoted as Q2W);
  • the dosage of docetaxel albumin nanoparticles for injection is 30, 40, 50 mg/m 2 , administered once a week, administered for 2 weeks, stopped for 1 week, and 3 weeks for One cycle (denoted as QW 2/3).
  • 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 docetaxel albumin administration. Time to dosing. The administration period is the same as that of docetaxel albumin nanoparticles for injection.
  • intravenous infusion of Nivolumab 360mg Q3W, 30min followed by intravenous infusion of docetaxel for injection (albumin-bound) 75mg/ m2 Q3W, 60min, or intravenous infusion of Nivolumab 360mg Q3W, 30min, Afterwards, docetaxel for injection (albumin-bound) 100 mg/m 2 Q3W was administered intravenously for 60 minutes.
  • the doses of the docetaxel albumin nanoparticles for injection described in this application are all calculated by anhydrous docetaxel.
  • the term "individual” refers to a mammal, such as a human being, but may also be other mammals, such as livestock or laboratory animals and the like.
  • treating means administering a compound or formulation described herein to ameliorate 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) remission of a disease or disease state, even if the disease or disease state regresses.
  • terapéuticaally effective amount means that 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” will vary 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 a person skilled in the art according to its own knowledge and this disclosure.
  • the present application also provides a composition containing docetaxel albumin nanoparticles, which comprises or is made of docetaxel and acid-denatured albumin.
  • composition described above optionally further comprises an osmotic pressure regulator, or a pH regulator.
  • the docetaxel is preferably anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate.
  • the acid-denatured albumin is obtained by denaturing human serum albumin after adding acid to an appropriate pH value.
  • the acid is selected from acidic amino acids or acidic polypeptides, organic acids, inorganic acids.
  • the acidic amino acid or acidic polypeptide includes but not limited to cysteine hydrochloride, glutathione, etc.; the organic acid includes but not limited to citric acid, tartaric acid, etc.; the inorganic acid includes but not limited to hydrochloric acid, sulfuric acid, etc.
  • Said acid is preferably cysteine hydrochloride, glutathione, hydrochloric acid, more preferably cysteine hydrochloride.
  • the appropriate pH value is 3.5-5.5, preferably 3.5-5.0, more preferably 3.8-4.7, more preferably 4.0-4.5.
  • the mass ratio of docetaxel to human serum albumin is 1:(2.0-10.0), preferably 1:(3.0-7.0), more preferably 1:( 4.0 ⁇ 6.0).
  • the content of sodium octanoate in the human serum albumin is not higher than 0.08mmol/g protein, preferably 0.03-0.08mmol/g protein, more preferably 0.04-0.08mmol/g protein, more preferably 0.04-0.07 mmol/g protein.
  • the particle size of the docetaxel albumin nanoparticles is about 60-200 nm, preferably 90-150 nm, more preferably 90-135 nm.
  • the composition containing docetaxel albumin nanoparticles described in the present application contains an osmotic pressure regulator, so as to adjust the osmotic pressure within an appropriate range.
  • the type of the osmotic pressure regulator is not particularly limited, for example, it may 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 conditions such as the type and dosage of the diluent or reconstitution medium used clinically.
  • 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, most preferably 2.25:1.
  • the docetaxel albumin compositions described herein comprise a pH regulator to adjust the pH to an appropriate
  • “Stable” as used herein means that no settling of nanoparticles or turbidity of the suspension occurs.
  • the kind of the pH adjuster is not particularly limited.
  • the pH range is 3.4-5.8; preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8.
  • a docetaxel albumin composition (eg, a composition comprising docetaxel albumin nanoparticles) described herein is a suspension.
  • 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 60-200 nm, preferably 90-150 nm, more preferably 90-135 nm.
  • the suspension contains 0-1.8% (w/v) of sodium chloride, preferably 0.45%-1.8% (w/v), more preferably 0.9%-1.8% (w/v).
  • the suspension contains 2-10 mg/ml of docetaxel, preferably 2-8 mg/ml.
  • the suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours; at 2-8°C for at least 7 days, preferably at least 10 days.
  • the docetaxel albumin composition (for example, the composition containing docetaxel albumin nanoparticles) described in the present application is a lyophilized powder.
  • the particle size of the docetaxel albumin nanoparticles is about 60-200nm, preferably 90-150nm, more preferably 90-135nm.
  • 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, most preferably 2.25:1.
  • the lyophilized powder is lyophilized 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 60-200 nm, preferably 90-150 nm, more preferably 90-135 nm.
  • the suspension contains 0-1.8% (w/v) of 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; at 2-8°C for at least 7 days, preferably at least 10 days.
  • the lyophilized powder is reconstituted into suspension using a reconstitution medium.
  • the reconstitution medium is selected from water for injection, sodium chloride solution or glucose solution, preferably water for injection.
  • the pH 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 the docetaxel albumin nanoparticles in the reconstituted suspension is about 60-200 nm, preferably 90-150 nm, more preferably 90-135 nm.
  • 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; at 2-8°C for at least 7 days, preferably at least 10 days.
  • the composition comprising docetaxel albumin nanoparticles described herein is stable at 25°C for at least 36 months after lyophilization.
  • the “stable” mentioned here includes, but is not limited to, no obvious degradation of docetaxel, no obvious aggregation of protein nanoparticles, no obvious increase in particle size, no significant increase in docetaxel content, moisture, acidity, osmotic pressure or molar concentration, etc. No significant changes.
  • the “stabilization” can be one or more of the situations listed above.
  • the “stable” means that there is no significant change in the content of 7-epidocetaxel and/or albumin multimers.
  • the present application also provides a medicament made from the above-mentioned composition containing docetaxel albumin nanoparticles.
  • the medicine is in a clinically acceptable dosage form, preferably an injection, more preferably a liquid injection or a freeze-dried powder injection.
  • the pH 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) of sodium chloride, Preferably 0.45%-1.8% (w/v), more preferably 0.9%-1.8% (w/v), more preferably 0.9% (w/v).
  • the liquid injection contains 2-10 mg/ml of docetaxel, preferably 2-8 mg/ml.
  • the liquid injection is stable at 25°C for at least 24 hours, preferably at least 30 hours; at 2-8°C for at least 7 days, preferably at least 10 days.
  • stable means that there is no precipitation of nanoparticles or turbidity of the liquid injection.
  • the freeze-dried powder injection when the injection is a freeze-dried 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, most preferably 2.25:1.
  • the particle size of docetaxel albumin nanoparticles is about 60-200 nm, preferably 90-150 nm, more preferably 90-135 nm.
  • the pH of the suspension for preparing the freeze-dried powder injection before freeze-drying is 3.4-5.8, preferably 3.6-5.6, or 3.8-5.0, more preferably 3.9-4.8.
  • the suspension is stable for at least 24 hours at 25°C, and at least 10 days at 2-8°C.
  • 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 resulting reconstituted suspension has a pH of 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 reconstituted suspension is about 60-200 nm, preferably 90-150 nm, more preferably 90-135 nm.
  • 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%.
  • the reconstituted suspension contains 2-10 mg/ml of docetaxel, preferably 2-8 mg/ml.
  • the reconstituted suspension is stable at 25°C for at least 24 hours, preferably at least 30 hours; at 2-8°C for at least 7 days, preferably at least 10 days.
  • the present application also provides a method for preparing the composition containing docetaxel albumin nanoparticles, which includes the following steps:
  • step (2) optionally includes the step of diluting the human serum albumin solution with water for injection to obtain albumin dilution before adding acid to adjust the pH value.
  • step (2) optionally includes an incubation step after adding acid to adjust the pH value.
  • step (3) optionally includes a cooling step after mixing and loading drugs.
  • step (4) optionally includes, before dialysis, concentrating the drug-loaded solution obtained in step (3) to obtain a concentrated solution.
  • a concentration or dilution step is optionally included to adjust the concentration of docetaxel in the suspension after dialysis.
  • step (4) optionally includes step (5) sterile filtration after step (4).
  • step (6) freeze-drying is optionally included after step (5).
  • the docetaxel in step (1) is in any form, preferably anhydrous docetaxel, docetaxel hemihydrate or docetaxel trihydrate.
  • the mass ratio of docetaxel to human serum albumin is 1:(2.0-10.0), preferably 1:(3.0-7.0), more preferably 1:(4.0-6.0).
  • the organic solvent in step (1) is selected from solvents miscible with water, such as ethanol, methanol, acetone, DMSO, etc., preferably ethanol.
  • the organic phase solution contains 45-90 mg/ml of docetaxel, preferably 45-70 mg/ml.
  • the content of sodium octanoate in the human serum albumin solution in step (2) is not higher than 0.12mmol/g protein, preferably not higher than 0.08mmol/g protein, preferably 0.045-0.08mmol/g protein.
  • the albumin dilution contains 6-25 mg/ml albumin, preferably 10-20 mg/ml, more preferably 12-18 mg/ml, more preferably 15 mg/ml.
  • the acid is selected from acidic amino acids or acidic polypeptides, organic acids, and inorganic acids.
  • the acidic amino acid or acidic polypeptide includes but not limited to cysteine hydrochloride, glutathione, etc.; the organic acid includes but not limited to citric acid, tartaric acid, etc.; the inorganic acid includes but not limited to hydrochloric acid, sulfuric acid, etc.
  • the acid is preferably cysteine hydrochloride, glutathione, hydrochloric acid, more preferably cysteine hydrochloride.
  • the pH value is preferably 3.5-5.5, preferably 3.5-5.0, more preferably 3.8-4.7, more preferably 4.0-4.5.
  • step (2) the incubation refers to raising the temperature to 35°C-42°C, preferably 38°C-42°C after adding acid to adjust the pH value; incubating for more than 30min, preferably 30min-60min.
  • the salt solution described in step (2) is selected from the aqueous solution of sodium chloride, potassium chloride, sodium sulfate or magnesium sulfate, preferably the aqueous solution of sodium chloride; the concentration of the salt solution is not less than 2%, preferably 2% ⁇ 35%, more preferably 10% to 20%.
  • the drug loading condition is to raise the temperature of 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.
  • the temperature reduction in step (3) refers to temperature reduction to below room temperature, preferably 0-20°C, more preferably 7-15°C.
  • the concentrated solution in step (4) contains about 4-10 mg/ml docetaxel, preferably 6-10 mg/ml, more preferably 7-9 mg/ml, more preferably 8 mg/ml.
  • step (4) is dialysis to remove excess small molecule compounds, there are no special restrictions on the type and amount of dialysate, and the molecular weight cut-off of the dialysis membrane, and those skilled in the art can make a choice based on common technical knowledge or experience.
  • 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.
  • sodium chloride solution is used as the dialysate for dialysis.
  • the molecular weight cut-off 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, preferably 3-10 times, more preferably 3-6 times that of the drug-carrying solution or concentrated solution.
  • the concentration of the sodium chloride solution is not higher than 1.8% (w/v), preferably 0.45%-1.8% (w/v), more preferably 0.9%-1.8% (w/v).
  • the human serum albumin solution used in step (2) needs to adjust the content of sodium octanoate in advance.
  • Those skilled in the art can select an appropriate method to adjust the content of sodium caprylate in the human serum albumin solution according to common technical knowledge or experience, including but not limited to dialysis.
  • the method for adjusting the content of sodium octanoate in the human serum albumin solution is:
  • the dilution factor of the human albumin solution is not less than 4 times, preferably 4-7 times.
  • the molecular weight cut-off of the dialysis membrane is 10-50KDa, preferably 10-30KDa, more preferably 10KDa or 30KDa.
  • the volume of the dialysate can be determined by those skilled in the art through routine tests according to the required sodium octanoate content.
  • the volume of the dialysate is about 3 times or more, preferably 3-10 times, more preferably 3-6 times the volume of the diluted albumin solution.
  • the human serum albumin solution with low sodium caprylate content contains sodium caprylate lower than 0.16mmol/g protein, preferably lower than 0.12mmol/g protein, preferably lower than 0.10mmol/g protein, preferably lower than 0.08 mmol/g protein.
  • the human serum albumin solution with low sodium octanoate content can be directly used to prepare the docetaxel albumin nanoparticle composition described in this application, and can also be mixed with other human serum albumin solutions with sodium octanoate content in proportion to obtain After reaching the desired content, it is used to prepare the docetaxel albumin nanoparticle composition.
  • the present application also provides a composition containing docetaxel albumin nanoparticles, which is prepared by the method described in the eighth aspect.
  • the dialysis step will remove excess small molecule compounds in the drug-carrying solution or concentrated solution.
  • 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 little free acidic Amino acids or acidic peptides.
  • Said "almost no free acidic amino acid or acidic polypeptide" means that the content of free acidic amino acid or acidic polypeptide in the composition is lower than 0.25% (w/w) of docetaxel.
  • acid-denatured albumin is prepared using cysteine hydrochloride or glutathione to adjust the pH, the content of free cysteine or glutathione in the composition is lower than that of Docey 0.25% (w/w) of his race.
  • the present application also provides a medicine, which is prepared from the composition containing docetaxel albumin nanoparticles prepared by the above method.
  • the medicine is in a clinically acceptable dosage form, preferably an injection, more preferably a liquid injection or a freeze-dried powder injection.
  • the present application also provides a method for adjusting the content of sodium octanoate in human serum albumin solution, comprising the following steps: taking a commercially available human serum albumin solution and diluting it with water for injection or normal saline to obtain the diluted Albumin solution: Diluted albumin solution is dialyzed with water for injection or normal saline as dialysate to partially remove sodium octanoate to obtain human serum albumin solution with low sodium octanoate content.
  • the dilution factor of the human albumin solution is not less than 4 times, preferably 4-7 times.
  • the molecular weight cut-off of the dialysis membrane is 10-50KDa, preferably 10-30KDa, more preferably 10KDa or 30KDa.
  • the volume of the dialysate can be determined by those skilled in the art through routine tests according to the required sodium octanoate content.
  • the volume of the dialysate is about 3 times or more, preferably 3-10 times, more preferably 3-6 times the volume of the diluted albumin solution.
  • the human serum albumin solution with low sodium caprylate content contains sodium caprylate lower than 0.16mmol/g protein, preferably lower than 0.12mmol/g protein, preferably lower than 0.10mmol/g protein, preferably lower than 0.08 mmol/g protein.
  • the human serum albumin solution with low sodium octanoate content can be directly used to prepare the docetaxel albumin nanoparticle composition described in the present application, and can also be combined with other human serum albumin solutions with sodium octanoate content. After the protein solution is mixed in proportion to obtain the desired content, it is used to prepare the docetaxel albumin nanoparticle composition.
  • the content of docetaxel described in this application is all calculated by anhydrous docetaxel.
  • the numerical values or numerical ranges described in the present application can fluctuate up and down within the range understood by those skilled in the art without affecting the implementation of the present application.
  • the floating range is for example ⁇ 20%, or ⁇ 17%, or ⁇ 15% %, or ⁇ 12%, or ⁇ 10%, or ⁇ 9%, or ⁇ 8%, or ⁇ 7%, or ⁇ 6%, or ⁇ 5%, or ⁇ 4%, or ⁇ 3%, or ⁇ 2% , or ⁇ 1%.
  • the inventors of the present application unexpectedly found that sodium octanoate, a heat stabilizer in albumin, has a greater impact on the physical stability of docetaxel albumin nanoparticles, because sodium octanoate can compete with drugs for binding to albumin Hydrophobic sites reduce the binding force of the drug to the protein, resulting in instability of the nanosuspension.
  • the nanoparticles When directly using commercially available human serum albumin solution as an auxiliary material (containing sodium caprylate 0.16mmol/g protein), the nanoparticles promptly settled within 10 hours; however, when dialysis was used to reduce the sodium caprylate content in the albumin solution, When it is lower than 0.08mmol/g protein, the stability of the prepared nanoparticles is greatly improved, and can be kept stable for at least 24 hours at 25°C, and can be placed stably for at least 10 days at 2-8°C.
  • human serum albumin solution containing sodium caprylate 0.16mmol/g protein
  • the inventors of the present application also found that the 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.
  • a large amount of organic acid or its salt must be added as a stabilizer, resulting in drug penetration
  • the pressure is high, and it will cause obvious pain during clinical use, and cause osmotic damage to the cells and tissues at the injection site.
  • the present application uses acid-denatured albumin to prepare docetaxel albumin nanoparticles, and controls the content of sodium octanoate in the albumin solution without additional addition of other salt stabilizers. Small.
  • CN103054798A teaches that the stability of nanoparticles prepared from anhydrous docetaxel is significantly better than that of docetaxel trihydrate and hemihydrate.
  • the crystallization water of docetaxel that is, anhydrous, hemihydrate, and trihydrate has no effect on the stability of the prepared docetaxel albumin nanoparticle composition. This greatly expands the selection range of the use form of docetaxel, and has great industrial application value.
  • 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 application show that 7-epidocetaxel and protein polymers have little change.
  • the completed accelerated stability test shows that the freeze-dried powder can be stored at 30°C and 25°C. Stable storage for 18 months. According to the existing data, it is predicted that it can be stored stably for at least 20 months at 30°C, and at least 36 months at temperatures below 25°C.
  • the static stability observation test shows that the composition provided by the application, whether it is a suspension before lyophilization or a reconstituted suspension after lyophilization, can be stable for at least 24 hours at room temperature, and can be stable for at least 10 hours under refrigerated conditions. Days, there will be no turbidity of the suspension or sedimentation of the nanoparticles; stability studies for longer periods of time are in progress. Compared with the case where the reconstituted suspension of the commercially available product is only stable for 8 hours, the product of the present application greatly reduces the limitations of clinical use.
  • docetaxel is encapsulated in human serum albumin.
  • the preparation does not contain Tween-80 (Tween-80) and ethanol, and does not need to use corticosteroids (such as dexamethasone) for pretreatment.
  • corticosteroids such as dexamethasone
  • the combination of the docetaxel albumin composition described in the present application and the immune checkpoint inhibitor can significantly improve the anti-tumor effect and produce a synergistic effect.
  • the composition of docetaxel albumin and PD-1 inhibitor or PD-L1 inhibitor can act synergistically to significantly inhibit the growth of various tumors such as colon cancer, liver cancer, melanoma or squamous cell carcinoma of the head and neck, Especially in the treatment of melanoma or head and neck squamous cell carcinoma has obvious synergistic effect.
  • Figure 1 shows the effect of dexamethasone (DEX) pretreatment on (TAXOTERE) inhibits the effect of mouse liver cancer Hepa1-6 xenograft tumor growth. Among them, compared with solvent control group, *P ⁇ 0.05, **P ⁇ 0.01; compared with DEX/TAXOTERE group, ###P ⁇ 0.001.
  • Figure 2A shows the effect of DEX on the efficacy of Opdivo (Nivolumab) in inhibiting the efficacy of colon cancer MC38 xenografts in mice.
  • Figure 2B shows the comparison of the curative effect of Opdivo combined with DTX-HSA or DEX/TAXOTERE in inhibiting colon cancer MC38 xenografts in mice. Among them, compared with solvent control group, **P ⁇ 0.01; compared with DEX/Opdivo or TAXOTERE/DEX/Opdivo group, ##P ⁇ 0.01.
  • Figure 3 shows the comparison of the efficacy of Keytruda (pembrolizumab) combined with docetaxel for injection (albumin-bound) (DTX-HSA) or DEX/TAXOTERE in inhibiting colon cancer MC38 xenografts in mice.
  • Keytruda pembrolizumab
  • docetaxel for injection albumin-bound
  • DEX/TAXOTERE DEX/TAXOTERE
  • Figure 4A shows the effects of DTX-HSA and DEX/TAXOTERE on the body weight of mice with liver cancer Hepa1-6 transplanted tumors. Among them, compared with solvent control group, *P ⁇ 0.05, **P ⁇ 0.01; compared with DEX/TAXOTERE group, ###P ⁇ 0.001.
  • Figure 4B shows the comparison of the efficacy of DTX-HSA and DEX/TAXOTERE in inhibiting mouse liver cancer Hepa1-6 homologous transplantation. Among them, compared with solvent control group, *P ⁇ 0.05, **P ⁇ 0.01; compared with DEX/TAXOTERE group, ###P ⁇ 0.001.
  • Figure 5A shows the effect of Keytruda combined with DTX-HSA or DEX/TAXOTERE on the body weight of liver cancer Hepa1-6 xenograft mice. Among them, compared with solvent control group, *P ⁇ 0.05; compared with DEX/TAXOTERE/Keytruda group, #P ⁇ 0.05.
  • Figure 5B shows the comparison of the efficacy of Keytruda combined with DTX-HSA or DEX/TAXOTERE regimens in inhibiting mouse liver cancer Hepa1-6 xenografts. Among them, compared with solvent control group, ***P ⁇ 0.001; compared with DEX/TAXOTERE/Keytruda group, ##P ⁇ 0.01.
  • Figure 6 shows the comparison of the efficacy of DTX-HSA, Keytruda and their combined administration regimens in inhibiting Hepa1-6 xenograft tumors in mice. Among them, compared with the solvent control group, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 7 shows the comparison of the curative effects of DTX-HSA, Keytruda and their combined regimens in inhibiting B16/F10 transplanted tumors in mice. Among them, compared with the solvent control group, **P ⁇ 0.01, ***P ⁇ 0.001.
  • the stable means that no sedimentation of nanoparticles or turbidity of the suspension occurs.
  • reconstituted suspensions described in the examples are isotonic suspensions.
  • docetaxel albumin nanoparticle composition is as follows:
  • step (6) Take the suspension before freeze-drying obtained in step (5), and freeze-dry to obtain freeze-dried powder.
  • the dialysis step reduced the levels of sodium octanoate.
  • the concentration of sodium chloride in the post-dialysis suspension is essentially the same as the initial concentration of sodium chloride in the dialysate.
  • the particle size of nanoparticles in the suspension was detected by dynamic light scattering method, and the suspension was left to observe the sedimentation phenomenon.
  • the results in Table 2 show that the content of sodium chloride in the dialysate has no significant effect on the particle size of docetaxel albumin nanoparticles in the suspension before freeze-drying and in the reconstituted suspension.
  • the suspension before freeze-drying and the reconstituted suspension of each formula were left to stand at 25°C for 24 hours, and no solution turbidity or precipitation was found; after standing at 2-8°C for 10 days, no solution was turbid or precipitated . It is suggested that the pre-lyophilized suspension and the reconstituted suspension of each formulation are stable for at least 24 hours at 25°C, and at least 10 days at 2-8°C. There was no significant change in stability before and after lyophilization.
  • the inventor also referred to the Chinese Pharmacopoeia 2020 Edition Sibu General Rule 0512, and used high performance liquid chromatography to detect the content of cysteine in the suspension after dialysis. The results showed that the post-dialysis suspension contained almost no free cysteine (less than 0.25% (w/w) of docetaxel).
  • the inventor also detected the pH value of the suspension before and after dialysis, and found that the pH value of the suspension before and after dialysis remained basically unchanged.
  • a The lyophilized powder is reconstituted with water for injection (isotonic suspension).
  • Formulation 1-1 was reconstituted as an isotonic suspension containing docetaxel 4 mg/ml;
  • Formulation 1-2 was reconstituted as an isotonic suspension containing docetaxel 2 mg/ml;
  • Formulation 1-3 was reconstituted as an isotonic suspension containing docetaxel Docetaxel 8mg/ml isotonic suspension; formulations 1-4 were reconstituted as an isotonic suspension containing docetaxel 4mg/ml.
  • docetaxel trihydrate (8g in terms of anhydrous docetaxel) and dissolve it in 160ml absolute ethanol to obtain an organic phase solution after dissolving; get the human serum albumin solution (sodium caprylate) containing 16g albumin content of 0.08mmol/g protein), diluted with water for injection to a solution containing albumin 10mg/ml, then added an appropriate amount of cysteine hydrochloride to adjust the pH to 4.5, and incubated at 40°C for 1 hour to obtain an acid-denatured albumin aqueous phase solution; sodium chloride is prepared with water for injection to prepare a saline solution with a concentration of 10%; the organic phase solution and the aqueous phase solution are heated to 40°C, the organic phase, the aqueous phase and the saline solution are mixed for drug loading, and the obtained material is placed in an ice-water bath to cool down to 18°C to obtain the drug-loaded solution; concentrate the drug-loaded solution to a docetaxel-containing concentration of about
  • the particle size of docetaxel albumin nanoparticles was 101.3nm. After standing at 25°C for 24 hours and at 2-8°C for 10 days, no turbidity or precipitation was found in the solution Appear. Reconstitute the freeze-dried powder with water for injection to obtain a reconstituted suspension (isotonic suspension). The particle size of docetaxel albumin nanoparticles has no significant change, which is 103.5nm; the reconstituted suspension is left standing at 25°C for 24 hours , Standing at 2-8°C for 10 days, no turbidity or precipitation was seen in the solution. It is suggested that the suspension before lyophilization and the reconstituted suspension are stable at 25°C for at least 24 hours, and at 2-8°C for at least 10 days.
  • the suspension before lyophilization and the lyophilized powder were reconstituted with water for injection to obtain the reconstituted suspension (isotonic suspension), and the particle size of docetaxel albumin nanoparticles had no significant difference. It was 121.3nm; after standing at 25°C for 24 hours, or at 2-8°C for 10 days, no turbidity or precipitation appeared in the suspension before freeze-drying or the reconstituted suspension. It is suggested that the suspension before lyophilization and the reconstituted suspension are stable at 25°C for at least 24 hours, and at 2-8°C for at least 10 days. Detection by high performance liquid chromatography showed that the suspension after dialysis hardly contained free cysteine (the content was about 0.21% (w/w) of docetaxel). The pH value of the suspension was basically unchanged before and after dialysis.
  • suspension before freeze-drying and the reconstituted suspension mentioned above 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 crystal water does not affect the implementation of the present application.
  • the acceptable limit used to define the stability of the pharmaceutical composition is "the percentage content of 7-epidocetaxel ⁇ 1.0%”.
  • N/A* indicates that the experiment has not yet reached that time point and therefore no data are available.
  • the results in Table 4 show that the production 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 this application can effectively control the generation of 7-form docetaxel.
  • the results in Table 4 show that under the above three test conditions, the content of 7-epidocetaxel in the product of the present application is within a controllable range (content ⁇ 1.0%), and the content of protein multimers has no obvious change.
  • the product of this application can be stored stably for at least 20 months at 30°C, and at least 36 months at below 25°C.
  • the API raw material is other forms of docetaxel, such as hemihydrate and trihydrate
  • its chemical stability (7-epidocetaxel percentage content and protein polymer content)
  • the change of is similar to that when anhydrous docetaxel is used as raw material.
  • Chinese patent CN106137969A adds arginine, proline, etc. as inhibitors to the formula. It is believed that the effect of using arginine as an inhibitor is the best, and it can be stored at 2-8°C for at least 24 months, or even up to 30 months.
  • the sodium caprylate content in the human serum albumin solution is lower than 0.08mmol/g protein, it can maintain a stable suspension state at 25°C for more than 24 hours, and the stability time is more than twice that of the sample containing 0.16mmol/g protein.
  • the content of sodium octanoate in the human serum albumin solution is lower than 0.08mmol/g protein, the suspension before freeze-drying and the reconstituted isotonic suspension can be placed stably for at least 10 days at 2-8°C.
  • a The lyophilized powder is reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension).
  • Embodiment 7 aqueous phase solution pH value influences on stability
  • the process formula and preparation method refer to the formula 1-1 of Example 1, and only use cysteine hydrochloride in step (2) to adjust the pH of the aqueous phase solution to 7.0, 6.5, 6.0, 5.5, 5.0, 4.7, 4.1, 3.8, 3.5. Observe the changes of nanoparticle size and suspension stability in the suspension before freeze-drying and the reconstituted suspension.
  • the pH of the aqueous phase solution is within the range of 3.5-5.5, the obtained suspension before freeze-drying and the reconstituted suspension can maintain a stable suspension state for more than 20 hours at 25°C, and maintain a stable suspension state for at least 7 days at 2-8°C.
  • the suspension before freeze-drying and the reconstituted suspension can still maintain a stable suspension state after standing at 25°C for 30 hours and at 2-8°C for 10 days. Freeze-drying and reconstitution had no significant effect on stability.
  • the pH of the aqueous phase solution is above 6.0, the particle diameter of the obtained nanoparticles increases significantly, and the particle diameter of the nanoparticles increases to 180nm at pH 6.0.
  • the pH of the aqueous phase solution is higher than 6.0, the stability of the suspension drops significantly, and turbidity occurs within 1 hour, and the state of stable suspension of nanoparticles cannot be maintained.
  • a The lyophilized powder is reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension).
  • Embodiment 8 prepares the human serum albumin solution of low sodium octanoate content
  • the human serum albumin solutions with low sodium octanoate content in the above Examples 8-1 to 8-3 can be further concentrated.
  • Example 1-1 the drug-loaded nano suspension and the blank suspension were prepared (step (1) without adding the drug docetaxel), and the human serum albumin solution and the blank suspension were investigated by circular dichroism method. The proportion of each secondary structure of the protein in the liquid and the drug-loaded nanosuspension, the results are shown in Table 7.
  • Example 10 is prepared with reference to Example 1 of Chinese Patent CN 106137969 B (201510157393.1)
  • the suspension is white without opalescence, turbid by visual inspection, and many precipitates can be seen, and precipitation occurs after standing for 10 minutes.
  • the technicians believe that the stability of the obtained suspension is poor, and it is impossible to continue the follow-up 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-mentioned patent, which is enough to prove that the stability of the product of the present application is obviously better than that of the product in the patent CN106137969B.
  • Embodiment 11 investigates the influence of protein drug ratio 1.5 (reducing protein concentration simultaneously is 5mg/ml)
  • the drug-loaded solution was concentrated to a docetaxel-containing concentration of about 6 mg/ml to obtain a concentrated solution; an isotonic sodium chloride (0.9%, w/v) solution was used as a dialysate to carry out the concentrated solution 5-fold dialysis, the molecular weight cut-off of the dialysis membrane is 30KDa, to obtain a suspension after dialysis; sterilizing and filtering through a 0.45 ⁇ m+0.2 ⁇ m membrane to obtain a suspension before lyophilization; freeze-drying to obtain a lyophilized powder.
  • the particle size of docetaxel albumin nanoparticles was 91.38nm, and it was completely turbid after standing at 25°C for 20 hours.
  • the lyophilized powder was reconstituted with water for injection to obtain a reconstituted suspension (isotonic suspension), and the reconstituted suspension was visually turbid.
  • Embodiment 12 investigates the impact of different dialysates
  • the particle size of docetaxel albumin nanoparticles was 102.2nm. After standing at 25°C for 16 hours, there were many precipitates at the bottom. rate drops. Reconstitute the lyophilized powder with water for injection to obtain a reconstituted suspension (isotonic suspension). The particle size of docetaxel albumin nanoparticles has no significant change, which is 103.3nm; the reconstituted suspension has become turbid at 25°C for 2.5 hours .
  • the particle size of docetaxel albumin nanoparticles is 92.43nm. After standing at 25°C for 16 hours, there is a slight precipitation at the bottom. Standing at 2-8°C for 24 hours, light transmission rate drops. Use water for injection to reconstitute the lyophilized powder to obtain a reconstituted suspension (isotonic suspension). The particle size of docetaxel albumin nanoparticles has no significant change, which is 90.91nm; Precipitate.
  • Example 13 Maximum tolerated dose (MTD) and toxicity comparison of docetaxel for injection (albumin-bound) (DTX-HSA) and docetaxel injection (TAXOTERE) in nude mice
  • Docetaxel for injection (albumin-bound) (DTX-HSA), which is prepared by formula 1-1 in Example 1 of the present application and by the preparation method of Example 1.
  • This test defines the maximum tolerated dose (MTD) of intravenous administration of drugs to mice as the observation period, the animal does not appear death and irreversible toxic reactions, or no body weight loss of more than 15% for 3 consecutive days, the dose is determined Considered the maximum tolerated dose for acute single administration.
  • MTD maximum tolerated dose
  • the isotonic concentration of DTX-HSA after reconstitution is 3.801mg/mL, according to the allowable dosage volume for different routes of drug administration or blood collection to animals jointly issued by the European Federation of Pharmaceutical Industry Associations and the European Alternative Method Validation Center in 2001
  • the maximum administration volume of slow intravenous injection in mice is 25mL/kg. Three administrations within 24 hours, the maximum dose can be given to 285.1mg/kg.
  • Docetaxel for injection (albumin-bound): 285.1, 228.1, 182.5, 146.0 mg/kg (gradient 1.25).
  • Docetaxel injection (TAXOTERE): 187.5, 150, 120, 96 mg/kg (gradient 1.25).
  • mice with small body weight differences, and divide them into 8 groups according to weight balance, with 5 mice in each group, respectively DTX-HSA285.1, 228.1, 182.5, 146.0mg/kg group and TAXOTERE 187.5, 150, 120 , 96mg/kg group.
  • Dosing frequency 3 times within 24 hours, with an interval of 4 hours.
  • Dosage Calculate the dosage based on the latest weighing.
  • Body weight All animals were weighed once before the experiment, and animals with appropriate body weight were selected for the experiment. The animals were weighed once a day at a fixed time.
  • Death and dying record the time of death for dead animals, pay attention to increase the frequency of observation for dying animals, and determine the time of death during the experiment.
  • mice During the observation period, no mice died, no irreversible symptoms of toxicity, and no body weight loss exceeding 15% of the maximum dose for 3 consecutive days can be considered as the maximum tolerated dose (MTD) of the experimental drug.
  • MTD maximum tolerated dose
  • mice in the DTX-HSA 285.1 and 228.1mg/kg groups started to have slight hindlimb tremors from D4 and D7 respectively, and the hindlimb tremor symptoms of the animals in the 228.1mg/kg group recovered on D20, and the animals in the 285.1mg/kg group recovered on D22. There was no obvious abnormality in DTX-HSA182.5 and 146.0mg/kg animals.
  • mice in the TAXOTERE 187.5 and 150mg/kg groups started to experience mild to moderate hindlimb tremors starting from D4 and D6 respectively.
  • Animals in TAXOTERE 120 and 96mg/kg groups had no obvious abnormalities.
  • mice had no death, no irreversible symptoms of toxicity, and no body weight loss exceeding 15% of the maximum dose for 3 consecutive days. Observation time: 24 days.
  • the MTD of DTX-HSA nude mice is 228.1 mg/kg; the MTD of TAXOTERE nude mice is 150.0 mg/kg.
  • DTX-HSA was prepared according to the formula and preparation method of Example 1-1 (the same below).
  • mice in group 5 had reduced activity and could not stand 1 hour after administration; all male and female animals in group 6 (TAXOTERE control group) had reduced activity and could not stand , Ear skin/coat reddened, salivation was seen in 2 female animals, vomiting was seen in 4 female animals, loose stool/watery stool was seen in 2 female animals, and the above animals were given a single intramuscular injection within 1 hour after administration Symptoms improved and animal activities returned to normal after treatment with dexamethasone sodium phosphate injection (source: Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd., batch number: 181116). Animals in other groups (groups 1-4) had no allergic reaction.
  • Example 15 Effect of dexamethasone on the curative effect of docetaxel (mouse liver cancer Hepa1-6 model)
  • mice were used to establish a mouse liver cancer Hepa1-6 syngeneic xenograft model to evaluate the effect of dexamethasone on the anti-tumor effect of docetaxel.
  • Test method 50 female C57BL/6 mice were subcutaneously inoculated with mouse liver cancer Hepa1-6 cells (4 ⁇ 10 6 /mouse/0.1mL) in the armpit of the right forelimb, and on the 6th day after inoculation (recorded as D0 on the day), selected The 32 animals with good tumor growth were evenly divided into 4 groups according to the tumor volume, with 8 animals in each group.
  • the grouping and administration methods are as follows: Groups 1, 2, and 3 were given distilled water by intragastric administration at the same frequency as group 4. . Dosing volume 10mL/kg.
  • Tumor volume 1/2 ⁇ long diameter ⁇ short diameter2 , use a vernier caliper to measure the long and short diameter of the tumor.
  • Example 16 Effect of dexamethasone on the curative effect of Opdivo and its combined regimen with docetaxel (mice colon cancer MC38 model)
  • Test method 43 female humanized B-hPD-1 mice were subcutaneously inoculated with mouse colon cancer MC38 cells (2 ⁇ 10 6 /mouse/0.1 mL) in the armpit of the right forelimb, and on the 5th day after inoculation (the day was recorded as D0), 40 animals with good tumor growth were selected, and the average tumor volume was about 110 mm 3 . According to the tumor volume, it was evenly divided into 5 groups, with 8 animals in each group.
  • the grouping and administration methods are as follows:
  • Groups 12 and 5 were given distilled water by intragastric administration at the same frequency as group 3 was given DEX.
  • DTX-HSA administration volume is 20mL/kg, others are 10mL/kg.
  • the TAXOTERE/DEX/Opdivo group did not enhance the tumor inhibitory effect, but even slightly increased the tumor volume.
  • the tumor volume was significantly reduced.
  • the effect of DTX-HSA/Opdivo on inhibiting the growth of mouse colon cancer MC38 xenografts was significantly enhanced (P ⁇ 0.01) (See Figure 2B).
  • test results showed that given the same dose of Opdivo and docetaxel, compared with common preparations of docetaxel, DTX-HSA preparations do not need to use DEX pretreatment, can effectively avoid the disadvantages of combined use of DEX to reduce the efficacy of Opdivo, and can be more effective inhibit tumor growth.
  • Example 17 Effect of dexamethasone on the curative effect of docetaxel combined with Keytruda (mice colon cancer MC38 model)
  • Test method 30 female humanized B-hPD-1 mice were subcutaneously inoculated with mouse colon cancer MC38 cells (2 ⁇ 10 6 /mouse/0.1 mL) in the armpit of the right forelimb, 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 68 mm 3 . According to the tumor volume, it was evenly divided into 3 groups, with 8 animals in each group.
  • the grouping and administration methods are as follows:
  • Groups 1 and 2 were intragastrically administered distilled water at the same frequency as group 3 was administered DEX. Dosing volume 10mL/kg.
  • mice Biocytogen B-hPD-1 mice were used to establish a mouse liver cancer Hepa1-6 homologous xenograft tumor model, and the tumor inhibitory effect of dexamethasone on DTX-HSA and the combination of TAXOTERE and PD-1 antibody (Keytruda) was evaluated The impact of the effect.
  • Test method 60 female humanized B-hPD-1 mice were subcutaneously inoculated with mouse liver cancer Hepa1-6 cells (5 ⁇ 10 6 /mouse/0.1mL) in the armpit of the right forelimb. D0), select 48 animals with good tumor growth, and divide them into 6 groups according to the tumor volume, with 8 animals in each group. Distilled water was administered intragastrically at the same frequency.
  • DTX-HSA administration volume is 20mL/kg, others are 10mL/kg.
  • Example 19 Synergistic curative effect of combination of Keytruda and docetaxel (mouse melanoma B16/F10 model)
  • Biocytogen B-hPD-1 mice were used to establish a mouse melanoma B16/F10 syngeneic xenograft model to evaluate the combination of docetaxel for injection (albumin-bound) and PD-1 antibody Keytruda in anti-tumor effect.
  • Test method 40 female humanized B-hPD-1 mice were divided into 4 groups according to body weight, 10 animals in each group, all mice were inoculated with B16/F10 cells (1 ⁇ 10 6 /mouse/0.1mL), The day of inoculation is recorded as D0, and the grouping and administration methods are as follows:
  • This trial is a single-arm, multi-center phase Ib/II clinical study for subjects with PD-L1 positive recurrent or metastatic head and neck squamous cell carcinoma who have progressed on platinum-containing regimens. Efficacy, safety and pharmacokinetics of docetaxel combined with nivolumab.
  • the dosing regimen of docetaxel for injection is: 75mg/m 2 or 100mg/m 2 , Q3W, iv 30-60min
  • Nivolumab (360mg, Q3W) combined with ipilimumab (Ipilimumab) and two cycles of platinum-containing double-drug chemotherapy for the treatment of metastatic or recurrent NSCLC without EGFR and ALK gene mutations (CheckMate-9LA)
  • Nivolumab (360mg, Q3W) combined with XELOX (oxaliplatin + capecitabine) in the treatment of advanced or metastatic gastric cancer/gastroesophageal junction adenocarcinoma.
  • XELOX oxaliplatin + capecitabine
  • docetaxel for injection 100mg/m 2 Q3W has no DLT (dose limit toxicity) observed in patients with solid tumors, and has entered 125mg/m 2 Q3W Dose ramping phase.
  • the MTD maximum tolerated dose
  • the commonly used clinical dosage regimen is 75-100 mg/m 2 Q3W.
  • docetaxel for injection was chosen to be 75mg/m 2 or 100mg/m 2 Q3W, iv60min.
  • the combination regimen of this clinical study is: Nivolumab 360mg Q3W, iv30min combined with docetaxel for injection (albumin bound) 75mg/ m2 Q3W, iv60min, or Nivolumab 360mg Q3W, iv30min combined with docetaxel for injection Race (albumin-bound) 100mg/m 2 Q3W, iv60min.
  • One of the dosing regimens was selected for Phase II study based on safety and efficacy results.
  • Dose-finding starts with a low dose and proceeds sequentially. 3 subjects were initially enrolled in each dose group, and observed for 21 days. If no DLT occurred, a higher dose level study (up to 100mg/m 2 ) was carried out; The dose level continues to enroll 3 subjects for safety observation, if ⁇ 2 of the 6 subjects have DLT, a higher dose level study (up to 100mg/m 2 ) will be conducted; if 2 of the 3 subjects have DLT If a DLT occurs, the investigator and the sponsor will discuss and decide on the follow-up study.
  • DLT is defined as: the following toxic reactions related to the test drug occurring within the first cycle (21 days):
  • Safety events in subsequent dosing cycles are also one of the influencing factors for selecting the dose of the combined regimen.
  • Phase II study Comprehensively analyze the safety, tolerability, efficacy and pharmacokinetic study results of Phase Ib, determine RP2D (recommended dose for Phase 2), and conduct Phase II study.
  • Objective Response Rate During the period from the initiation of the trial drug to withdrawal from the study, the best overall response in the study as assessed by the investigator according to RECIST v1.1 (Evaluation Criteria for Efficacy and Response in Solid Tumors Based on Single Path Measurement) was a complete response Proportion of subjects with (CR) or partial response (PR) (ie, CR+PR). Subjects who are evaluated for PR or CR for the first time need to confirm the efficacy after 4 weeks.
  • Phase II trial adopts Simon's two-stage (Optimum) design.
  • the first stage needs to enroll 30 subjects. If the number of cases of remission (PR+CR) does not exceed 5, the trial will be terminated. Otherwise, the trial will advance to the second stage and continue to enroll 52 subjects. If If the total number of cases of two-stage remission exceeds 17, it is considered that the combined program has subsequent development value in this indication. A total of 82 subjects need to be enrolled in the two phases.
  • Phase Ib and Phase II received docetaxel for injection (albumin-bound) RP2D combined with Nivolumab until they met the criteria for termination of treatment or withdrawal from the trial, and the trial drug was used for a maximum of 2 years.
  • Tumor assessment was performed every 6 weeks (42 days ⁇ 7 days) and was confirmed by an independent imaging evaluation center (IRC).
  • IRC independent imaging evaluation center
  • Phase Ib PD-L1 test results from previous level 3 and above hospitals or central laboratories with CAP, CLIA or other equivalent qualification certification are acceptable, and the test results need to be reviewed by the sponsor's biomarker team. Or provide fresh or archived tumor tissue within 3 years for PD-L1 immunohistochemistry (IHC) detection.
  • IHC immunohistochemistry
  • Phase II fresh or archived tumor tissue within 3 years can be provided for PD-L1 immunohistochemistry (IHC) detection.
  • Oropharyngeal cancer subjects need to provide previous HPV p16 immunohistochemical test results, or qualified tumor tissue samples for testing HPV status.
  • At least one measurable lesion confirmed by CT or MRI previously received radiotherapy, disease progression or persistent tumor ⁇ 3 months after radiotherapy can be used as a measurable lesion.
  • the main organ function meets the following criteria within 7 days before treatment (no component blood transfusion, human granulocyte colony-stimulating factor (G-CSF), thrombopoietin (TPO) within 2 weeks before the administration of the test drug , interleukin-11 and erythropoietin (EPO) and other medical supportive treatment):
  • the serum pregnancy test is negative within 7 days before the first use of the test drug, and the subjects and their spouses must agree to take adequate contraception from signing the informed consent form to 6 months after the last dose During this period, women are not breastfeeding and men avoid sperm donation.
  • Active brain metastases and leptomeningeal metastases Those with brain metastases need to reach at least 8 weeks after treatment and within 28 days before the first use of the test drug, and those with MRI evidence showing no PD can be enrolled; at least 2 weeks before the first use of the test drug, they do not need to receive systemic therapy.
  • Cortisol hormone therapy prednisone > 10mg/day or equivalent dose of similar drugs
  • skull base lesions without definite evidence of dura mater or brain parenchymal involvement need to be considered after discussion with the sponsor's medical monitor No entry.
  • autoimmune diseases within 2 years before the first use of the investigational drug, except for the following conditions: a. Well-controlled type I diabetes; b. Well-controlled hypothyroidism that only needs hormone replacement therapy; c. Skin diseases that do not require systemic treatment (such as vitiligo, psoriasis or alopecia); d. Subjects who are not expected to relapse in the absence of external triggers.
  • Serious heart rhythm or conduction abnormalities such as ventricular arrhythmia, third-degree atrioventricular block, etc. that require clinical intervention;
  • HCV antibody (+) positive during the screening period HCV RNA negative can be included, and anti-HCV treatment other than interferon is allowed
  • active hepatitis B HCV DNA ⁇ 500IU/ml can be included, anti-HBV treatment other than interferon is allowed
  • known HIV positive or known acquired immunodeficiency syndrome AIDS
  • Previously received T cell co-stimulation or drugs acting on immune checkpoint pathways including PD-1, PD-L1/2, CTLA-4 inhibitors, etc.).
  • Treatment with drugs, etc. take Chinese medicine with anti-tumor indications within 2 weeks before the first use of the test drug.
  • a strong inhibitor or strong inducer of CYP3A4 has been used within 2 weeks before the first use of the investigational drug.
  • Subjects can continue to use the investigational drug until they meet the treatment termination/withdrawal criteria, or the treatment reaches 2 years.
  • the subject is tolerant to the test drug
  • docetaxel for injection (albumin-bound) combined with Nivolumab was used to study its clinical efficacy in recurrent or metastatic head and neck squamous cell carcinoma, without dexamethasone pretreatment, to further improve immune checkpoint inhibitors
  • the curative effect on patients with recurrent or metastatic head and neck squamous cell carcinoma can improve the response rate, prolong the overall survival period OS, reduce the risk of death, and benefit the patients.

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Abstract

多西他赛白蛋白组合物和免疫检查点抑制剂在治疗肿瘤，特别是在治疗结肠癌、肝癌、黑色素瘤和头颈鳞癌中的应用，包括制药用途、药物组合物或治疗方法。多西他赛白蛋白组合物在临床使用时无需皮质类固醇预处理，从而可与免疫检查点抑制剂高效联用，产生协同的抗瘤效果。

Description

多西他赛白蛋白组合物和免疫检查点抑制剂的组合及用途
相关申请
本申请要求于2021年5月26日提交的第20210577701.1号中国专利申请以及于2021年8月12日提交的第202110921678.3号中国专利申请的优先权，通过引用的方式将上述申请的全部内容整体并入本文，用于所有目的。
技术领域
本申请属于药物领域，具体涉及多西他赛白蛋白组合物和免疫检查点抑制剂在制备用于治疗肿瘤的药物中的用途。
背景技术
多西他赛是由红豆杉的针叶中提取的非细胞毒性前体(10-去乙酰浆果赤霉素Ⅲ)半合成生产所得，是紫杉醇类似物，较紫杉醇有更强的抗肿瘤活性。
多西他赛水溶性较差，目前市售制剂为其普通注射液，最早由赛诺菲开发，商品名为
Figure PCTCN2022094859-appb-000001
常规给药剂量为每3周给药一次，静脉输注1h，剂量为75mg/m 2。然而以
Figure PCTCN2022094859-appb-000002
Figure PCTCN2022094859-appb-000003
为代表的多西他赛注射液，由于配方中使用乙醇和吐温80，容易引发严重的过敏反应，需要地塞米松预处理，患者顺应性差；此外，乙醇会影响中枢神经系统，需要降低输液速度来减缓中毒症状。
由于人血白蛋白是人体内源性物质，具有很好的生物相容性，可作为疏水性药物的天然载体，增加难溶性药物溶解度。美国Abraxis公司以人血白蛋白作为辅料，采用乳化法开发的注射用紫杉醇(白蛋白结合型)(商品名
Figure PCTCN2022094859-appb-000004
)于2005年经FDA批准上市，用于治疗联合化疗失败的转移性乳腺癌或辅助化疗6个月内复发的乳腺癌，之后相继获批用于治疗非小细胞肺癌、胰腺癌及胃癌(日本)。
Figure PCTCN2022094859-appb-000005
与紫杉醇注射液
Figure PCTCN2022094859-appb-000006
相比，由于配方中不含可导致严重过敏反应的溶剂聚氧乙烯蓖麻油，因此不需要预先给予抗过敏药物，并且可以高浓度快速给药，将输液时间缩短至30分钟以内，显著提高了患者的顺应性；由于安全性的提高，紫杉醇的给药剂量从175mg/m 2可提高到260～300mg/m 2；
Figure PCTCN2022094859-appb-000007
中的聚氧乙烯蓖麻油会抑制紫杉醇与白蛋白的结合，配方中不含有聚氧乙烯蓖麻油，可以更充分利用白蛋白独特的“gp60-窖蛋白-SPARC”通道使药物向肿瘤区富集，从而增加疗效。目前各国科研人员正在尝试使用人血白蛋白开发其他药物的白蛋白纳米粒，如多西他赛等。
提高多西他赛白蛋白产品质量，需要同时考虑其物理稳定性(悬浮液稳定性)和化学稳定性(多西他赛降解情况和白蛋白多聚体增加情况)。现有技术公开的多西他赛白蛋白组合物，一种是加入大量螯合剂作为稳定剂，以提高产品的物理稳定性，但是未考察对其化学稳定性的影响；另外，大量的螯合剂提高稳定性的同时形成高渗溶液，临床使用不便。另外一种是需要在产品中加入大量的氨基酸，可以抑制多西他赛的降解及白蛋白多聚体的增加，但未对物理稳定性的影响进行深入研究，仅提及复溶后悬浮液室温可稳定8小时以上。
因此，找到可以同时保证多西他赛白蛋白组合物的物理稳定性和化学稳定性的方法是本领域亟待解决的问题。
目前开展了多项针对免疫检查点阻断疗法与化疗相结合的联合给药(Chemotherapy in combination with immunotherapy，CIT)治疗的临床研究，化疗药物包括顺铂、卡铂、紫杉醇、多西他赛、培美曲塞、吉西他滨、伊立替康等。然而，以
Figure PCTCN2022094859-appb-000008
为代表的多西他赛注射液，由于严重的过敏反应，临床上必须使用地塞米松进行预处理，推测这将影响其与PD-1抗体联用效果。
因此，制备无需地塞米松预处理的多西他赛制剂与免疫检查点抑制剂联用，具有广泛意义。
发明内容
本申请提供了一种多西他赛新剂型，临床使用时无需皮质类固醇(如地塞米松)预处理，从而可与免疫检查点抑制剂高效联用。具体地，本申请的多西他赛白蛋白组合物和免疫检查点抑制剂联用具有显著的抗肿瘤疗效，产生了协同的抗瘤作用。
第一方面，本申请提供多西他赛白蛋白组合物和免疫检查点抑制剂在制备用于治疗肿瘤的药物中的用途。在具体的实施方案中，所述免疫检查点抑制剂为PD-1抑制剂或PD-L1抑制剂。
第二方面，本申请提供多西他赛白蛋白组合物在制备改善免疫检查点抑制剂治疗肿瘤的效果的药物中的用途。在具体的实施方案中，所述免疫检查点抑制剂为PD-1抑制剂或PD-L1抑制剂。
第三方面，本申请提供一种治疗肿瘤的复方药物或药物组合产品，其包含(1)多西他赛白蛋白组合物，和(2)免疫检查点抑制剂。在实施方案中，所述免疫检查点抑制剂为PD-1抑制剂或PD-L1抑制剂。
第四方面，本申请提供一种治疗个体(例如患者或受试者)的肿瘤的方法，其包括向所述个体施用治疗有效量的(1)多西他赛白蛋白组合物和(2)免疫检查点抑制剂，或者如第三方面 所述的复方药物或药物组合产品。在具体的实施方案中，所述免疫检查点抑制剂为PD-1抑制剂或PD-L1抑制剂。
在第五方面，本申请提供用于改善免疫检查点抑制剂对肿瘤的疗效的方法，其包括向已经接受免疫检查点抑制剂治疗的患肿瘤的个体施用治疗有效量的多西他赛白蛋白组合物。在具体的实施方案中，所述免疫检查点抑制剂为PD-1抑制剂或PD-L1抑制剂。
在上述第一方面至第五方面中，所述肿瘤为实体瘤。优选地，所述实体瘤为唾液腺癌、食管癌和食管胃部结合部癌、未分化甲状腺癌、卵巢癌、头颈癌、结直肠癌、肝癌、黑色素瘤、非小细胞肺癌、乳腺癌、胃癌、头颈部鳞状细胞癌、肾细胞癌、胆管癌、膀胱癌及尿路肿瘤、宫颈癌、小细胞肺癌、胰腺癌、子宫肿瘤、鼻咽癌、口咽癌、喉咽癌、喉癌、口腔癌、唇癌、上颌窦肿瘤、筛窦肿瘤、骨肿瘤。在优选的实施方案中，所述实体瘤为结肠癌、肝癌、黑色素瘤或头颈部鳞状细胞癌(简称头颈鳞癌)，在更优选的实施方案中，所述实体瘤为黑色素瘤或头颈部鳞状细胞癌。
在上述第一方面至第五方面中，所述多西他赛白蛋白组合物为多西他赛白蛋白纳米粒组合物，其中所述多西他赛白蛋白纳米粒的粒径优选为约60-200nm，更优选为90-150nm，例如为90-135nm。
在上述第一方面至第五方面中，所述药物或者所述复方药物或药物组合产品被配制成临床可接受的制剂，优选为注射剂型，包括液体注射剂、注射用粉剂、注射用片剂。在一些实施方案中，多西他赛白蛋白组合物和免疫检查点抑制剂存在于同一制剂中，或者分别独立制剂。
例如，在一些实施方案中，含多西他赛白蛋白纳米粒的组合物和PD-1抑制剂或PD-L1抑制剂存在于同一制剂中。在另一些实施方案中，含多西他赛白蛋白纳米粒的组合物和PD-1抑制剂或PD-L1抑制剂分别独立制剂。
在一些实施方案中，当所述多西他赛白蛋白组合物(例如，含多西他赛白蛋白纳米粒的组合物)被制成液体注射剂时，以无水多西他赛计，多西他赛活性成分含量为3.3-4.3mg/ml，优选4mg/ml。
在一些实施方案中，所述PD-1抑制剂或PD-L1抑制剂选自Nivolumab等重组人源化抗PD-1单克隆抗体注射液、Pembrolizumab等重组抗PD-1全人源单克隆抗体注射液(PD-1单抗)，Atezolizumab等重组人源化抗PD-L1单克隆抗体注射液、Avelumab、Durvalumab等全人源抗PD-L1抗体注射液，优选Nivolumab。
在上述第一方面至第五方面中，任选地，所述药物或者所述复方药物或药物组合产品中进一步包含其他治疗所述肿瘤的药物。
在一些实施方案中，本申请还提供多西他赛白蛋白纳米粒联合PD-1抑制剂或PD-L1抑制剂在制备用于治疗肿瘤的药物中的用途。所述PD-1抑制剂或PD-L1抑制剂选自Nivolumab。在优选的实施方案中，所述肿瘤为头颈鳞癌。优选地，所述头颈鳞癌为复发或转移性头颈鳞癌。
本申请所述的复发或转移性头颈癌鳞癌包括无法再次接受局部根治性治疗的患者和远处转移性的，原发于口腔、口咽、下咽和喉的头颈部鳞癌。
在一些实施方案中，本申请提供包含多西他赛白蛋白纳米粒和Nivolumab的治疗肿瘤的药物。
在一些实施方案中，所述药物中可进一步包含其他治疗复发或转移性头颈部鳞癌的一线、二线药物，所述药物是指中国或其它国家和地区(例如美国、欧盟、日本、韩国等等)药物管理部门批准用于复发或转移性头颈部鳞癌治疗的一线、二线药物。
在优选的实施方案中，所述药物为注射剂型，包括液体注射剂、注射用粉剂、注射用片剂等等。当所述多西他赛白蛋白纳米粒为注射用粉剂时，以无水多西他赛计，含活性成分80mg/支，含活性成分3.3-4.3mg/ml，优选4mg/ml。当Nivolumab为注射液时，含Nivolumab100mg/支。
在一些实施方案中，本申请提供了一种治疗肿瘤的联合方案，其包括对肿瘤患者施用治疗有效量的注射用多西他赛白蛋白纳米粒和Nivolumab。所述施用优选为注射给药。
在一些实施方案中，本申请还提供了一种改善Nivolumab对肿瘤的疗效的方法，其包括在对肿瘤患者施用Nivolumab的基础上，进一步联合施用治疗有效量的多西他赛白蛋白纳米粒。
在优选的实施方案中，注射用多西他赛白蛋白纳米粒的施用方式为静脉给药。优选地，给药周期为每3周给药一次。给药剂量范围25-200mg/m 2。在具体实施方案中，每次静脉给药，所述白蛋白药物制剂的滴注给药时间为30min-60min，优选为60±10min。
在一些实施方案中，注射用多西他赛白蛋白纳米粒的给药剂量为50、75、100、125、150mg/m 2，每3周给药一次(记为Q3W)；
在一些实施方案中，注射用多西他赛白蛋白纳米粒的给药剂量为30、40、50mg/m 2，每周给药1次，给药3周，停药1周，4周为1个周期(记为QW 3/4)；
在一些实施方案中，注射用多西他赛白蛋白纳米粒的给药剂量为50、60、75mg/m 2，每2周给药一次(记为Q2W)；
在一些实施方案中，注射用多西他赛白蛋白纳米粒的给药剂量为30、40、50mg/m 2，每周给药1次，给药2周，停药1周，3周为一个周期(记为QW 2/3)。
在一些实施方案中，以Nivolumab计，Nivolumab的给药方式为240mg Q2W，或者360mg Q3W，或者480mg Q4W，优选为静脉给药，可在多西他赛白蛋白给药前、中、后的任意时间给药。给药周期与注射用多西他赛白蛋白纳米粒相同。
在优选的实施方案中，静脉滴注Nivolumab 360mg Q3W，30min，之后静脉滴注注射用多西他赛(白蛋白结合型)75mg/m 2Q3W，60min，或静脉滴注Nivolumab 360mg Q3W，30min，之后静脉滴注注射用多西他赛(白蛋白结合型)100mg/m 2Q3W，60min。
本申请所述的注射用多西他赛白蛋白纳米粒的剂量均以无水多西他赛计。
本文所使用的术语“个体”是指哺乳动物，如人类，但也可以是其它哺乳动物，如家畜或实验动物等。
术语“治疗”意为将本申请所述化合物或制剂进行给药以改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：(i)抑制疾病或疾病状态，即遏制其发展；(ii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“治疗有效量”意指(i)治疗特定疾病、病况或障碍，或(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状的本申请化合物的用量。构成“治疗有效量”的本申请化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
第六方面，本申请还提供一种含多西他赛白蛋白纳米粒的组合物，其包含多西他赛和酸变性白蛋白或者由多西他赛和酸变性白蛋白制成。
在一些实施方案中，上述组合物还任选地包含渗透压调节剂、或者pH调节剂。
在一些实施方案中，所述多西他赛优选无水型多西他赛、多西他赛半水合物或多西他赛三水合物。
在一些实施方案中，所述酸变性白蛋白由人血白蛋白加酸调至适当的pH值后变性得到。在优选的实施方案中，所述酸选自酸性氨基酸或酸性多肽、有机酸、无机酸。所述酸性氨基酸或酸性多肽包括但不限于盐酸半胱氨酸、谷胱甘肽等；所述有机酸包括但不限于柠檬酸、酒石酸等；所述无机酸包括但不限于盐酸、硫酸等。所述酸优选盐酸半胱氨酸、谷胱甘肽、盐酸，更优选盐酸半胱氨酸。在优选的实施方案中，所述适当的pH值为3.5～5.5，优选3.5～5.0，更优选3.8-4.7，更优选4.0～4.5。
在一些实施方案中，以无水多西他赛计，多西他赛和人血白蛋白的质量比为1:(2.0～10.0)，优选1:(3.0～7.0)，更优选1：(4.0～6.0)。
在一些实施方案中，所述人血白蛋白中辛酸钠的含量不高于0.08mmol/g蛋白，优选0.03-0.08mmol/g蛋白，进一步优选0.04-0.08mmol/g蛋白，更优选0.04-0.07mmol/g蛋白。
在一些实施方案中，所述多西他赛白蛋白纳米粒的粒径为约60-200nm，优选90-150nm，更优选90-135nm。
在任选的实施方案中，本申请所述的含多西他赛白蛋白纳米粒的组合物包含渗透压调节剂，以便将渗透压调节在适当的范围内。所述渗透压调节剂的类型并不受特别限制，例如可选自氯化钠、葡萄糖、磷酸盐或枸橼酸盐等，优选氯化钠。其种类、用量可以由本领域技术人员根据临床使用的稀释剂或复溶介质的种类、用量等具体情况确定。在一些实施方案中，所述渗透压调节剂为氯化钠，氯化钠与多西他赛的重量比为(0.75-9):1，优选(1-7):1，优选(1.5-4.5):1，最优选2.25:1。
在任选的实施方案中，本申请所述的多西他赛白蛋白组合物(例如，含多西他赛白蛋白纳米粒的组合物)包含pH调节剂，以便将pH值调节在适当的范围内，使制备本申请所述组合物的悬浮液和/或本申请组合物的重建悬浮液在25℃条件下至少稳定24小时，优选至少稳定30小时；和/或在2～8℃下至少稳定7天，优选至少稳定10天。此处所述的“稳定”，是指没有发生纳米粒沉降或悬浮液浑浊。所述pH调节剂的种类并不受特别的限制。优选地，所述pH值范围为3.4-5.8；优选3.6-5.6、或者3.8-5.0、更优选3.9-4.8。
在一些实施方案中，本申请所述的多西他赛白蛋白组合物(例如，含多西他赛白蛋白纳米粒的组合物)为悬浮液。其中，所述悬浮液的pH值为3.4-5.8，优选3.6-5.6，或者3.8-5.0，更优选3.9-4.8。所述悬浮液中多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm。所述悬浮液含氯化钠0-1.8％(w/v)，优选0.45％-1.8％(w/v)，更优选0.9％-1.8％(w/v)。在一些实施方案中，所述悬浮液含多西他赛2-10mg/ml，优选2-8mg/ml。所述悬浮液在25℃条件下至少稳定24小时，优选至少稳定30小时；在2～8℃下至少稳定7天，优选至少稳定10天。
在另一些实施方案中，本申请所述的多西他赛白蛋白组合物(例如，含多西他赛白蛋白纳米粒的组合物)为冻干粉。所述多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm。在一些实施方案中，所述冻干粉含氯化钠，氯化钠与多西他赛的重量比为(0.75-9):1，优选(1-7):1，优选(1.5-4.5):1，最优选2.25:1。
在一些实施方案中，所述冻干粉由含多西他赛白蛋白纳米粒的悬浮液冻干而成。其中，所述悬浮液的pH值为3.4-5.8，优选3.6-5.6，或者3.8-5.0，更优选3.9-4.8。所述悬浮液中多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm。所述悬浮液含氯化钠0-1.8％(w/v)，优选0.45％-1.8％(w/v)，更优选0.9％-1.8％(w/v)。所述悬浮液在25℃条件下至少稳定24小时，优选至少稳定30小时；在2～8℃下至少稳定7天，优选至少稳定10天。
在一些实施方案中，所述冻干粉使用复溶介质重建悬浮液。所述复溶介质选自注射用水、氯化钠溶液或葡萄糖溶液，优选注射用水。所述重建悬浮液的pH值为3.4-5.8，优选3.6-5.6，或者3.8-5.0，更优选3.9-4.8。重建悬浮液中多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm。所述重建悬浮液含氯化钠0-1.8％(w/v)，优选0.45％-1.8％(w/v)，更优选0.9％-1.8％(w/v)。重建的等渗悬浮液在25℃条件下至少稳定24小时，优选至少稳定30小时；在2～8℃下至少稳定7天，优选至少稳定10天。
在一些实施方案中，本申请所述的含多西他赛白蛋白纳米粒的组合物冻干后在25℃条件下至少稳定36个月。此处所述的“稳定”包括但不仅限于多西他赛无明显降解，蛋白纳米粒未发生明显聚合，粒度未明显增大，多西他赛含量、水分、酸度、渗透压或者摩尔浓度等无明显变化。所述“稳定”可以是上述列举的情形中的一种或多种。在一些实施方案中，所述“稳定”表现为7-表多西他赛和/或白蛋白多聚体的含量无明显变化。
第七方面，本申请还提供了一种药物，其由上述含多西他赛白蛋白纳米粒的组合物制成。所述药物为临床可接受的剂型，优选注射剂，进一步优选液体注射剂或冻干粉针剂。
在一些实施方案中，当所述注射剂为液体注射剂时，所述液体注射剂的pH值为3.4-5.8，优选3.6-5.6，或者3.8-5.0，更优选3.9-4.8。所述液体注射剂中多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm；所述液体注射剂含氯化钠0-1.8％(w/v)，优选0.45％-1.8％(w/v)，更优选0.9％-1.8％(w/v)，更优选0.9％(w/v)。在一些实施方案中，所述液体注射剂含多西他赛2-10mg/ml，优选2-8mg/ml。所述液体注射剂在25℃条件下至少稳定24小时，优选至少稳定30小时；在2～8℃下至少稳定7天，优选至少稳定10天。此处所述的“稳定”，是指没有发生纳米粒沉降或液体注射剂浑浊。
在一些实施方案中，当所述注射剂为冻干粉针剂时，所述冻干粉针剂含氯化钠，氯化钠：多西他赛的重量比为(0.75-9):1，优选(1-7)：1；优选(1.5-4.5):1，最优选2.25:1。多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm。制备所述冻干粉针剂的悬浮液在冻干前的pH为3.4-5.8，优选3.6-5.6，或者3.8-5.0，更优选3.9-4.8。所述悬浮液在25℃条件下至少稳定24小时，在2～8℃下至少稳定10天。
在一些实施方案中，所述冻干粉针剂使用复溶介质重建悬浮液。所述复溶介质选自注射用水、氯化钠溶液或葡萄糖溶液，优选注射用水。所得重建悬浮液的pH值为3.4-5.8，优选3.6-5.6，或者3.8-5.0，更优选3.9-4.8。重建悬浮液中多西他赛白蛋白纳米粒的粒径约60-200nm，优选90-150nm，更优选90-135nm。所述重建悬浮液含0-1.8％(w/v)氯化钠，优选0.45％-1.8％(w/v)，更优选0.9％-1.8％(w/v)，更优选0.9％。在一些实施方案中，所述重建悬 浮液含多西他赛2-10mg/ml，优选2-8mg/ml。重建悬浮液25℃条件下至少稳定24小时，优选至少稳定30小时；在2～8℃下至少稳定7天，优选至少稳定10天。
第八方面，本申请还提供所述含多西他赛白蛋白纳米粒的组合物的制备方法，其包括如下步骤：
(1)将多西他赛溶解于有机溶剂中，溶解后得到有机相溶液；
(2)取人血白蛋白溶液，加酸调节pH值，得酸变性白蛋白水相溶液；另取一种盐溶液；
(3)将有机相溶液、水相溶液及盐溶液混合载药，得到载药液；
(4)透析，得到透析后悬浮液；
其中，步骤(2)在加酸调节pH值前，任选地包含用注射用水稀释人血白蛋白溶液得白蛋白稀释液的步骤。
其中，步骤(2)在加酸调节pH值后，任选地包含孵育步骤。
其中，步骤(3)在混合载药后，任选地包含降温步骤。
其中，步骤(4)在透析前，任选地包含，将步骤(3)所得载药液浓缩得到浓缩液。
其中，步骤(4)透析后，任选地包含浓缩或稀释步骤，以调节透析后悬浮液中多西他赛的浓度。
其中，步骤(4)后任选地含有步骤(5)除菌过滤。
其中，步骤(5)后任选地含有步骤(6)冻干。
其中，步骤(1)中所述多西他赛为任意形式，优选为无水型多西他赛、多西他赛半水合物或多西他赛三水合物。以无水多西他赛计，多西他赛和人血白蛋白的质量比为1：(2.0～10.0)，优选1:(3.0～7.0)，更优选1:(4.0～6.0)。
其中，步骤(1)中所述有机溶剂选自可与水互溶的溶剂，例如乙醇、甲醇、丙酮、DMSO等，优选乙醇。以无水多西他赛计，所述有机相溶液含多西他赛45-90mg/ml，优选45-70mg/ml。
其中，步骤(2)中所述人血白蛋白溶液中辛酸钠的含量为不高于0.12mmol/g蛋白，优选不高于0.08mmol/g蛋白，优选0.045-0.08mmol/g蛋白。
其中，步骤(2)中，白蛋白稀释液含白蛋白6-25mg/ml，优选10-20mg/ml，更优选12-18mg/ml，更优选15mg/ml。
其中，步骤(2)中，所述酸选自酸性氨基酸或酸性多肽、有机酸、无机酸。所述酸性氨基酸或酸性多肽包括但不仅限于盐酸半胱氨酸、谷胱甘肽等；所述有机酸包括但不仅限于柠檬酸、酒石酸等；所述无机酸包括但不仅限于盐酸、硫酸等。所述酸优选盐酸半胱氨酸、 谷胱甘肽、盐酸，更优选盐酸半胱氨酸。所述pH值优选3.5～5.5，优选3.5～5.0，更优选3.8-4.7，更优选4.0～4.5。
其中，步骤(2)中，所述孵育是指在加酸调节pH值后，升温至35℃-42℃，优选38℃-42℃；孵育30min以上，优选30min-60min。
其中，步骤(2)中所述的盐溶液选自氯化钠、氯化钾、硫酸钠或硫酸镁的水溶液，优选氯化钠的水溶液；盐溶液浓度不低于2％，优选2％～35％，更优选10％～20％。
其中，步骤(3)中，载药条件为将有机相溶液、水相溶液升温至35℃-42℃，优选38℃-42℃，将三者混合载药。
其中，步骤(3)中所述降温，指降温至低于室温，优选0-20℃，更优选7-15℃。
其中，步骤(4)中所述浓缩液含多西他赛约4-10mg/ml，优选6-10mg/ml，更优选7-9mg/ml，更优选8mg/ml。
其中，步骤(4)透析以去除多余的小分子化合物，对于透析液的种类和用量、透析膜的截留分子量没有特别限制，本领域技术人员可以根据普通技术知识或经验作出选择。为方便进一步制剂和临床使用，透析液优选临床可接受的渗透压调节剂的水溶液，例如氯化钠、葡萄糖、磷酸盐或枸橼酸盐的水溶液。在一些实施方案中，步骤(4)中使用氯化钠溶液作为透析液进行透析。透析膜的截留分子量为10-50KDa，优选10-30KDa，更优选10KDa或者30KDa。透析液体积不低于载药液或浓缩液的3倍，优选3-10倍，更优选3-6倍。所述氯化钠溶液的浓度不高于1.8％(w/v)，优选0.45％-1.8％(w/v)，更优选0.9％-1.8％(w/v)。
在一些实施方案中，步骤(2)使用的人血白蛋白溶液，需事先调整所含辛酸钠的含量。本领域技术人员可以根据普通技术知识或经验选取适宜的方法调节人血白蛋白溶液中辛酸钠的含量，包括但不仅限于透析。
在一些实施方案中，调节人血白蛋白溶液中辛酸钠的含量的方法为：
取市售的人血白蛋白溶液，用注射用水或者生理盐水稀释，得到稀释的白蛋白溶液；用注射用水或者生理盐水作为透析液对稀释的白蛋白溶液进行透析，以部分去除辛酸钠，得到低辛酸钠含量的人血白蛋白溶液。
在一些实施方案中，人血白蛋白溶液的稀释倍数不低于4倍，优选4-7倍。所述透析，透析膜的截留分子量为10-50KDa，优选10-30KDa，更优选10KDa或者30KDa。透析液的体积是本领域技术人员根据需要的辛酸钠含量通过常规试验可以确定的。优选地，透析液的体积约为稀释的白蛋白溶液的体积的3倍以上，优选3-10倍，更优选3-6倍。
在一些实施方案中，所述低辛酸钠含量的人血白蛋白溶液含辛酸钠低于0.16mmol/g蛋白，优选低于0.12mmol/g蛋白，优选低于0.10mmol/g蛋白，优选低于0.08mmol/g蛋白。所 述低辛酸钠含量的人血白蛋白溶液可以直接用于制备本申请所述的多西他赛白蛋白纳米粒组合物，也可与其它辛酸钠含量的人血白蛋白溶液按比例混合得到期望含量后，再用于制备多西他赛白蛋白纳米粒组合物。
第九方面，本申请还提供了一种含多西他赛白蛋白纳米粒的组合物，其由第八方面所述的方法制备得到。
在所述制备方法中，透析步骤将去除载药液或浓缩液中多余的小分子化合物。例如，在一些实施方案中，步骤(2)使用酸性氨基酸或酸性多肽调节pH来制备酸变性白蛋白；透析步骤基本去除了多余的酸性氨基酸或酸性多肽；所述组合物几乎不含有游离的酸性氨基酸或酸性多肽。所述“几乎不含有游离的酸性氨基酸或酸性多肽”是指游离的酸性氨基酸或酸性多肽在组合物中的含量低于多西他赛的0.25％(w/w)。例如，在一些实施方案中，使用盐酸半胱氨酸或谷胱甘肽调节pH以制备酸变性白蛋白，所述组合物中游离的半胱氨酸或谷胱甘肽的含量低于多西他赛的0.25％(w/w)。
进一步地，本申请还提供一种药物，其由上述方法制得的含多西他赛白蛋白纳米粒的组合物制备得到。所述药物为临床可接受的剂型，优选注射剂，进一步优选液体注射剂或冻干粉针剂。
第十方面，本申请还提供了一种调节人血白蛋白溶液中辛酸钠的含量的方法，包含以下步骤：取市售的人血白蛋白溶液，用注射用水或者生理盐水稀释，得到稀释的白蛋白溶液；用注射用水或者生理盐水作为透析液对稀释的白蛋白溶液进行透析，以部分去除辛酸钠，得到低辛酸钠含量的人血白蛋白溶液。
在一些实施方案中，人血白蛋白溶液的稀释倍数不低于4倍，优选4-7倍。所述透析，透析膜的截留分子量为10-50KDa，优选10-30KDa，更优选10KDa或者30KDa。透析液的体积是本领域技术人员根据需要的辛酸钠含量通过常规试验可以确定的。优选地，透析液的体积约为稀释的白蛋白溶液的体积的3倍以上，优选3-10倍，更优选3-6倍。
在一些实施方案中，所述低辛酸钠含量的人血白蛋白溶液含辛酸钠低于0.16mmol/g蛋白，优选低于0.12mmol/g蛋白，优选低于0.10mmol/g蛋白，优选低于0.08mmol/g蛋白。
在一些实施方案中，所述低辛酸钠含量的人血白蛋白溶液可以直接用于制备本申请所述的多西他赛白蛋白纳米粒组合物，也可与其它辛酸钠含量的人血白蛋白溶液按比例混合得到期望含量后，再用于制备多西他赛白蛋白纳米粒组合物。本申请所述的多西他赛，其含量均以无水多西他赛计。
本申请所述的数值或数值范围，在不影响本申请实施的前提下，可在本领域技术人员理解的范围内上下浮动，所述浮动范围例如±20％，或±17％，或±15％，或±12％，±10％，或±9％，或±8％，或±7％，或±6％，或±5％，或±4％，或±3％，或±2％，或±1％。
本申请的发明人出乎意料地发现，白蛋白中的热稳定剂辛酸钠对多西他赛白蛋白纳米粒的物理稳定性影响较大，其原因是辛酸钠可以与药物竞争性结合白蛋白的疏水位点，降低药物与蛋白的结合力，导致纳米悬浮液不稳定。当直接使用市售的人血白蛋白溶液作为辅料时(含辛酸钠0.16mmol/g蛋白)，10h小时内纳米粒即发生沉降；然而，当采用透析手段降低白蛋白溶液中的辛酸钠含量，使其低于0.08mmol/g蛋白时，制得的纳米粒稳定性大大提高，在25℃条件下至少可保持稳定24h以上，在2-8℃至少可稳定放置10天以上。
本申请的发明人还发现pH值也是影响多西他赛白蛋白纳米粒物理稳定性的重要因素。现有技术制备的多西他赛白蛋白纳米粒悬浮液pH值均在白蛋白等电点以上，为维持悬浮液的稳定性，需加入大量的有机酸或其盐作为稳定剂，导致药物渗透压较高，临床使用时会引起明显的疼痛，并引起注射局部细胞和组织的渗透性损伤。而本申请以酸变性白蛋白制备多西他赛白蛋白纳米粒，并控制白蛋白溶液中辛酸钠的含量，无需额外加入其它盐类稳定剂，重建得稳定的等渗悬浮液，对血管刺激小。
此外，CN103054798A教导无水型多西他赛制备的纳米粒稳定性明显优于多西他赛三水合物和半水合物。然而，在本申请技术方案中，多西他赛的结晶水情况，即无水型、半水合物、三水合物对所制备的多西他赛白蛋白纳米粒组合物稳定性无影响。这大大拓展了多西他赛使用形式的选择范围，有极大的产业应用价值。
本申请提供了一种物理和化学均稳定的多西他赛白蛋白组合物。本申请提供的组合物，加速及长期稳定性考察显示，7-表多西他赛、蛋白多聚体变化很少，已完成的加速稳定性试验显示冻干粉可在30℃和25℃条件稳定储存18个月，根据现有数据预测可在30℃条件下稳定储存至少20个月，25℃以下的条件下稳定储存至少36个月。已完成的静置稳定性观察试验显示，本申请提供的组合物不论是冻干前悬浮液，还是冻干后重建悬浮液，在室温条件下可稳定至少24小时，冷藏条件下可稳定至少10天，不会出现悬浮液浑浊或纳米粒沉降；放置更长时间的稳定性考察正在进行中。相对于市售产品重建悬浮液仅稳定8h的情况相比，本申请的产品大大减轻了临床使用限制。
本申请将多西他赛包裹于人血白蛋白中，与普通注射剂相比，制剂中不含有吐温80(Tween-80)和乙醇，无需使用皮质类固醇(如地塞米松)预处理，不仅安全耐受性好，而且能有效避免地塞米松预处理削弱多西他赛和/或PD-1抑制剂抑瘤作用的弊端。
本申请所述的多西他赛白蛋白组合物与免疫检查点抑制剂的联合用药能够显著提升抑瘤效果，产生了协同作用。例如，多西他赛白蛋白组合物与PD-1抑制剂或PD-L1抑制剂能够协同作用，显著抑制结肠癌、肝癌、黑色素瘤或头颈部鳞状细胞癌等多种肿瘤的生长，特别是在治疗黑色素瘤或头颈部鳞状细胞癌方面具有明显的协同效应。
以上所述的实施方案代表了本申请的示例性实施方案，但是本申请并不限于以上实施方案。另外，本申请的以上实施方案中的各个技术特征可以相互组合，从而构成一个或多个新的技术方案，这些新的技术方案也落在本申请的范围内，只要这样的新的技术方案是在技术上可行即可。
附图说明
图1显示了地塞米松(DEX)预处理对
Figure PCTCN2022094859-appb-000009
(TAXOTERE)抑制小鼠肝癌Hepa1-6移植瘤生长作用的影响。其中，与溶剂对照组比较，*P<0.05，**P<0.01；与DEX/TAXOTERE组比较，###P<0.001。
图2A显示了DEX对Opdivo(Nivolumab)抑制小鼠结肠癌MC38移植瘤的疗效的影响。其中，与溶剂对照组比较，*P<0.05,**P<0.01；与DEX/Opdivo或TAXOTERE/DEX/Opdivo组比较，#P<0.05。
图2B显示了Opdivo联合DTX-HSA或DEX/TAXOTERE抑制小鼠结肠癌MC38移植瘤的疗效比较。其中，与溶剂对照组比较，**P<0.01；与DEX/Opdivo或TAXOTERE/DEX/Opdivo组比较，##P<0.01。
图3显示了Keytruda(pembrolizumab)联合注射用多西他赛(白蛋白结合型)(DTX-HSA)或DEX/TAXOTERE抑制小鼠结肠癌MC38移植瘤的疗效比较。其中，与溶剂对照组比较，***P<0.001；与DEX/TAXOTERE/Keytruda组比较，###P<0.001。
图4A显示了DTX-HSA与DEX/TAXOTERE对肝癌Hepa1-6移植瘤小鼠体重的影响。其中，与溶剂对照组比较，*P<0.05,**P<0.01；与DEX/TAXOTERE组比较，###P<0.001。
图4B显示了DTX-HSA与DEX/TAXOTERE抑制小鼠肝癌Hepa1-6同源移植瘤的疗效比较。其中，与溶剂对照组比较，*P<0.05,**P<0.01；与DEX/TAXOTERE组比较，###P<0.001。
图5A显示了Keytruda联合DTX-HSA或DEX/TAXOTERE对肝癌Hepa1-6移植瘤小鼠的体重的影响。其中，与溶剂对照组比较，*P<0.05；与DEX/TAXOTERE/Keytruda组比较，#P<0.05。
图5B显示了Keytruda联合DTX-HSA或DEX/TAXOTERE方案抑制小鼠肝癌 Hepa1-6移植瘤的疗效比较。其中，与溶剂对照组比较，***P<0.001；与DEX/TAXOTERE/Keytruda组比较，##P<0.01。
图6显示了DTX-HSA、Keytruda及其联合给药方案抑制小鼠Hepa1-6移植瘤的疗效比较。其中，与溶剂对照组比较，**P<0.01,***P<0.001。
图7显示了DTX-HSA、Keytruda及其联合给药方案抑制小鼠B16/F10移植瘤的疗效比较。其中，与溶剂对照组比较，**P<0.01，***P<0.001。
实施例
以下实施例是对本申请的具体说明，不应对本申请的范围构成限制。
除非特别说明，实施例中所述的“稳定”，是指没有发生纳米粒沉降或悬浮液浑浊。
除非特别说明，实施例中所述的“重建悬浮液”是等渗悬浮液。
实施例1多西他赛和白蛋白的组合物的制备
多西他赛白蛋白纳米粒组合物的配方如下：
表1多西他赛白蛋白纳米粒组合物的配方
配方号 1-1 1-2 1-3 1-4
无水多西他赛 8g 8g 8g 8g
人血白蛋白 36g 36g 36g 80g
氯化钠 a 18g 36g 9g 18g
注：a经换算，冻干前悬浮液中氯化钠的含量。相应透析液中氯化钠的浓度(w/v)分别为：配方1-10.9％；配方1-21.8％；配方1-30.45％；配方1-40.9％。
(1)称取8g无水多西他赛溶解于120ml乙醇中，溶解后得到有机相溶液；
(2)取配方量的人血白蛋白溶液(辛酸钠的含量为0.08mmol/g蛋白)，用注射用水将其稀释为含白蛋白15mg/ml的溶液，再加入适量盐酸半胱氨酸调节pH至4.1，42℃孵育30min，得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为20％的盐溶液。
(3)将有机相溶液和水相溶液升温至42℃，有机相、水相及盐溶液混合载药，所得物料置于冰水浴中降温至15℃，得到载药液；
(4)将载药溶液浓缩至含多西他赛浓度约8mg/ml，得浓缩液；使用氯化钠溶液作为透析液对浓缩液进行5倍透析，透析膜截留分子量为30KDa，得到透析后悬浮液；然后，加适量透析液调整悬浮液浓度至含多西他赛4mg/ml；
(5)经0.45μm+0.2μm膜片进行除菌过滤，得冻干前悬浮液；
(6)取步骤(5)所得冻干前悬浮液，冻干，得冻干粉。
结果显示，透析步骤会降低辛酸钠的含量。在透析步骤结束时，透析后悬浮液中氯化钠的浓度与透析液中氯化钠的初始浓度基本相同。使用动态光散射法方法检测悬浮液中纳米粒的粒径，并静置悬浮液观察沉降现象。表2结果显示，透析液中氯化钠的含量对冻干前悬浮液、重建悬浮液中多西他赛白蛋白纳米粒的粒径无显著影响。将各配方的冻干前悬浮液、重建悬浮液在25℃条件下静置24小时，未见溶液浑浊或沉淀出现；在2～8℃下静置10天，也未见溶液浑浊或沉淀出现。提示，各配方的冻干前悬浮液、重建悬浮液在25℃条件下至少稳定24小时，在2～8℃下至少稳定10天。冻干前后稳定性无显著变化。
比较配方1-1和1-4的结果可见，调整多西他赛与白蛋白的用量比例，也未对纳米粒的粒径、悬浮液的稳定性产生显著影响。
此外，发明人还参照中国药典2020年版四部通则0512，使用高效液相色谱法检测透析后悬浮液中半胱氨酸的含量。结果显示透析后悬浮液中几乎不含有游离的半胱氨酸(含量低于多西他赛的0.25％(w/w))。
发明人还检测了透析前后悬浮液的pH值，发现透析前后悬浮液的pH值基本不变。
表2不同配方多西他赛白蛋白纳米粒的粒径与稳定性
Figure PCTCN2022094859-appb-000010
注：a冻干粉使用注射用水重建悬浮液(等渗混悬液)。配方1-1重建为含多西他赛4mg/ml的等渗悬浮液；配方1-2重建为含多西他赛2mg/ml的等渗悬浮液；配方1-3重建为含多西他赛8mg/ml的等渗悬浮液；配方1-4重建为含多西他赛4mg/ml的等渗悬浮液。
实施例2
称取多西他赛三水合物(以无水多西他赛计为8g)溶解于160ml无水乙醇中，溶解后得到有机相溶液；取含16g白蛋白的人血白蛋白溶液(辛酸钠的含量为0.08mmol/g蛋白)，用注射用水稀释成含白蛋白10mg/ml的溶液，再加入适量盐酸半胱氨酸调节pH至4.5，40℃孵育1小时,得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为10％的盐溶液；将有机相溶液和水相溶液升温至40℃，有机相、水相及盐溶液混合载药，所得物料置于冰水浴中降温至18℃，得到载药液；将载药液浓缩至含多西他赛浓度约10mg/ml，得浓缩液；使用等渗氯化钠(0.9％，w/v)溶液作为透析液对浓缩液进行6倍透析，透析膜截留分子量为30KDa，得到透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得到冻干前悬浮液；冻干，得冻干粉。
冻干前悬浮液中，多西他赛白蛋白纳米粒的粒径为101.3nm，在25℃条件下静置24小时，在2～8℃下静置10天，均未见溶液浑浊或沉淀出现。使用注射用水复溶冻干粉得到重建悬浮液(等渗悬浮液)，多西他赛白蛋白纳米粒的粒径无明显变化，为103.5nm；重建悬浮液在25℃条件下静置24小时，在2～8℃下静置10天，均未见溶液浑浊或沉淀出现。提示，冻干前悬浮液、重建悬浮液在25℃条件下至少稳定24小时，在2～8℃下至少稳定10天。
高效液相色谱法检测显示，透析后悬浮液中几乎不含有游离的半胱氨酸(含量约为多西他赛的0.13％(w/w))。透析前后悬浮液的pH值基本不变。
实施例3
称取多西他赛半水合物(以无水多西他赛计为8g)溶解于80ml 96％乙醇中，溶解后得到有机相溶液；取含50g白蛋白的人血白蛋白溶液(辛酸钠的含量为0.08mmol/g蛋白)，用注射用水稀释成含白蛋白20mg/ml溶液，再加入适量盐酸半胱氨酸调节pH至3.9，得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为2％的盐溶液；将有机相溶液和水相溶液升温至35℃，将有机相溶液和水相溶液及盐溶液混合载药，所得物料置于冰水浴中降温至12℃，得到载药液；将载药液浓缩至含多西他赛浓度约7mg/ml，得浓缩液，使用等渗氯化钠溶液(0.9％，w/v)作为透析液对浓缩液进行5倍透析，透析膜截留分子量为10KDa，得到透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得冻干前悬浮液；冻干，得冻干粉。
冻干前悬浮液和冻干粉经注射用水复溶后所得重建悬浮液(等渗悬浮液)，多西他赛白蛋白纳米粒的粒径无明显差异，冻干前为119.7nm，重建后为121.3nm；在25℃条件下静置24小时，或者在2～8℃下静置10天，冻干前悬浮液、重建悬浮液均未见溶液浑浊或沉淀出现。提示，冻干前悬浮液、重建悬浮液在25℃条件下至少稳定24小时，在2～8℃下至少稳定10 天。高效液相色谱法检测显示，透析后悬浮液中几乎不含有游离的半胱氨酸(含量约为多西他赛的0.21％(w/w))。透析前后悬浮液的pH值基本不变。
实施例4多西他赛的使用形式对组合物稳定性的影响
称取无水多西他赛、多西他赛半水合物、多西他赛三水合物(以无水多西他赛计，均为8g)溶解于120ml乙醇中，溶解后得到有机相溶液；取含36g白蛋白的人血白蛋白溶液(辛酸钠的含量为0.08mmol/g蛋白)，用注射用水稀释成含白蛋白20mg/ml的溶液，再加入适量盐酸半胱氨酸调节pH至4.3，42℃孵育60min，得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为10％的盐溶液；有机相溶液和水相溶液升温至42℃，将有机相溶液和水相溶液及盐溶液混合载药，所得物料置于冰水浴中降温至12℃，得到载药液；将载药液浓缩至含多西他赛浓度约7mg/ml，得浓缩液；使用等渗氯化钠溶液作为透析液对浓缩液进行5倍透析，透析膜截留分子量为30KDa，得透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得冻干前悬浮液；冻干，得冻干粉。
由下表3结果可以看出，分别以无水多西他赛、多西他赛半水合物、多西他赛三水合物作为原料，使用相同方法制备多西他赛白蛋白纳米粒，所得纳米粒的粒径无明显差异，均在110nm左右，且冻干前后无明显变化。并且，冻干前悬浮液、冻干粉经注射用水复溶的重建悬浮液(含多西他赛4mg/ml)的稳定性也无明显差异，在25℃条件下静置24小时，在2～8℃下静置10天，均未见溶液浑浊或沉淀出现。提示，上述冻干前悬浮液、重建悬浮液可在25℃条件下至少稳定24小时，在2～8℃下至少稳定10天。可见，多西他赛是否含有结晶水不影响本申请的实施。
高效液相色谱法检测显示，透析后悬浮液中几乎不含有游离的半胱氨酸(含量低于多西他赛的0.25％(w/w))。
表3多西他赛的使用形式对组合物稳定性的影响
Figure PCTCN2022094859-appb-000011
实施例5化学稳定性考察
将实施例1中配方1-1所得的含多西他赛白蛋白纳米粒的冻干粉置于西林瓶中，压上胶塞和铝盖，在不同储存条件下储存18个月，考察产品中7-表多西他赛的百分比含量及蛋白多聚体含量与时间的关系，结果见表4。
表4实施例1配方1-1产品的稳定性
Figure PCTCN2022094859-appb-000012
注：用于定义药物组合物稳定性的可接受极限是“7-表多西他赛的百分比含量≤1.0％”。
N/A*表示实验尚未达到该时间点，因此暂无数据。
表4结果表明，7-表多西他赛的产生与储存温度相关，储存温度越高，7-表多西他赛的含量增长越快。本申请产品可有效控制7-表多西他赛的生成。表4结果显示，在上述三种试验条件下，本申请产品中7-表多西他赛含量均在可控范围内(含量≤1.0％)，且蛋白多聚体含量无明显变化。根据现有数据预测，本申请产品可以在30℃条件下稳定储存至少20个月，25℃以下的条件下稳定储存至少36个月。
当API原料为多西他赛的其他形式，如半水合物和三水合物时，在上述试验条件下，其化学稳定性(7-表多西他赛的百分比含量及蛋白多聚体含量)的变化情况与以无水多西他赛为原料时类似。
中国专利CN106137969A在配方中加入精氨酸、脯氨酸等作为抑制剂，认为采用精氨酸作为抑制剂的效果最佳，可以在2～8℃的条件下贮存至少24个月，甚至可达30个月。该专利提供的数据显示所述产品在2～8℃的条件下贮存12个月时，7-表多西他赛的含量在0.5-0.61％之间；而本申请在25℃条件下储存12个月，7-表多西他赛的含量仅约为0.41％，在2～8℃的条件下贮存12个月时，7-表多西他赛的含量仅0.14％，足以说明本申请相较于CN106137969A，可更有效保证多西他赛的化学稳定性。
实施例6辛酸钠的含量对组合物稳定性的影响
称取8g无水多西他赛溶解于120ml乙醇中，溶解后得到有机相溶液；
取含不同浓度辛酸钠的人血白蛋白溶液(含人血白蛋白36克)，用注射用水稀释至含白蛋白15mg/ml，再加入适量盐酸半胱氨酸调节pH至4.2，42℃孵育30min，得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为15％的盐溶液；
将有机相溶液和水相溶液升温至42℃，将有机相溶液和水相溶液及盐溶液混合载药，所得物料置于冰水浴中降温至15℃，得到载药液；将载药液浓缩至含多西他赛浓度约8mg/ml，得浓缩液；使用等渗氯化钠溶液作为透析液对浓缩液进行5倍透析(透析膜截留分子量30KDa)，得到透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得冻干前悬浮液；冻干，得冻干粉。
由表5结果可知，原料中辛酸钠的含量对多西他赛白蛋白纳米粒的粒径无明显影响。但直接以市售人血白蛋白溶液(含辛酸钠0.16mmol/g蛋白)为原料制备多西他赛白蛋白纳米粒，25℃放置10h即出现浑浊，冻干前悬浮液和重建等渗悬浮液的物理稳定性不佳。降低人血白蛋白溶液中辛酸钠的含量有助于改善纳米粒悬浮液的物理稳定性。当人血白蛋白溶液中辛酸钠含量低于0.08mmol/g蛋白时，可在25℃保持稳定悬浮状态24h以上，稳定时间是含辛酸钠0.16mmol/g蛋白所得样品的2倍以上。当人血白蛋白溶液中辛酸钠含量低于0.08mmol/g蛋白时，所述冻干前悬浮液和重建等渗悬浮液可在2-8℃至少稳定放置10天以上。
表5辛酸钠的含量对组合物稳定性的影响
Figure PCTCN2022094859-appb-000013
注：a冻干粉经注射用水复溶得到重建悬浮液(等渗悬浮液)。
实施例7水相溶液pH值对稳定性影响
比较水相溶液pH值对产品稳定性的影响。工艺配方和制备方法参照实施例1配方1-1，仅在步骤(2)中使用盐酸半胱氨酸将水相溶液的pH分别调至7.0、6.5、6.0、5.5、5.0、4.7、4.1、3.8、3.5。观察冻干前悬浮液、重建悬浮液中纳米粒粒径及悬浮液稳定性的变化。
研究发现，水相溶液pH在3.5-5.5范围内，所得纳米粒的粒径随pH增高略有增加，但冻干复溶对粒径无明显影响。水相溶液pH在3.5-5.5范围内，所得冻干前悬浮液、重建悬浮液均可在25℃保持稳定悬浮状态20h以上，在2-8℃至少保持7天的稳定悬浮状态。进一步的，当水相溶液pH在3.8-4.7范围内时，冻干前悬浮液、重建悬浮液在25℃静置30h，在2-8℃静置10天，仍能保持稳定的悬浮状态。冻干复溶对稳定性无明显影响。
但当水相溶液pH为6.0以上时，所得纳米粒的粒径显著增加，pH6.0时纳米粒的粒径增至180nm。并且，当水相溶液pH高于6.0时，悬浮液的稳定性明显下降，1h以内即出现浑浊，不能保持纳米粒稳定悬浮的状态。
表6水相蛋白pH对多西他赛白蛋白纳米粒的影响
Figure PCTCN2022094859-appb-000014
注：a冻干粉用注射用水复溶得到重建悬浮液(等渗悬浮液)。
实施例8制备低辛酸钠含量的人血白蛋白溶液
实施例8-1
取市售的人血白蛋白溶液(辛酸钠的含量为0.16mmol/g蛋白)，用注射用水稀释成含白蛋白15mg/ml溶液，得到白蛋白稀释液；用注射用水作为透析液对白蛋白稀释液进行透析(膜包截留分子量为30KDa)，透析倍数6倍，部分去除蛋白中的辛酸钠，得到低辛酸钠含量的人血白蛋白溶液。
参照中国药典收录的人血白蛋白溶液中辛酸钠的测定方法(中国药典2020年版四部3111)检测所得蛋白溶液中辛酸钠的含量，结果显示处理后的蛋白溶液中含辛酸钠约0.045mmol/g蛋白。
实施例8-2
取市售的人血白蛋白溶液(辛酸钠的含量为0.16mmol/g蛋白)，用生理盐水稀释成含白蛋白12mg/ml溶液，得到白蛋白稀释液；用生理盐水作为透析液对白蛋白稀释液进行透析(膜包截留分子量为10KDa)，透析倍数5倍，部分去除蛋白中的辛酸钠，得到低辛酸钠含量的人血白蛋白溶液。经检测，所得低辛酸钠含量的人血白蛋白溶液中含辛酸钠约0.060mmol/g蛋白。
实施例8-3
取市售的人血白蛋白溶液(辛酸钠的含量为0.16mmol/g蛋白)，用注射用水稀释成含白蛋白20mg/ml溶液，得到白蛋白稀释液；用注射用水作为透析液对白蛋白稀释液进行透析(膜包截留分子量为30KDa)，透析倍数3倍，部分去除蛋白中的辛酸钠，得到低辛酸钠含量的人血白蛋白溶液。经检测，所得低辛酸钠含量的人血白蛋白溶液中含辛酸钠约0.080mmol/g蛋白。
为保存方便或使用需要，以上实施例8-1至8-3的低辛酸钠含量的人血白蛋白溶液可进一步进行浓缩处理。
实施例9工艺过程对蛋白二级结构的影响
按照实施例1-1的配方和制备方法，制备载药纳米悬浮液和空白悬浮液(步骤(1)不加药物多西他赛)，采用圆二色谱法考察人血白蛋白溶液、空白悬浮液和载药纳米悬浮液中蛋白各二级结构的占比，结果见表7。
结果表明，经过整个工艺流程制备的空白悬浮液和载药纳米悬浮液，从蛋白二级结构α-螺旋、β-折叠、转角及无规卷曲上看与人血白蛋白溶液相比，存在差异，说明该工艺改变了白蛋白的二级结构。
研究结果提示，本申请即使在透析步骤中除去酸，酸变性白蛋白的二级结构仍不能恢复至变性前的状态。并且，结合多西他赛之后，载药白蛋白的二级结构相对于未载药的酸变性白蛋白又进一步发生了变化。
表7蛋白各二级结构占比
  人血白蛋白溶液 空白悬浮液 a 载药纳米悬浮液 a
α-螺旋(％) 39.5 23.9 27.9
β-折叠(％) 15.1 11.7 4.4
转角(％) 16.8 26.2 30.1
无规卷曲(％) 28.6 38.2 37.6
注：a：实施例9所述的空白悬浮液和载药纳米悬浮液均为冻干后重建的等渗悬浮液。
实施例10参考中国专利CN 106137969 B(201510157393.1)的实施例1进行制备
称取无水多西他赛1.5g溶解于100ml无水乙醇中，超声溶解后得到油相溶液；取含7.5g白蛋白的人血白蛋白溶液(浓度为200mg/ml)，用注射用水稀释成含白蛋白6mg/ml溶液，水相体积共1250ml。水相中加入500mgL-谷胱甘肽，并在70℃下孵育6min。将油相在1000rpm的高剪切下均匀分散至水相中，得到混悬液。
该混悬液白色无乳光，目测浑浊，可见较多析出物，放置10分钟后出现沉淀，技术人员认为所得混悬液稳定性较差，无法按照专利内容继续后续操作。因此，采用本申请方法制得的产品的储存稳定性数据与上述专利中提供的储存稳定性数据进行对比，足以证明本申请产品的稳定性明显优于专利CN106137969B中产品的稳定性。
表8
Figure PCTCN2022094859-appb-000015
N/A：稳定性研究中暂无该时间点
实施例11考察蛋白药物比1.5的影响(同时降低蛋白浓度为5mg/ml)
称取无水多西他赛1.6g溶解于23.7ml无水乙醇中，溶解后得到有机相溶液，浓度为67.5mg/ml；取含2.4g白蛋白的人血白蛋白溶液(辛酸钠的含量为0.08mmol/g蛋白)，用注射用水稀释成含白蛋白5mg/ml的溶液，再加入适量盐酸半胱氨酸调节pH至4.0，40℃孵育0.5h，得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为14.4％的盐溶液；将有机相溶液和水相溶液升温至40℃，有机相、水相及盐溶液混合载药，所得物料置于冰水浴中降温至18℃，得到载药液；将载药液浓缩至含多西他赛浓度约6mg/ml，得浓缩液；使用等渗氯化钠(0.9％，w/v)溶液作为透析液对浓缩液进行5倍透析，透析膜截留分子量为30KDa，得到透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得到冻干前悬浮液；冻干，得冻干粉。
冻干前悬浮液中，多西他赛白蛋白纳米粒的粒径为91.38nm，在25℃条件下静置20小时，完全浑浊。使用注射用水复溶冻干粉得到重建悬浮液(等渗悬浮液)，重建悬浮液目测浑浊。
实施例12考察不同透析液的影响
称取无水多西他赛1.8g溶解于26.7ml无水乙醇中，溶解后得到有机相溶液，浓度为67.5mg/ml；取含8.1g白蛋白的人血白蛋白溶液(辛酸钠的含量为0.08mmol/g蛋白)，用注射用水稀释成含白蛋白15mg/ml的溶液，再加入适量盐酸半胱氨酸调节pH至4.0，40℃孵育0.5h，得酸变性白蛋白水相溶液；氯化钠用注射用水配制浓度为14.4％的盐溶液；将有机相溶液和水相溶液升温至40℃，有机相、水相及盐溶液混合载药，所得物料置于冰水浴中降温至18℃，得到载药液；将载药液浓缩至含多西他赛浓度约6mg/ml，得浓缩液；之后分别用以下两种方法处理并进行测量。
(1)使用等渗葡萄糖(5％，w/v)溶液作为透析液对浓缩液进行5倍透析，透析膜截留分子量为30KDa，得到透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得到冻干前悬浮液；冻干，得冻干粉。
冻干前悬浮液中，多西他赛白蛋白纳米粒的粒径为102.2nm，在25℃条件下静置16小时，底部较多沉淀，在2～8℃下静置16小时，透光率下降。使用注射用水复溶冻干粉得到重建悬浮液(等渗悬浮液)，多西他赛白蛋白纳米粒的粒径无明显变化，为103.3nm；重建悬浮液在25℃条件下2.5h已浑浊。
(2)使用等渗PBS缓冲液(pH为7.31，渗透压320mOsmol/Kg)作为透析液对浓缩液进行5倍透析，透析膜截留分子量为30KDa，得到透析后悬浮液；经0.45μm+0.2μm膜片进行除菌过滤，得到冻干前悬浮液；冻干，得冻干粉。
冻干前悬浮液中，多西他赛白蛋白纳米粒的粒径为92.43nm，在25℃条件下静置16小时，底部略有沉淀，在2～8℃下静置24小时，透光率下降。使用注射用水复溶冻干粉得到重建悬浮液(等渗悬浮液)，多西他赛白蛋白纳米粒的粒径无明显变化，为90.91nm；重建悬浮液在25℃条件下2.5h可见有析出。
实施例13注射用多西他赛(白蛋白结合型)(DTX-HSA)与多西他赛注射液(TAXOTERE)在裸小鼠中的最大耐受剂量(MTD)和毒性比较
1.药品及试验材料
1.1供试品
注射用多西他赛(白蛋白结合型)(DTX-HSA)，其采用本申请实施例1中的配方1-1、通过实施例1的制备方法制备得到。
1.2对照药
多西他赛注射液(商品名：泰索帝)
Figure PCTCN2022094859-appb-000016
2.实验动物
Figure PCTCN2022094859-appb-000017
3.实验设计
3.1实验原理
本试验定义药物对小鼠静脉给药的最大耐受剂量(MTD)为在观察期内，动物没有出现死亡和不可恢复的毒性反应，或者无体重减轻连续3天超过15％，该剂量即被认为是急性单次给药的最大耐受剂量。
3.2给药剂量设定
DTX-HSA复溶后等渗浓度为3.801mg/mL，参照2001年欧洲制药工业协会联合会和欧洲替代方法验证中心联合发布的关于对动物不同途径给药或采血时所能允许的给药体积和采血体积指导原则，小鼠缓慢静脉注射的最大给药容积为25mL/kg。24h内3次给药，剂量最大可给到285.1mg/kg。
本试验预选用剂量：
注射用多西他赛(白蛋白结合型)：285.1，228.1，182.5，146.0mg/kg(梯度1.25)。
多西他赛注射液(TAXOTERE)：187.5，150，120，96mg/kg(梯度1.25)。
4.实验方法
4.1动物分组及标记
挑选健康、体重差异较小的小鼠，按体重均衡分为8组，每组5只小鼠，分别为DTX-HSA285.1，228.1，182.5，146.0mg/kg组及TAXOTERE 187.5，150，120，96mg/kg组。
4.2给药
给药方式：静脉注射
给药频次：24h内给药3次，给药间隔4h。
给药容积：25mL/kg。
给药速度：缓慢推注。
给药量：以最近1次称重计算给药量。
4.3指标观察
一般状态观察：所有动物于试验期每天观察，观察指标包括但不限于皮肤、被毛、眼、耳、鼻、口腔、胸部、腹部、泌尿生殖部、四肢等部位，以及呼吸、运动、排尿、排便和行为改变等。动物不良反应参见下表。
体重：所有动物于试验前称重1次，挑选合适体重动物用于试验。每天固定时间称量动物体重1次。
死亡和濒死：死亡动物记录死亡时间，濒死动物注意增加观察频率，试验期间确定死亡时间。
4.4评价指标
在观察期内，小鼠无死亡，无不可恢复的毒性症状，无体重减轻连续3天超过15％的最大剂量即可认为是本实验药物的最大耐受剂量(MTD)。
5.实验结果
5.1死亡情况
DTX-HSA和TAXOTERE各给药组均未见动物死亡。
5.2临床观察
DTX-HSA 285.1和228.1mg/kg组动物分别自D4和D7开始，可见轻微后肢震颤，228.1mg/kg组动物D20后肢震颤症状恢复，285.1mg/kg组动物D22后肢震颤症状恢复。DTX-HSA182.5和146.0mg/kg组动物未见明显异常。
TAXOTERE 187.5，150mg/kg组动物分别自D4和D6开始，可见轻中度后肢震颤，150mg/kg组动物D19后肢震颤症状恢复，187.5mg/kg组动物D24后肢震颤症状恢复。TAXOTERE 120和96mg/kg组动物未见明显异常。
5.3体重
DTX-HSA 285.1mg/kg组1/5只动物D5～D8体重降低超过15％，1/5只动物在D10，D12，D13体重降低超过15％，其他DTX-HSA给药组未见体重降低超过15％的动物。
TAXOTERE 187.5mg/kg组1/5只动物D4～D14体重降低超过15％，其他TAXOTERE给药组未见体重降低超过15％的动物。
表9给药后小鼠毒性情况(n＝5)
Figure PCTCN2022094859-appb-000018
MTD：在观察期内，小鼠无死亡，无不可恢复的毒性症状，无体重减轻连续3天超过15％的最大剂量。观察时间：24天。
6.结论
本试验条件下，DTX-HSA裸小鼠MTD为228.1mg/kg；TAXOTERE裸小鼠MTD为150.0mg/kg。
实施例14 DTX-HSA与TAXOTERE对犬单次给药的副作用比较
36只Beagle犬，分为6组，每组6只动物(雌性各半)，分组及给药方式如下表：
表10犬给药剂量与给药方法
Figure PCTCN2022094859-appb-000019
注：组1给予试验动物的溶媒为0.9％氯化钠注射液，组2给予试验动物与组5制剂相等剂量的人血白蛋白溶液。DTX-HSA按照实施例1-1的配方和和制备方法制得(下同)。
结果：给药第1天：组5(DTX-HSA高剂量组)1只雄性动物给药后1小时可见活动减少和不能站立；组6(TAXOTERE对照组)所有雌雄动物可见活动减少、不能站立、耳朵皮肤/被毛变红、2只雌性动物可见流涎、4只雌性动物可见呕吐、2只雌性动物可见稀便/水样便，上述动物于给药后1小时内单次肌肉注射给予地塞米松磷酸钠注射液(来源：广州白云山天心制药股份有限公司，批号：181116)治疗可见征状好转，动物活动恢复正常。其余组(组1-4)动物均未出现过敏反应。
可见，相对于TAXOTERE，DTX-HSA这一剂型引起过敏反应的几率和严重程度均明显降低。
实施例15地塞米松对多西他赛疗效的影响(小鼠肝癌Hepa1-6模型)
试验模型：采用C57BL/6小鼠建立小鼠肝癌Hepa1-6同源移植瘤模型，评价地塞米松对多西他赛抗肿瘤作用的影响。
试验方法：50只雌性C57BL/6小鼠，右前肢腋部皮下接种小鼠肝癌Hepa1-6细胞(4×10 6/只/0.1mL)，接种后第6天(当天记为D0)，挑选肿瘤生长良好的32只动物，按肿 瘤体积均衡分为4组，每组8只动物，分组及给药方式如下表：①、②、③组按照与④组给予DEX相同的频次灌胃给予蒸馏水。给药体积10mL/kg。
表11给药剂量与给药方法
Figure PCTCN2022094859-appb-000020
D15结束试验。数据采用SPSS 19.0统计软件分析。
试验结果：由表12、图1可知，本试验条件下，与溶剂对照组相比，DTX-HSA和TAXOTERE均能显著抑制Hepa1-6移植瘤的生长(P<0.01)，且效果相当。而DEX的加入极大地影响了TAXOTERE的抑瘤效果(DTX-HSA或TAXOTERE组与TAXOTERE/DEX组相比，P<0.001)，TAXOTERE/DEX组的肿瘤体积甚至显著高于溶剂对照组(D15，P<0.05)，提示为预防过敏反应和体液潴留联合使用DEX，会促进肿瘤生长，极大地削弱多西他赛的抗肿瘤作用。DTX-HSA不需要地塞米松预处理，避免了DEX促进肿瘤生长的不良影响，对于改善多西他赛的抗肿瘤作用有重要意义。
注：肿瘤体积＝1/2×长径×短径 2，使用游标卡尺测量肿瘤长短径。
表12地塞米松对多西他赛疗效的影响
组别 平均瘤体积(mean±sd)
①溶剂对照组 484.6±193.1
②DTX-HSA 217.9±76.2 **###
③TAXOTERE 233.4±84.6 **###
④TAXOTERE/DEX 794.6±379.4 *
注：与溶剂对照组比较， *P<0.05； **P<0.01；与TAXOTERE/DEX组比较， ###P<0.001
实施例16地塞米松对Opdivo及其与多西他赛联用方案疗效的影响(小鼠结肠癌MC38模型)
试验模型：采用百奥赛图B-hPD-1小鼠建立小鼠结肠癌MC38同源移植瘤模型，评价DEX对单用Opdivo的抑瘤作用的影响，以及DTX-HSA或DEX/TAXOTERE与PD-1抗体(Opdivo)联用的抑瘤效果。
试验方法：43只雌性人源化B-hPD-1小鼠，右前肢腋部皮下接种小鼠结肠癌MC38细胞(2×10 6/只/0.1mL)，接种后第5天(当天记为D0)，挑选肿瘤生长良好的40只动物，平均肿瘤体积约110mm 3。按肿瘤体积均衡分为5组，每组8只动物，分组及给药方式如下表：
①②、⑤组按照与③组给予DEX相同的频次灌胃给予蒸馏水。
DTX-HSA给药体积为20mL/kg，其他均为10mL/kg。
表13给药剂量与给药方法
Figure PCTCN2022094859-appb-000021
D19结束试验。数据采用SPSS 19.0统计软件分析。以肿瘤体积(mm 3)为疗效指标。
表14各组瘤体积比较
组别 平均瘤体积(mean±sd)
①溶剂对照组 1325.2±787.8
②DTX-HSA/Opdivo 478.1±249.9 **##
③TAXOTERE/DEX/Opdivo 1151.3±580.4
④DEX/Opdivo 733.0±260.3 *
⑤Opdivo 505.7±191.6 **#
注：与溶剂对照组比较， *P<0.05， **P<0.01；与DEX/Opdiv组比较， #P<0.05；与TAXOTERE/DEX/Opdivo组比较， ##P<0.01。
试验结果：由表14、图2A可知，本试验条件下，与溶剂对照组相比，Opdivo组肿瘤体积明显减小(P<0.01)，说明单用Opdivo30mg/kg具有显著的抑瘤效果；进一步使用DEX处理，DEX/Opdivo虽然体现出一定的抑瘤作用(与溶剂对照组相比，P<0.05)，但与单用Opdivo相比，抑制鼠结肠癌MC38移植瘤生长的作用显著降低(P<0.05)，表明DEX预处理会影响PD-1抗体Opdivo的疗效。
进一步联合TAXOTERE(76mg/kg)，TAXOTERE/DEX/Opdivo组与DEX/Opdivo组相比，抑瘤效果不仅没有增强，肿瘤体积甚至略有增大。而使用DTX-HSA代替TAXOTERE，省略DEX处理，肿瘤体积显著减小，与TAXOTERE/DEX/Opdivo组相比，DTX-HSA/Opdivo抑制鼠结肠癌MC38移植瘤生长的作用显著增强(P<0.01)(见图2B)。试验结果表明，给予相同剂量的Opdivo和多西他赛，与多西他赛普通制剂相比，DTX-HSA制剂无需使用DEX预处理，能有效避免联用DEX降低Opdivo疗效的弊端，能更有效地抑制肿瘤生长。
实施例17地塞米松对多西他赛与Keytruda联用方案疗效的影响(小鼠结肠癌MC38模型)
试验模型：采用百奥赛图B-hPD-1小鼠建立小鼠结肠癌MC38同源移植瘤模型，评价DTX-HSA或DEX/TAXOTERE与PD-1抗体(Keytruda)联用的抑瘤效果。
试验方法：30只雌性人源化B-hPD-1小鼠，右前肢腋部皮下接种小鼠结肠癌MC38细胞(2×10 6/只/0.1mL)，接种后第5天(当天记为D0)，挑选肿瘤生长良好的24只动物，平均肿瘤体积约68mm 3。按肿瘤体积均衡分为3组，每组8只动物，分组及给药方式如下表：
①、②组按照与③组给予DEX相同的频次灌胃给予蒸馏水。给药体积10mL/kg。
表15给药剂量与给药方法
Figure PCTCN2022094859-appb-000022
D20结束试验。数据采用SPSS 19.0统计软件分析。以肿瘤体积(mm 3)为疗效指标。
表16各组瘤体积比较
Figure PCTCN2022094859-appb-000023
注：与溶剂对照组比较， ***P<0.001；与TAXOTERE/DEX/Keytruda组比较， ###P<0.001。
试验结果：由图3可知，本试验条件下，与溶剂对照组相比，TAXOTERE/DEX/Keytruda组未表现出显著地抑瘤功效，提示DEX预处理，显著削弱了多西他赛和Keytruda的抗肿瘤作用。而将普通多西他赛制剂(TAXOTERE)替换成本申请所述的DTX-HSA，无需联用DEX，DTX-HSA/Keytruda组显著抑制MC38移植瘤的生长，抗肿瘤作用显著增强(与溶剂对照组比较，P＜0.001；与TAXOTERE/DEX/Keytruda组比较，P<0.001)。试验结果表明DTX-HSA与Keytruda联用，省略DEX，能有效避免DEX预处理削弱多西他赛与PD-1抗体Keytruda抑瘤作用的不良影响。
实施例18地塞米松对Keytruda、多西他赛及联用方案疗效的影响(小鼠肝癌Hepa1-6模型)
试验模型：采用百奥赛图B-hPD-1小鼠建立小鼠肝癌Hepa1-6同源移植瘤模型，评价地塞米松对DTX-HSA和TAXOTERE与PD-1抗体(Keytruda)联用的抑瘤效果的影响。 试验方法：60只雌性人源化B-hPD-1小鼠，右前肢腋部皮下接种小鼠肝癌Hepa1-6细胞(5×10 6/只/0.1mL)，接种后第5天(当天记为D0)，挑选肿瘤生长良好的48只动物，按肿瘤体积均衡分为6组，每组8只动物，分组及给药方式如下表：①、②、④、⑤组按照与③组给予DEX相同的频次灌胃给予蒸馏水。DTX-HSA给药体积为20mL/kg，其他均为10mL/kg。
表17给药剂量与给药方法
Figure PCTCN2022094859-appb-000024
D20结束试验。数据采用SPSS 19.0统计软件分析。
试验结果：由图4A可知，DTX-HSA组小鼠的体重与溶剂对照组无显著差异，提示DTX-HSA的耐受性较好。而经DEX预处理的TAXOTERE组小鼠，体重与溶剂对照组和DTX-HSA相比明显降低(P＜0.05)，耐受性较DTX-HSA组差。由图4B可知，TAXOTERE给予DEX预处理后，显著促进Hepa1-6肿瘤生长，D20肿瘤体积显著高于溶剂对照组(P＜0.01)。而DTX-HSA不需要DEX预处理，能够显著抑制肿瘤的生长(P＜0.05)。试验结果表明DTX-HSA不仅耐受性较TAXOTERE/DEX好，而且能有效避免DEX预处理降低多西他赛抗肿瘤效果的不良影响。
由图5A可知，DTX-HSA与Keytruda联用后动物耐受良好，体重与溶剂对照组相比无明显差异。TAXOTERE给予DEX预处理后再与Keytruda联用，体重明显减低(与溶剂 对照组相比，P＜0.05；与DTX-HSA/Keytruda组相比，P<0.05)，动物耐受性较DTX-HSA与Keytruda联用差。
由图5B可知，DTX-HSA与Keytruda联合给药，可显著抑制肿瘤的生长(P＜0.001)。而TAXOTERE给予DEX预处理后，与Keytruda联合给药后几乎无抗肿瘤活性(与溶剂对照组相比，P>0.05)。进一步表明DEX预处理会影响多西他赛与PD-1抗体联用的抗肿瘤效果。
试验过程中，DEX/TAXOTERE/Keytruda组死亡1只小鼠，其余各组无死亡。由图6可知，单独使用DTX-HSA和Keytruda能显著抑制Hepa1-6移植瘤的生长，DTX-HSA与Keytruda联合给药，无需地塞米松预处理，对Hepa1-6移植瘤取得了更好的抑制作用。
表18各组瘤重比较
Figure PCTCN2022094859-appb-000025
注：与溶剂对照组比较， *P<0.05； **P<0.01；与DEX/TAXOTERE组比较， #P<0.05， ###P<0.001。
实施例19 Keytruda和多西他赛联合用药的协同疗效(小鼠黑色素瘤B16/F10模型)
试验模型：采用百奥赛图B-hPD-1小鼠建立小鼠黑色素瘤B16/F10同源移植瘤模型，评价注射用多西他赛(白蛋白结合型)与PD-1抗体Keytruda联合抗肿瘤作用。
试验方法：40只雌性人源化B-hPD-1小鼠，按体重均衡分为4组，每组10物，全部小鼠接种B16/F10细胞(1×10 6/只/0.1mL)，接种当天记为D0，分组及给药方式如下表：
表19给药剂量与给药方法
Figure PCTCN2022094859-appb-000026
Figure PCTCN2022094859-appb-000027
D15结束试验。数据采用SPSS 19.0统计软件，Repeated Measure过程分析随时间变化多次测量间肿瘤体积的变化。采用Burgi修正公式：Q＝E(a+b)/[E(a)+E(b)-E(a)*E(b)(其中E(a+b)：a、b两药联合给药后的抑瘤率，E(a)：a单独给药时的抑瘤率，E(b)：b单独给药时的抑瘤率。Q<0.85具有拮抗作用，0.85≤Q≤1.15具有相加作用，Q>1.15具有协同作用)判断两种药物的联合作用。
试验结果：试验期间，溶剂对照组死亡3只小鼠，其余各组小鼠无死亡。由图7和表20可知，本试验条件下，与溶剂对照组相比，单独使用DTX-HSA能显著抑制B16/F10移植瘤的生长(P＜0.001)，Keytruda对B16/F10移植瘤也有一定的抑制作用。DTX-HSA与Keytruda联合给药后对B16/F10移植瘤的抑制作用具有协同作用(P＜0.001)(Q＝0.713/((0.474+0.177)-0.474*0.177)＝1.26)。
表20各组瘤重比较
Figure PCTCN2022094859-appb-000028
注：与溶剂对照组比较， ***P<0.001。
实施例20 DTX-HSA与Opdivo联合治疗的临床研究
一、试验设计
1.1总体设计
本试验为一项针对含铂方案治疗进展的PD-L1表达阳性的复发或转移性头颈部鳞癌受试者的单臂、多中心Ib/II期临床研究，旨在评价注射用多西他赛(白蛋白结合型)联合Nivolumab的有效性、安全性和多西他赛的药代动力学特征。
1.2剂量选择依据
给药方案：
Nivolumab的给药方案为：Nivolumab 360mg Q3W(每3周给药一次)，i.v.30min
注射用多西他赛(白蛋白结合型)的给药方案为：75mg/m 2或100mg/m 2，Q3W，i.v.30～60min
制定依据：
参照Nivolumab(360mg，Q3W)联合伊匹木单抗(Ipilimumab)及两个周期的含铂双药化疗治疗无EGFR和ALK基因突变的转移或复发性NSCLC方案(CheckMate-9LA)、Nivolumab(360mg，Q3W)联合XELOX(奥沙利铂+卡培他滨)治疗晚期或转移性胃癌/胃食管结合部腺癌方案，本研究中Nivolumab采用360mg Q3W的给药方式。
目前正在开展的临床I期研究显示，注射用多西他赛(白蛋白结合型)100mg/m 2Q3W在实体瘤患者中未观察到DLT(剂量限值毒性)，已进入125mg/m 2Q3W剂量爬坡阶段。结合已上市制剂泰索帝临床研究，实体瘤患者中MTD(最大耐受剂量)为115mg/m 2Q3W，临床常用给药方案为75～100mg/m 2Q3W。综上本研究Ib期注射用多西他赛(白蛋白结合型)选择75mg/m 2or 100mg/m 2Q3W，i.v.60min。
综上，此临床研究联合给药方案为：Nivolumab 360mg Q3W,i.v.30min联合注射用多西他赛(白蛋白结合型)75mg/m 2Q3W,i.v.60min，或Nivolumab 360mg Q3W,i.v.30min联合注射用多西他赛(白蛋白结合型)100mg/m 2Q3W,i.v.60min。根据安全性和疗效结果选择其中一个给药方案进行II期研究。
1.3试验设计
Ib期：
探索75mg/m 2和100mg/m 2两个剂量水平的注射用多西他赛(白蛋白结合型)联合Nivolumab 360mg的安全性和耐受性。
剂量探索从低剂量开始，依次进行。每个剂量组初始入组3例受试者，观察21天，若无DLT发生，则进行更高剂量水平研究(最高100mg/m 2)；若3例中有1例发生DLT，则在该剂量水平继续入组3例受试者进行安全性观察，若6例受试者中<2例发生DLT，则进行更高剂量水平研究(最高100mg/m 2)；若3例中有2例发生DLT，则研究者与申办方共同讨论决定后续研究。
DLT定义为：第一周期内(21天)发生的与试验药物有关的以下毒性反应：
(1)非血液学毒性：
4级(含)以上毒性作用。
3级毒性作用，以下除外：
经最佳支持治疗后3天内恢复至≤2级；
3级肝功能异常，但经治疗后7天内恢复至1级或基线；
经研究者判断无症状且无需干预的单纯性实验室检查异常。
(2)血液学毒性：
4级中性粒细胞减少，持续>5天。
发热性中性粒细胞减少(ANC<1.0×10 9/L，一次体温达到38.3℃或体温持续在38℃超过一小时)，持续>3天。
3级血小板减少伴2级出血。
4级血小板减少。
4级贫血。
死亡。
(3)其他经研究者判断应永久停用试验用药品的毒性反应。
后续给药周期发生的安全性事件也是选择联合方案剂量的影响因素之一。
综合分析Ib期的安全性、耐受性、有效性及药代动力学研究结果，确定RP2D(2期推荐剂量)，进行II期研究。
II期：
根据Ib期研究确定的RP2D，开展注射用多西他赛(白蛋白结合型)联合Nivolumab的II期研究，以观察联合方案的有效性，主要研究终点为ORR。
客观缓解率(ORR)定义：开始使用试验药物至退出研究期间，研究者根据RECIST v1.1(基于单径测量基础上实体瘤疗效反应的评价标准)评估的研究中最佳总体反应为完全缓解(CR)或部分缓解(PR)(即CR+PR)的受试者比例。首次评估PR或CR的受试者需要在4周后进行疗效确认。
II期试验采用Simon二阶段(Optimum)设计。第一阶段需要入组受试者30例，若缓解(PR+CR)例数不超过5例，则试验终止，否则，试验推进至第二阶段，再继续入组52例受试者，若两阶段缓解总例数超过17例，则认为该联合方案在该适应症有后续开发价值。两阶段共需入组受试者82例。
Ib期和II期所有受试者接受注射用多西他赛(白蛋白结合型)RP2D联合Nivolumab治疗，直至符合试验终止治疗或退出标准，试验用药品最长使用2年。
每6周(42天±7天)进行1次肿瘤评估，需由独立的影像评估中心(IRC)进行确认。
为评估注射用多西他赛(白蛋白结合型)在联合Nivolumab后的药代动力学特征，Ib期和II期所有受试者均需要在第1和3周期进行PK血样采集，用于检测游离和总的多西他赛含量。
二、试验人群：
2.1入选标准
受试者必须符合以下所有标准，才有资格入组本试验：
1.年龄≥18周岁(以签署知情同意书当天为准)且自愿签署知情同意书者。
2.组织学或细胞学确诊的头颈部鳞癌(原发性肿瘤位于口腔、口咽、喉或下咽部)，PD-L1表达阳性(表达PD-L1的肿瘤细胞≥1％)，且不适合接受局部根治性治疗者。
3.含铂方案治疗失败，定义为：
a).复发或转移性头颈部鳞癌，接受含铂方案治疗期间或之后疾病进展；
b).局部晚期头颈癌，既往多模式治疗中，含铂方案治疗之后6个月内复发或转移；
4.有既往或合格的肿瘤组织样本用于检测PD-L1：
a)Ib期：可接受既往3级及以上医院或有CAP、CLIA或其他同等资质认证的中心实验室的PD-L1检测结果，检测结果需经申办方生物标志物团队审核。或提供新鲜或3年内存档的肿瘤组织用于PD-L1免疫组织化学(IHC)检测。
b)II期：可提供新鲜或3年内存档的肿瘤组织用于PD-L1免疫组织化学(IHC)检测。
5.口咽癌受试者需提供既往HPV p16免疫组化检测结果，或合格的肿瘤组织样本用于检测HPV状态。
6.根据RECIST v1.1，至少有一个CT或MRI确认的可测量病灶(既往接受过放疗，疾病进展者或放疗后≥3个月肿瘤持续存在者可作为可测量病灶)。
7.ECOG评分体能状态为0-1的受试者。
8.预计生存期≥3个月者。
9.主要器官功能在治疗前7天内，符合下列标准(在试验用药品给药前2周内未接受过成分输血、人粒细胞集落刺激因子(G-CSF)、促血小板生成素(TPO)、白介素-11和促红细胞生成素(EPO)等医学支持治疗)：
Figure PCTCN2022094859-appb-000029
Figure PCTCN2022094859-appb-000030
10.有生育潜力的妇女在首次使用试验用药品前7天内的血清妊娠试验为阴性，受试者及其配偶必须同意从签署知情同意书开始至末次给药后6个月内采取足够的避孕措施，此期间女性为非哺乳期且男性避免捐精。
2.2排除标准
受试者符合下列任何一项排除标准，则没有资格参与本项研究：
1.组织学或细胞学确诊的复发或转移性鼻咽癌、原发病灶不明的头颈部鳞癌、唾液腺癌或非鳞状组织癌(如粘膜黑色素瘤)。
2.活动性脑转移和软脑膜转移。有脑转移的，需要达到治疗后至少8周和首次使用试验用药品前28天内，核磁共振证据显示未PD的，可以入组；在首次使用试验用药品前至少2周，不需要接受系统性皮质醇激素治疗(强的松>10mg/天或等价剂量的同类药品)可以入组；没有硬脑膜或脑实质受累确切证据的颅底病变，需要与申办方医学监察员讨论后才能考虑能否入组。
3.首次使用试验用药品前5年内有其他恶性肿瘤病史，除外以下情况：a.任何其他侵袭性恶性肿瘤(受试者因其进行过充分治疗)无病状态持续>3年，研究者评估不会影响肿瘤疗效评估；b.已被治愈的基底细胞或鳞状细胞皮肤癌、浅表膀胱癌、前列腺癌、宫颈癌或乳腺癌原位癌等局部可治愈的癌症。
4.首次使用试验用药品前2年内患有已知或疑似自身免疫疾病，除外以下情况：a.控制良好的I型糖尿病；b.只需接受激素替代治疗且控制良好的甲状腺功能减退症；c.无需进行全身治疗的皮肤疾病(如白癜风、银屑病或脱发)；d.预计在无外部触发因素的状态下病情不会复发的受试者。
5.有难以控制的第三腔隙积液(eg.胸腔积液、腹水或心包积液)，经研究者判断不适合入组。
6.首次使用试验用药品前6个月内有严重的心血管疾病史，包括但不限于：
a).有严重的心脏节律或传导异常，如需要临床干预的室性心律失常、Ⅲ度房室传导阻滞等；
b).有心肌梗塞、心绞痛、血管成形术、冠状动脉架桥外科病史；
c).心力衰竭，纽约心脏病学会(NYHA)分级为III级及以上；
d).控制不良的高血压(尽管使用了最优治疗，收缩压≥150mmHg和/或舒张压≥95mmHg)。
7.筛选期心电图QT/QTc间期延长者(QTcF>480ms，Fridericia公式：QTcF＝QT/RR0.33，RR＝60/心率)和/或筛选期超声心动图(ECHO)或多门控采集(MUGA)技术显示左室射血分数(LVEF)≤50％；
8.筛选期HCV抗体(+)阳性(HCV RNA阴性可以纳入，允许干扰素以外的抗HCV治疗)、活动性乙型肝炎(HBV DNA≤500IU/ml可以纳入，允许干扰素以外的抗HBV治疗)、已知HIV阳性或已知获得性免疫缺陷综合症(AIDS)。
9.在首次使用试验用药品前4周内接受过主要脏器外科手术(不包括穿刺活检、气切和胃造瘘)，或需要在试验期间接受择期手术者。
10.既往抗肿瘤治疗毒性反应未恢复至1级及以下(CTCAE V5.0)，除外以下情况：2级神经病变、脱发，既往抗肿瘤治疗引起的甲状腺功能减退(含激素替代治疗者)以及研究者判断无安全风险的毒性。
11.既往接受过T细胞协同刺激或作用于免疫检查点通路的药物(包括PD-1、PD-L1/2、CTLA-4抑制剂等)。
12.既往接受其他免疫治疗且发生过≥3级的irAE(免疫相关不良反应)。
13.既往接受过紫杉类药物治疗者(既往接受含紫杉类诱导化疗，且6个月后进展的可以入组)。
14.在首次使用试验用药品前4周内接受过化疗、放疗、靶向治疗、免疫治疗和其他临床研究药物等抗肿瘤治疗，其他情况如下：
首次使用试验用药品前2周内进行的局部姑息放疗；首次使用试验用药品前2周内或已知药物的5个半衰期内(以时间长的为准)的口服氟尿嘧啶类、小分子靶向药物等的治疗；首次使用试验用药品前2周内服用具有抗肿瘤适应症的中药。
15.在首次使用试验用药品前2周内接受皮质类固醇(强的松>10mg/天或等价剂量的同类药物)或其他免疫抑制剂治疗，除外以下情况：a.使用局部、眼部、关节腔内、鼻内和吸入型糖皮质激素治疗；b.短期使用糖皮质激素进行预防治疗(例如预防造影剂过敏)。
16.在首次使用试验用药品前2周内或计划在研究期间接受减毒活疫苗。
17.在首次使用试验用药品前2周内使用过CYP3A4的强效抑制剂或强效诱导剂。
18.已知对白蛋白或单克隆抗体存在≥3级超敏反应和/或禁忌症者。
19.其他研究者认为不适合参加临床试验的情况，包括但不限于：受试者并发严重或不受控制的医学病症，干扰研究结果的解读，影响试验依从性等情况。
2.3终止治疗/退出标准
受试者可以持续使用试验用药品直至符合终止治疗/退出标准，或者治疗满2年。
终止治疗标准：
●撤回知情同意书；
●妊娠或计划妊娠；
●对研究干预的依从性差；
●发生临床不良事件(AE)、实验室检查异常或其他医疗状况，继续参与研究将不符合受试者的最佳获益；
●疾病进展，若符合以下情况，可继续治疗：
√受试者对试验药物耐受；
√研究者评估临床稳定；
√后续治疗不会延迟其他严重并发症的干预措施；
●严重的方案偏离影响本研究评价；
●已接受试验用药品满2年；
●其他研究者决定的终止；
退出标准：
●撤回知情同意书并拒绝接受后续随访；
●失访；
●死亡；
三、研究结果
本实施例采用注射用多西他赛(白蛋白结合型)联合Nivolumab，研究其在复发或转移性头颈部鳞癌的临床疗效，不需要地塞米松预处理，进一步改善免疫检查点抑制剂对复发或转移性头颈部鳞癌患者的疗效，提高应答率，延长总生存期OS，降低死亡风险，使患者受益。
本研究的结果表明，两药联合后不仅没有产生新的安全性问题，而且对头颈部鳞癌的治疗产生了显著的协同效应。这表明本申请的多西他赛白蛋白组合物和免疫检查点抑制剂联用在治疗肿瘤时能够产生协同作用，显著提升了抗肿瘤的效果。

Claims (13)

  1. 多西他赛白蛋白组合物和免疫检查点抑制剂在制备用于治疗肿瘤的药物中的用途，其中所述免疫检查点抑制剂优选为PD-1抑制剂或PD-L1抑制剂。
  2. 多西他赛白蛋白组合物在制备用于改善免疫检查点抑制剂治疗肿瘤的疗效的药物中的用途，其中所述免疫检查点抑制剂优选为PD-1抑制剂或PD-L1抑制剂。
  3. 复方药物或药物组合产品，其包含多西他赛白蛋白组合物和免疫检查点抑制剂，其中所述免疫检查点抑制剂优选为PD-1抑制剂或PD-L1抑制剂，和/或优选地，所述复方药物或药物组合产品用于治疗肿瘤。
  4. 多西他赛白蛋白组合物，其用于改善免疫检查点抑制剂治疗肿瘤的疗效，其中所述免疫检查点抑制剂优选为PD-1抑制剂或PD-L1抑制剂。
  5. 用于治疗个体的肿瘤的方法，其包括向所述个体施用治疗有效量的多西他赛白蛋白组合物和免疫检查点抑制剂，或者权利要求3所述的复方药物或药物组合产品。
  6. 用于改善免疫检查点抑制剂对肿瘤的疗效的方法，其包括向已经接受免疫检查点抑制剂治疗的患肿瘤的个体施用治疗有效量的多西他赛白蛋白组合物。
  7. 如权利要求1或2所述的用途、权利要求3所述的复方药物或药物组合产品、权利要求4所述的多西他赛白蛋白组合物，权利要求5或6所述的方法，其中所述多西他赛白蛋白组合物为多西他赛白蛋白纳米粒组合物，其中所述多西他赛白蛋白纳米粒的粒径优选为约60-200nm，更优选为90-150nm，例如为90-135nm。
  8. 如权利要求7所述的用途、复方药物或药物组合产品、多西他赛白蛋白组合物或者方法，其中所述多西他赛白蛋白纳米粒组合物中的白蛋白为人血白蛋白，优选为酸变性白蛋白，以及优选地，所述白蛋白中辛酸钠的含量不高于0.12mmol/g蛋白，更优选不高于0.08mmol/g蛋白，例如为0.045-0.08mmol/g蛋白。
  9. 如权利要求1-8任一项所述的用途、复方药物或药物组合产品、多西他赛白蛋白组合物或者方法，其中所述肿瘤为实体瘤，优选地，所述实体瘤选自唾液腺癌、食管癌和食管胃部结合部癌、未分化甲状腺癌、卵巢癌、头颈癌、结直肠癌、肝癌、黑色素瘤、非小细胞肺癌、乳腺癌、胃癌、肾细胞癌、胆管癌、膀胱癌及尿路肿瘤、小细胞肺癌、胰腺癌、子宫肿瘤、鼻咽癌、口咽癌、喉咽癌、喉癌、口腔癌、唇癌、上颌窦肿瘤、筛窦肿瘤或骨 肿瘤，更优选地，所述实体瘤选自结肠癌、肝癌、黑色素瘤或头颈部鳞状细胞癌，优选为黑色素瘤或头颈部鳞状细胞癌。
  10. 如权利要求1-9任一项所述的用途、复方药物或药物组合产品、多西他赛白蛋白组合物或者方法，其中所述药物、所述复方药物或药物组合产品或者所述多西他赛白蛋白组合物被配制成临床可接受的制剂，优选为注射剂型，更优选为液体注射剂、注射用粉剂或注射用片剂；以及任选地，所述多西他赛白蛋白组合物和免疫检查点抑制剂存在于同一制剂中，或者分别配制为不同的制剂。
  11. 如权利要求10所述的用途、复方药物或药物组合产品、多西他赛白蛋白组合物或者方法，其中当所述制剂为液体注射剂时，以无水多西他赛计，多西他赛活性成分含量为3.3-4.3mg/ml。
  12. 如权利要求1-11任一所述的用途、复方药物或药物组合产品、多西他赛白蛋白组合物或者方法，其中所述PD-1抑制剂或PD-L1抑制剂选自重组人源化抗PD-1单克隆抗体注射液、重组抗PD-1全人源单克隆抗体注射液(PD-1单抗)，Atezolizumab、Avelumab、Durvalumab或全人源抗PD-L1抗体注射液。
  13. 如权利要求1-12任一所述的用途、复方药物或药物组合产品或者方法，其中所述药物或者所述复方药物或药物组合产品还包含其他的肿瘤治疗剂。
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