WO2023109904A1 - 多西他赛白蛋白组合物与vegf抑制剂或vegfr抑制剂的组合及用途 - Google Patents

多西他赛白蛋白组合物与vegf抑制剂或vegfr抑制剂的组合及用途 Download PDF

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WO2023109904A1
WO2023109904A1 PCT/CN2022/139315 CN2022139315W WO2023109904A1 WO 2023109904 A1 WO2023109904 A1 WO 2023109904A1 CN 2022139315 W CN2022139315 W CN 2022139315W WO 2023109904 A1 WO2023109904 A1 WO 2023109904A1
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docetaxel
cancer
albumin
drug
solution
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PCT/CN2022/139315
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English (en)
French (fr)
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李春雷
张晓君
姚雪坤
赫晓林
祁欢欢
张丽轩
赵媛媛
陈东健
邢倩斌
王世霞
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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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Publication of WO2023109904A1 publication Critical patent/WO2023109904A1/zh

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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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

Definitions

  • the application belongs to the field of medicine, and specifically relates to the use of a docetaxel albumin composition and a VEGF inhibitor or a VEGFR 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 has poor patient compliance.
  • ethanol can affect the central nervous system, and the infusion rate needs to be reduced to slow down the symptoms of poisoning.
  • 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 high-concentration rapid administration shortens 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.
  • 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.
  • VEGF or VEGFR inhibitors which prevent tumor angiogenesis by blocking VEGF or VEGFR signaling pathways, thereby inhibiting tumor growth.
  • approved VEGF or VEGFR inhibitors include bevacizumab, ranibizumab, aflibercept, pegaptanib, rivoceranib, pyrotinib dimaleate, ponatinib, tivozanib, nintedanib, plitidepsin, surufatinib, sorafenib, lenvatinib Ni, sunitinib, pazopanib, cabozantinib, regorafenib, vandetanib, fruquintinib, axitinib, etc.
  • the approved indications cover colorectal cancer, non-small A variety of malignant tumors in cell lung cancer, ovarian cancer, cervical cancer, liver cancer, glioma, kidney cancer
  • docetaxel that can be used in tumor therapy without pretreatment with corticosteroids (such as dexamethasone) when used.
  • This application provides a new dosage form of docetaxel, which does not require corticosteroid (such as dexamethasone (DEX)) pretreatment during clinical use, improves the convenience and safety of clinical administration, and is compatible with VEGF inhibitors or VEGFR inhibitors
  • corticosteroid such as dexamethasone (DEX)
  • DEX dexamethasone
  • the combination of drugs can further improve the anti-tumor effect and produce a synergistic anti-tumor effect.
  • the present application provides the use of a docetaxel albumin composition and a VEGF inhibitor or a VEGFR inhibitor in the preparation of a drug for treating tumors.
  • the present application provides the use of the docetaxel albumin composition in the preparation of a drug for improving the curative effect of a VEGF inhibitor or a VEGFR inhibitor in treating tumors.
  • the present application provides a compound drug or drug combination product for treating tumors, which comprises (1) a docetaxel albumin composition, and (2) a VEGF inhibitor or a VEGFR inhibitor.
  • the present application provides a drug for improving the efficacy of VEGF inhibitors or VEGFR inhibitors in treating tumors, which comprises a docetaxel albumin composition.
  • 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) VEGF inhibitor or VEGFR inhibitor, or compound drug or drug combination product as described in the third aspect.
  • the present application provides a method for improving the efficacy of a VEGF inhibitor or a VEGFR inhibitor in treating tumors, which includes administering a therapeutically effective amount of multiple Sitaxel albumin composition. .
  • 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 a gynecological tumor, such as ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, endometrial cancer, more preferably epithelial ovarian cancer, fallopian tube cancer or primary peritoneal cancer.
  • a gynecological tumor such as ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, endometrial cancer, more preferably epithelial ovarian cancer, fallopian tube cancer or primary peritoneal cancer.
  • 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 an injection form, including liquid injection, powder for injection, and tablet for injection.
  • the albumin nanoparticles of docetaxel and the VEGF inhibitor or VEGFR inhibitor can be present in the same pharmaceutical preparation, or can be formulated independently to make the medicine in the form of a combination product.
  • the active ingredient when the composition containing docetaxel albumin nanoparticles is made into a liquid injection, based on anhydrous docetaxel, the active ingredient is 3.3-4.3 mg/ml, preferably 4 mg/ml .
  • the docetaxel albumin nanoparticles are injection powders, the active ingredient is 80 mg/bottle based on anhydrous docetaxel.
  • the VEGF inhibitor or VEGFR inhibitor is selected from anti-VEGF monoclonal antibodies such as bevacizumab and its biosimilars, VEGFR TKI inhibitors such as anlotinib and apatinib, preferably Bevacizumab.
  • the drug or the compound drug or drug combination product further includes other drugs for treating the tumor.
  • the tumor is preferably ovarian cancer, fallopian tube cancer, and primary peritoneal cancer.
  • the present application provides the use of docetaxel albumin nanoparticles and bevacizumab in the preparation of a drug for treating tumors.
  • the present application provides the use of docetaxel albumin nanoparticles in the preparation of a drug for improving the effect of bevacizumab in treating tumors.
  • the present application provides a drug for treating tumors, which comprises docetaxel albumin nanoparticles and bevacizumab.
  • the present application provides a drug for improving the effect of bevacizumab in treating tumors, which comprises docetaxel albumin nanoparticles.
  • the drug may further include other drugs for the treatment of ovarian cancer, fallopian tube cancer, and primary peritoneal cancer.
  • 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 injection powders
  • the active ingredient is 80 mg/bottle based on anhydrous docetaxel.
  • the present application provides a combination regimen for treating tumors, which includes administering therapeutically effective doses of docetaxel albumin nanoparticles for injection and bevacizumab to tumor/cancer patients.
  • the administration is preferably injection administration.
  • the present application also provides a method for improving the curative effect of bevacizumab on tumors, which comprises further administering a therapeutically effective amount in combination with bevacizumab to tumor patients docetaxel albumin nanoparticles.
  • 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 dosage of docetaxel albumin nanoparticles for injection is 50, 75, 100, 125, 150 mg/m 2 , once every 3 weeks (denoted as Q3W).
  • the administration of bevacizumab is 15 mg/kg, Q3W, preferably intravenous administration, and bevacizumab is given first and then docetaxel white protein.
  • the administration period is the same as that of docetaxel albumin nanoparticles for injection.
  • bevacizumab is infused intravenously, 15 mg/kg Q3W, and the time for the first infusion of bevacizumab is about 90 minutes. If the first infusion is well tolerated, the time for the second infusion The time can be shortened to about 60 minutes. If the patient also has good tolerance to the infusion of about 60 minutes, all subsequent infusions can be completed in about 30 minutes. Afterwards, docetaxel for injection (albumin-bound) 100 mg/m 2 Q3W intravenously for about 30 minutes, or docetaxel for injection (albumin-bound) 75 mg/m 2 Q3W for about 30 minutes.
  • 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 docetaxel and acid-denatured albumin or is made of docetaxel and acid-denatured albumin.
  • 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 preferably 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 include a pH regulator to adjust the pH to an appropriate
  • a pH regulator to adjust the pH to an appropriate
  • stable 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 for at least 24 hours at 25°C, 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 freeze-dried 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.
  • Said 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 the 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%-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 dialysate can be determined by those skilled in the art according to the required sodium octanoate content through routine tests.
  • the volume of the dialysate is more than 3 times the volume of the diluted albumin solution, preferably 3-10 times, more preferably 3-6 times.
  • 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 ninth 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.
  • the 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: take a commercially available human serum albumin solution and dilute it with water for injection or normal saline to obtain the diluted albumin solution; use water for injection or normal saline as the dialysate to dialyze the diluted albumin solution to partially remove sodium octanoate to obtain a 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 more than 3 times the volume of the diluted albumin solution, preferably 3-10 times, more preferably 3-6 times.
  • 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 great influence on the physical stability of docetaxel albumin nanoparticles.
  • the reason is that sodium octanoate can compete with drugs to bind to the hydrophobic site of albumin, reducing the binding force between drugs and proteins, resulting in the instability of the nanosuspension.
  • the nanoparticles When directly using commercially available human albumin solution as auxiliary material (containing sodium octanoate 0.16mmol/g protein), the nanoparticles promptly settled within 10 hours; yet, when dialysis was used to reduce the sodium octanoate 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.
  • 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 to reconstitute a stable isotonic suspension, which has no effect on blood vessel stimulation. 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 at room temperature for at least 24 hours, 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 a longer period 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 limitation 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 be pretreated with corticosteroids (such as dexamethasone). It is safe and well tolerated, and can effectively avoid the drawbacks of dexamethasone pretreatment weakening the tumor inhibitory effect of docetaxel and/or VEGF inhibitors or VEGFR inhibitors.
  • the combination of the docetaxel albumin composition described in this application and a VEGF inhibitor or a VEGFR inhibitor can significantly improve the tumor-suppressing effect and produce a synergistic effect, which provides a basis for clinical combined drug regimens.
  • the docetaxel albumin composition and bevacizumab can act synergistically to significantly inhibit the growth of various tumors such as gynecological tumors, especially in the treatment of ovarian cancer, fallopian tube cancer or primary peritoneal cancer. Synergy.
  • Figure 1 shows the effect of dexamethasone (DEX) pretreatment on (TAXOTERE) Inhibits the Influence of Hepa1-6 Xenograft Tumor Growth in Mice. Among them, compared with solvent control group, *P ⁇ 0.05, **P ⁇ 0.01; compared with DEX/TAXOTERE group, ###P ⁇ 0.001.
  • the stable means that no sedimentation of nanoparticles or turbidity of the suspension occurred.
  • the "reconstituted suspension” described in the examples is an isotonic suspension.
  • 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 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 103.5nm; 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 was found 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).
  • Example 7 The pH value of the aqueous phase solution affects the 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).
  • human serum albumin solution (the content of sodium octanoate is 0.16mmol/g protein), dilute it into a solution containing albumin 12mg/ml with normal saline, and obtain the albumin dilution; use normal saline as the dialysate to dilute the albumin
  • the solution was dialyzed (the molecular weight cut-off of the membrane bag was 10KDa), the dialysis multiple was 5 times, and sodium octanoate in the protein was partially removed to obtain a human serum albumin solution with low sodium octanoate content.
  • the obtained human serum albumin solution with low sodium caprylate content contains about 0.060 mmol/g protein of sodium caprylate.
  • the human serum albumin solutions with low sodium octanoate content in the above Examples 8-1 to 8-3 can be further concentrated.
  • Embodiment 9 The impact of technical process on protein secondary structure
  • 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 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 10 minutes of standing.
  • the technicians believe that the stability of the obtained suspension is poor, and the follow-up operation cannot be continued according to the content of the patent. 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.
  • 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 was 92.43nm. After standing at 25°C for 16 hours, there was a slight precipitation at the bottom. After standing at 2-8°C for 24 hours, the 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 Comparison of maximum tolerated dose (MTD) and toxicity 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.
  • DTX-HSA 182.5 and 146.0mg/kg group animals had no obvious abnormalities.
  • 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 prescription and preparation method of Example 1-1 (the same below).
  • 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.
  • This trial is a single-arm, multicenter phase II clinical study for patients with epithelial ovarian cancer, fallopian tube cancer or primary peritoneal cancer (especially platinum-resistant and/or recurrent patients), aiming to evaluate the Efficacy, safety and pharmacokinetics of docetaxel combined with bevacizumab (albumin-bound).
  • the dosing regimen of bevacizumab is: 15mg/kg Q3W, i.v administration
  • the dosing regimen of docetaxel for injection is: 100mg/m 2 Q3W, about iv30min; or 75mg/ m2 Q3W, about iv30min.
  • 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 evaluable cases need to be enrolled in the two phases.
  • Imaging examinations were performed every 6 weeks (42 days ⁇ 7 days) to evaluate the anti-tumor efficacy, and imaging examinations were performed every 9 weeks (63 days ⁇ 7 days) after 3 imaging evaluations (about 18 weeks).
  • PK blood collection points are as follows: within 30 minutes before the administration of the first cycle and the third cycle, ⁇ 5 minutes immediately after the end of the infusion, 2 hours ⁇ 1 hour after the end of the infusion, and 24 hours ⁇ 2 hours after the end of the infusion.
  • the previous platinum-containing therapy was effective, and the disease relapsed or progressed between 28 days and 6 months (184 calendar days) after the last platinum-containing therapy (platinum-resistant type), and the number of previous platinum-containing chemotherapy lines is not limited, but No more than two lines of non-platinum chemotherapy.
  • 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) , interleukin-11 and erythropoietin (EPO) and other medical supportive treatment):
  • the subject must agree to take effective contraceptive measures from the signing of the informed consent form to the last 6 months after the last dose, and the serum pregnancy test of women of childbearing age (WOCBP, see Appendix III) within 7 days before the first use of the test drug is negative.
  • WOCBP serum pregnancy test of women of childbearing age
  • Mucinous ovarian cancer or less malignant ovarian tumors (such as low-grade serous ovarian cancer).
  • CNS central nervous system
  • Severe cardiac rhythm or conduction abnormalities including but not limited to ventricular arrhythmias requiring clinical intervention, third-degree atrioventricular block, etc.;
  • HCV antibody (+) positive in the screening period HCV RNA negative can be included, and anti-HCV treatment other than interferon is allowed
  • active hepatitis B HCV DNA ⁇ 2000IU/ml can be included, anti-HBV treatment other than interferon is allowed
  • known HIV positive or known acquired immunodeficiency syndrome AIDS
  • a strong inhibitor or strong inducer of CYP3A4 has been used within 2 weeks before the first use of the investigational drug.
  • Protocol analysis set Including patients with measurable lesions at baseline in FAS, no major protocol deviations, and patients with at least one tumor assessment or disease progression at any time after treatment.
  • PK analysis set (Pharmacokinetic Analysis Set, PKS): including subjects in the FAS who have at least one evaluable blood drug concentration after administration.
  • Tumor marker analysis set including subjects with at least one post-dose CA125 assessment in the FAS.
  • the primary efficacy endpoint is the objective response rate (ORR) based on IRC evaluation, and subjects who are first evaluated as CR or PR need to confirm the efficacy after at least 4 weeks.
  • ORR objective response rate
  • the tumor response status of the subjects was summarized, the confirmed ORR was calculated, and the estimated 95% confidence interval (CI) was calculated using the Clopper-Pearson exact method.
  • ORR and DCR evaluated by the investigator Calculate the proportion of subjects with CR+PR+SD, and use Clopper-Pearson to calculate its 95% CI.
  • CA125 remission rate Calculate the proportion of subjects whose best response is CA125 remission, and use Clopper-Pearson to calculate its 95% CI.
  • the laboratory examination (blood routine, urine routine, blood biochemistry, etc.), vital signs, physical examination, etc. were statistically described by visit. Provide a detailed list of laboratory tests, vital signs, physical examination, etc. before and after dosing.
  • Efficacy was evaluated according to RECIST v1.1 criteria, which were divided into complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD).
  • DCR Disease control rate
  • At least one post-enrolment follow-up measurement must meet the SD criteria, and the follow-up and enrollment must be separated by at least 6-8 weeks.
  • Case 1 After the diagnosis of high-grade serous ovarian cancer, bilateral ovarian tumors were surgically removed. Postoperatively, 6 cycles of TP (paclitaxel + cisplatin) chemotherapy were performed, and PR was evaluated for efficacy, followed by 10 cycles of paclitaxel chemotherapy, and the disease was stable during reexamination. A follow-up CT showed multiple peritoneal metastases, and 7 cycles of TP (paclitaxel + cisplatin) chemotherapy was given, and SD was re-examined. Patients were enrolled in this trial after being given ARIELI targeted therapy and evaluated for PD. The regimen of docetaxel for injection (albumin-bound) combined with bevacizumab was given for 5 cycles. The best efficacy evaluation was SD, and the target lesions shrunk.
  • TP paclitaxel + cisplatin
  • Case 2 After the diagnosis of high-grade serous ovarian cancer, extensive hysterectomy, right adnexa, omentum and appendectomy + pelvic lymph node dissection were performed. To give TP (paclitaxel + cisplatin) regimen chemotherapy for 8 courses. After completing the treatment, the subject participated in the maintenance treatment of the olaparib clinical trial. After taking the drug for more than 2 months, he asked to withdraw from the trial due to severe side effects. Tumor reduction surgery was performed after recurrence, and 5 courses of TP (paclitaxel + cisplatin) chemotherapy were given after the operation.
  • TP paclitaxel + cisplatin
  • TEAE Treatment Emergent Adverse Event
  • CTCAE grades were mostly grade 1-2, and no grade 5 TEAEs related to docetaxel for injection (albumin-bound) occurred.
  • docetaxel for injection (albumin-bound) combined with bevacizumab was used to study its clinical efficacy in platinum-resistant recurrent ovarian cancer.
  • Dexamethasone pretreatment was not required, and the platinum
  • the curative effect in patients with drug-resistant recurrent ovarian cancer, the ORR rate is higher than 40%, improves the response rate, prolongs the overall survival period OS, reduces the risk of death, and benefits patients.

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Abstract

提供多西他赛白蛋白组合物与VEGF抑制剂或VEGFR抑制剂在治疗肿瘤例如实体瘤，特别是在治疗妇科肿瘤，如卵巢癌、宫颈癌、子宫内膜癌中的各种应用，包括相关的制药用途、药物组合物或治疗方法等。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，与VEGF抑制剂或VEGFR抑制剂联合用药能显著提升抑瘤效果，产生了协同作用。

Description

多西他赛白蛋白组合物与VEGF抑制剂或VEGFR抑制剂的组合及用途
相关申请
本申请要求于2021年12月16日提交的第202111561157.8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VEGF抑制剂或VEGFR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 PCTCN2022139315-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 PCTCN2022139315-appb-000002
Figure PCTCN2022139315-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 PCTCN2022139315-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 PCTCN2022139315-appb-000005
与紫杉醇注射液
Figure PCTCN2022139315-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 PCTCN2022139315-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。
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。
抗血管生成疗法以VEGF或VEGFR抑制剂为主要代表，通过阻断VEGF或VEGFR信号通路阻止肿瘤血管生成，从而抑制肿瘤生长。目前已获批上市的VEGF或VEGFR抑制剂包括贝伐珠单抗、ranibizumab、aflibercept、pegaptanib、rivoceranib、pyrotinib dimaleate、ponatinib、tivozanib、nintedanib、plitidepsin、索凡替尼、索拉非尼、仑伐替尼、舒尼替尼、帕唑帕尼、卡博替尼、瑞戈非尼、凡德他尼、呋喹替尼、阿西替尼等，获批的适应症涵盖结直肠癌、非小细胞肺癌、卵巢癌、宫颈癌、肝癌、神经胶质瘤、肾癌、胃癌等内的多种恶性肿瘤。
但目前仍没有使用时无需皮质类固醇(如地塞米松)预处理，即可用于肿瘤治疗的多西他赛制剂。
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(DEX))预处理，提高临床给药的便利性和安全性，并且与VEGF抑制剂或VEGFR抑制剂联用可进一步提高抗肿瘤疗效，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与VEGF抑制剂或VEGFR抑制剂在制备用于治疗肿瘤的药物中的用途。
第二方面，本申请提供多西他赛白蛋白组合物在制备用于改善VEGF抑制剂或VEGFR抑制剂治疗肿瘤的疗效的药物中的用途。
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)VEGF抑制剂或VEGFR抑制剂。
第四方面，本申请提供一种用于改善VEGF抑制剂或VEGFR抑制剂治疗肿瘤的疗效的药物，其包含多西他赛白蛋白组合物。
第五方面，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)VEGF抑制剂或VEGFR抑制剂，或者如第三方面所述的复方药物或药物组合产品。
第六方面，本申请提供一种用于改善VEGF抑制剂或VEGFR抑制剂治疗肿瘤的疗效的方法，其包括向已经接受VEGF抑制剂或VEGFR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。。
在上述第一方面至第六方面中，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，所述实体瘤为妇科肿瘤，如卵巢癌、输卵管癌、原发性腹膜癌、宫颈癌、子宫内膜癌，更优选为上皮性卵巢癌、输卵管癌或原发性腹膜癌。
在上述第一方面至第六方面中，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六方面中，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，多西他赛白蛋白纳米粒和VEGF抑制剂或VEGFR抑制剂可以存在于同一药物制剂中，也可分别独立制剂，以组合产品的形式制成所述药物。在一些实施方案中，当所述含多西他赛白蛋白纳米粒的组合物制成液体注射剂时，以无水多西他赛计，含活性成分3.3-4.3mg/ml，优选4mg/ml。在一些实施方案中，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。
在一些实施方案中，所述VEGF抑制剂或VEGFR抑制剂选自贝伐珠单抗及其生物类似物等抗VEGF单克隆抗体，安罗替尼、阿帕替尼等VEGFR TKI抑制剂，优选贝伐珠单抗。
在上述第一方面至第六方面中，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，所述肿瘤优选为卵巢癌、输卵管癌和原发性腹膜癌。
在一些实施方案中，本申请提供多西他赛白蛋白纳米粒和贝伐珠单抗在制备用于治疗肿瘤的药物中的用途。
在一些实施方案中，本申请提供多西他赛白蛋白纳米粒在制备用于改善贝伐珠单抗治疗肿瘤的效果的药物中的用途。
在一些实施方案中，本申请提供一种治疗肿瘤的药物，其包含多西他赛白蛋白纳米粒和贝伐珠单抗。
在一些实施方案中，本申请提供一种用于改善贝伐珠单抗治疗肿瘤的效果的药物，其包含多西他赛白蛋白纳米粒。
在一些实施方案中，所述药物中可进一步包含其他治疗卵巢癌、输卵管癌、原发性腹膜癌的药物，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)药物管理部门批准用于卵巢癌、输卵管癌、原发性腹膜癌的治疗的药物。
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。
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/癌患者施用治疗有效量的注射用多西他赛白蛋白纳米粒和贝伐珠单抗。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用于改善贝伐珠单抗对肿瘤的疗效的方法，其包括在对肿瘤患者施用贝伐珠单抗的基础上，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。给药剂量范围为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)。
在优选的实施方案中，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，以贝伐珠单抗计，贝伐珠单抗的给药方式为15mg/kg，Q3W，优选为静脉给药，先给予贝伐珠单抗再给予多西他赛白蛋白。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，静脉滴注贝伐珠单抗，15mg/kg Q3W，贝伐珠单抗首次输注时间为90min左右，如果首次输注耐受性良好，则第二次输注的时间可以缩短到60min左右。如果患者对60min左右的输注也具有良好的耐受性，随后进行的所有输注都可以用30min左右的时间完成。之后静脉滴注注射用多西他赛(白蛋白结合型)100mg/m 2Q3W，30min左右，或静脉滴注注射用多西他赛(白蛋白结合型)75mg/m 2Q3W，30min左右。
本申请所述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。
第七方面，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，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。在优选的实施方案中，所述适当的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。
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)。在一些实施例中，所述悬浮液含多西他赛2-10mg/ml，优选2-8mg/ml。所述悬浮液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。
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。
第八方面，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。
第九方面，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。
在一些实施例中，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。优选的，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。
第十方面，本申请还提供了一种含多西他赛白蛋白纳米粒的组合物，其由第九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。
第十一方面，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。优选的，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)，10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VEGF抑制剂或VEGFR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VEGF抑制剂或VEGFR抑制剂的联合用药能显著提升抑瘤效果，产生了协同作用，为临床联合用药方案提供了依据。例如，多西他赛白蛋白组合物与贝伐珠单抗能够协同作用，显著抑制妇科肿瘤等多种肿瘤的生长，特别是在治疗卵巢癌、输卵管癌或原发性腹膜癌方面具有明显的协同效应。
附图说明
图1显示了地塞米松(DEX)预处理对
Figure PCTCN2022139315-appb-000008
(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。
具体实施方式
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1-1 0.9％；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 PCTCN2022139315-appb-000009
Figure PCTCN2022139315-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 PCTCN2022139315-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 PCTCN2022139315-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 PCTCN2022139315-appb-000013
Figure PCTCN2022139315-appb-000014
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。进一步地，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 PCTCN2022139315-appb-000015
Figure PCTCN2022139315-appb-000016
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 PCTCN2022139315-appb-000017
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 PCTCN2022139315-appb-000018
2.实验动物
Figure PCTCN2022139315-appb-000019
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-HSA 182.5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 PCTCN2022139315-appb-000020
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犬单次给药副作用比较
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 PCTCN2022139315-appb-000021
Figure PCTCN2022139315-appb-000022
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的处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 PCTCN2022139315-appb-000023
Figure PCTCN2022139315-appb-000024
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，对于保障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 DTX-HSA与贝伐珠单抗联合治疗的临床研究
一、试验设计
1.1 总体设计
本试验为一项针对上皮性卵巢癌、输卵管癌或原发性腹膜癌患者(尤其是铂耐药和/或复发患者)的单臂、多中心II期临床研究，旨在评价注射用多西他赛(白蛋白结合型)联合贝伐珠单抗的有效性、安全性和多西他赛的药代动力学特征。
1.2 剂量及给药方案：
贝伐珠单抗的给药方案为：15mg/kg Q3W,i.v给药
注射用多西他赛(白蛋白结合型)的给药方案为：100mg/m 2Q3W，i.v.30min左右；或75mg/m 2Q3W，i.v.30min左右。
1.3 试验设计
本研究采用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例可评估病例。
所有受试者均接受注射用多西他赛(白蛋白结合型)100mg/m 2Q3W联合贝伐珠单抗15mg/kg Q3W治疗，或注射用多西他赛(白蛋白结合型)75mg/m 2Q3W联合贝伐珠单抗15mg/kg Q3W治疗，直至疾病进展、发生不可耐受的毒性、受试者退出试验或开始新的抗肿瘤治疗或其他符合终止治疗或退出标准的情况。
每6周(42天±7天)进行一次影像学检查以评估抗肿瘤疗效，进行3次影像学评估(约18周)之后每9周(63天±7天)进行一次影像学检查。
为评估注射用多西他赛(白蛋白结合型)联合贝伐珠单抗后的药代动力学特征，所有受试者均需要在第1周期及第3周期进行稀疏PK血样采集，用于检测游离和总的多西他赛含量。PK采血点如下：第1周期及第3周期给药前30min内、输注结束即刻±5min、输注结束后2h±1h和输注结束后24h±2h。
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18-78(含)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上皮性卵巢癌、输卵管癌或原发性腹膜癌。
3.前一线含铂治疗有效，末次含铂治疗后28天至6个月(184个公历日)之间疾病复发或进展(铂耐药型)，既往含铂化疗线数不受限制，但非铂化疗线数不超过两线。
4.根据RECIST v1.1，至少有一个可测量病灶。
5.ECOG评分体能状态为0-1的受试者。
6.预计生存期≥3个月者。
7.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 PCTCN2022139315-appb-000025
8.受试者必须同意从签署知情同意书开始至末次给药后6个月内采取有效的避孕措施，育龄女性(WOCBP，见附录三)在首次使用试验用药品前7天内的血清妊娠试验为阴性。
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.既往使用过贝伐珠单抗治疗或首次使用试验用药品前18周内使用过其他抗血管生成治疗(包括但不限于阿帕替尼、安罗替尼等)。
2.含铂治疗期间(首次用药至末次用药后28天内)疾病进展(原发性铂难治型)。
3.粘液性卵巢癌或恶性程度较低的卵巢肿瘤(如低级别浆液性卵巢癌)。
4.有症状的中枢神经系统(CNS)转移或癌性脑膜炎(如果受试者在试验筛选前完成了CNS转移的放疗或手术，且放疗或术后受试者的神经系统稳定≥4周(即筛选时未发现因脑转移导致的新的神经功能缺损、中枢神经系统影像学检查未发现新的病灶、并且不需要进行治疗)，则可以参与本研究)。
5.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：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。
6.首次使用试验用药品前6个月内有过血栓栓塞、脑梗、出血性疾病史有出血倾向的患者。
7.首次使用试验用药品前6个月内有肠梗阻、胃肠穿孔、腹腔瘘或腹腔脓肿病史。
8.首次使用试验用药品前5年内接受过腹部或盆腔放疗。
9.筛选检查尿蛋白>2+，或尿蛋白为2+且24小时尿蛋白定量＞1g。
10.已知对试验用药品或其主要辅料过敏者。
11.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。
12.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，包括但不限于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、冠状动脉搭桥外科病史；
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)；
e)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)；
f)筛选期超声心动图显示左室射血分数(LVEF)≤50％。
13.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≤2000IU/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)。
14.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)，或需要在试验期间接受择期手术者。
15.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、脱发以及研究者判断无安全风险的毒性。
16.在首次使用试验用药品前4周内接受过化疗、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周内或已知的药物5个半衰期内的口服氟尿嘧啶类、小分子靶向药物等的治疗；首次使用试验用药品前2周内的服用具有抗肿瘤活性的中药。
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.在首次使用试验用药品前4周内接受过减毒活疫苗。
19.正在服用或首次使用试验用药品前10天内服用过阿司匹林(＞325mg/天)或其他可能影响血小板功能的非甾体类抗炎药。
20.哺乳期女性。
21.有需要接受静脉抗生素治疗的活动性感染(由研究者判断)。
22.酒精或药物滥用或依赖史。
23.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：受试者并发严重或无法控制的医学病症，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
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，研究者判断继续参与研究将不符合受试者的最佳获益；
●严重的方案偏离影响本研究评价；
●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、观察指标与统计分析
1.分析数据集
1)全分析集(FAS，Full Analysis Set)：包括所有成功入组且至少接受过一次试验用药品的受试者。
2)符合方案分析集(PPS，Per Protocol Set)：包括FAS中基线有可测量病灶，未发生重大方案偏离且在用药后至少有一次肿瘤评估或任何时候出现疾病进展的患者。
3)PK分析集：(Pharmacokinetic Analysis Set，PKS)：包括FAS中至少有一个用药后可评价的血药浓度的受试者。
4)肿瘤标志物分析集：包括FAS中有至少一次用药后CA125评估的受试者。
最终的人群划分将在数据库锁定之前的数据审核会上讨论并确定。
2.主要疗效终点分析
主要疗效终点为基于IRC评价的客观缓解率(ORR)，首次评估为CR或PR的受试者需在至少4周后确认疗效。汇总描述受试者的肿瘤应答情况，计算经确认的ORR，并计算采用Clopper-Pearson精确法估计95％置信区间(CI)。
3.次要疗效终点分析
(1)研究者评估的ORR、DCR：计算CR+PR+SD的受试者所占的比例，并采用Clopper-Pearson计算其95％CI。
(2)PFS、DOR和OS：采用Kaplan-Meier法估计PFS、DOR、OS的中位时间及其95％置信区间。
(3)CA125缓解率：计算最佳反应为CA125缓解的受试者所占的比例，并采用Clopper-Pearson计算其95％CI。
4.安全性分析
基于FAS，对不良事件、严重不良事件、实验室检查指标、生命体征、体格检查、ECG、ECOG评分等进行汇总分析。
按系统器官分类及首选术语、严重程度对所有AE和严重不良事件等进行分析汇总。列出所有AE、与试验用药品相关AE、SAE等的详细列表。
对实验室检查(血常规、尿常规、血生化等)、生命体征、体格检查等按访视进行统计描述。列出给药前后实验室检查、生命体征、体格检查等的详细列表。
5.药代动力学及药效动力学分析
基于药代动力学浓度集，对多西他赛(白蛋白结合型)静脉给药后的游离型和总的多西他赛浓度绘制药物浓度-时间曲线，并进行描述性统计分析。如果数据足够，上述全部受 试者的数据将利用非线性混合效应模型(NONMEM)进行群体药代动力学(PPK)分析，分析详情及结果参见单独报告。群体药动学/药效学分析将用于探索药物的暴露量与药效学(临床结果、安全性指标)的关系。如果有足够的数据可用，将进行PK-PD建模，分析详情及结果参见单独报告。
四、研究结果
疗效评价
按照RECIST v1.1标准进行疗效评价，分为完全缓解(CR)、部分缓解(PR)、疾病稳定(SD)和疾病进展(PD)。
总缓解率(ORR)＝(CR+PR)/总的可评价病例数*100％。
疾病控制率(DCR)＝(CR+PR+SD)/总的可评价病例数*100％。
在SD病例中，至少必须有一次入组后随访测量符合SD标准，并且随访与入组至少间隔6-8周。
截至2022年12月2日，本项目共入组5例卵巢癌患者，3例为75mg/m 2组，2例为100mg/m 2组。2例受试者进行过至少一次疗效评估，其中，1例受试者(75mg/m 2组)评估SD，靶病灶缩小；1例受试者(75mg/m 2组)评估PR，ORR＝50％，DCR＝100％。
上述结果表明，注射用多西他赛(白蛋白结合型)联合贝伐珠单抗的方案对患者疾病控制有效。
典型病例
病例1：确诊高级别浆液性卵巢癌后，手术切除双侧卵巢肿瘤。术后行TP(紫杉醇+顺铂)方案化疗6周期，疗效评估PR，后行紫杉醇化疗10周期，期间复查疾病稳定。后复查CT显示腹膜多发转移，予以TP(紫杉醇+顺铂)方案化疗7周期，复查SD。给与艾瑞颐靶向治疗，评估PD后入组本试验。给与注射用多西他赛(白蛋白结合型)联合贝伐珠单抗方案治疗5周期，最佳疗效评估为SD，靶病灶缩小。
病例2：确诊高级别浆液性卵巢癌后，行广泛全子宫、右侧附件、大网膜及阑尾切除+盆腔淋巴结清扫术。予以TP(紫杉醇+顺铂)方案化疗8疗程。完成治疗后受试者参加奥拉帕利临床试验维持治疗，服药2月余，因副反应重，要求退出试验。复发后行减瘤术，术后予TP(紫杉醇+顺铂)方案化疗5疗程，复查进展后参加多美素+塞伐珠单抗临床试验，最佳疗效评估PR，因副反应重，要求退出试验。复查进展后给与紫杉醇治疗7周期，因左耳听力损伤，要求退出治疗。之后入组本试验，给与注射用多西他赛(白蛋白结合型)联合贝伐珠单抗方案治疗4周期，最佳疗效评估为PR。出现了例如脱发、全身乏力等不良 事件，CTCAE(常见不良事件评价标准，Common Terminology Criteria for Adverse Events)分级均为1-2级。
安全性数据
已过DLT(剂量限制性毒性，Dose-limiting toxicity)观察期的3例75mg/m 2组和1例100mg/m 2组受试者均未观察到DLT。
所有受试者中，多于20％受试者未发生与注射用多西他赛(白蛋白结合型)相关的TEAE(治疗期间出现的不良事件，Treatment Emergent Adverse Event)，另有部分受试者发生了与注射用多西他赛(白蛋白结合型)相关的TEAE。所有TEAE中，CTCAE分级多为1-2级，没有与注射用多西他赛(白蛋白结合型)相关的5级TEAE发生。
本实施例采用注射用多西他赛(白蛋白结合型)联合贝伐珠单抗，研究其在铂耐药复发性卵巢癌中的临床疗效，不需要地塞米松预处理，进一步提高了铂耐药复发性卵巢癌患者的疗效，ORR率高于40％，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，而且对卵巢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VEGF抑制剂或VEGFR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与VEGF抑制剂或VEGFR抑制剂在制备用于治疗肿瘤的药物中的用途。
  2. 多西他赛白蛋白组合物在制备用于改善VEGF抑制剂或VEGFR抑制剂治疗肿瘤的疗效的药物中的用途。
  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VEGF抑制剂或VEGFR抑制剂，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VEGF抑制剂或VEGFR抑制剂治疗肿瘤的疗效。
  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VEGF抑制剂或VEGFR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VEGF抑制剂或VEGFR抑制剂对肿瘤的疗效的方法，其包括向已经接受VEGF抑制剂或VEGFR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；和/或更优选地，所述多西他赛白蛋白纳米粒组合物由多西他赛和酸变性白蛋白制成。
  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、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、筛窦肿瘤和骨肿瘤，更优选地，所述实体瘤选自妇科肿瘤，如卵巢癌、输卵管癌、原发性腹膜癌、宫颈癌和子宫内膜癌，优选为上皮性卵巢癌、输卵管癌或原发性腹膜癌。
  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与VEGF抑制剂或VEGFR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VEGF抑制剂或所述VEGFR抑制剂选自抗VEGF单克隆抗体，例如贝伐珠单抗及其生物类似物；和VEGFR TKI抑制剂，例如安罗替尼和阿帕替尼，优选地，所述VEGF抑制剂或所述VEGFR抑制剂为贝伐珠单抗。
  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。
PCT/CN2022/139315 2021-12-16 2022-12-15 多西他赛白蛋白组合物与vegf抑制剂或vegfr抑制剂的组合及用途 WO2023109904A1 (zh)

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CN101883578A (zh) * 2007-08-20 2010-11-10 百时美施贵宝公司 Vefr-2抑制剂用于治疗转移癌的用途
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