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WO2025185024A1 - Formulation de liposome de docétaxel, son procédé de préparation et son utilisation - Google Patents

Formulation de liposome de docétaxel, son procédé de préparation et son utilisation

Info

Publication number
WO2025185024A1
WO2025185024A1 PCT/CN2024/103795 CN2024103795W WO2025185024A1 WO 2025185024 A1 WO2025185024 A1 WO 2025185024A1 CN 2024103795 W CN2024103795 W CN 2024103795W WO 2025185024 A1 WO2025185024 A1 WO 2025185024A1
Authority
WO
WIPO (PCT)
Prior art keywords
docetaxel
cancer
oil
preparation
phospholipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/103795
Other languages
English (en)
Chinese (zh)
Other versions
WO2025185024A8 (fr
Inventor
曹震
潘斌
张高薪
李泽民
支泽仑
周畅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wecarelife Biotech Co Ltd
Original Assignee
Wecarelife Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wecarelife Biotech Co Ltd filed Critical Wecarelife Biotech Co Ltd
Publication of WO2025185024A1 publication Critical patent/WO2025185024A1/fr
Publication of WO2025185024A8 publication Critical patent/WO2025185024A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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 present invention relates to the field of pharmaceutical technology, in particular to a docetaxel fat body preparation and a preparation method and application thereof.
  • Docetaxel is a chemotherapy drug used to treat various types of cancer, including breast cancer, non-small cell lung cancer, prostate cancer, gastric cancer, and head and neck cancer. It binds to microtubules, preventing their depolymerization, thereby inhibiting cell division and leading to cell death.
  • Docetaxel is a hydrophobic drug with a logP (oil-water partition coefficient) of 1.6 and an extremely low solubility in water of only 0.006 mg/mL.
  • Docetaxel is currently used in clinical practice in the form of an injection. To improve the solubility of docetaxel, polysorbate 80 (Tween 80) and ethanol are often added.
  • nanocarriers are widely used in the research and development of new docetaxel formulations.
  • Polymer carriers can provide better drug protection and reduce drug distribution in the body, thereby reducing its toxic side effects.
  • these methods also have some shortcomings.
  • liposomes have a hydrophilic core, and only the hydrophobic environment between their phospholipid bilayers can be used to carry docetaxel, but their encapsulation capacity is low.
  • the stability of liposomes may be poor, and fusion, rupture or drug leakage may occur easily, which may affect their distribution in the body and the release of the drug.
  • the amount of polymer carrier encapsulated docetaxel is also low.
  • PEG and PLGA are not naturally present in the body, and can cause the body to produce corresponding antibodies to resist their effects. Therefore, these novel preparations, while improving the solubility and effect of docetaxel, are also faced with problems such as no targeted modification, low encapsulation rate and potential safety.
  • the novel nanoparticle liposomes consist of a hydrophobic core composed of neutral lipids and are encapsulated by a monomolecular phospholipid membrane. Similar in structure to naturally occurring lipid droplets and lipoproteins, they can efficiently dissolve and encapsulate hydrophobic small molecules. Furthermore, the components of liposomes are naturally present in the body, resulting in excellent biocompatibility. Furthermore, their preparation is simple and efficient. If liposomes are used to deliver docetaxel or other hydrophobic drugs, they are expected to enhance drug solubility, improve bioavailability, enhance efficacy and safety, and enable targeted delivery, greatly facilitating treatment for patients.
  • the technical problem to be solved by the present invention is to provide a docetaxel fat body preparation and a preparation method and application thereof, wherein the fat body preparation has good bioavailability and safety and can carry targeting molecules.
  • the docetaxel fat body preparation provided by the present invention comprises: a single molecule phospholipid membrane and docetaxel and neutral lipid wrapped in the single molecule phospholipid membrane.
  • the monomolecular phospholipid membrane comprises one or more of phospholipids, functional polar lipids and cationic lipids;
  • the phospholipid is selected from one or more of 2-bis-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC), egg yolk lecithin, soybean lecithin, dioleoylphosphatidylethanolamine, distearoylphosphatidylcholine, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid, sodium distearoylphosphatidylglycerol, dimyristoylphosphatidylcholine, 1-stearoyl-lysophosphatidylcholine, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, phosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphati
  • the functional polar lipid is selected from one or more of polyethylene glycol-modified sterols, biotin-modified sterols, amino acid-modified sterols, polypeptide-modified sterols, polysaccharide-modified sterols, nucleic acid-modified sterols, polyethylene glycol-modified phospholipids, biotin-modified phospholipids, amino acid-modified phospholipids, polypeptide-modified phospholipids, polysaccharide-modified phospholipids and nucleic acid-modified phospholipids;
  • the cationic lipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) and 3 ⁇ -[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.
  • the neutral lipid is selected from one or more of fish oil, corn oil, tricaprylin, triolein, retinol ester, wax ester, sterol ester, sterol ester, castor oil, sunflower oil, soybean oil, peanut oil, clove oil, simethicone, cinnamon oil, tea oil, liquid paraffin, star anise oil, mixed fatty acid glycerides (stearin), hydrogenated vegetable oil, refined olive oil and fat-soluble vitamins.
  • the present invention further screens and optimizes the materials of the single-molecule phospholipid membrane and the neutral phospholipids.
  • the material of the monomolecular phospholipid membrane is phospholipid
  • the neutral lipid is at least one of fish oil, corn oil, tricaprylin, and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is fish oil.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipid is corn oil
  • the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is tricaprylin.
  • the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is triolein.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipids are fish oil and corn oil.
  • the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are fish oil and tricaprylin.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipids are fish oil and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are corn oil and tricaprylin.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipids are corn oil and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are tricaprylin and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipids are fish oil, tricaprylin and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipids are corn oil, tricaprylin and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipid is fish oil, corn oil and triolein.
  • the material of the monomolecular phospholipid membrane is DOPC
  • the neutral lipids are fish oil, tricaprylin and corn oil.
  • the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil.
  • the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is tricaprylin.
  • the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil and corn oil.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil and tricaprylin.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil and tricaprylin.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil and triolein.
  • the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipids are tricaprylin and triolein.
  • the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil, tricaprylin and triolein.
  • the material of the monomolecular phospholipid membrane is phosphatidylcholine
  • the neutral lipid is fish oil, corn oil and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidylcholine
  • the neutral lipid is fish oil, tricaprylin and corn oil.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is corn oil.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is tricaprylin.
  • the material of the monomolecular phospholipid membrane is phosphatidic acid, and the neutral lipid is triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil and corn oil.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are fish oil and tricaprylin.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are corn oil and tricaprylin.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are corn oil and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are tricaprylin and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil, tricaprylin and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are corn oil, tricaprylin and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid
  • the neutral lipid is fish oil, corn oil and triolein.
  • the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are fish oil, tricaprylin and corn oil.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is fish oil.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is corn oil.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is tricaprylin.
  • the material of the monomolecular phospholipid membrane is DOPC and phosphatidic acid, and the neutral lipid is glycerol Oil trioleate.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil and corn oil.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil and tricaprylin.
  • the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil and triolein.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are corn oil and tricaprylin.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are corn oil and triolein.
  • the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are tricaprylin and triolein.
  • the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil, tricaprylin and triolein.
  • the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are corn oil, tricaprylin and triolein.
  • the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil, corn oil and triolein.
  • the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil, tricaprylin and corn oil.
  • the volume ratio of fish oil to tricaprylin in the neutral lipid is 1:(0.1-10). In some specific embodiments, the volume ratio of fish oil to tricaprylin is 1:(0.5-5). More specifically, the volume ratio of fish oil to tricaprylin is 1:(0.8-2). For example, the volume ratio of fish oil to tricaprylin is 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.0.
  • the mass ratio of docetaxel, neutral lipid and phospholipid is 1:(20-200):(10:100).
  • the mass ratio of docetaxel, neutral lipid and phospholipid is 1: (20 ⁇ 100):(10 ⁇ 50).
  • the mass ratio of docetaxel, neutral lipid and phospholipid is 1:(20-50):(10-25).
  • the mass ratio of docetaxel, neutral lipid and phospholipid is 1:(30-40):(15-20).
  • the mass ratio of docetaxel, neutral lipid and phospholipid is 1:30:(15-20), or 1:31:(15-20), or 1:32:(15-20), or 1:33:(15-20), or 1:34:(15-20), or 1:35:(15-20), or 1:36:(15-20), or 1:37:(15-20), or 1:38:(15-20), or 1:39:(15-20), or 1:40:(15-20).
  • the mass ratio of docetaxel, neutral lipid and phospholipid is 1:33:15, or 1:33:16, or 1:33:17, or 1:33:18, or 1:33:19, or 1:33:20, or 1:34:15, or 1:34:16, or 1:34:17, or 1:34:18, or 1:34:19, or 1:34:20, or 1:35:15, or 1:35:16, or 1:35:17, or 1:35:18, or 1:35:19, or 1:35:20.
  • the neutral lipid is fish oil and tricaprylin with a volume ratio of 1:1
  • the single-molecule phospholipid membrane is DOPC, which can achieve a higher encapsulation efficiency, an average particle size within 200nm, and good stability even for long-term placement.
  • the docetaxel fat body preparation of the present invention further comprises a targeting molecule, which targets and recognizes organs, tissues or cells.
  • the organs, tissues or cells are from the human body or animal body;
  • the organs are endocrine organs, digestive organs, circulatory organs, urinary organs, reproductive organs, locomotor organs, the nervous system, and sensory organs.
  • Endocrine organs include the thyroid gland and pancreas.
  • Digestive organs include the stomach, liver, gallbladder, spleen, pancreas, small intestine, and large intestine.
  • Respiratory organs include the lungs.
  • Circulatory organs include the heart and blood vessels.
  • Urinary organs include the kidneys, ureters, and bladder.
  • Reproductive organs include the uterus and ovaries.
  • Locomotor organs include muscles and bones.
  • the nervous system includes the cerebrum and cerebellum.
  • Sensory organs include the skin, eyes, and ears.
  • the tissue or cell is derived from a tumor in a human or animal body.
  • the tumor includes: lung cancer, kidney cancer of the throat, liver, muscle tissue, blood, bone, brain, breast, neck, oral or nasal mucosa, bladder, central nervous system, cervix, head and neck, colon, endometrium, external genitalia, esophagus, gallbladder, gastrointestinal tract, genitourinary tract, head, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, melanoma, testicle, and/or thyroid.
  • the targeting molecule can be embedded in the monomolecular phospholipid membrane, or it can be bound to the phospholipids on the monomolecular phospholipid membrane through the avidin-biotin system, or it can be connected to a substance that specifically targets phospholipids to bind to the monomolecular phospholipid membrane, or it can be a combination of any two or more of the above methods.
  • the targeting molecule is at least one of LTA-P33, ApoE, BCMA antibody, Nrp-B, Trf-B, LDLR-B, ErbB2-B, CXCR4-B, GRP78-B or Soma-B.
  • the targeting molecule is linked to biotin, and a phospholipid molecule labeled with streptavidin is added during the preparation of the adipocytes.
  • the targeting molecule is linked to streptavidin, and a phospholipid molecule labeled with biotin is added during the preparation of the adipocytes.
  • the targeting molecule is connected to a peptide segment that targets and recognizes a single molecule of phospholipid membrane, which includes at least one of AAMB, ALDI, CYB5R3-N, LDAMP1, HSD17B13-N28, MDT-28-P, MLDS-P, DHS-3-P, HSD17B11-N28, PspA-H1, Vipp1-H1, Snf7-H1, Chmp1B-H1, PB, PE, and PF.
  • the targeting molecule and the peptide segment that targets and recognizes a single molecule of phospholipid membrane can be connected via a linker or directly connected without a linker, which is not limited by the present invention.
  • the linker is a cleavable linker or a self-cleaving linker.
  • the amino acid sequence of the cleavable linker is LEAGCKNFFPRSFTSCGSLE, and the self-cleaving linker is P2A, T2A, or E2A.
  • the neutral lipid is selected from at least one of fish oil, tricaprylin and triolein
  • the phospholipid is DOPC
  • the phospholipid may further include a phospholipid labeled with biotin.
  • the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(20-200):(10-100):(2-20).
  • the docetaxel, neutral lipids, phospholipids and targeting molecules The mass ratio is 1:(20 ⁇ 100):(10 ⁇ 50):(2 ⁇ 15).
  • the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(20-50):(10-25):(2-10).
  • the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(30-40):(15-20):(2-5).
  • the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:30:(15-20):(2-5), or 1:31:(15-20):(2-5), or 1:32:(15-20):(2-5), or 1:33:(15-20):(2-5), or 1:34:(15-20):(2-5), or 1:35:(15-20):(2-5), or 1:36:(15-20):(2-5), or 1:37:(15-20):(2-5), or 1:38:(15-20):(2-5), or 1:39:(15-20):(2-5), or 1:40:(15-20):(2-5).
  • the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:33:15:(2-5), or 1:33:16:(2-5), or 1:33:17:(2-5), or 1:33:18:(2-5), or 1:33:19:(2-5), or 1:33:20:(2-5), or 1:34:15:(2-5), or 1:34:16:(2-5), or 1:34:17:(2 ⁇ 5), or 1:34:18:(2 ⁇ 5), or 1:34:19:(2 ⁇ 5), or 1:34:20:(2 ⁇ 5), or 1:35:15:(2 ⁇ 5), or 1:35:16:(2 ⁇ 5), or 1:35:17:(2 ⁇ 5), or 1:35:18:(2 ⁇ 5), or 1:35:19:(2 ⁇ 5), or 1:35:20:(2 ⁇ 5).
  • the solution in the embodiment of the present invention is more conducive to improving the encapsulation efficiency and stability of the preparation. It can also ensure that the efficacy of the preparation is not affected and the targeting is better.
  • the targeting molecule is LTA-P33;
  • the neutral lipids are fish oil and tricaprylin;
  • the monomolecular phospholipid membrane is 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine;
  • the volume ratio of the fish oil to tricaprylin is 1:(0.1-10). Preferably, the volume ratio of the fish oil to tricaprylin is 1:1.
  • the targeting molecule is ApoE
  • the neutral lipids are fish oil and tricaprylin;
  • the monomolecular phospholipid membrane is 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine;
  • the volume ratio of the fish oil to tricaprylin is 1:(0.1-10). Preferably, the volume ratio of the fish oil to tricaprylin is 1:1.
  • the targeting molecule is a BCMA antibody, and the BCMA is labeled with streptavidin;
  • the neutral lipid is triolein
  • the monomolecular phospholipid membrane is a phospholipid labeled with 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine and biotin, wherein the phospholipid labeled with biotin is one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and sphingomyelin.
  • the biotin-labeled phospholipid is biotin-labeled phosphatidylcholine (18:1 Biotinyl Cap PE, referred to as Bio-PE).
  • the mass ratio of the 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC) to the biotin-labeled phospholipid is (0-200):(0.05-200).
  • the mass ratio of DOPC to biotin-labeled phospholipid is (100-200):(0.05-10) or (150-200):(0.05-0.1). In some specific embodiments, the mass ratio of DOPC to biotin-labeled phospholipid is (100-150):(0.05-1). Preferably, the mass ratio of DOPC to biotin-labeled phospholipid is 145:0.08.
  • the present invention provides a method for preparing the aforementioned docetaxel fat body preparation without the targeting molecule, comprising:
  • Step 1 mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel;
  • Step 2 The neutral lipids and phospholipids containing docetaxel are mixed, and the docetaxel-loaded fat bodies are obtained after repeated vortexing and centrifugation.
  • the present invention provides a method for preparing the docetaxel fat body preparation containing the targeting molecule as described above, comprising:
  • Step A mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel;
  • Step B mixing the targeting molecule solution with the phospholipid, and removing the solvent to obtain the phospholipid containing the targeting molecule;
  • Step C The neutral lipid containing docetaxel and the phospholipid containing the targeting molecule are mixed, and the docetaxel-encapsulated fat body is obtained after repeated vortexing and centrifugation.
  • the present invention provides a method for preparing the docetaxel fat body preparation containing the targeting molecule as described above, comprising:
  • Step a mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel;
  • Step b mixing the neutral lipid containing docetaxel with a buffer and phospholipids, and repeatedly vortexing and centrifuging;
  • Step c mixing with targeting molecules and incubating to obtain docetaxel-loaded fat bodies.
  • the solvent is an organic solvent.
  • the organic solvent is any one or two of anhydrous ethanol, chloroform, methanol, benzene, toluene, xylene, butanol, isopropanol, ether, acetone, cyclohexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, cyclohexanone or petroleum ether.
  • the above combination preferably anhydrous ethanol.
  • the solvent in the targeting molecule solution is an organic solvent.
  • the organic solvent is a mixture of methanol and at least one of the following solvents: the solvent includes anhydrous ethanol, chloroform, benzene, toluene, xylene, butanol, isopropanol, ether, acetone, cyclohexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, cyclohexanone or petroleum ether, preferably a mixture of methanol and chloroform;
  • the buffer solution is PBS buffer, HEPES buffer, sucrose solution, NaCl solution, KCl solution, MgCl2 solution, etc.
  • the repeated vortexing and centrifugation include:
  • the parameters of the vortex include: vortexing at 3000-4000 rpm for 3-7 minutes, operating for 1-10 seconds, and resting for 1-10 seconds.
  • the vortex speed is 3000, 3200, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 rpm.
  • the vortex duration is 3 minutes, 4 minutes, 5 minutes, 6 minutes, or 7 minutes.
  • the vortex stops for 1 second every working 1 second, or stops for 2 seconds every working 2 seconds, or stops for 2 seconds every working 2 seconds, or stops for 3 seconds every working 3 seconds, or stops for 4 seconds every working 4 seconds, or stops for 5 seconds every working 5 seconds, or stops for 6 seconds every working 6 seconds, or stops for 7 seconds every working 7 seconds, or stops for 8 seconds every working 8 seconds, or stops for 9 seconds every working 9 seconds, or stops for 10 seconds every working 10 seconds, or stops for 6 seconds every working 5 seconds, or stops for 7 seconds every working 5 seconds, or stops for 8 seconds every working 5 seconds, or stops for 9 seconds every working 5 seconds, or stops for 10 seconds every working 6 seconds, or stops for 7 seconds every working 6 seconds, or stops for 8 seconds every working 6 seconds, or stops for 9 seconds every working 6 seconds, or stops for 10 seconds every working 6 seconds, or stops for 7 seconds every working 6 seconds, or stops for 8 seconds every working 6 seconds, or stops for 9 seconds every working 6 seconds, or stops for 10 seconds every working 6 seconds, or stops for 8 seconds every working 7 seconds, or stops
  • the vortex parameters in this step include 4000 rpm, vortexing for 10 seconds, stopping for 10 seconds, and vortexing for 4 minutes.
  • the vortex parameters in this step include 4000 rpm, vortexing for 10 seconds, stopping for 5 seconds, and vortexing for 3 minutes.
  • the mixture 1 was centrifuged to collect the lower layer solution, and then vortexed again to obtain a mixture 2.
  • the vortexing parameters include 1000-4000 rpm
  • the centrifugation parameters include: centrifugation at 800-1200 g for 3-7 min at room temperature.
  • the centrifugation speed is 800 g, 900 g, 1000 g, 1100 g, or 1200 g
  • the centrifugation time is 3 min, 4 min, 5 min, 6 min, or 7 min.
  • the centrifugation conditions include centrifugation at 1000 g for 5 min at room temperature.
  • the vortexing parameters include 1000-4000 rpm
  • the centrifugation parameters include: in actual application, centrifugation at 18000-22000g for 3-7 minutes.
  • the centrifugation speed is 18000g, 19000g, 20000g, 21000g or 22000g
  • the centrifugation time is 3 minutes, 4 minutes, 5 minutes, 6 minutes or 7 minutes.
  • the centrifugation conditions include centrifugation at 20000g for 5 minutes at room temperature.
  • the mixture 3 is centrifuged to collect the lower layer solution, and vortexed again to obtain the mixture 4 containing the fat body.
  • the vortex parameters include 1000-4000 rpm
  • the centrifugation parameters include: in actual application, centrifugation at 800-1200g for 3-7 minutes.
  • the centrifugation speed is 800g, 900g, 1000g, 1100g, or 1200g
  • the centrifugation time is 3 minutes, 4 minutes, 5 minutes, 6 minutes, or 7 minutes.
  • the centrifugation conditions include centrifugation at 1000g for 5 minutes at room temperature.
  • the preparation method provided by the present invention is simple and easy to operate, and the resulting preparation has good encapsulation efficiency and stability. It has been verified that the non-targeted docetaxel fat body can inhibit the growth of various cancer cells, such as hematological tumor cells, breast cancer cells, and liver cancer cells, with an inhibitory effect superior to the clinically used albumin paclitaxel and a safety superior to the clinically used docetaxel drug (docetaxel injection). Targeted docetaxel fat body inhibits the growth of lung cancer with greater effectiveness than the clinically used docetaxel drug (docetaxel injection).
  • various cancer cells such as hematological tumor cells, breast cancer cells, and liver cancer cells
  • the above-mentioned preparation or the preparation obtained by the above-mentioned method is used in the preparation of drugs or vaccines for preventing and treating tumors.
  • the tumor includes: lung cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, blood tumor, bone cancer, brain cancer, breast cancer, cervical cancer, oral or nasal mucosal cancer, bladder cancer, central nervous system cancer, cervical cancer, head and neck cancer, colon cancer, endometrial cancer, external genital cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genitourinary tract cancer, head cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, melanoma, testicular cancer and/or thyroid cancer.
  • the present invention provides a medicine or vaccine, which includes the preparation as described above, or the preparation prepared by the method as described above.
  • the medicine or vaccine of the present invention also includes pharmaceutically acceptable excipients.
  • the dosage form of the medicine or vaccine of the present invention is oral preparation, inhalant or injection.
  • the drug or vaccine is in the form of an oral preparation, for example, a tablet, a pill, an oral solution, a capsule, a syrup, a dropper or a granule.
  • the capsule is a hard capsule or a soft capsule.
  • the tablet is an oral tablet or buccal tablet.
  • Oral tablets are tablets for oral administration. Most of the drugs in these tablets are absorbed through the gastrointestinal tract to exert their effects, while some drugs in other tablets exert their effects locally in the gastrointestinal tract.
  • the oral tablets are conventional compressed tablets, dispersible tablets, effervescent tablets, chewable tablets, coated tablets, or sustained-release tablets.
  • the medicine or vaccine is inhaled, and optionally, it is an inhalation aerosol, an inhalation powder, or a liquid preparation for use in a nebulizer.
  • the medicine or vaccine is an injection, for example, an injection solution or an injection powder.
  • the medicine or vaccine of the present invention further comprises an effective amount of a tumor inhibitor
  • the tumor suppressors include: cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU), methotrexate, daunorubicin, dactinomycin-D, irinotecan (CPT-11), mitoxantrone, estramustine, vincristine, dexamethasone, prednisone, lomustine, methotrexate, pirarubicin, doxorubicin, gemcitabine, quizartinib or bevacizumab.
  • the present invention also provides a method for preventing and treating tumors, comprising administering the aforementioned drug or vaccine, wherein the administration method includes oral administration, inhalation and/or injection.
  • the subject of the method is a human or, the subject of the method is a primate or a non-primate mammal.
  • the present invention provides novel nanoparticle fat bodies (having a hydrophobic core) that encapsulate docetaxel to construct docetaxel fat bodies, which exhibit excellent anti-tumor effects and are safer than the clinically used docetaxel drug (docetaxel injection). Furthermore, the docetaxel fat bodies undergo targeted surface modification, effectively inhibiting the growth of lung cancer, liver cancer, and hematologic tumors, with significantly better inhibitory effects than the clinically used docetaxel drug (docetaxel injection), and also possessing superior safety.
  • FIG. 1 shows that fat bodies have good biocompatibility.
  • Mice treated with saline or fat bodies Serum biochemical indicators, body weight changes, and liver tissue sections after 50 days: A, body weight changes; B, alanine aminotransferase (ALT); C, aspartate aminotransferase (AST); D, creatinine (Cre); E, urea (Urea); F, hemoglobin (HGB); G, red blood cell count (RBC); H, low-density lipoprotein cholesterol (LDL-C); I, triglycerides (TAG).
  • A body weight changes
  • B alanine aminotransferase
  • C aspartate aminotransferase
  • D creatinine
  • Cre AST
  • E urea
  • F hemoglobin
  • G red blood cell count
  • H low-density lipoprotein cholesterol
  • I triglycerides
  • Figure 2 Preparation of non-targeted docetaxel fat bodies, including: A, preparation process of docetaxel fat bodies; B, HPLC detection of DTX free standard in ethanol and DTX in docetaxel fat bodies; C, TLC detection of DTX signal in docetaxel fat bodies, the dotted box indicates the location of DTX standard; D, optical microscopy observation of the morphology of blank fat bodies and docetaxel fat bodies, scale bar is 5 ⁇ m;
  • Figure 3 shows that docetaxel fat bodies have good structural stability, where: A, dynamic light scattering detection of the change in average particle size of docetaxel fat bodies with different contents after being placed for different time periods; B, dynamic light scattering detection of the change in PDI (polydispersity index) of docetaxel fat bodies with different contents after being placed for different time periods; C, change in drug loading (DL) of docetaxel fat bodies with different contents after being placed for different time periods; D, change in encapsulation efficiency (EE) of docetaxel fat bodies with different contents after being placed for different time periods; E, change in the number of DTX molecules (Molecules/Adiposome, M/A) in each fat body after being placed for different time periods; F, optical microscopy observation of the morphological structure of docetaxel fat bodies with low DTX content, with a scale bar of 2 ⁇ m; G, optical microscopy observation of the morphological structure of docetaxel fat bodies with high DTX content, with a
  • Figure 4 shows that docetaxel adipsomes have a stable structure and a slow release rate, including: A, DTX retention of docetaxel adipsomes and docetaxel injection after different dialysis times; B, average particle size and PDI of docetaxel adipsomes after different dialysis times; C, optical micrographs of docetaxel adipsomes after different dialysis times, scale bar is 5 ⁇ m; ** indicates p ⁇ 0.01, *** indicates p ⁇ 0.001.
  • DTX-Ad is docetaxel adipsomes
  • Commercial DTX is the commercialized docetaxel drug docetaxel injection.
  • Figure 5 The clearance of docetaxel fat bodies in vivo is lower than that of docetaxel injection, where: A, Photos of centrifuged blood collected from mice after docetaxel adipocytes and docetaxel injection were administered at different times. The white portion indicated by the red arrow is the docetaxel adipocyte. B, Plasma was separated from mice after docetaxel adipocytes and docetaxel injection were administered at different times, and the DTX content was detected. ** indicates p ⁇ 0.01, and *** indicates p ⁇ 0.001.
  • DTX-Ad is docetaxel adipocytes
  • Commercial DTX is the commercialized docetaxel drug docetaxel injection.
  • Figure 6 shows that docetaxel fat bodies have anti-tumor activity, wherein: docetaxel fat bodies and ethanol (control group) were treated with the corresponding cells at the indicated concentrations, and the cell survival rate was measured by CCK8; A, breast cancer cell 4T1; B, human prostate cancer cell PC3; C, human hematologic malignancy cell H929; D, human hematologic malignancy cell Raji; *** indicates p ⁇ 0.001;
  • Figure 7 shows that docetaxel adipocytes are more effective in killing cancer cells than the clinical drug albumin-paclitaxel.
  • the prepared docetaxel adipocytes and the clinical drug albumin-paclitaxel were used to treat hematological tumor cells H929 (A), liver cancer cells Hepa1-6 (B), breast cancer cells 4T1 (C), and colorectal cancer cells CT26 (D) at the concentrations shown; the cell survival rates were measured using CCK8 assay; ** indicates p ⁇ 0.01, and *** indicates p ⁇ 0.001.
  • Figure 8 shows that docetaxel fat bodies inhibit the growth of breast cancer in mice, with a safety profile superior to the clinical drug docetaxel injection.
  • A changes in tumor volume in mice treated with saline, docetaxel injection, and docetaxel fat bodies
  • B body weight change in mice treated with saline, docetaxel injection, and docetaxel fat bodies
  • C hemolysis rate of erythrocytes treated with docetaxel injection and docetaxel fat bodies
  • *** indicates p ⁇ 0.001, **** indicates p ⁇ 0.0001.
  • Figure 9 shows that lung-targeted docetaxel adipocytes are more effective in inhibiting lung cancer than docetaxel injection, wherein: A, tissue distribution of fluorescently labeled lung-targeted docetaxel adipocytes (Lu-DTX-Ad); B, changes in lung tumor volume and tumor inhibition rate in mice after normal saline, docetaxel injection, and lung-targeted docetaxel adipocytes; *** indicates p ⁇ 0.001;
  • Figure 10 shows the preparation of liver-targeted docetaxel fat bodies, where: A, staining results of purified recombinant protein ApoE expressed in a prokaryotic system; B, optical microscopy results of liver-targeted docetaxel fat bodies, scale bar is 5 ⁇ m; C, expression of LDLR in mouse liver, hepatoma cell lines HepG2 and Hepa1-6, and embryonic kidney epithelial cells HEK293; D, fluorescent labeling The tissue distribution of liver-targeted docetaxel after fat body injection into mice at different times;
  • Figure 11 shows that the effect of liver-targeted docetaxel fat body in inhibiting liver cancer is better than that of docetaxel injection, wherein: A, the size, weight and inhibition rate of liver tumor volume after treatment of liver cancer mice with normal saline group, docetaxel injection group, docetaxel fat body group and liver-targeted docetaxel fat body group; B, the body weight change rate after treatment of liver cancer mice with normal saline group, docetaxel injection group, docetaxel fat body group and liver-targeted docetaxel fat body group; C, the biochemical indicators of liver function and renal function after treatment of liver cancer mice with normal saline group, docetaxel injection group, docetaxel fat body group and liver-targeted docetaxel fat body group; wherein, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01;
  • Figure 12 shows the preparation and targeting of hematologic tumor cell-targeted adipomes, wherein: A, preparation of hematologic tumor cell-targeted adipomes; B, expression of BCMA in hematologic tumor cells Raji, hematologic tumor cells H929, and human embryonic kidney epithelial cells HEK293; C, flow cytometry results of fluorescently labeled non-targeted adipomes, hematologic tumor cell-targeted adipomes, and inhibition of endocytosis of targeted adipomes by hematologic tumor cells H929; D, optical microscopy results of fluorescently labeled non-targeted adipomes, hematologic tumor cell-targeted adipomes, and inhibition of endocytosis of targeted adipomes by hematologic tumor cells H929. Scale bar is 5 ⁇ m.
  • Figure 13 shows that hematologic malignancy cell-targeted docetaxel adipomes are more effective than docetaxel injection: H929 cells were treated with the same concentration of 20 ng/ml of DTX, including untargeted docetaxel adipomes, hematologic malignancy cell-targeted docetaxel adipomes, and the clinical drug docetaxel injection, for 2, 4, and 16 hours. The culture medium was removed and replaced with drug-free culture medium. Cell survival was measured using CCK8 assay. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, and *** indicates p ⁇ 0.001.
  • the present invention provides a docetaxel fat body preparation and its preparation method and application.
  • Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify the method and application of this article without departing from the content, spirit and scope of the present invention. Modifications or appropriate changes and combinations can be made to implement and apply the technology of the present invention.
  • a and/or B can mean: A exists alone, A and B exist at the same time, and B exists alone.
  • a and B can be singular or plural.
  • At least one means one or more, and “more than one” means two or more. “At least one of the following” or similar expressions refers to any combination of these items, including any combination of single or plural items.
  • drug herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition is administered.
  • prevention herein includes prevention and/or treatment.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) unwanted physiological changes or lesions in the treated subject, such as cancer and tumors.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable.
  • Subjects in need of treatment include subjects who already have a condition or disease, as well as subjects who are susceptible to a condition or disease or subjects for whom a condition or disease is to be prevented.
  • slowing down, alleviating, weakening, alleviating, and alleviating their meaning also includes situations such as elimination, disappearance, and non-occurrence.
  • administered refers to an organism that receives treatment for a particular disease or condition as described herein.
  • the organism receiving treatment for a disease or condition is a mammal, such as a human, A primate (eg, monkey) or non-primate mammal.
  • subject herein refers to an organism that is being treated for a particular disease or condition as described herein.
  • a “subject” includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.
  • the term "effective amount” refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount” also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplasm” refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer” and “tumor” are not mutually exclusive when used herein.
  • IC50 in this article refers to the half-inhibitory concentration of the antagonist being measured. It can be understood that a certain concentration of a drug induces 50% tumor cell death. This concentration is called the 50% inhibitory concentration, that is, the concentration corresponding to the ratio of dead cells to total cells is equal to 50%.
  • the IC50 value can be used to measure the ability of a drug to induce death. That is, the stronger the induction ability, the lower the value.
  • the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
  • test materials used in the present invention are all common commercial products and can be purchased in the market.
  • the present invention uses a novel nano-carrier fat body with a hydrophobic core structure to encapsulate the docetaxel drug, which has a 100-fold increase in solubility in water and has biological activity, significantly killing multiple cancer cells, with better effectiveness than the clinical drug albumin paclitaxel and better safety than the clinical drug docetaxel injection.
  • the present invention uses different methods to target the surface of the docetaxel fat body, including lung targeting, liver targeting, and blood cancer cell targeting, effectively inhibiting the growth of lung cancer, liver cancer, and blood tumors.
  • the inhibitory effect is significantly better than the clinically used docetaxel drug (docetaxel injection), and its safety is also better than docetaxel injection.
  • the present invention is further described below in conjunction with the examples.
  • Example 1 Fat body has good biocompatibility
  • Neutral lipids Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1/1.
  • step 3 Add 100 ⁇ l of PBS and 5 ⁇ l of the neutral lipid from step 1) to a microcentrifuge tube and vortex for 4 minutes (vortex for 10 seconds, rest for 10 seconds) (in actual application, vortex for 3-7 minutes, vortexing conditions are 3000-4000 rpm, and 4000 rpm is used in this example) to obtain a milky white lipid mixture 1.
  • the lipid mixture 1 is centrifuged at 1000 g for 5 minutes (in actual application, centrifugation at 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system presents two layers. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 2.
  • step 3 The lipid mixture 2 obtained in step 3) was centrifuged at 20000g for 5 min (in practical applications After centrifugation at 18,000-22,000 g for 3-7 min, remove the precipitate at the bottom of the microcentrifuge tube and vortex to obtain a milky white lipid mixture 3.
  • step 5 The lipid mixture 3 obtained in step 4) is centrifuged at 1000 g for 5 minutes (in actual application, 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system presents two layers. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 4, which is the final fat body.
  • the construction method is as follows:
  • Neutral lipids Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1/1.
  • step 5 Add 100 ⁇ l PBS and 5 ⁇ l of the solution prepared in step 3) to a microcentrifuge tube.
  • the neutral lipid of DTX was vortexed for 4 minutes (vortexed for 10 seconds and stopped for 10 seconds) (in actual application, vortexed for 3-7 minutes, the vortexing condition was 3000-4000 rpm, and 4000 rpm was used in this embodiment) to obtain a milky white lipid mixture 1.
  • the lipid mixture 1 was centrifuged at 1000 g for 5 minutes (in actual application, centrifuged at 800-1200 g for 3-7 minutes). After centrifugation, the liquid phase system showed two layers.
  • the lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 2.
  • step 5 The lipid mixture 2 obtained in step 5) was centrifuged at 20,000 g for 5 minutes (in actual application, 18,000-22,000 g for 3-7 minutes is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.
  • step 6) The lipid mixture 3 obtained in step 6) was centrifuged at 1000 g for 5 minutes (in actual application, 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system showed two layers. The lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 4, which was the final fat body carrying the hydrophobic small molecule compound docetaxel (docetaxel fat body).
  • the presence of docetaxel in the docetaxel liposomes was determined by HPLC (high-performance liquid chromatography) and TLC (thin-layer chromatography), respectively.
  • the TLC detection method was as follows: the constructed docetaxel liposomes were added with an equal volume of methanol and two volumes of chloroform to extract lipids. The organic phase was collected and dried with nitrogen to obtain total lipids.
  • the obtained total lipids were added to 100 ⁇ l of chloroform, and 10 ⁇ l was loaded onto a silica gel plate.
  • the plate was developed in a solvent of n-hexane:diethyl ether:glacial acetic acid (volume ratio 80:20:1) to separate TAGs.
  • the silica gel plate was then developed in a solvent of chloroform:methanol:glacial acetic acid:water (volume ratio 75:13:9:3) to separate DOPC and DTX.
  • the elution time of DTX in docetaxel fat body is consistent with the elution time of DTX standard substance free in ethanol, and is all 7.5min (B among Fig. 2).
  • the signal of whether there is DTX in fat body is judged according to the position of DTX standard substance (shown in the dotted line frame).
  • DTX standard substance shown in the dotted line frame.
  • Dynamic light scattering instrument detects that the average particle size of blank fat body is 110nm, and PDI (polydispersity index) is 0.15.
  • the average particle size of docetaxel fat body is 118nm and PDI is 0.11.
  • Figure 2D optical Microscopic observation of the morphology of blank and docetaxel-containing adipocytes revealed uniform spherical structures without contamination by other membrane impurities.
  • Docetaxel fat bodies have good stability
  • the morphological structure of the docetaxel fat body after 1 day and 42 days of storage was observed by optical microscopy. Both were uniform spherical structures and were not contaminated by other membrane impurities (F and G in Figure 3). This shows that the docetaxel fat body has high purity, good uniformity, and good structural stability.
  • mice were injected with equal amounts of docetaxel injection and docetaxel fat body respectively, at a concentration of 15 mg/kg, with 4 mice in each group, a total of 24 mice. After 10, 30 and 60 minutes after injection respectively, mice were killed, the plasma of mice was separated, and the content change of DTX was detected. As shown in Figure 5, after injection at different times, docetaxel fat body structure (A in Figure 5, the white fat body-like structure shown by the red arrow) can still be observed in the mouse serum. In addition, after injection for 60 minutes, the concentration of docetaxel in the mouse plasma processed by docetaxel fat body was significantly higher than that of docetaxel injection (B in Figure 5). The above results show that docetaxel fat body is stable in blood, and its clearance rate in vivo is lower than that of docetaxel injection, significantly prolonging the time when the drug exists in the body.
  • Docetaxel fat bodies have anti-tumor activity
  • docetaxel fat bodies have biological activity and can kill tumor cells.
  • the non-targeted docetaxel fat bodies prepared in the previous example were selected to treat breast cancer cells 4T1 ( Figure 6 A), human prostate cancer cells PC3 ( Figure 6 B), human blood tumor cells.
  • Figure 6 A the concentration of docetaxel increased
  • Figure 6 B human blood tumor cells
  • Cell viability assay 3000 cells/well of the cells to be treated were plated in a 96-well plate. After overnight attachment, the cells were treated with the indicated drug concentrations and incubated for 72 hours. Subsequently, the original culture medium was replaced with culture medium containing 10% CCK8. After 1 hour of incubation, the absorbance at 450 nm was read using a microplate reader. Cell viability was calculated using the following formula:
  • Ae represents the absorbance value of the well containing cells after drug treatment.
  • Ac represents the absorbance value of the wells containing cells without drug treatment.
  • Ab represents the absorbance value of blank wells containing only culture medium and CCK8 reagent, which is used to correct background noise.
  • the prepared docetaxel fat bodies and the clinical drug albumin-paclitaxel were used to treat hematological tumor cells H929 (Figure 7A), liver cancer cells Hepa1-6 (Figure 7B), breast cancer cells 4T1 (Figure 7C), and colorectal cancer cells CT26 ( Figure 7D) at the indicated concentrations.
  • docetaxel fat bodies were significantly more effective than albumin-paclitaxel in killing various cancer cells.
  • the IC50 of docetaxel fat bodies was significantly lower than that of albumin-paclitaxel (Table 1).
  • Table 1 IC50 of docetaxel fat body and albumin paclitaxel in killing various cancer cells
  • mice were administered intravenously at an equivalent dose of 15 mg/kg of docetaxel once weekly for three times. Tumor volume and body weight changes were measured during the dosing period. As shown in the figure, docetaxel fat body significantly inhibited subcutaneous tumor growth compared to the saline group, demonstrating an effect comparable to that of the clinical drug docetaxel injection (Figure 8A). However, the results of weight change showed that docetaxel fat bodies significantly reduced mouse body weight compared to docetaxel injection. The weight change rate of mice treated with docetaxel injection was -16.64 ⁇ 2.77%, while that of mice treated with docetaxel fat bodies was -9.58 ⁇ 8.34% (Figure 8B).
  • step (2) Take 500 ⁇ l of the red blood cell suspension obtained in step (1) and add it to 500 ⁇ l of pure water as a positive control, and add it to 500 ⁇ l of PBS as a negative control, and mix well.
  • step (3) Take 500 ⁇ l of the diluted solution in step (3) and add 500 ⁇ l of red blood cell suspension
  • the final concentrations of DTX were 6 ⁇ g/ml, 15 ⁇ g/ml, and 30 ⁇ g/ml.
  • step (3) Take 500 ⁇ l of the diluted solution in step (3) and add it to 500 ⁇ l PBS, mix well, and use it as the background tube for each concentration in the experimental group.
  • Hemolysis rate of erythrocytes [(absorbance of experimental group - absorbance of background tube) / absorbance of positive control] ⁇ 100%.
  • Example 3 Lung-targeted docetaxel fat body is more effective in inhibiting lung cancer than docetaxel injection
  • Neutral lipids Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1/1.
  • LTA-P33 (peptide sequence: MELTIFILRLAIYILTFPLYLLNFLGLWCRGDK, SEQ ID NO: 4) was dissolved in a mixture of methanol and chloroform (1:1, v/v) to a final concentration of 0.5 mg/ml.
  • step 5 Take 20 ⁇ l of the solution in step 4) and 80 ⁇ l of 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine solution (containing 2 mg DOPC), mix them evenly, and add the total volume of 100 ⁇ l into a microcentrifuge tube. Blow dry the solvent with high-purity nitrogen gas.
  • step 5 Add 100 ⁇ l PBS and 5 ⁇ l neutral lipid containing DTX prepared in step 3) into a microcentrifuge tube, vortex for 4 minutes (vortex for 10 seconds, stop for 10 seconds) (in actual application, vortex for 3-7 minutes, vortex conditions are 3000-4000 rpm, and 4000 rpm is used in this embodiment) to obtain a milky white lipid mixture 1, and centrifuge the lipid mixture 1 at 1000 g for 5 minutes (in actual application, centrifuge at 800-1200 g for 3-7 minutes). After centrifugation, the liquid phase system presents two layers. Collect the lower milky white solution by extraction and vortex to obtain a milky white lipid mixture 2.
  • step 5 The lipid mixture 2 obtained in step 5) was centrifuged at 20,000 g for 5 minutes (in actual application, 18,000-22,000 g for 3-7 minutes is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.
  • step 6) The lipid mixture 3 obtained in step 6) was centrifuged at 1000 g for 5 min (in actual application, 800-1200 g for 3-7 min is acceptable). After centrifugation, the liquid phase system showed two layers. The lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 4, which was the final docetaxel adiposome with LTA-P33.
  • the docetaxel adiposome with LTA-P33 was recorded as lung-targeted docetaxel adiposome (Lung-Targeted Docetaxel Adiposome, Lu-DTX-Ad), and targeting verification and efficacy verification were performed.
  • the dosage was equivalent to 15 mg/kg of docetaxel, administered intravenously once a week for a total of two times.
  • the changes in the lung tumor volume of the mice were detected.
  • the results are shown in the figure.
  • the docetaxel injection group The lung tumor signal in the treated mice was smaller than that in the saline group, with an average tumor inhibition rate of 45.3%.
  • Example 4 Liver-targeted docetaxel fat bodies are more effective in inhibiting liver cancer than docetaxel injection
  • liver-targeted docetaxel adiposome Lv-DTX-Ad
  • liver-targeted docetaxel fat bodies The process for constructing liver-targeted docetaxel fat bodies is as follows:
  • Non-targeted docetaxel fat bodies were prepared according to the method in Example 2.
  • the docetaxel adiposomes with ApoE are recorded as liver-targeted docetaxel adiposomes (Liver-Targeted Docetaxel Adiposome, Lv-DTX-Ad), and target verification and efficacy verification are performed.
  • LDL-receptor Lv-DTX-Ad
  • the ApoE-Flag gene (a fusion gene of ApoE and Flag obtained by removing the signal peptide sequence of ApoE and fusing the Flag tag to the C-terminus of ApoE, SEQ ID NO: 1) was constructed into the vector pET28a-SMT3, and the resulting recombinant vector with the correct sequence was named pET28a-SMT3-ApoE-Flag.
  • the constructed recombinant vector pET28a-SMT3-ApoE-Flag was introduced into E. coli Rosetta to obtain recombinant bacteria, which were recorded as E-pET28a-SMT3-ApoE-Flag.
  • the cells were then disrupted using a JG-1A high-pressure cell disruptor to obtain a bacterial lysate.
  • the resulting cell lysate was ultracentrifuged at 30,000 g for 60 minutes, and the supernatant was collected. 50 ⁇ l of the supernatant was added to an equal volume of 2x Sample Buffer, and the resulting mixture was used as sample 1 (supernatant fraction). The remaining supernatant was incubated with Chelating Sepharose Fast Flow, a filler chelated with nickel ions, and incubated at 4°C for 2 hours before being transferred to a 4 ml column.
  • the flow-through liquid (i.e., the flow-through liquid) was collected, and 50 ⁇ l of the flow-through liquid was added to an equal volume of 2xSample Buffer.
  • the resulting mixed solution was used as sample 2 (flow-through fraction).
  • Nonspecific bands were first washed with 40 mM imidazole to resuspend the filler in the column, the outflowing wash liquid was collected, 50 ⁇ l of the wash liquid was added to an equal volume of 2xSample Buffer, and the resulting mixed solution was used as sample 3 (40 mM fraction).
  • sample 5 Take 50 ⁇ l of the recombinant protein ApoE solution and add 2xSample Buffer. The resulting mixture is used as sample 5 (ApoE component). The resulting mixture is placed in a dialysis bag, and then the dialysis bag is placed in a beaker containing 100 times the volume of buffer A of the dialysis bag and dialyzed overnight. Take 50 ⁇ l of the solution in the dialysis bag and add an equal volume of 2xSample Buffer. The resulting mixture is used as sample 6 (dialysis component). The above samples 1-6 are analyzed by SDS-PAGE and then subjected to staining analysis and identification.
  • Lanes 1 and 2 contained numerous nonspecific bands, and the target protein, ApoE-Flag, was primarily concentrated in sample 1.
  • Lane 4 contained the fusion protein at a molecular weight of 55 kDa
  • lanes 5 and 6 contained the recombinant ApoE-Flag protein, cleaved from the HIS-SMT3 tag, at a molecular weight of 35 kDa.
  • ApoE nucleotide sequence (removing the ApoE signal peptide sequence, and at the C-terminus of ApoE Fusion Flag tag)
  • ApoE amino acid sequence (removing the ApoE signal peptide sequence and fusing the Flag tag to the C-terminus of ApoE)
  • liver-targeted docetaxel adiposomes The morphology of the obtained liver-targeted docetaxel adiposomes (Lv-DTX-Ad) was observed under optical microscopy.
  • Lv-DTX-Ad was a uniformly sized spherical structure that stained red with the neutral lipid-specific dye LipidTox Red ( Figure 10, B).
  • Western blot analysis revealed high LDLR expression in the mouse liver and in the hepatocellular carcinoma cell lines HepG2 and Hepa1-6, while expression was very low in embryonic kidney epithelial HEK293 cells ( Figure 10, C).
  • the prepared fluorescently labeled liver-targeted docetaxel adiposomes were injected into mice via the tail vein.
  • liver-targeted docetaxel adiposomes were consistently enriched in the liver. Twenty-four hours after injection, a strong signal was still detected in the liver (Figure 10, D). This indicates that we have successfully constructed liver-targeted docetaxel fat bodies and further evaluated their efficacy and safety in the treatment of liver cancer.
  • mice were randomly divided into three groups, each containing five mice: a saline group, a docetaxel injection group, a docetaxel adipocyte group, and a liver-targeted docetaxel adipocyte group. Docetaxel equivalent to 15 mg/kg was administered intravenously once a week for three times. Thirty-one days after cancer cell injection, the mice were sacrificed, their livers isolated, and liver tumor size measured. Blood was also collected for liver and kidney function tests.
  • the liver tumor volume in the docetaxel injection group was significantly smaller, with an average tumor inhibition rate of 47.4%.
  • the liver tumor inhibition rate in the docetaxel adipocyte group was 84.5%, significantly better than that in the docetaxel injection group.
  • the liver-targeted docetaxel adipocyte group achieved the highest inhibition rate, reaching 97.9% ( Figure 11A).
  • the average weight loss of mice in the liver-targeted docetaxel fat body treatment group was 7%
  • the average weight loss of mice in the docetaxel fat body treatment group was 14.4%
  • the average weight loss of mice in the docetaxel injection treatment group was 38.9% (Figure 11B).
  • liver function inflammation indicator AST aspartate aminotransferase
  • Example 5 The effect of hematological tumor cells targeting docetaxel fat bodies is better than docetaxel injection
  • H929-Targeted Docetaxel Adiposome (H929-DTX-Ad) targeting hematologic malignancy cells and performed targeting and efficacy verification.
  • Phospholipid without biotin (DOPC), named 18:1( ⁇ 9-Cis)PC
  • biotin-modified phosphatidylethanolamine Bio-PE
  • 18:1Biotinyl Cap PE neutral lipid is glyceryl trioleate (TAG). All of the above products were purchased from Sigma.
  • the lipid mixture 2 is centrifuged at 20,000 g for 5 min (in actual application, 18,000-22,000 g for 3-7 min is acceptable). After centrifugation, the sediment at the bottom of the tube is removed, and the remaining emulsion in the tube is resuspended to obtain a milky white lipid mixture 3.
  • the lipid mixture 3 was centrifuged at 1000 g for 5 min. After centrifugation, the upper white band was removed and the remaining emulsion in the tube was resuspended to obtain a milky white lipid mixture 4, which is the final biotin-labeled fat body.
  • Hematologic tumor cell targeting Adipocyte targeting of hematologic tumor cells H929
  • Relapsed myeloma is caused by hematologic malignant cells H929, which highly express BCMA (Tumor necrosis factor receptor superfamily member 17).
  • BCMA Tumor necrosis factor receptor superfamily member 17
  • Western blot revealed that only hematologic malignant cells H929 highly expressed BCMA protein, while another hematologic malignant cell type Raji and human embryonic kidney epithelial cells HEK293 did not express BCMA protein ( Figure 12B).
  • the blank group no Do any treatment
  • non-targeted fat body treatment group hematologic tumor cell targeted fat body group
  • hematologic tumor cell targeted fat body group fat body with BCMA antibody
  • inhibition of targeted fat body group additional of BCMA protein to block fat body with BCMA antibody
  • the peak of the hematologic tumor cell targeted fat body group was significantly shifted to the right (peak 3 in C in Figure 12), indicating that H929 cells phagocytized more fat bodies, while the peaks of the non-targeted fat body treatment group and the inhibition of targeted fat body group did not shift to the right (peak 2 and peak 4 in C in Figure 12).
  • the statistical results also showed that the fluorescence signal of the hematologic tumor cell targeted fat body group was the strongest (column 3 in C in Figure 12), which was significantly higher than that of the non-targeted fat body treatment group (column 2 in C in Figure 12), while the inhibition of targeted fat body group inhibited the endocytosis of fat bodies by H929 cells.
  • H929 cells showed the strongest internalization of the hematologic tumor cell-targeted adipocytes, while the addition of BCMA protein inhibited the internalization of the hematologic tumor cell-targeted adipocytes by H929 cells ( Figure 12D). These results demonstrate that the prepared hematologic tumor cell-targeted adipocytes successfully target the hematologic tumor H929 cells.
  • Neutral lipids Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1/1.
  • step 5 The lipid mixture 2 obtained in step 5) was centrifuged at 20,000 g for 5 minutes (in actual application, 18,000-22,000 g for 3-7 minutes is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.
  • step 6) The lipid mixture 3 obtained in step 6) was centrifuged at 1000 g for 5 min (in actual application, 800-1200 g for 3-7 min can be used). After centrifugation, the liquid phase system showed two layers. The lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 4.
  • step 8) 30ul of lipid mixture 4 obtained in step 7) was then added with 20ul of avidin-labeled BCMA antibody at a final concentration of 0.25mg/ml, incubated at room temperature for 1 hour, and the lipid mixture was centrifuged at 21000g for 6min. After centrifugation, the sediment at the bottom of the tube was removed, and the remaining emulsion in the tube was suspended to obtain a milky white lipid mixture 5. The lipid mixture was centrifuged at 1000g for 6min. After centrifugation, the upper white band was removed and the remaining emulsion in the tube was resuspended. The above steps were repeated three times to obtain hematologic tumor cell-targeted docetaxel fat bodies.
  • hematologic tumor cell-targeted docetaxel fat bodies have biological activity and can kill tumor cells.
  • Human hematologic tumor cells H929 were treated with commercial docetaxel injection, the non-targeted docetaxel fat bodies prepared in Example 2, and hematologic tumor cell-targeted docetaxel fat bodies, respectively. After treating the cells with the same concentration of 20 ng/ml of DTX for 2 hours, 4 hours, and 16 hours, the culture medium was removed, and then drug-free culture medium was added to measure cell survival.

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Abstract

L'invention concerne une formulation de liposome de docétaxel, son procédé de préparation et son utilisation. La formulation de liposome de docétaxel comprend une membrane phospholipidique monomoléculaire,ainsi que du docétaxel et un lipide neutre qui sont enveloppés dans la membrane phospholipidique monomoléculaire. Une modification ciblée est en outre effectuée sur la surface du liposome de docétaxel. La formulation de liposome présente une bonne biodisponibilité et une bonne sécurité, et l'effet anticancéreux de la formulation est significativement supérieur à celui d'une injection de docétaxel cliniquement utilisée.
PCT/CN2024/103795 2024-03-05 2024-07-05 Formulation de liposome de docétaxel, son procédé de préparation et son utilisation Pending WO2025185024A1 (fr)

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