WO2019170061A1 - Procédé d'enrobage de doxorubicine dans hfn et produit associé - Google Patents

Procédé d'enrobage de doxorubicine dans hfn et produit associé Download PDF

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WO2019170061A1
WO2019170061A1 PCT/CN2019/076897 CN2019076897W WO2019170061A1 WO 2019170061 A1 WO2019170061 A1 WO 2019170061A1 CN 2019076897 W CN2019076897 W CN 2019076897W WO 2019170061 A1 WO2019170061 A1 WO 2019170061A1
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hfn
doxorubicin
loaded
solution
urea
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PCT/CN2019/076897
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阎锡蕴
范克龙
朱京东
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昆山新蕴达生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to a method of encapsulating doxorubicin (DOX) of HFn (human H ferritin, a ferritin formed by self-assembly of the H subunit of human ferritin) and a product thereof.
  • DOX doxorubicin
  • HFn human H ferritin, a ferritin formed by self-assembly of the H subunit of human ferritin
  • the present invention relates to methods and products for depolymerizing, refolding HFn and entraining doxorubicin using urea or guanidine hydrochloride.
  • Ferritin is an important functional protein involved in and maintain iron metabolism balance. It is a kind of protein containing high iron content widely distributed in animals, plants and microbial cells. From bacteria to humans, although the ferritin amino acid sequences of different organisms are greatly different, their structures are similar.
  • the typical ferritin structure is composed of a protein shell and an iron core.
  • the protein shell is a cage structure formed by self-assembly of 24 subunits (outer diameter 12 nm, inner diameter 8 nm).
  • the main component of the iron core is ferrihydrite. (5Fe 2 O 3 ⁇ 9H 2 O).
  • the ferritin shell is usually composed of two protein subunits (H and L). In different tissues and organs of the body, the proportion of H and L subunits in the ferritin molecule is different.
  • Human H ferritin refers to ferritin formed by self-assembly of the H subunit of human iron eggs.
  • the H subunit of human ferritin can self-assemble to form a cage protein, and the number of H subunits of human ferritin is usually 24.
  • the full length amino acid sequence of human H ferritin is shown in SEQ ID NO: 1.
  • the drug loading of ferritin from different sources depends mainly on two ways: 1.
  • the drug enters the inside of the protein shell through ion channels or hydrophobic channels on ferritin under specific treatment conditions; 2.
  • a high concentration of urea the protein shell is depolymerized, and the drug to be entrapped is added during the subsequent recombination process, thereby realizing the loading of the drug inside the protein shell.
  • HFn entrapped doxorubicin is particularly efficient under high-concentration urea conditions, and 33 molecules of doxorubicin are loaded per molecule of HFn; the brief procedure of the method is: adding human HFn to a final concentration greater than 6M In the urea, react at room temperature for 30 minutes, then add appropriate amount of doxorubicin reagent, and protect from light for 10 minutes, then use dialysis method to gradually reduce the urea concentration in the reaction system to 0, thus achieving doxorubicin Envelope in HFn.
  • Doxorubicin is an anti-tumor antibiotic that inhibits the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA, has a broad anti-tumor spectrum, and has a role in various tumors. It is a non-specific drug for various growth. Periodic tumor cells have a killing effect. It is mainly used for acute leukemia and is effective for acute lymphoblastic leukemia and granulocyte leukemia. It is generally used as a second-line drug, that is, it can be considered when the drug is the drug of choice. For malignant lymphoma, it can be used as the drug of choice for alternate use. It has a certain effect on breast cancer, sarcoma, lung cancer, bladder cancer and other various cancers, and is often used in combination with other anticancer drugs.
  • doxorubicin when used directly in the injection, doxorubicin has a wide range of biochemical effects on the body due to its diffuse distribution throughout the blood, and has a strong cytotoxic effect.
  • the main toxic reactions are: white blood cells and thrombocytopenia, about 60% to 80% of patients can occur; 100% of patients have varying degrees of hair loss, can resume growth after stopping the drug; cardiotoxicity, manifested as arrhythmia, ST- T changes, more often 1 to 6 months after withdrawal; nausea, loss of appetite; drug spillage outside the blood vessels can cause tissue ulcers and necrosis.
  • urine can appear red after administration.
  • a first aspect of the invention relates to a method of HFn-loaded doxorubicin comprising the steps of:
  • the additive in which the dissolution of doxorubicin is promoted is selected from the group consisting of glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine or a mixture thereof.
  • amino acid sequence of the H subunit comprising HFn is set forth in SEQ ID NO.
  • the additive that promotes doxorubicin dissolution is selected from the group consisting of glycerin or sucrose.
  • the mass ratio of HFn to doxorubicin in the solution is from 1:2 to 5:1, preferably from 1:1 to 4:1, more preferably from 8:2.5 to 8:3.5.
  • the removal of urea in solution, the additive to promote doxorubicin dissolution, and residual doxorubicin are carried out by desalting, preferably using a desalting column.
  • the mass ratio of HFn-loaded doxorubicin is 5% to 60%, preferably, at least 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher, more preferably at least 20%, 25%, 30% or 35%.
  • the HFn content in the solution ranges from 0.1 to 100 mg/ml, preferably from 0.2 to 10 mg/ml, more preferably from 0.3 to 5 mg/ml, more preferably from 0.5 to 2 mg/ml, more preferably , 0.7 to 1.4 mg/ml; and/or, the content of doxorubicin ranges from 0.1 to 50 mg/ml, preferably from 0.2 to 10 mg/ml, more preferably from 0.3 to 5 mg/ml, more preferably from 0.5 to 2 mg/ml, more preferably 0.7 to 1.4 mg/ml.
  • the concentration of urea is 8 M, and/or depolymerization is 9-20 h, more preferably 10-14 h, and/or the incubation temperature is 35 °C to 48 °C, preferably 39 °C to 45 °C.
  • the solution is Tris-HCl buffer, citric acid-sodium citrate buffer or acetic acid-sodium acetate buffer, preferably, the pH of the solution is 4-10, more preferably 7-9, More preferably, 7.5 to 8.5; and/or, preferably, the concentration of the solution is 20 to 200 mM, more preferably 25 to 100 mM, more preferably 25 to 50 mM.
  • a second aspect of the invention relates to an adriamycin-encapsulated HFn obtained by the method of HFn-loaded doxorubicin described in the above first aspect.
  • a third aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising doxorubicin-containing HFn obtained by the method of HFn-loaded doxorubicin described in the above first aspect.
  • the method for enrolling doxorubicin in the HFn of the present invention can greatly increase the entrapment amount of doxorubicin, and the clinical administration dose will be greatly reduced under the premise of achieving the same therapeutic effect.
  • Figure 1 AKTA map of a separate HFn sample.
  • the HFn of the present invention refers to a fully human heavy chain (H subunit) ferritin having an amino acid sequence as shown in SEQ ID NO.
  • the depolymerization of the present invention means that the tight closed spherical structure of the ferritin natural 24-mer is opened, and the visual representation is that the peak time on the gel exclusion column is significantly delayed, so that the depolymerized protein peak and the unagglomerated protein peak occur. Separation.
  • the depolymerization ratio is at least 50%, such as at least 55%, 60%, 65%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, up to achievable 100%.
  • Repolymerization means that after the substance causing depolymerization is removed or reduced to a sufficiently low concentration, the depolymerized ferritin returns to the tightly closed globular structure of the natural 24-mer, which is visually represented as a gel-exhaust column.
  • the peak time is reduced to a peak time similar to that of natural ferritin.
  • the polycondensation ratio is at least 50%, such as 55%, 60%, 65%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, up to 100%.
  • the HFn-loaded doxorubicin in the present invention refers to a process in which doxorubicin is carried in a cage structure of HFn, and is in the same solution system in the process of HFn depolymerization and recombination in a solution system.
  • the doxorubicin loaded will be encased in a cage structure that is re-formed after recombination.
  • the upper limit of the doxorubicin encapsulation in the HFn of the present invention is the maximum amount of drug that can be contained in the internal volume of the HFn cage structure, i.e., the amount of drug that can be contained in a volume of about 268 cubic nanometers.
  • the drug loading is 5 to 60%, preferably 10 to 50%, 20 to 40%, 25 to 35%, such as at least 8%, 10%, 15%, in terms of mass ratio. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher.
  • 50-500 doxorubicin molecules are entrained per molecule of HFn, such as 60, 80, 90, 100, 110, 120, 130, 140, 150, 170, 190, 200, 220, 240, 260 , 280, 240, 260, 280, 300, 350, 400, 450, 500 or more.
  • the method for enrolling doxorubicin in the HFn of the present invention can greatly increase the entrapment amount of doxorubicin, and the clinical administration dose will be greatly reduced under the premise of achieving the same therapeutic effect.
  • the buffer system of the doxorubicin-containing solution of the present invention may include, but is not limited to, Tris-HCl, citric acid-sodium citrate, acetic acid-sodium acetate buffer.
  • the buffer concentration is in the range of 20 mM to 200 mM, preferably, 30 mM to 150 mM, more preferably 40 mM to 80 mM, and the pH is in the range of 6.9.0 to 9.0, preferably 7.2 to 8.5, more preferably 7.5 to 8.3. .
  • the doxorubicin-containing buffer system of the present invention also contains an additive which promotes the dissolution of doxorubicin, and any additive known in the art for promoting the dissolution of doxorubicin can be used in the method of the present invention.
  • the additive is selected from the group consisting of glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine, or mixtures thereof, and in some embodiments, the additive is selected from the group consisting of glycerin or sucrose. Or a mixture of glycerin and sucrose, in some embodiments, the additive has a volume ratio or weight ratio of from 8 to 40%, preferably from 10 to 25%, more preferably from 12 to 20%.
  • the doxorubicin-encapsulated HFn of the present invention may be provided in the form of a pharmaceutical composition, i.e., in addition to the HFn encapsulating doxorubicin, which also contains a pharmaceutically acceptable carrier.
  • a pharmaceutical composition i.e., in addition to the HFn encapsulating doxorubicin, which also contains a pharmaceutically acceptable carrier.
  • These pharmaceutical compositions may be formulated according to conventional techniques using pharmaceutically acceptable carriers or diluents, as well as any other known excipients, such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Gennaro, Mack Publishing Co., 2013.
  • the doxorubicin-encapsulated HFn obtained by the method of the present invention can be used to treat and/or prevent a disease or condition in a subject.
  • the disease or condition in which the doxorubicin-coated HFn of the present invention can treat and/or prevent depends on the doxorubicin contained, i.e., it is known in the art to be treated and/or prevented by doxorubicin.
  • the disease or condition can be treated and/or prevented by the doxorubicin-containing HFn of the present invention.
  • the doxorubicin-encapsulated HFn of the present invention can be targeted to a tumor to release the entrained doxorubicin at the tumor site after administration to achieve prevention and/or treatment of the tumor.
  • the disease or condition is selected from the group consisting of acute leukemia (lymphocytic and granulocyte), malignant lymphoma, breast cancer, bronchial lung cancer (undifferentiated small cell and non-small cell), ovarian cancer, Soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, stomach cancer, liver cancer.
  • acute leukemia lymphocytic and granulocyte
  • malignant lymphoma breast cancer
  • bronchial lung cancer undifferentiated small cell and non-small cell
  • ovarian cancer Soft tissue sarcoma, osteo
  • compositions of this invention may be administered by any suitable route and mode, including, without limitation, intravenous or subcutaneous injection or infusion.
  • compositions of the invention may also be used in combination with other drugs, such as ABVD, CAF, CAOP, FAM, AC, AOP, ACP, CY-VA-DIC, MACC, or in combination with radiation therapy.
  • drugs such as ABVD, CAF, CAOP, FAM, AC, AOP, ACP, CY-VA-DIC, MACC, or in combination with radiation therapy.
  • HFn the subunit sequence is as shown in SEQ ID NO. 1, a cage structure composed of 24 subunits, Nature Nanotechnology, 2012, 7(7), 459-464.
  • 50 mM Tris-HCl pH 7.2 Concentrated to 25-30 mg/mL to make it a HFn mother liquor.
  • Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved.
  • Each sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using the same buffer as the sample pH.
  • the sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin, manufacturer Gene Company Limited Gene Co., Ltd., model ND2000), BCA protein assay (the amount of HFn can be obtained, kit manufacturer) : Thermo Fisher Scientific (China) Co., Ltd., Item No. Prod #23227), HPLC (received monomer after HFn-loaded doxorubicin, manufacturer: Agilent Technologies (China) Co., Ltd., Model High Performance Liquid Chromatography 1260Infinity ) Determination.
  • the results are shown in Table 1.
  • Figures 1 and 2 show the AKTA spectra of individual HFn and DOX-enhanced HFn, respectively, which clearly demonstrate the case of confirming whether HFn encloses DOX.
  • the HFn solution (50 mM Tris-HCl pH 8.0) was first concentrated to 25-30 mg/mL to make it a HFn mother liquor. Prepare 20 mg/mL DOX mother liquor for use in ultrapure water. Since many samples need to be configured, the configuration of the samples is recorded in the form of Table 2.
  • Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved.
  • the sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using 50 mM Tris-HCl pH 8.0 buffer solution.
  • the sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin), BCA protein assay (the amount of HFn can be obtained), and HPLC (the monomer after HFn-loaded doxorubicin) Case) Determination.
  • SDS-PAGE determination of the presence of protein
  • NANODROP measured by the value of doxorubicin
  • BCA protein assay the amount of HFn can be obtained
  • HPLC the monomer after HFn-loaded doxorubicin
  • the HFn solution (50 mM Tris-HCl pH 8.0) was first concentrated to 25-30 mg/mL to make it a HFn mother liquor. Prepare 20 mg/mL DOX mother liquor for use in ultrapure water.
  • each sample was 0.960 g of urea, 16 mg of HFn was added to each urea, and 0.3 mL of DOX mother liquor was added separately. Finally, each sample was separately added to a buffer solution to a volume of 2 mL.
  • the sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using 50 mM Tris-HCl pH 8.0 buffer solution.
  • the sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin), BCA protein assay (the amount of HFn can be obtained), and HPLC (the monomer after HFn-loaded doxorubicin) Case) Determination.
  • SDS-PAGE determination of the presence of protein
  • NANODROP measured by the value of doxorubicin
  • BCA protein assay the amount of HFn can be obtained
  • HPLC the monomer after HFn-loaded doxorubicin
  • the incubation temperature was determined to be 42 ° C and the incubation time was determined to be 12 h.
  • the present invention also investigates the effect of additives on HFn entrapped DOX.
  • the present inventors have discovered that the additive may primarily address the problem of aggregation of HFn upon entrapment, reducing the amount of HFn entrapped DOX when the additive inhibits aggregation, and increases the amount of HFn entrapped DOX when the additive promotes aggregate generation.
  • the present invention tested various additives including, but not limited to, glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine or mixtures thereof, and the additives which were found to be more effective were glycerin and sucrose.
  • Incubation buffer 50 mM Tris-HCl pH: 8.0 (containing 1%, 5%, 10%, 15%, 20% glycerol).
  • HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally HFn was concentrated to about 20 mg/mL for use.
  • Incubation buffer 50 mM Tris-HCl pH: 8.0 (containing 1%, 3%, 5%, 10% sucrose)
  • HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally HFn was concentrated to about 20 mg/mL for use.
  • Incubation buffer 50 mM Tris-HCl pH: 8.0 (containing 0.01%, 0.05%, 0.10%, 0.50% Tween)
  • HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally HFn was concentrated to about 20 mg/mL for use.
  • Incubation buffer 50 mM Tris–HCl pH: 8.0 (containing 1%, 2%, 4% arginine)
  • the purified HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally the HFn was concentrated to about 25-30 mg/mL for use.
  • the HFn solution (50 mM Tris-HCl pH 8.0) was first concentrated to 25-30 mg/mL to make it a HFn mother liquor.
  • Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved.
  • Each sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using 50 mM Tris-HCl pH 8.0 buffer.
  • the sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin), BCA protein assay (the amount of HFn can be obtained), and HPLC (the monomer after HFn-loaded doxorubicin) Case) Determination.
  • SDS-PAGE determination of the presence of protein
  • NANODROP measured by the value of doxorubicin
  • BCA protein assay the amount of HFn can be obtained
  • HPLC the monomer after HFn-loaded doxorubicin
  • the ionic strength of the selected system was 30 mM Tris-HCl.

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Abstract

L'invention concerne un procédé d'enrobage de doxorubicine dans HFn et son produit associé. L'invention concerne spécifiquement l'exploration des conditions optimales utilisant spécifiquement différentes concentrations d'urée, différents additifs, différents rapports de HFn/doxorubicine et/ou différentes conditions d'incubation, telles que la température d'incubation, la durée d'incubation, la solution tampon d'incubation et la valeur pH. Grâce aux conditions d'enrobage ainsi obtenues, l'enrobage de la doxorubicine dans HFn peut être rapide, convenable, efficace et suffisante.
PCT/CN2019/076897 2018-03-07 2019-03-04 Procédé d'enrobage de doxorubicine dans hfn et produit associé WO2019170061A1 (fr)

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