WO2022206796A1 - 一种peg化雷帕霉素化合物及其制备方法与应用 - Google Patents

一种peg化雷帕霉素化合物及其制备方法与应用 Download PDF

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WO2022206796A1
WO2022206796A1 PCT/CN2022/083907 CN2022083907W WO2022206796A1 WO 2022206796 A1 WO2022206796 A1 WO 2022206796A1 CN 2022083907 W CN2022083907 W CN 2022083907W WO 2022206796 A1 WO2022206796 A1 WO 2022206796A1
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pegylated
rapamycin
pegylated rapamycin
rapamycin compound
freeze
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PCT/CN2022/083907
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English (en)
French (fr)
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郭圣荣
沈园园
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杭州宜生医药科技发展有限公司
沈阳三生制药有限责任公司
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Priority to JP2023523642A priority Critical patent/JP2023545563A/ja
Priority to EP22778978.1A priority patent/EP4316523A1/en
Priority to US18/252,309 priority patent/US20240009228A1/en
Publication of WO2022206796A1 publication Critical patent/WO2022206796A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/56—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the technical field of biomedicine, and relates to a PEGylated rapamycin compound and a preparation method and application thereof.
  • Immunosuppressive drugs refer to drugs that inhibit the immune response of the body, which can inhibit the proliferation and function of cells related to the immune response (macrophages such as T cells and B cells), thereby reducing the antibody immune response.
  • Immunosuppressants are mainly used for organ transplantation to resist rejection and autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, dermatomycosis, membranous nephritis, inflammatory bowel disease and autoimmune hemolytic anemia.
  • Rapamycin also known as sirolimus, is a lipophilic triene nitrogen-containing macrolide antibiotic immunosuppressant produced by Streptomyces hygroscopicus, which can be used for the treatment of cancer, anti-graft Adjuvant therapy for rejection and other immune diseases. Rapamycin plays an immunosuppressive role by blocking the signal transduction involved in mammalian target of rapamycin (mTOR), preventing T cell differentiation and dendritic cell maturation.
  • mTOR mammalian target of rapamycin
  • the first object of the present invention is to provide a PEGylated rapamycin compound, which has suitable hydrophobic rapamycin end and hydrophilic PEG chain end, and is a good carrier for preparing nanoparticles.
  • the second object of the present invention is to provide a method for preparing the above compound, with mild reaction conditions and simple operation.
  • the third object of the present invention is to provide an application of the above-mentioned compound to prepare a nanomedicine, a freeze-dried preparation, a pharmaceutical composition and the use in the preparation of a medicine for reducing immune response.
  • the invention also discloses a method for preparing the above-mentioned PEGylated rapamycin compound.
  • the mPEG-COOH is dissolved in an organic solvent, catalysts EDC ⁇ HCl, MAP and RAPA are added, and the mixture is stirred at 10-40° C. in the dark. reaction.
  • the reaction temperature of the preparation method is preferably 20-30°C.
  • the organic solvent is one or both of dichloromethane or chloroform, preferably dichloromethane.
  • the molar ratio of mPEG-COOH to RAPA is 5:1-1:5, preferably 3:1-1:3.
  • a separation and purification step after the reaction is also included, and the separation and purification step is a dialysis purification method or a silica gel column chromatography method.
  • the dialysis purification method uses a dialysis bag and a dialysis solvent for separation and purification.
  • the molecular weight cut-off of the dialysis bag is 500-5000, preferably 1500.
  • the dialysis solvent is one or more of DMSO, halogenated hydrocarbon, tetrahydrofuran and ultrapure water, preferably one or both of DMSO and ultrapure water, more preferably DMSO and ultrapure water.
  • the silica gel column chromatography elution mode is isocratic elution or gradient elution, preferably gradient elution.
  • the eluent used in the gradient elution is two or more of dichloromethane, ethyl acetate and anhydrous methanol, preferably dichloromethane and anhydrous methanol mixed solvent.
  • the ratio of dichloromethane to anhydrous methanol (v/v) is 100:1-10:1, preferably 50:1-20:1.
  • the present invention also discloses a nano-drug, which comprises the above-mentioned PEGylated rapamycin compound with effective drug loading, preferably, the above-mentioned PEGylated rapamycin compound with effective drug loading and free rapamycin element composition.
  • the particle size of the PEGylated rapamycin nanoparticles is 5-1000 nm, preferably 50-200 nm.
  • the drug loading of the PEGylated rapamycin nanoparticles is 15%-100%, preferably 25%-85%.
  • the present invention also discloses a method for preparing the above nanomedicine, which is prepared by a bottom-up method.
  • the emulsification/solvent evaporation method is to dissolve the PEGylated rapamycin compound, or the PEGylated rapamycin compound and rapamycin in an organic solvent to form an organic phase, and then Add it to the water phase containing polyvinyl alcohol, and form an oil-in-water emulsion by high-speed stirring, ultrasonication, vortex vibration and/or high-pressure homogenizer, and finally evaporate and evaporate the organic solvent to obtain PEGylated rapamycin nanometers. Granule solution.
  • the organic solvent used in the emulsification/solvent evaporation method is one or both of chloroform and CH 2 Cl 2 , preferably CH 2 Cl 2 .
  • the concentration of the PEGylated rapamycin in the organic phase of the emulsification/solvent evaporation method is 0.1-10 mg/mL, preferably 0.5-5 mg/mL.
  • the emulsification/solvent evaporation method also includes free rapamycin, and the ratio of the free rapamycin/the PEGylated rapamycin is 0-20/1 ( w/w), preferably 1/5-5/1 (w/w).
  • the ratio of the organic phase/the aqueous phase of the emulsification/solvent evaporation method is 1/1-1/100 (v/v), preferably 1/2-1 /10(v/v).
  • the concentration of the polyvinyl alcohol in the water phase of the emulsification/solvent evaporation method is 0-5% (w/v), preferably 0.5%-2% ( w/v).
  • the invention also discloses a freeze-dried preparation, which is prepared by freeze-drying the above-mentioned aqueous solution of PEGylated rapamycin nanoparticles and a freeze-drying protective agent.
  • the freeze-drying protective agent is one or more of sucrose, lactose, mannitol, glucose, trehalose, and maltose.
  • the concentration of the lyophilized protective agent in the aqueous solution of the PEGylated rapamycin nanoparticles is 0.1%-20% (w/v), preferably 2%- 8% (w/v).
  • the pre-freezing temperature used in preparing the freeze-dried preparation is ⁇ -10°C, preferably -30°C--50°C.
  • the pre-freezing methods are quick freezing and slow freezing, preferably quick freezing.
  • the present invention also discloses a pharmaceutical composition, which comprises a therapeutically effective dose of the above-mentioned PEGylated rapamycin compound and a pharmaceutically acceptable carrier.
  • biological drugs are also included.
  • the biological drug is one or more of urate oxidase, enzymes and coenzyme drugs, nucleic acid and its degradation products and derivatives, cell growth factors or cytokines, Uric acid oxidase is preferred.
  • the present invention also discloses the use of the above-mentioned PEGylated rapamycin compound, the above-mentioned nanomedicine, the above-mentioned freeze-dried preparation or the above-mentioned pharmaceutical composition in preparing a medicine for reducing immune response.
  • the PEGylated rapamycin of the present invention has clear structural composition, high purity, suitable hydrophobic rapamycin end and hydrophilic PEG chain end, and is a good carrier for preparing nanoparticles.
  • the PEGylated rapamycin nanoparticle of the present invention is composed of PEGylated rapamycin and rapamycin, and takes the PEGylated rapamycin as a carrier, and the rapamycin part in the PEGylated rapamycin molecule It forms the hydrophobic core of the nanoparticle with the free rapamycin molecule, and the surface of the nanoparticle has a hydrophilic PEG segment, which has high dispersion stability in water.
  • the PEGylated rapamycin nanoparticles of the present invention have immune targeting properties, which can enrich and release drugs in immune organs such as the spleen, effectively inhibit the generation of anti-drug antibodies of biological drugs, and generate immune tolerance; the present invention
  • the high content of rapamycin in PEGylated rapamycin nanoparticles has a better effect on inhibiting the generation of anti-drug antibodies than rapamycin PLGA nanoparticles, and also avoids long-term multi-dose administration of rapamycin bands Toxic and side effects, does not contain PLGA and other pharmaceutical excipients.
  • the hydrophilic polyethylene glycol is linked to the hydrophobic rapamycin through esterification reaction to obtain the amphiphilic PEGylated rapamycin, and then the PEGylated rapamycin alone or PEGylated rapamycin is Together with rapamycin, PEGylated rapamycin nanoparticles with good water solubility can be prepared, which can be effectively used to inhibit the generation of anti-drug antibodies of biological drugs such as uric acid oxidase, etc., and has a good clinical application prospect.
  • Figure 1 shows the 1 H NMR and 13 C NMR spectra of mPEG-COOH (CDCl 3 is the solvent), wherein Figure 1(a) is the entire 1 H NMR spectrum, and Figure 1(b) is the 1 H NMR spectrum The assignment of each peak on the spectrum of , Figure 1(c) is the full picture of the 13 C NMR spectrum, Figure 1(d) is a partial enlarged view of the 13 C NMR spectrum, and Figure 1(e) is the 13 C NMR spectrum. The assignment of each peak on the spectrum (CDCl 3 is the solvent);
  • Fig. 2 is the 1 H NMR and 13 C NMR spectra of PEGylated rapamycin (CDCl 3 is the solvent), wherein Fig. 2(a) is the whole picture of the 1 H NMR spectrum, Fig. 2(b)-Fig. 2 (c) is a partial enlarged view of the 1 H NMR spectrum, FIG. 2(d) is the full image of the 13 C NMR spectrum, and FIGS. 2(e)-2(h) are the partially enlarged views of the 13 C NMR spectrum;
  • Fig. 3 is the structural formula (I) of the PEGylated rapamycin compound of the embodiment of the present invention.
  • the raw materials, reagents, instruments, etc. used in the present invention can be purchased through commercial channels.
  • the abbreviation terms involved are as follows, EDC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP: dimethylaminopyridine; DMSO: dimethylsulfoxide Sulfone; PVA: Polyvinyl Alcohol; DLS: Dynamic Laser Light Dispersion.
  • mPEG-COOH (referred to as PEG, with an average molecular weight of about 2000, 0.2 g, 0.1 mmol) was dissolved in CH 2 Cl 2 (8 mL), RAPA (0.1 g, 0.1 mmol), EDC.HCl (0.04 g, 0.2 mmol) were added.
  • mPEG-COOH PEG for short, average molecular weight is about 2000, 0.3007g, 0.15mmol
  • CH 2 Cl 2 50mL
  • RAPA 0.2722g, 0.30mmol
  • EDC.HCl 0.0289g, 0.15mmol
  • DMAP 0.0020 g, 0.15 mmol
  • mPEG-COOH (PEG for short, average molecular weight is about 2000, 1.8027g, 0.90mmol) was dissolved in CH2Cl2 ( 20mL), RAPA (0.2758g, 0.30mmol), EDC.HCl (0.1727g, 0.90mmol) were added ) and DMAP (0.0114 g, 0.90 mmol), shake/stir for complete dissolution, stir at room temperature for 24 hours in the dark, and concentrate the reaction solution to obtain a PEGylated rapamycin-containing concentrated solution with a crude yield of 59.1%.
  • mPEG-COOH (PEG for short, average molecular weight is about 2000, 1.8011 g, 0.90 mmol) was dissolved in CH 2 Cl 2 (10 mL), RAPA (0.2754 g, 0.30 mmol), EDC.HCl (0.1720 g, 0.90 mmol) were added ) and DMAP (0.0105g, 0.09mmol), shake/stir to dissolve completely, stir at room temperature in the dark for 18 hours, and concentrate the reaction solution to obtain a concentrated solution containing PEGylated rapamycin with a crude yield of 62.1%.
  • the PEGylated rapamycin-containing concentrate was purified by column chromatography, see Examples 6-8 below.
  • the ratio of each component is PEG-RAPA: RAPA: PEGylated by-product of rapamycin is 52:15:9 (w/w), using silica gel column chromatography, The eluent is dichloromethane/anhydrous methanol (50:1/30:1-20:1), elution time 25min/40min/120min, PEG-RAPA yield 85.3%, PEG-RAPA purity 95.9%, RAPA The removal rate was 93.2%, and the removal rate of rapamycin PEGylated by-products was 90.0%.
  • the ratio of each component is PEG-RAPA:RAPA:rapamycin PEGylated by-product is 48:13:12 (w/w), using silica gel column chromatography, The eluent is dichloromethane/anhydrous methanol (50:1/30:1/10:1), elution time 25min/50min/25min, PEG-RAPA yield 80.8%, PEG-RAPA purity 95.1%, RAPA The removal rate was 92.8%, and the removal rate of PEGylated by-products of rapamycin was 90.7%.
  • the ratio of each component is PEG-RAPA:RAPA:rapamycin PEGylated by-product is 41:11:16 (w/w), use silica gel column chromatography, wash
  • the removal agent is dichloromethane/anhydrous methanol (50:1/40:1/30:1), the elution time is 25min/70min/200min, the yield of PEG-RAPA is 63.1%, the purity of PEG-RAPA is 98.5%, and RAPA is removed.
  • the removal rate of rapamycin PEGylated by-products was 96.4%, and the removal rate was 100.0%.
  • the present invention adopts mPEG-COOH with the structure shown in the following formula (II) and the hydroxyl group on the 40th carbon atom on the rapamycin molecule to obtain the PEGylated rapamycin with the structure shown in the following formula (I) through esterification reaction
  • the results of structural characterization are shown in Figures 1 and 2.
  • the 1 H NMR spectrum of mPEG-COOH corresponds to the hydrogen atom (a) on the methylene group in the -CH 2 -COOH structure in the mPEG-COOH molecule for calibration (peak area calibration is 2.00) , the area of the peak corresponding to the hydrogen atom (b) in the repeating structural unit of the mPEG-COOH molecule is 183.86, the degree of polymerization of mPEG-COOH is calculated to be 46, and the molecular weight of mPEG-COOH is 2114.
  • PEGylated rapamycin nanoparticle solutions were determined by DLS method.
  • the particle size and PDI of PEGylated rapamycin nanoparticle solution were determined by DLS method.
  • the particle size and PDI of PEGylated rapamycin nanoparticle solution were determined by DLS method.
  • the PEGylated rapamycin nanoparticle solution prepared according to the method of Example 10-12 above was centrifuged at 12,000 r/min for 45 minutes, the supernatant was discarded, the PVA was washed away, and the precipitate was resuspended in ultrapure water to obtain a concentrated nanoparticle aqueous solution. 2 mL of the nanoparticle aqueous solution was respectively taken into a 10 mL vial, and a freeze-drying protective agent (mass fraction 5%) was added for freeze-drying.
  • the mass fraction of lyoprotectant was 5%. Take 0.1 g of the lyophilized protective agent and put it in a 10 mL vial, dissolve it with 2 mL of PEGylated rapamycin nanoparticle aqueous solution, and then freeze-dry it. The appearance, reconstitution speed and clarity of the prepared nanoparticle freeze-dried powder for injection were observed, and the particle size and PDI of the nanoparticles after reconstitution were determined.
  • Table 4 adopts different lyophilized protective agents to prepare PEGylated rapamycin nanoparticle lyophilized powder for injection
  • the PEGylated rapamycin nanoparticle freeze-dried powder injection was prepared when the pre-freezing temperature was -35°C and -45°C, respectively. The appearance, reconstitution speed and clarity of the prepared nanoparticle freeze-dried powder for injection were observed, and the particle size and PDI of the nanoparticles after reconstitution were determined.
  • the freeze-dried powder injection of PEGylated rapamycin nanoparticles was prepared by quick freezing method and slow freezing method respectively.
  • the appearance, reconstitution speed and clarity of the prepared nanoparticle freeze-dried powder for injection were observed, and the particle size and PDI of the nanoparticles after reconstitution were determined.
  • the PEGylated rapamycin nanoparticles (nanoparticle size 160.4nm, rapamycin content: 663.8 ⁇ g/bottle) prepared by the present invention; rapamycin PLGA nanoparticles (nanoparticle size 170.5nm, rapamycin 294.8 ⁇ g/bottle); Recombinant Candida urate oxidase (content: 0.72mg/mL*7mL/bottle), Shenyang R&D Center of Shenyang Sunshine Pharmaceutical Co., Ltd.
  • mice 45 mice were randomly divided into 3 groups according to body weight, 15 mice in each group. The groups and doses are shown in Table 7.
  • Group 1, group 2, and group 3 were administered twice a week for 4 consecutive weeks (according to the test results of anti-urate oxidase antibody, the administration period may be extended), and each group was administered intravenously to mice.
  • Urate oxidase was mixed with nanoparticles before administration in groups 2 and 3 before administration.
  • mice whole blood was collected in a non-anticoagulant tube, and the serum was separated and frozen for detection of anti-urate oxidase antibodies.
  • mice in each group were administered by tail vein injection (the gray background was the intraperitoneal injection when the tail vein could not be administered, 1 to 2 times for each mouse) for 4 weeks (8 times), group 2 (PEGylated rapamycin polymerization The titer of anti-urate oxidase antibody was the smallest, and group 3 (PLGA-loaded rapamycin nanoparticles + urate oxidase) was not significantly better than control group 1 (urate oxidase control group).
  • mice in each group were administered by tail vein injection (the gray background was the intraperitoneal injection when the tail vein could not be administered, each no more than 5 times) for 6 weeks (12 times), and the results were basically the same as the administration for 4 weeks, and the group 3 There were 3 mice with increased antibody titers.
  • the combination of urate oxidase protein and PEGylated rapamycin nanoparticles can effectively prevent the production of anti-urate oxidase antibodies in mice, and the effect is significantly better than the combination of urate oxidase protein and rapamycin PLGA nanoparticles.

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Abstract

一种PEG化雷帕霉素化合物及其制备方法与应用，PEG化雷帕霉素化合物如式(I)所示，其中，n为10-150。制备方法为将mPEG-COOH溶于有机溶剂中，加入催化剂EDC•HCl、DMAP以及RAPA，在10-40℃避光下搅拌反应。所述PEG化雷帕霉素化合物及其药物制剂能用于减少免疫应答的药物，可有效抑制生物药物如尿酸氧化酶等的抗药抗体生成，适合制备成纳米药物。

Description

一种PEG化雷帕霉素化合物及其制备方法与应用 技术领域
本发明属于生物医药技术领域，涉及一种PEG化雷帕霉素化合物及其制备方法与应用。
背景技术
2018年全球最畅销与销售额增长最大的TOP10药品中有8种为单克隆抗体。全球生物医药产业快速增长，但与小分子化药相比，大多数生物药物都具有免疫原性，进入机体后会引起免疫应答，产生抗药抗体，从而中和或改变生物药物的药物动力学和生物分布，更严重地可能诱导机体出现超敏反应等危及生命的毒副作用。因此，抑制生物药物治疗时抗药抗体的生成对提高生物药物的安全性和有效性非常关键。
免疫抑制剂是指对机体的免疫反应具有抑制作用的药物，能抑制与免疫反应有关细胞(T细胞和B细胞等巨噬细胞)的增殖和功能，从而能降低抗体免疫反应。免疫抑制剂主要用于器官移植抗排斥反应和自身免疫病如类风湿性关节炎、红斑狼疮、皮肤真菌病、膜肾球肾炎、炎性肠病和自身免疫性溶血贫血等。
雷帕霉素(rapamycin，RAPA)，又称西罗莫司，是吸水链霉菌所产生的一种亲脂性三烯含氮大环内酯抗生素类免疫抑制剂，可用于治疗癌症、抗移植物排斥反应及其它免疫性疾病的辅助治疗。雷帕霉素可通过阻断哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)参与的信号转导，阻止T细胞分化和树突状细胞成熟，从而发挥免疫抑制的作用。
Takashi K.Kishimoto首次报道负载免疫抑制剂-雷帕霉素的聚乳酸-羟基乙酸(poly(lactic acid-co-glycolic acid)，PLGA)纳米粒具有免疫耐受性，可有效抑制生物药物的抗药抗体的生成(Nature nanotechnology，2016，11(10):890-899)，并将其定义为免疫耐受性纳米粒。上述文献报道将雷帕霉素包埋到 PLGA载体上制备的雷帕霉素PLGA纳米粒具有诱导免疫耐受、抑制生物药物的抗药抗体生成的功能。PLGA为人工合成的高分子量化合物，其本身不具有药理作用，为一种药用辅料产品。
发明内容
本发明的第一目的是提供了一种PEG化雷帕霉素化合物，该化合物具有适宜的疏水雷帕霉素端和亲水PEG链端，是制备纳米粒的良好载体。
本发明的第二目的是提供了一种制备上述化合物的方法，反应条件温和、操作简单。
本发明的第三目的是提供了一种上述化合物的应用，将其制备成纳米药物、冻干制剂、药物组合物以及在制备用于减少免疫应答的药物中的用途。
一种如式(I)所示的PEG化雷帕霉素化合物，其中，n为10-150。
Figure PCTCN2022083907-appb-000001
本发明还公开了一种上述的PEG化雷帕霉素化合物的制备方法，将mPEG-COOH溶于有机溶剂中，加入催化剂EDC·HCl和MAP，以及RAPA，在10～40℃避光下搅拌反应。
在本发明的一实施例中，该制备方法的反应温度优选为20～30℃。
在本发明的一实施例中，所述的有机溶剂为二氯甲烷或氯仿中的一种或 两种，优选为二氯甲烷。
在本发明的一实施例中，所述的mPEG-COOH与RAPA的摩尔比为5:1～1:5，优选为3:1-1:3。
在本发明的一实施例中，还包括反应后的分离纯化步骤，所述的分离纯化步骤为透析纯化法或硅胶柱层析法。
在本发明的一实施例中，所述的透析纯化法使用透析袋和透析溶剂进行分离纯化。其中，所述的透析袋的截留分子量为500-5000，优选为1500。所述的透析溶剂为DMSO、卤代烃、四氢呋喃、超纯水中的一种或多种，优选为DMSO、超纯水中的一种或两种，更优选为DMSO和超纯水。
在本发明的一实施例中，所述的硅胶柱层析法洗脱方式为等度洗脱或梯度洗脱，优选为梯度洗脱。
在本发明的一实施例中，所述的梯度洗脱所使用的洗脱剂为二氯甲烷、乙酸乙酯、无水甲醇中的两种或多种，优选为二氯甲烷和无水甲醇的混合溶剂。其中，二氯甲烷与无水甲醇(v/v)的比为100:1-10:1，优选为50:1-20:1。
本发明还公开了一种纳米药物，其包括有效载药量的上述的PEG化雷帕霉素化合物，优选为，由有效载药量的上述的PEG化雷帕霉素化合物与游离雷帕霉素组成。
在本发明的一实施例中，所述的PEG化雷帕霉素纳米粒的粒径为5-1000nm，优选为50-200nm。
在本发明的一实施例中，所述的PEG化雷帕霉素纳米粒的载药量为15％-100％，优选为25％-85％。
本发明还公开了一种上述的纳米药物的制备方法，采用自下而上的方法制得，所述的PEG化雷帕霉素纳米粒的制备方法包括乳化/溶剂挥发法、纳米沉淀法、薄膜分散法、自组装法或SPG膜法乳化，优选为乳化/溶剂挥发法。
在本发明的一实施例中，所述的乳化/溶剂挥发法为将PEG化雷帕霉素化合物，或PEG化雷帕霉素化合物和雷帕霉素溶解在有机溶剂中形成有机相，然后加入到含有聚乙烯醇的水相中，通过高速搅拌、超声、漩涡振荡和/或用 高压匀质机制成水包油乳状液，最后经蒸发、挥干有机溶剂得到PEG化雷帕霉素纳米粒溶液。
在本发明的一实施例中，该乳化/溶剂挥发法所用的有机溶剂为氯仿、CH 2Cl 2中的一种或两种，优选为CH 2Cl 2。
在本发明的一实施例中，该乳化/溶剂挥发法的所述的PEG化雷帕霉素在所述的有机相中的浓度为0.1-10mg/mL，优选为0.5-5mg/mL。
在本发明的一实施例中，该乳化/溶剂挥发法还包括游离雷帕霉素，所述的游离雷帕霉素/所述的PEG化雷帕霉素的比例为0-20/1(w/w)，优选为1/5-5/1(w/w)。
在本发明的一实施例中，该乳化/溶剂挥发法的所述的有机相/所述的水相的比例为1/1-1/100(v/v)，优选为1/2-1/10(v/v)。
在本发明的一实施例中，该乳化/溶剂挥发法的所述的聚乙烯醇在所述的水相中的浓度为0-5％(w/v)，优选为0.5％-2％(w/v)。
本发明还公开一种冻干制剂，其由上述的PEG化雷帕霉素纳米粒的水溶液和冻干保护剂冻干制得。
在本发明的一实施例中，所述的冻干保护剂为蔗糖、乳糖、甘露醇、葡萄糖、海藻糖、麦芽糖中的一种或多种。
在本发明的一实施例中，所述的冻干保护剂在所述的PEG化雷帕霉素纳米粒的水溶液中的浓度为0.1％-20％(w/v)，优选为2％-8％(w/v)。
在本发明的一实施例中，在制备冻干制剂所用的预冻温度为<-10℃，优选为-30℃--50℃。预冻方式为快冻和慢冻，优选为快冻。
本发明还公开了一种药物组合物，其包括治疗有效剂量的上述的PEG化雷帕霉素化合物及可药用的载体。
在本发明的一实施例中，还包括生物药物。
在本发明的一实施例中，所述的生物药物为尿酸氧化酶、酶和辅酶类药物、核酸及其降解物和衍生物类药物、细胞生长因子或细胞因子中的一种或多种，优选为尿酸氧化酶。
本发明还公开了上述的PEG化雷帕霉素化合物，上述的纳米药物，上述的冻干制剂或上述的药物组合物在制备用于减少免疫应答的药物中的用途。
与现有技术相比，本发明的有益效果如下：
1.本发明的PEG化雷帕霉素结构组成明确，纯度高，具有适宜的疏水雷帕霉素端和亲水PEG链端，是制备纳米粒良好载体。
2.本发明的PEG化雷帕霉素纳米粒由PEG化雷帕霉素和雷帕霉素组成，以PEG化雷帕霉素为载体，PEG化雷帕霉素分子中雷帕霉素部分与游离雷帕霉素分子构成纳米粒疏水的核，纳米粒表面有亲水PEG链段，在水中分散稳定性高。
3.本发明的PEG化雷帕霉素纳米粒具有免疫靶向性，可在免疫器官如脾脏富集并缓释药物，有效抑制生物药的抗药抗体生成，产生免疫耐受性；本发明PEG化雷帕霉素纳米粒中雷帕霉素的含量高，具有比雷帕霉素PLGA纳米粒更好地抑制抗药抗体生成的功效，同时也避免长期多剂量给药雷帕霉素带来的毒副作用，不含有PLGA等药用辅料。
本发明将亲水性聚乙二醇通过酯化反应连接到疏水性雷帕霉素上制得两亲性PEG化雷帕霉素，然后将PEG化雷帕霉素单独或PEG化雷帕霉素与雷帕霉素一起制成具有良好水溶性的PEG化雷帕霉素纳米粒，能够有效用于抑制生物药如尿酸氧化酶等的抗药抗体生成，具有良好的临床应用前景。
附图说明
图1为mPEG-COOH的的 1H NMR和 13C NMR谱图(CDCl 3为溶剂)，其中，图1(a)为 1H NMR图谱的全图，图1(b)为 1H NMR图谱的谱图上各峰的归属，图1(c)为 13C NMR谱图的全图，图1(d)为 13C NMR谱图的局部放大图，图1(e)为 13C NMR图谱的谱图(CDCl 3为溶剂)上各峰的归属；
图2为PEG化雷帕霉素的 1H NMR和 13C NMR谱图(CDCl 3为溶剂)，其中，图2(a)为 1H NMR图谱的全图，图2(b)-图2(c)为 1H NMR图 谱的局部放大图，图2(d)为 13C NMR谱图的全图，图2(e)-图2(h)为 13C NMR谱图的局部放大图；
图3为本发明实施例的PEG化雷帕霉素化合物的结构式(I)。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应该理解，这些实施例仅用于说明本发明，而不用于限定本发明的保护范围。在实际应用中本领域技术人员根据本发明做出的改进和调整，仍属于本发明的保护范围。
本发明所用的原料、试剂、仪器等均可通过市售渠道购买。其中，涉及的缩写术语如下，EDC.HCl：1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐；DMAP：二甲基氨基吡啶；DMSO：二甲基亚砜；PVA：聚乙烯醇；DLS：动态激光光散色法。
一、PEG化雷帕霉素的制备与纯化
实施例1
将mPEG-COOH(简称PEG，平均分子量约为2000，0.2g，0.1mmol)溶于CH 2Cl 2(8mL)中，加入RAPA(0.1g，0.1mmol)，EDC.HCl(0.04g，0.2mmol)和DMAP(0.024g，0.2mmol)，振荡/搅拌使完全溶解，室温避光反应36小时，将反应液浓缩得浓缩液，浓缩液用DMSO(2mL)溶解，将所得溶液置于透析袋(截留分子量:1500Da)中，先后用DMSO、超纯水分别透析1d和2d，每4h换一次透析介质，透析液冷冻干燥得到0.074g PEG化雷帕霉素，收率23.2％。
实施例2
将mPEG-COOH(简称PEG，平均分子量约为2000，0.3007g，0.15mmol)溶于CH 2Cl 2(50mL)中，加入RAPA(0.2722g，0.30mmol)，EDC.HCl(0.0289g，0.15mmol)和DMAP(0.0020g，0.15mmol)，振荡/搅拌使完全溶解，室温避光搅拌反应8小时，将反应液浓缩得到含PEG化雷帕霉素浓缩液，粗产率12.1％。
实施例3
将mPEG-COOH(简称PEG，平均分子量约为2000，1.196g，0.60mmol)溶于CH 2Cl 2(50mL)中，加入RAPA(0.2754g，0.30mmol)，EDC.HCl(0.1157g，0.60mmol)和DMAP(0.0070g，0.06mmol)，振荡/搅拌使完全溶解，室温避光搅拌反应4小时，将反应液浓缩得到含PEG化雷帕霉素浓缩液，粗产率39.8％。
实施例4
将mPEG-COOH(简称PEG，平均分子量约为2000，1.8027g，0.90mmol)溶于CH 2Cl 2(20mL)中，加入RAPA(0.2758g，0.30mmol)，EDC.HCl(0.1727g，0.90mmol)和DMAP(0.0114g，0.90mmol)，振荡/搅拌使完全溶解，室温避光搅拌反应24小时，将反应液浓缩得到含PEG化雷帕霉素浓缩液，粗产率59.1％。
实施例5
将mPEG-COOH(简称PEG，平均分子量约为2000，1.8011g，0.90mmol)溶于CH 2Cl 2(10mL)中，加入RAPA(0.2754g，0.30mmol)，EDC.HCl(0.1720g，0.90mmol)和DMAP(0.0105g，0.09mmol)，振荡/搅拌使完全溶解，室温避光搅拌反应18小时，将反应液浓缩得到含PEG化雷帕霉素浓缩液，粗产率62.1％。使用柱层析法纯化含PEG化雷帕霉素浓缩液，见下述实施例6～8。
实施例6
在含PEG化雷帕霉素的浓缩液中，各组分比例为PEG-RAPA:RAPA:雷帕霉素PEG化副产物为52:15:9(w/w)，采用硅胶柱层析，洗脱剂为二氯甲烷/无水甲醇(50:1/30:1-20:1)，洗脱时间25min/40min/120min，PEG-RAPA产率85.3％，PEG-RAPA纯度95.9％，RAPA去除率93.2％，雷帕霉素PEG化副产物去除率90.0％。
实施例7
在含PEG化雷帕霉素的浓缩液中，各组分比例为PEG-RAPA:RAPA:雷帕 霉素PEG化副产物为48:13:12(w/w)，采用硅胶柱层析，洗脱剂为二氯甲烷/无水甲醇(50:1/30:1/10:1)，洗脱时间25min/50min/25min，PEG-RAPA产率80.8％，PEG-RAPA纯度95.1％，RAPA去除率92.8％，雷帕霉素PEG化副产物去除率90.7％。
实施例8
在PEG化雷帕霉素的浓缩液中，各组分比例为PEG-RAPA:RAPA:雷帕霉素PEG化副产物为41:11:16(w/w)，采用硅胶柱层析，洗脱剂为二氯甲烷/无水甲醇(50:1/40:1/30:1)，洗脱时间25min/70min/200min，PEG-RAPA产率63.1％，PEG-RAPA纯度98.5％，RAPA去除率96.4％，雷帕霉素PEG化副产物去除率100.0％。
二、PEG化雷帕霉素的结构表征
实施例9
本发明采用具有如下式(II)所示结构的mPEG-COOH和雷帕霉素分子上第40位碳原子上的羟基通过酯化反应得到如下式(I)所示结构的PEG化雷帕霉素，结构表征结果如图1和图2所示。
图1中，以mPEG-COOH的 1H NMR谱图中对应mPEG-COOH分子中-CH 2-COOH结构中的亚甲基上的氢原子(a)进行定标(峰面积定标为2.00)，mPEG-COOH分子的重复结构单元中氢原子(b)对应的峰的面积为183.86，计算得到mPEG-COOH的聚合度为46，mPEG-COOH分子量为2114。
图2中，结合mPEG-COOH，雷帕霉素和PEG化雷帕霉素的核磁共振谱，发现雷帕霉素10-Cδ97.47，未发生较大改变；28-Cδ75.70，未发生较大改变；40-Cδ79.31，发生较大改变，说明PEG化雷帕霉素为雷帕霉素分子中与40-C相连的OH和mPEG-COOH发生酯化产物。
PEG化雷帕霉素的分子结构，如图3所示：
Figure PCTCN2022083907-appb-000002
mPEG-COOH的结构简式：
Figure PCTCN2022083907-appb-000003
二、PEG化雷帕霉素纳米粒溶液的制备
实施例10
称取20mg PEG-RAPA和2mg RAPA，加入5mLCH 2Cl 2使其充分溶解得有机相，取50mL含0.5％PVA的水溶液为水相，采用探头超声，在冰浴条件下，边超声边用注射器将有机相滴入水相中，超声乳化10min得白色乳剂。40℃条件下采用旋转蒸发仪除去有机溶剂，4000r/min离心5min以去除未包封的药物和粒径较大的粒子，取上清液，即得PEG化雷帕霉素纳米粒溶液。
采用DLS法测定PEG化雷帕霉素纳米粒溶液的粒径和多分散指数(PDI，polydispersity index)。
取100μL PEG化雷帕霉素纳米粒溶液，加入乙腈至1mL，超声20min 破坏纳米粒结构，使雷帕霉素游离释放到溶液中，12000r/min离心10min，取上清液，0.22μm微孔滤膜过滤后，采用HPLC分析测定RAPA浓度，测定PEG化雷帕霉素纳米粒的包封率和载药量。
检测结果如表1所示。
表1 PEG化雷帕霉素纳米粒的检测结果
Figure PCTCN2022083907-appb-000004
实施例11
称取20mg PEG-RAPA和4mg RAPA，加入20mLCH 2Cl 2使其充分溶解得有机相，取40mL含1％PVA的水溶液为水相，采用探头超声，在冰浴条件下，边超声边用注射器将有机相滴入水相中，超声乳化20min得白色乳剂。40℃条件下采用旋转蒸发仪除去有机溶剂，4000r/min离心5min以去除未包封的药物和粒径较大的粒子，取上清液，即得PEG化雷帕霉素纳米粒溶液。
采用DLS法测定PEG化雷帕霉素纳米粒溶液的粒径和PDI。
取100μL PEG化雷帕霉素纳米粒溶液，加入乙腈至1mL，超声20min破坏纳米粒结构，使雷帕霉素游离释放到溶液中，12000r/min离心10min，取上清液，0.22μm微孔滤膜过滤后，采用HPLC分析测定RAPA浓度，测定PEG化雷帕霉素纳米粒的包封率和载药量。
检测结果如表2所示。
表2 PEG化雷帕霉素纳米粒的检测结果
Figure PCTCN2022083907-appb-000005
实施例12
称取20mg PEG-RAPA和8mg RAPA，加入40mLCH 2Cl 2使其充分溶解得有机相，取120mL含2％PVA的水溶液为水相，采用探头超声，在冰浴条件下，边超声边用注射器将有机相滴入水相中，超声乳化30min得白色乳剂。40℃条件下采用旋转蒸发仪除去有机溶剂，4000r/min离心5min以去除未包 封的药物和粒径较大的粒子，取上清液，即得PEG化雷帕霉素纳米粒溶液。
采用DLS法测定PEG化雷帕霉素纳米粒溶液的粒径和PDI。
取100μL PEG化雷帕霉素纳米粒溶液，加入乙腈至1mL，超声20min破坏纳米粒结构，使雷帕霉素游离释放到溶液中，12000r/min离心10min，取上清液，0.22μm微孔滤膜过滤后，采用HPLC分析测定RAPA浓度，测定PEG化雷帕霉素纳米粒的包封率和载药量。
检测结果如表3所示。
表3 PEG化雷帕霉素纳米粒的检测结果
Figure PCTCN2022083907-appb-000006
三、PEG化雷帕霉素纳米粒冻干粉针剂的制备
实施例13
将按照上述实施例10-12方法制备的PEG化雷帕霉素纳米粒溶液12000r/min离心45min，弃上清，洗去PVA，沉淀用超纯水重悬后，得浓缩纳米粒水溶液。分别取2mL纳米粒水溶液于10mL西林瓶中，加入冻干保护剂(质量分数5％)，进行冻干。
(1)采用不同冻干保护剂制备PEG化雷帕霉素纳米粒冻干粉针剂
冻干保护剂质量分数为5％。取冻干保护剂0.1g置于10mL西林瓶中，分别用2mL PEG化雷帕霉素纳米粒水溶液溶解后，进行冻干。观察所制备纳米粒冻干粉针剂的外观、复溶速度、澄明度，测定复溶后纳米粒粒径和PDI。
采用不同冻干保护剂制备PEG化雷帕霉素纳米粒冻干粉针剂质量评价如表4所示。
表4采用不同冻干保护剂制备PEG化雷帕霉素纳米粒冻干粉针剂
Figure PCTCN2022083907-appb-000007
注：1.外观+萎缩、塌陷严重，不能完全脱落；++部分萎缩、塌陷，稍有黏壁；+++没有萎缩、塌陷，可整块脱落。2.复溶速度+需要超声1min完全复溶，++需要超声30s完全复溶，+++立即复溶。3.澄明度+乳光性较差，浊度明显；++有一定乳光，但稍有浊度感；+++乳光明显，无浑浊现象
(2)采用不同预冻温度制备PEG化雷帕霉素纳米粒冻干粉针剂
分别在预冻温度为-35℃和-45℃时制备PEG化雷帕霉素纳米粒冻干粉针剂。观察所制备纳米粒冻干粉针剂的外观、复溶速度、澄明度，测定复溶后纳米粒粒径和PDI等。
采用不同预冻温度制备PEG化雷帕霉素纳米粒冻干粉针剂质量评价如表5所示。
表5不同预冻温度制备PEG化雷帕霉素纳米粒冻干粉针剂
Figure PCTCN2022083907-appb-000008
注：1.外观+萎缩、塌陷严重，不能完全脱落；++部分萎缩、塌陷，稍有黏壁；+++没有萎缩、塌陷，可整块脱落。2.复溶速度+需要超声1min完全复溶，++需要超声30s完全复溶，+++立即复溶。3.澄明度+乳光性较差，浊度明显；++有一定乳光，但稍有浊度感；+++乳光明显，无浑浊现象
(3)采用不同预冻方式制备PEG化雷帕霉素纳米粒冻干粉针剂
分别采用快冻法和慢冻法制备PEG化雷帕霉素纳米粒冻干粉针剂。观察所制备纳米粒冻干粉针剂的外观、复溶速度、澄明度，测定复溶后纳米粒粒径和PDI等。
采用不同预冻方式制备PEG化雷帕霉素纳米粒冻干粉针剂质量评价如表6所示。
表6不同预冻方式制备PEG化雷帕霉素纳米粒冻干粉针剂
Figure PCTCN2022083907-appb-000009
注：1.外观+萎缩、塌陷严重，不能完全脱落；++部分萎缩、塌陷，稍有黏壁；+++没有萎缩、塌陷，可整块脱落。2.复溶速度+需要超声1min完全复溶，++需要超声30s完全复溶，+++立即复溶。3.澄明度+乳光性较差，浊度明显；++有一定乳光，但稍有浊度感；+++乳光明显，无浑浊现象
四、两种雷帕霉素纳米粒与尿酸氧化酶联合给药对降低小鼠体内抗尿酸氧化酶抗体效果的试验
实施例14
本发明制备的PEG化雷帕霉素纳米粒(纳米粒粒径160.4nm，雷帕霉素含量：663.8μg/瓶)；雷帕霉素PLGA纳米粒(纳米粒粒径170.5nm,雷帕霉素含量：294.8μg/瓶)；重组假丝酵母尿酸氧化酶(含量：0.72mg/mL*7mL/瓶)，沈阳三生制药有限责任公司沈阳研发中心。
将45只小鼠，按体重随机分为3组，每组15只。分组及给药剂量见表7。
表7药效试验剂量表
Figure PCTCN2022083907-appb-000010
组1、组2、组3为每周给药2次，连续给药4周(根据抗尿酸氧化酶抗体检测结果，可能延长给药周期)，各组均为小鼠静脉注射给药。组2和组3给药前将尿酸氧化酶与纳米粒混合后给药。
最后一次给药之后第5天左右取小鼠全血0.5mL于非抗凝管内，分离血清后冻存检测抗尿酸氧化酶抗体。
抗尿酸氧化酶抗体检测结果如表8、表9所示：
表8给药4周抗尿酸氧化酶抗体滴度
Figure PCTCN2022083907-appb-000011
各组小鼠尾静脉注射给药(灰色背景为尾静脉无法给药时改为腹腔注射给药，每只1～2次)4周(8次)，组2(PEG化雷帕霉素聚合物纳米粒+尿酸氧化酶)抗尿酸氧化酶抗体滴度最小，组3(PLGA载雷帕霉素纳米粒+尿酸氧化酶)未见明显优于对照组1(尿酸氧化酶对照组)。
表9给药6周抗尿酸氧化酶抗体滴度
Figure PCTCN2022083907-appb-000012
各组小鼠尾静脉注射给药(灰色背景为尾静脉无法给药时改为腹腔注射给药，每只不超过5次)6周(12次)，结果基本同给药4周，且组3有3只小鼠抗体滴度增加。
尿酸氧化酶蛋白与PEG化雷帕霉素纳米粒联合用药能有效阻止小鼠体内 抗尿酸氧化酶抗体的产生，且效果明显优于尿酸氧化酶蛋白与雷帕霉素PLGA纳米粒联合用药。
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节，也不限制该发明仅为所述的具体实施方式。显然，根据本说明书的内容，可作很多的修改和变化。本说明书选取并具体描述这些实施例，是为了更好地解释本发明的原理和实际应用，从而使所属技术领域技术人员能很好地理解和利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。

Claims (16)

  1. 一种如式(I)所示的PEG化雷帕霉素化合物，其中，n为10-150。
    Figure PCTCN2022083907-appb-100001
  2. 一种如权利要求1所述的PEG化雷帕霉素化合物的制备方法，其特征在于，将mPEG-COOH溶于有机溶剂中，加入催化剂EDC·HCl和DMAP，以及RAPA，在10～40℃避光下搅拌反应。
  3. 根据权利要求2所述的PEG化雷帕霉素化合物的制备方法，其特征在于，所述的有机溶剂为二氯甲烷或氯仿中的一种或两种。
  4. 根据权利要求2所述的PEG化雷帕霉素化合物的制备方法，其特征在于，所述的mPEG-COOH与RAPA的摩尔比为5:1～1:5。
  5. 根据权利要求2所述的PEG化雷帕霉素化合物的制备方法，其特征在于，还包括反应后的分离纯化步骤，所述的分离纯化步骤为透析纯化法或硅胶柱层析法。
  6. 一种纳米药物，其包括如权利要求1所述的PEG化雷帕霉素化合物。
  7. 根据权利要求6所述的纳米药物，其特征在于，所述的PEG化雷帕霉素化合物的有效载药量为15％-100％。
  8. 根据权利要求6所述的纳米药物，其特征在于，还包括雷帕霉素。
  9. 一种如权利要求6～8任一项所述的纳米药物的制备方法，其特征在于，将PEG化雷帕霉素化合物，或PEG化雷帕霉素化合物和雷帕霉素溶解在有机溶剂中形成有机相，然后加入到含有聚乙烯醇的水相中，通过高速搅拌、超声、漩涡振荡和/或用高压匀质机制成水包油乳状液，最后经蒸发、挥干有机溶剂得到PEG化雷帕霉素纳米粒溶液。
  10. 根据权利要求9所述的纳米药物的制备方法，其特征在于，所述的PEG化雷帕霉素在所述的有机相中的浓度为0.1-10mg/mL；所述的聚乙烯醇在所述的水相中的浓度为0-5％(w/v)，所述的有机相/所述的水相的比例为1/1-1/100(v/v)。
  11. 一种冻干制剂，其特征在于，其由如权利要求9所述的PEG化雷帕霉素纳米粒的水溶液和冻干保护剂冻干制得。
  12. 根据权利要求11所述的冻干制剂，其特征在于，所述的冻干保护剂为蔗糖、乳糖、甘露醇、葡萄糖、海藻糖、麦芽糖中的一种或多种。
  13. 一种药物组合物，其包括治疗有效剂量的如权利要求1所述的PEG化雷帕霉素化合物及可药用的载体。
  14. 根据权利要求13所述的药物组合物，其特征在于，还包括生物药物。
  15. 根据权利要求14所述的药物组合物，其特征在于，所述的生物药物为尿酸氧化酶、酶和辅酶类药物、核酸及其降解物和衍生物类药物、细胞生长因子或细胞因子中的一种或多种。
  16. 权利要求1所述的PEG化雷帕霉素化合物，权利要求6～8任一项所述的纳米药物，权利要求11所述的冻干制剂或权利要求13～15任一项所述的药物组合物在制备用于减少免疫应答的药物中的用途。
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