WO2023160631A1 - 用于关节镇痛的纳米制剂及其制备方法和应用 - Google Patents

用于关节镇痛的纳米制剂及其制备方法和应用 Download PDF

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WO2023160631A1
WO2023160631A1 PCT/CN2023/077964 CN2023077964W WO2023160631A1 WO 2023160631 A1 WO2023160631 A1 WO 2023160631A1 CN 2023077964 W CN2023077964 W CN 2023077964W WO 2023160631 A1 WO2023160631 A1 WO 2023160631A1
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nano
preparation
castor oil
polyoxyethylene
hel
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French (fr)
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雷光华
邓彩凤
曾超
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Xiangya Hospital of Central South University
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Xiangya Hospital of Central South University
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Priority to JP2024549556A priority Critical patent/JP2025506762A/ja
Priority to US18/841,369 priority patent/US20250170069A1/en
Priority to EP23759253.0A priority patent/EP4470532A4/en
Publication of WO2023160631A1 publication Critical patent/WO2023160631A1/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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • 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
    • A61K9/1277—Preparation processes; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biomedicine, in particular to a nano-preparation for joint pain relief and its preparation method and application.
  • Osteoarthritis is a multifactorial aging disease with joint pain as the main symptom. It has a high disability rate and has caused a heavy burden to the country and society. Analgesia is the most important appeal of OA patients.
  • Glucocorticoid GC
  • GC Glucocorticoid
  • GC has poor solubility and has an analgesic effect.
  • the clinically used triamcinolone acetonide immediate-release suspension injection has an average residence time of 3.8 days in the knee joint of patients with osteoarthritis, and the frequency of intra-articular injection is relatively high. Therefore, it is of great significance to increase the solubility of GC, reduce the frequency of GC administration, and prolong its analgesic effect in osteoarthritis.
  • encapsulating drugs in liposomes or nano-carriers of polymer materials can improve the solubility of drugs.
  • liposomes have less drug loading on GC and are prone to drug precipitation in a short time; encapsulating GC in polymers
  • the solubility of GC can also be improved in nanocarriers prepared from materials, but there are few polymer materials available for human injection, and it is difficult to achieve long-term drug release after intra-articular injection.
  • polymer-based sustained-release microspheres have gradually become one of the research hotspots for new pharmaceutical preparations.
  • the polymer sustained-release microspheres on the market are mainly polylactic-glycolic acid (PLGA) sustained-release microspheres.
  • GC such as triamcinolone acetonide, dexamethasone, dexamethasone palmitate, etc.
  • the residence time of the drug in the joint cavity and the analgesic time are related to the particle size of the microspheres, but when the particle size of the microspheres is larger than 25 ⁇ m, there is a risk of promoting the development of joint inflammation.
  • the preparation process of PLGA sustained-release microspheres is complicated, terminal sterilization is difficult, and the lactic acid and glycolic acid produced during the degradation process may cause a decrease in the pH of the joint cavity, thereby promoting the development of an inflammatory response.
  • the technical problem to be solved by this invention is to overcome the deficiencies and defects mentioned in the above background technology, and to provide a nano-preparation for joint analgesia made of glucocorticoids, phospholipids and polyoxyethylene castor oil, which can also Long-term sustained drug release (more than 4 weeks), but its preparation process is simple and easy to control, and easy to sterilize, not only solves the problem of poor solubility of GC, but also the excipients used will not cause risks such as local pH reduction.
  • a nano-preparation for joint pain relief comprises glucocorticoids and nano-carriers, the nano-carriers are mainly composed of phospholipids and auxiliary agents, the auxiliary agents include polyoxyethylene castor oil.
  • the present invention finds that stable nanoparticles cannot be formed when only phospholipids are used or only polyoxyethylene castor oil is used to coat glucocorticoids.
  • glucocorticoids When glucocorticoids are loaded into nanoparticles composed of phospholipids and polyoxyethylene castor oil, their ability to inhibit the expression of inflammatory factors in macrophages is stronger (as shown in Figure 1, the degree of intra-articular inflammation is closely related to joint pain); and
  • nano-formulations composed of phospholipids and polyoxyethylene castor oil can achieve long-term sustained release in arthritic inflammatory joints (as shown in Figure 2), so further, these nanoparticles loaded with glucocorticoids Long-term analgesia can be achieved (as shown in Figure 3).
  • the glucocorticoids include clobetasol propionate, diflurasone acetate, dexamethasone propionate, difluprednate, mometasone furoate, diflucorolone valerate , betamethasone butyrate propionate, fluocinolone acetate, hydrocortisone butyrate propionate, beclomethasone dipropionate, diprone propionate, betamethasone valerate, dexamethasone valerate, prednisolone Triamcinolone Acetate Valerate, Fluocinolone, Hydrocortisone Butyrate, Clobetasone Butyrate, Alclomethasone Propionate, Triamcinolone Acetonide, Flumethasone Pivalate, Prednisolone, Hydrocortisone , dexamethasone, dexamethasone palmitate, triamcinolone acet
  • the phospholipids include one or more of natural soybean phospholipids, natural egg yolk phospholipids, and synthetic phospholipids, more preferably phospholipids E80, S100, PC98-T, and HSPC.
  • the polyoxyethylene castor oil includes polyoxyethylene 35 castor oil (EL-35), polyoxyethylene 40 castor oil (EL-40), polyoxyethylene 40 hydrogenated castor oil (HEL-40), polyoxyethylene One or more of ethylene 60 hydrogenated castor oil (HEL-60). More preferably solid polyoxyethylene castor The combined use of sesame oil (at least one of HEL-40 and HEL-60) and liquid polyoxyethylene castor oil (at least one of EL-35 and EL-40) can further increase the drug loading.
  • the auxiliary agent also includes polyethylene glycol 15-hydroxystearate (trade name: Kolliphor HS15), and the drug loading is higher after adding polyethylene glycol 15-hydroxystearate.
  • polyethylene glycol 15-hydroxystearate trade name: Kolliphor HS15
  • said adjuvant comprises polyoxyethylene 40 hydrogenated castor oil (HEL-40), polyoxyethylene 35 castor oil (EL-35) and polyethylene glycol 15-hydroxystearate .
  • the auxiliary agent is composed of polyoxyethylene 40 hydrogenated castor oil (HEL-40), polyoxyethylene 35 castor oil (EL-35) and polyethylene glycol 15-hydroxystearate.
  • the auxiliary agent comprises polyoxyethylene 60 hydrogenated castor oil (HEL-60), polyoxyethylene 35 castor oil (EL-35) and polyethylene glycol 15-hydroxystearate. More preferably, the auxiliary agent is composed of polyoxyethylene 60 hydrogenated castor oil (HEL-60), polyoxyethylene 35 castor oil (EL-35) and polyethylene glycol 15-hydroxystearate.
  • the adjuvant comprises polyoxyethylene 40 hydrogenated castor oil (HEL-40), polyoxyethylene 40 castor oil (EL-40) and polyethylene glycol 15-hydroxystearate.
  • the auxiliary agent is composed of polyoxyethylene 40 hydrogenated castor oil (HEL-40), polyoxyethylene 40 castor oil (EL-40) and polyethylene glycol 15-hydroxystearate.
  • the auxiliary agent comprises polyoxyethylene 60 hydrogenated castor oil (HEL-60), polyoxyethylene 40 castor oil (EL-40) and polyethylene glycol 15-hydroxystearate. More preferably, the auxiliary agent is composed of polyoxyethylene 60 hydrogenated castor oil (HEL-60), polyoxyethylene 40 castor oil (EL-40) and polyethylene glycol 15-hydroxystearate.
  • the three auxiliary agents may exist in any mass ratio.
  • said adjuvant comprises polyoxyethylene 60 hydrogenated castor oil (HEL-60) and polyoxyethylene 35 castor oil (EL-35). More preferably, the auxiliary agent is composed of polyoxyethylene 60 hydrogenated castor oil (HEL-60) and polyoxyethylene 35 castor oil (EL-35).
  • said adjuvant comprises polyoxyethylene 60 hydrogenated castor oil (HEL-60) and polyethylene glycol 15-hydroxystearate. More preferably, the auxiliary agent is made of polyoxyethylene Composition of ene 60 hydrogenated castor oil (HEL-60) and polyethylene glycol 15-hydroxystearate.
  • the two auxiliary agents may exist in any mass ratio.
  • the mass ratio of the phospholipid to the auxiliary agent is (1:20)-(20:1), preferably (1:10)-(10:1), more preferably (1:5)-(5:1 ), more preferably (1:2)-(2:1). Most preferably, the mass ratio of the phospholipid to the auxiliary agent is 3:5.
  • the drug loading amount of the glucocorticoid is 0.5-20%.
  • the particle size of the nano-preparation is less than 200nm.
  • the dosage form of the nano-preparation is injection or freeze-dried powder.
  • the present invention also correspondingly provides a method for preparing the nano-preparation for joint pain relief, comprising the following steps: dissolving glucocorticoids, phospholipids, and auxiliary agents in an organic solvent, The organic reagent is removed by evaporation, the film is removed by adding water, and then the microporous film is obtained by ultrasonic probe, high-pressure homogenization or extrusion.
  • the present invention also correspondingly provides an application of the nano-preparation in the preparation of a drug for joint pain relief of osteoarthritis.
  • the nano-preparation of the present invention can be used for joint analgesia, has a large drug load, and can stay in the joint cavity for at least 4 weeks. week.
  • the preparation method of the present invention has a simple preparation process, and the particle size is less than 200nm. Terminal sterilization can be achieved through probe ultrasound, high-pressure homogenization, or microporous membrane sterilization, and the preparation cost is relatively lower.
  • Fig. 1 is the in vitro biological activity investigation experiment result (after drug treatment 24h) of the nano-preparation that embodiment 2 prepares;
  • b) cell coding IL-1 ⁇ gene The relative expression level of the mRNA;
  • f relative expression of mRNA encoding iNOS gene in cells.
  • Fig. 2 is the in vivo distribution experiment result of the nano-preparation that embodiment 1 and embodiment 2 prepare;
  • a) NPs (HEL-40) and NPs (HEL-60) distribute in vivo fluorescence of small animals in OA model rat Imaging image;
  • b) Semi-quantitative histogram of distribution of NPs (HEL-40) and NPs (HEL-60) in inflammatory joints of OA model rats (n 3).
  • Fig. 3 is the result of the pharmacodynamic experiment of the nano-preparation prepared in Example 2;
  • a) OA model rat right hindlimb mechanical paw withdrawal threshold change figure (n 7), ****P ⁇ 0.0001, same as commercially available Injection (TA IR) compared with;
  • b) OA model rats bipedal weight difference chart (n 7), **P ⁇ 0.01, compared with commercially available injection (TA IR).
  • RV10 Rotary Evaporator German IKA Group
  • JY92-II Ultrasonic Cell Pulverizer Nevaporator
  • EmulsiFlex-C5 High Pressure Milk Homogenizer Canada AVESTIN Company
  • MS105DU Electronic Balance US METTLER TOLEDO company
  • Zetasizer Nano zsu3100 laser particle size analyzer Mealvern, UK
  • ModulyoD freeze dryer Thermo, USA
  • Lumina 3 small animal live imager Perkin Elmer, USA.
  • Triamcinolone acetonide (Aladdin Reagent (Shanghai) Co., Ltd.); Dexamethasone (Aladdin Reagent (Shanghai) Co., Ltd.); Dexamethasone palmitate (Aladdin Reagent (Shanghai) Co., Ltd.); Prednisolone ( Aladdin Reagent (Shanghai) Co., Ltd.); egg yolk lecithin E80 (German Lipoid company); soybean lecithin S100 (Germany Lipoid company); polyoxyethylene 35 castor oil (EL-35) (Germany BASF company); polyoxyethylene 40 castor oil (EL-40) (Germany BASF company); polyoxyethylene 40 hydrogenated castor oil (HEL-40) (Germany BASF company); polyoxyethylene 60 hydrogenated castor oil (HEL-60) (Germany BASF company); 15-hydroxystearic acid polyethylene glycol ester (HS15) (Germany BASF Company); DiD (Biotium, USA), tri
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipid E80, and HEL-40, and the dosage form is injection.
  • TA triamcinolone acetonide
  • TA triamcinolone acetonide
  • phospholipid E80 phospholipid E80
  • HEL-40 triamcinolone acetonide
  • the particle size is 133 nm.
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipid E80, and HEL-60, and the dosage form is injection.
  • TA triamcinolone acetonide
  • TA triamcinolone acetonide
  • phospholipid E80 phospholipid E80
  • HEL-60 triamcinolone acetonide
  • the particle size is 120nm.
  • a nanoparticle loaded with dexamethasone palmitate for joint pain relief the formulation of which comprises dexamethasone palmitate, phospholipid S100, and HEL-40, and the dosage form is injection. Its preparation method is as follows:
  • the particle size is 144nm.
  • Dexamethasone palmitate nanoparticles for joint pain relief the formulation of which contains dexamethasone Pine palmitate, phospholipid S100, HEL-60, the dosage form is injection. Its preparation method is as follows:
  • the particle size is 128nm.
  • a dexamethasone-loaded nanoparticle for joint pain relief the formulation of which comprises dexamethasone, phospholipid S100, and HEL-60, and the dosage form is injection. Its preparation method is as follows:
  • the particle size is 118nm.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • a prednisolone-loaded nanoparticle for joint pain relief the formulation of which comprises prednisolone, phospholipid PC98-T, and HEL-60, and the dosage form is an injection. Its preparation method is as follows:
  • the particle size is 129 nm.
  • Embodiment 7 is a diagrammatic representation of Embodiment 7:
  • a dexamethasone-loaded nanoparticle for joint pain relief the formulation of which comprises dexamethasone, phospholipid HSPC, and HEL-60, and the dosage form is injection. Its preparation method is as follows:
  • Embodiment 8 is a diagrammatic representation of Embodiment 8
  • the invention relates to dexamethasone palmitate nanoparticles for joint pain relief, the formulation of which comprises dexamethasone palmitate, phospholipid E80, and HEL-60, and the dosage form is injection. Its preparation method is as follows:
  • the particle size is 92nm.
  • Embodiment 9 is a diagrammatic representation of Embodiment 9:
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipids E80, HS15, and HEL-60, and the dosage form is injection.
  • TA triamcinolone acetonide
  • phospholipids E80, HS15, and HEL-60 triamcinolone acetonide
  • HEL-60 triamcinolone acetonide
  • the particle size is 124nm.
  • a dexamethasone-loaded nanoparticle for joint pain relief the formulation of which comprises dexamethasone, phospholipids E80, HS15, and EL-35, and the dosage form is injection. Its preparation method is as follows:
  • the particle size is 118nm.
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipid E80, EL-35, and HEL-40, and the dosage form is injection.
  • TA triamcinolone acetonide
  • the particle size is 136 nm.
  • a dexamethasone-loaded nanoparticle for joint pain relief the formulation of which comprises dexamethasone, phospholipid E80, EL-35, and HEL-60, and the dosage form is injection. Its preparation method is as follows:
  • the particle size is 132nm.
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipid E80, and HEL-40, and the dosage form is freeze-dried powder injection.
  • TA triamcinolone acetonide
  • phospholipid E80 phospholipid E80
  • HEL-40 triamcinolone acetonide
  • a dexamethasone nanoparticle for joint pain relief the formulation of which comprises dexamethasone, phospholipid E80, and HEL-60, and the dosage form is freeze-dried powder injection. Its preparation method is as follows:
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipid E80, and HEL-40, and the dosage form is injection.
  • TA triamcinolone acetonide
  • TA triamcinolone acetonide
  • phospholipid E80 phospholipid E80
  • HEL-40 triamcinolone acetonide
  • the invention relates to triamcinolone acetonide (TA)-loaded nanoparticles for joint pain relief, the formulation of which comprises triamcinolone acetonide (TA), phospholipid E80, and HEL-40, and the dosage form is injection.
  • TA triamcinolone acetonide
  • TA triamcinolone acetonide
  • phospholipid E80 phospholipid E80
  • HEL-40 triamcinolone acetonide
  • the present invention uses the same nanoparticle preparation method (thin film hydration method) of the embodiment to use alone Phospholipids or polyoxyethylene castor oil prepared glucocorticoid-loaded nanoparticles; at the same time, by fixing the ratio of phospholipids to polyoxyethylene castor oil, the composition of different polyoxyethylene castor oils and whether HS15 was added or not had an effect on the preparation of sugar-loaded nanoparticles. Effect of corticosteroid nanoparticle properties.
  • triamcinolone acetonide-loaded nanoparticles prepared by using phospholipid E80 or HEL-60 alone are very unstable or unable to entrap drugs well; it is necessary to use phospholipid E80 and HEL-60 in combination at a certain ratio to prepare Nanoparticles claimed by the inventors.
  • HEL-60 is used in combination with EL-35 or HEL-60 is used in combination with HS15, nanoparticles with higher drug loading can be prepared.
  • the mouse-derived macrophage RAW264.7 was inoculated in a 12-well cell culture plate, and after it adhered to the wall, it was stimulated with LPS for 24 hours, and the medium was replaced with a drug-containing medium, respectively TA solution (denoted as TA IR) and TA-loaded NPs (the concentration of TA is 4 ⁇ g/mL, the culture medium without drug treatment is used as a control, and the TA-loaded NPs are prepared according to Example 2, denoted as TA-NPs), after incubation for 24 hours, use real-time fluorescence to quantify The concentrations of IL-1 ⁇ , TNF- ⁇ , IL-6, IL-10, IL-4 and iNOS were determined by PCR method. Cells not treated with LPS and cells treated with LPS but not treated with TA were designated as "Control" group and "LPS" group, respectively.
  • the OA rat model was established by intra-articular injection of sodium iodoacetate into the knee joints of SD rats, and the OA rat model was obtained when the knee joints of the rats were swollen and the pain threshold was significantly reduced.
  • NPs (HEL-40) and NPs (HEL-60) were injected into the knee joint cavity of OA rats, respectively, on the 0d, 1d, 2d, 3d, 7d, 14d, 21d and On the 28th day, the fluorescence intensity at the inflammatory joints of the rats was examined by a small animal in vivo imager.
  • NPs HEL-60
  • HEL-40 NPs
  • OA model rats were treated with physiological saline (referred to as Saline), commercially available triamcinolone acetonide solution injection (referred to as TA IR), and NPs loaded with TA prepared in Example 2 (referred to as TA IR).
  • TA IR physiological saline
  • -NPs) and non-drug-loaded NPs prepared in Example 2 for treatment; in addition, an equal volume of normal saline was injected into the knee joint cavity of normal rats as a control (referred to as Control).
  • the dosage of TA was 60 ⁇ g for each knee joint, administered on the 7th day after modeling, and administered once in total. From the first day of administration, the mechanical paw withdrawal threshold and the weight difference between the two feet of the rats in each group were measured once a week. On the 35th day, the behavioral test of the rats was carried out.

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Abstract

本发明公开了一种用于关节镇痛的纳米制剂，所述纳米制剂包含糖皮质激素和纳米载体，所述纳米载体主要由磷脂和助剂组成，所述助剂包括聚氧乙烯蓖麻油。本发明的纳米制剂，可用于关节镇痛，载药量大，能在关节腔内滞留至少4周，其对OA的镇痛效果显著优于市售GC注射液，且可维持至少4周。还公开了该纳米制剂的制备方法和应用，工艺简单，且粒径小于200nm，可通过探头超声、高压均质或微孔滤膜除菌等方式实现终端灭菌，制备成本相对更低。

Description

用于关节镇痛的纳米制剂及其制备方法和应用 技术领域
本发明属于生物医药领域，特别是一种用于关节镇痛的纳米制剂及其制备方法和应用。
背景技术
骨关节炎(OA)是一种多因素引起的以关节疼痛为主要症状的衰老性疾病，致残率高，给国家和社会造成了沉重负担。镇痛是OA患者就诊的最主要诉求。糖皮质激素(glucocorticoid，GC)是一类药理作用多样的甾体类小分子药物，可通过抑制炎症反应而发挥镇痛作用，已被广泛用于OA镇痛；但GC溶解性差，存在镇痛效果维持时间较短、效果不够显著以及存在安全性隐患等亟待解决的重要问题。如临床使用的曲安奈德速释混悬注射剂，在骨关节炎患者的膝关节平均滞留时间为3.8天，关节腔注射频率较高。因此，提高GC溶解度，降低GC给药频率，延长其骨关节炎镇痛效果，具有重要意义。
通常将药物包裹在脂质体或高分子材料的纳米载体中可提高药物的溶解性，然而脂质体对GC的载药量少，且容易短时间内有药物析出；将GC包裹在高分子材料制备的纳米载体中，也可以提高GC的溶解性，但可供人体注射用的高分子材料较少，且关节腔注射后难以实现长时间释药。随着生物高分子聚合物材料在医药学领域的广泛应用，基于高分子聚合物的缓释微球逐渐成为新药物制剂的研究热点之一。目前，上市的高分子聚合物缓释微球主要是聚乳酸-羟基乙酸(PLGA)缓释微球。将GC(如曲安奈德、地塞米松、地塞米松棕榈酸酯等)包载在PLGA微球，将其进行关节腔注射后，GC对OA的镇痛效果可持续4-12周，且药物在关节腔中的滞留时间和镇痛时间与微球的粒径相关，但当微球的粒径大于25μm时，有促进关节炎症发展的风险。另外，PLGA缓释微球的制备工艺复杂，末端灭菌困难，且降解过程中产生的乳酸和羟基乙酸可能造成关节腔内pH降低，从而促进炎症反应的发展。
发明内容
本发明所要解决的技术问题是，克服以上背景技术中提到的不足和缺陷，提供一种由糖皮质激素、磷脂和聚氧乙烯蓖麻油制成的用于关节镇痛的纳米制剂，同样可以实现药物长时间缓释(4周以上)，但其制备工艺简单易控，且灭菌容易，不仅解决了GC的溶解性差的问题，而且所用辅料不会造成局部pH降低等风险。
为解决上述技术问题，本发明提出的技术方案为：
一种用于关节镇痛的纳米制剂，所述纳米制剂包含糖皮质激素和纳米载体，所述纳米载体主要由磷脂和助剂组成，所述助剂包括聚氧乙烯蓖麻油。
本发明研究发现，只使用磷脂、或只使用聚氧乙烯蓖麻油包裹糖皮质激素时，不能形成稳定的纳米粒。当糖皮质激素装载到由磷脂、聚氧乙烯蓖麻油组成的纳米粒后，其抑制巨噬细胞炎性因子表达的能力更强(如图1，关节内炎症程度与关节疼痛密切相关)；而且我们意外地发现由磷脂与聚氧乙烯蓖麻油组成的纳米制剂在关节炎的炎性关节中可实现长时间的缓释作用(如图2)，因此进一步地，这些装载糖皮质激素的纳米粒可实现长效镇痛(如图3)。
上述的纳米制剂，优选的，所述糖皮质激素包括丙酸氯倍他索、醋酸双氟拉松、丙酸地塞米松、二氟泼尼酯、糠酸莫米松、戊酸双氟可龙、倍他米松丁酸丙酸酯、醋酸氟轻松、丙酸丁酸氢可的松、丙酸倍氯米松、丙酸地泼罗酮、戊酸倍他米松、戊酸地塞米松、泼尼松龙醋酸戊酸酯、氟轻松、丁酸氢化可的松、丁酸氯倍他松、丙酸阿氯米松、曲安奈德、氟米松新戊酸酯、泼尼松龙、氢化可的松、地塞米松、地塞米松棕榈酸酯、曲安奈德棕榈酸酯中的一种或多种。更优选地塞米松、地塞米松棕榈酸酯、曲安奈德、泼尼松龙的一种或多种。
优选的，所述磷脂包括天然大豆磷脂、天然蛋黄磷脂、合成磷脂中的一种或多种，更优选磷脂E80、S100、PC98-T、HSPC。
优选的，所述聚氧乙烯蓖麻油包括聚氧乙烯35蓖麻油(EL-35)、聚氧乙烯40蓖麻油(EL-40)、聚氧乙烯40氢化蓖麻油(HEL-40)、聚氧乙烯60氢化蓖麻油(HEL-60)中的一种或几种。更优选固体聚氧乙烯蓖 麻油(HEL-40和HEL-60中的至少一种)与液体聚氧乙烯蓖麻油(EL-35和EL-40中的至少一种)进行联合使用，可以进一步提高载药量。
优选的，所述助剂还包括15-羟基硬脂酸聚乙二醇酯(商品名为Kolliphor HS15)，加入15-羟基硬脂酸聚乙二醇酯后载药量更高。
因此，根据一个优选的实施方案，所述助剂包含聚氧乙烯40氢化蓖麻油(HEL-40)、聚氧乙烯35蓖麻油(EL-35)和15-羟基硬脂酸聚乙二醇酯。更优选地，所述助剂由聚氧乙烯40氢化蓖麻油(HEL-40)、聚氧乙烯35蓖麻油(EL-35)和15-羟基硬脂酸聚乙二醇酯组成。
根据另一个优选的实施方案，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)、聚氧乙烯35蓖麻油(EL-35)和15-羟基硬脂酸聚乙二醇酯。更优选地，所述助剂由聚氧乙烯60氢化蓖麻油(HEL-60)、聚氧乙烯35蓖麻油(EL-35)和15-羟基硬脂酸聚乙二醇酯组成。
根据另一个优选的实施方案，所述助剂包含聚氧乙烯40氢化蓖麻油(HEL-40)、聚氧乙烯40蓖麻油(EL-40)和15-羟基硬脂酸聚乙二醇酯。更优选地，所述助剂由聚氧乙烯40氢化蓖麻油(HEL-40)、聚氧乙烯40蓖麻油(EL-40)和15-羟基硬脂酸聚乙二醇酯组成。
根据另一个优选的实施方案，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)、聚氧乙烯40蓖麻油(EL-40)和15-羟基硬脂酸聚乙二醇酯。更优选地，所述助剂由聚氧乙烯60氢化蓖麻油(HEL-60)、聚氧乙烯40蓖麻油(EL-40)和15-羟基硬脂酸聚乙二醇酯组成。
当所述助剂为由上述三种助剂联合使用时，所述三种助剂可以以任何质量比存在。
另外，当聚氧乙烯60氢化蓖麻油(HEL-60)和聚氧乙烯35蓖麻油(EL-35)联合使用或者聚氧乙烯60氢化蓖麻油(HEL-60)和15-羟基硬脂酸聚乙二醇酯联合使用时，可以制备更高载药量的纳米粒。
因此，根据一个最优选的实施方案，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)和聚氧乙烯35蓖麻油(EL-35)。更优选地，所述助剂由聚氧乙烯60氢化蓖麻油(HEL-60)和聚氧乙烯35蓖麻油(EL-35)组成。
根据另一个最优选的实施方案，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)和15-羟基硬脂酸聚乙二醇酯。更优选地，所述助剂由聚氧乙 烯60氢化蓖麻油(HEL-60)和15-羟基硬脂酸聚乙二醇酯组成。
当所述助剂为由上述两种助剂联合使用时，所述两种助剂可以以任何质量比存在。
优选的，所述磷脂与助剂的质量比为(1:20)-(20:1)，优选(1:10)-(10:1)，更优选(1:5)-(5:1)，更优选(1:2)-(2:1)。最优选地，所述磷脂与助剂的质量比为3:5。
优选的，所述糖皮质激素的载药量为0.5-20％。
优选的，所述纳米制剂的粒径小于200nm。
优选的，所述纳米制剂的剂型为注射液或冻干粉针。
基于一个总的发明构思，本发明还相应提供一种所述用于关节镇痛的纳米制剂的制备方法，包括如下步骤：将糖皮质激素、磷脂、助剂溶于有机溶剂中，通过减压蒸发法除去有机试剂，加水脱膜，然后通过探头超声、高压均质或挤压过微孔膜，即得。
基于一个总的发明构思，本发明还相应提供一种所述的纳米制剂在制备用于骨关节炎关节镇痛的药物中的应用。
与现有技术相比，本发明的有益效果为：
1、本发明的纳米制剂，可用于关节镇痛，载药量大，能在关节腔内滞留至少4周，其对OA的镇痛效果显著优于市售GC注射液，且可维持至少4周。
2、本发明的制备方法制备工艺简单，且粒径小于200nm，可通过探头超声、高压均质或微孔滤膜除菌等方式实现终端灭菌，制备成本相对更低。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是实施例2制备得到的纳米制剂的体外生物活性考察实验结果(药物处理24h后)；其中，a)细胞编码TNF-α基因的mRNA的相对表达量；b)细胞编码IL-1β基因的mRNA的相对表达量；c)细胞编码IL-6基因的 mRNA的相对表达量；d)细胞编码IL-4基因的mRNA的相对表达量；e)细胞编码IL-10基因的mRNA的相对表达量；f)细胞编码iNOS基因的mRNA的相对表达量。*P<0.05，***P<0.001，****P<0.0001。
图2是实施例1和实施例2制备得到的纳米制剂的体内分布实验结果；其中，a)NPs(HEL-40)和NPs(HEL-60)在OA模型大鼠体内分布的小动物活体荧光成像图；b)NPs(HEL-40)及NPs(HEL-60)在OA模型大鼠炎性关节分布的半定量柱状图(n＝3)。
图3是实施例2制备得到的纳米制剂的药效学实验结果；其中，a)OA模型大鼠右后肢机械缩足阈值变化图(n＝7)，****P<0.0001，同市售注射液(TA IR)相比；b)OA模型大鼠双足负重差值变化图(n＝7),**P<0.01，同市售注射液(TA IR)相比。
具体实施方式
为了便于理解本发明，下文将结合说明书附图和较佳的实施例对本发明做更全面、细致地描述，但本发明的保护范围并不限于以下具体实施例。
除非另有定义，下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的，并不是旨在限制本发明的保护范围。
除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
本发明所使用的仪器和试药:
RV10型旋转蒸发仪(德国IKA集团)，JY92-Ⅱ型超声波细胞粉碎仪(宁波新芝科学仪器研究所)，EmulsiFlex-C5高压乳匀机(加拿大AVESTIN公司)，MS105DU型电子天平(美国METTLER TOLEDO公司)，Zetasizer Nano zsu3100型激光粒度分析仪(英国Malvern公司)，ModulyoD冷冻干燥机(美国Thermo公司)，Lumina 3小动物活体成像仪(美国Perkin Elmer公司)。
曲安奈德(阿拉丁试剂(上海)有限公司)；地塞米松(阿拉丁试剂(上海)有限公司)；地塞米松棕榈酸酯(阿拉丁试剂(上海)有限公司)；泼尼松龙(阿拉丁试剂(上海)有限公司)；蛋黄卵磷脂E80(德国Lipoid 公司)；大豆卵磷脂S100(德国Lipoid公司)；聚氧乙烯35蓖麻油(EL-35)(德国BASF公司)；聚氧乙烯40蓖麻油(EL-40)(德国BASF公司)；聚氧乙烯40氢化蓖麻油(HEL-40)(德国BASF公司)；聚氧乙烯60氢化蓖麻油(HEL-60)(德国BASF公司)；15-羟基硬脂酸聚乙二醇酯(HS15)(德国BASF公司)；DiD(美国Biotium公司)，曲安奈德注射液(昆明积大制药股份有限公司)。
实施例1：
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、HEL-40，剂型为注射液。其制备方法如下：
将4mg曲安奈德、125mg磷脂E80、70mg HEL-40溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经冰水浴探头超声即得。粒径为133nm。
实施例2：
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、HEL-60，剂型为注射液。其制备方法如下：
将4mg曲安奈德、100mg磷脂E80、100mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经冰水浴探头超声即得。粒径为120nm。
实施例3：
一种用于关节镇痛的载地塞米松棕榈酸酯纳米粒，其配方包含地塞米松棕榈酸酯、磷脂S100、HEL-40，剂型为注射液。其制备方法如下：
将4mg地塞米松棕榈酸酯、125mg磷脂S100、70mg HEL-40溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经冰水浴探头超声即得。粒径为144nm。
实施例4：
一种用于关节镇痛的载地塞米松棕榈酸酯纳米粒，其配方包含地塞米 松棕榈酸酯、磷脂S100、HEL-60，剂型为注射液。其制备方法如下：
将4mg地塞米松棕榈酸酯、100mg磷脂S100、100mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经冰水浴探头超声即得。粒径为128nm。
实施例5：
一种用于关节镇痛的载地塞米松纳米粒，其配方包含地塞米松、磷脂S100、HEL-60，剂型为注射液。其制备方法如下：
将2mg地塞米松、50mg磷脂S100、10mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为118nm。
实施例6：
一种用于关节镇痛的载泼尼松龙纳米粒，其配方包含泼尼松龙、磷脂PC98-T、HEL-60，剂型为注射液。其制备方法如下：
将1mg泼尼松龙、40mg磷脂PC98-T、4mg HEL-60溶于氯仿与乙醇的混合液中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为129nm。
实施例7：
一种用于关节镇痛的载地塞米纳米粒，其配方包含地塞米松、磷脂HSPC、HEL-60，剂型为注射液。其制备方法如下：
将4mg地塞米松、30mg磷脂HSPC、150mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为105nm。
实施例8：
一种用于关节镇痛的载地塞米棕榈酸酯纳米粒，其配方包含地塞米松棕榈酸酯、磷脂E80、HEL-60，剂型为注射液。其制备方法如下：
将2mg地塞米松棕榈酸酯、15mg磷脂E80、150mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为92nm。
实施例9：
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、HS15、HEL-60，剂型为注射液。其制备方法如下：
将20mg曲安奈德、100mg磷脂E80、40mg HS15、60mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为124nm。
实施例10：
一种用于关节镇痛的载地塞米松纳米粒，其配方包含地塞米松、磷脂E80、HS15、EL-35，剂型为注射液。其制备方法如下：
将20mg地塞米松、100mg磷脂E80、40mg HS15、60mg EL-35溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为118nm。
实施例11：
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、EL-35、HEL-40，剂型为注射液。其制备方法如下：
将20mg曲安奈德、100mg磷脂E80、50mg EL35、50mg HEL-40溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为136nm。
实施例12：
一种用于关节镇痛的载地塞米松纳米粒，其配方包含地塞米松、磷脂E80、EL-35、HEL-60，剂型为注射液。其制备方法如下：
将20mg地塞米松、100mg磷脂E80、50mgEL-35、50mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经高压均质即得。粒径为132nm。
实施例13：
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、HEL-40，剂型为冻干粉针。其制备方法如下：
将4mg曲安奈德、125mg磷脂E80、70mg HEL-40溶于氯仿中，减压浓缩除去有机试剂，加入纯水水化，经冰水浴探头超声，然后加入质量体积比为10％的甘露醇，冷冻干燥即得。
实施例14：
一种用于关节镇痛的地塞米松纳米粒，其配方包含地塞米松、磷脂E80、HEL-60，剂型为冻干粉针。其制备方法如下：
将4mg地塞米松、100mg磷脂E80、100mg HEL-60溶于氯仿中，减压浓缩除去有机试剂，加入纯水水化，经冰水浴探头超声，然后加入质量体积比为10％的甘露醇，冷冻干燥即得。
对比例1：
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、HEL-40，剂型为注射液。其制备方法如下：
将4mg曲安奈德、125mg磷脂E80、4mg HEL-40溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经冰水浴探头超声即得。所得制剂不透明且有沉淀物。
对比例2:
一种用于关节镇痛的载曲安奈德(TA)纳米粒，其配方包含曲安奈德(TA)、磷脂E80、HEL-40，剂型为注射液。其制备方法如下：
将4mg曲安奈德、4mg磷脂E80、125mg HEL-40溶于氯仿中，减压浓缩除去有机试剂，加入生理盐水水化，经冰水浴探头超声即得。粒径为203nm。所得制剂在30min内便有沉淀析出。
为进一步证明本发明的纳米制剂的效果，开展了以下实验：
1)组成成分对纳米制剂载药量的影响
本发明通过实施例相同的纳米粒制备方法(薄膜水化法)，单独使用 磷脂或者聚氧乙烯蓖麻油制备了载糖皮质激素纳米粒；同时，通过固定磷脂与聚氧乙烯蓖麻油的比例，考察不同聚氧乙烯蓖麻油组成成分以及添加HS15与否对纳米粒制备载糖皮质激素纳米粒性质的影响。如表1所示，单独使用磷脂E80或HEL-60制备的载曲安奈德纳米粒非常不稳定或无法较好包载药物；需要磷脂E80和HEL-60在一定比例下联合使用才能制备出符合发明人要求的纳米粒。另外，当HEL-60与EL-35联合使用或者HEL-60与HS15联合使用时，可以制备更高载药量的纳米粒。
表1：不同组成成分对纳米粒制备载糖皮质激素纳米粒性质的影响
2)体外生物学活性考察
将小鼠源巨噬细胞RAW264.7接种在12孔细胞培养板中，待其贴壁后，以LPS刺激24h，将培养基更换为含药培养基，分别为TA溶液(记为TA IR)和载TA的NPs(TA的浓度为4μg/mL，不加药的培养基处理作为对照，载TA的NPs按照实施例2制得，记为TA-NPs)，孵育24h后，使用实时荧光定量PCR法测定其中IL-1β、TNF-α、IL-6、IL-10、IL-4以及iNOS的浓度。未经LPS处理的细胞和经LPS处理但未经TA处理的细胞分别记为“Control”组和“LPS”组。
由图1可知，相对于其他处理方法，TA-NPs处理可以显著降低巨噬细胞炎症因子基因的表达。
3)OA大鼠模型
对SD大鼠膝关节的关节腔注射碘乙酸钠构建OA大鼠模型，待大鼠膝关节出现肿胀，痛阈显著降低，即得OA大鼠模型。
4)体内分布实验
将糖皮质激素用荧光标记物DiD代替，按照实施1和实施例2制备纳米制剂，分别记为NPs(HEL-40)和NPs(HEL-60)。将DiD溶液(记为DiD)、NPs(HEL-40)和NPs(HEL-60)分别注射至OA大鼠膝关节腔内，分别于第0d、1d、2d、3d、7d、14d、21d及28d通过小动物活体成像仪考察大鼠炎性关节处的荧光强度。
由图2可知，相对于DiD，NPs(HEL-60)和NPs(HEL-40)可以在OA模型大鼠关节腔内的滞留至少可达28天。这些结果表明，NPs在炎性关节腔内具有良好的缓释性。
5)药效学研究
通过关节腔注射方式，对OA模型大鼠分别使用生理盐水(记为Saline)、市售曲安奈德溶液注射液(记为TA IR)、由实施例2制得载TA的NPs(记为TA-NPs)以及由实施例2制得未载药的NPs(记为Vehicle)进行治疗；另外，在正常大鼠膝关节腔内注射等体积的生理盐水作为对照(记为Control)。TA的给药剂量为每个膝关节60μg，于造模后第7天给药，共给药1次。从给药第一天起，每周测量一次各组大鼠的机械缩足阈值及双足负重差值。于第35天，进行大鼠行为学检测。
由图3可知，相对于其他方式来说，NPs治疗可以更有效的缓解OA大鼠的关节炎症。这些结果充分说明实现了关节炎滑膜双细胞靶向递送药物对OA治疗的有效性。也就是说，本发明制备得到的纳米制剂在制备用于骨关节炎关节镇痛的药物方面具有很好的应用前景。

Claims (13)

  1. 一种用于关节镇痛的纳米制剂，其特征在于，所述纳米制剂包含糖皮质激素和纳米载体，所述纳米载体主要由磷脂和助剂组成，所述助剂包括聚氧乙烯蓖麻油。
  2. 根据权利要求1所述的纳米制剂，其特征在于，所述糖皮质激素包括丙酸氯倍他索、醋酸双氟拉松、丙酸地塞米松、二氟泼尼酯、糠酸莫米松、戊酸双氟可龙、倍他米松丁酸丙酸酯、醋酸氟轻松、丙酸丁酸氢可的松、丙酸倍氯米松、丙酸地泼罗酮、戊酸倍他米松、戊酸地塞米松、泼尼松龙醋酸戊酸酯、氟轻松、丁酸氢化可的松、丁酸氯倍他松、丙酸阿氯米松、曲安奈德、氟米松新戊酸酯、泼尼松龙、氢化可的松、地塞米松、地塞米松棕榈酸酯、曲安奈德棕榈酸酯中的一种或多种。
  3. 根据权利要求1所述的纳米制剂，其特征在于，所述磷脂包括天然大豆磷脂、天然蛋黄磷脂、合成磷脂中的一种或多种。
  4. 根据权利要求1所述的纳米制剂，其特征在于，所述聚氧乙烯蓖麻油包括聚氧乙烯35蓖麻油、聚氧乙烯40蓖麻油、聚氧乙烯40氢化蓖麻油、聚氧乙烯60氢化蓖麻油中的一种或几种。
  5. 根据权利要求1所述的纳米制剂，其特征在于，所述助剂还包括15-羟基硬脂酸聚乙二醇酯。
  6. 根据权利要求1所述的纳米制剂，其特征在于，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)、聚氧乙烯35蓖麻油(EL-35)和15-羟基硬脂酸聚乙二醇酯。
  7. 根据权利要求1所述的纳米制剂，其特征在于，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)和聚氧乙烯35蓖麻油(EL-35)。
  8. 根据权利要求1所述的纳米制剂，其特征在于，所述助剂包含聚氧乙烯60氢化蓖麻油(HEL-60)和15-羟基硬脂酸聚乙二醇酯。
  9. 根据权利要求1所述的纳米制剂，其特征在于，所述磷脂与助剂的质量比为(1:20)-(20:1)。
  10. 根据权利要求9所述的纳米制剂，其特征在于，所述磷脂与助剂的质量比为(1:10)-(10:1)。
  11. 根据权利要求1-10中任一项所述的纳米制剂，其特征在于，所述糖皮质激素的载药量为0.5-20％，所述纳米制剂的粒径小于200nm，所述纳米制剂的剂型为注射液或冻干粉针。
  12. 一种如权利要求1-11中任一项所述用于关节镇痛的纳米制剂的制备方法，其特征在于，包括如下步骤：将糖皮质激素、磷脂、聚氧乙烯蓖麻油溶于有机溶剂中，通过减压蒸发法除去有机试剂，加水脱膜，然后通过探头超声、高压均质或挤压过微孔膜，即得。
  13. 一种如权利要求1-11中任一项所述的纳米制剂或由权利要求12所述制备方法制备得到的纳米制剂在制备用于骨关节炎关节镇痛的药物中的应用。
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