WO2020143688A1 - 一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法 - Google Patents

一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法 Download PDF

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WO2020143688A1
WO2020143688A1 PCT/CN2020/070987 CN2020070987W WO2020143688A1 WO 2020143688 A1 WO2020143688 A1 WO 2020143688A1 CN 2020070987 W CN2020070987 W CN 2020070987W WO 2020143688 A1 WO2020143688 A1 WO 2020143688A1
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autophagy
nasal cavity
inducer
neurodegenerative diseases
nasal
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PCT/CN2020/070987
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English (en)
French (fr)
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刘刚
刘静怡
文磊
张金凤
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纳菲(深圳)制药科技有限公司
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Priority to US17/372,403 priority Critical patent/US20220000804A1/en

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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
    • A61K31/12—Ketones
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K9/0043—Nose
    • 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
    • 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
    • A61K9/12—Aerosols; Foams
    • 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
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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, and relates to a nasal cavity autophagy inducer and a preparation method thereof. Specifically, it is a nasal cavity autophagy inducer for preventing and treating early neurodegenerative diseases and a preparation method thereof.
  • Autophagy is the process of phagocytosing cytoplasmic proteins or organelles and coating them into vesicles, fusing with lysosomes and degrading the contents of the envelope, thereby achieving the metabolic needs of the cells and organelles Update.
  • Neurons are heavily dependent on autophagy.
  • the destruction of the autophagy pathway can lead to the accumulation of ubiquitinated protein aggregates in neurons, inducing neuronal degeneration, which in turn leads to neurodegenerative diseases.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • tremor motor dysfunction
  • tremor tremor
  • the defects of the lysosomal pathway are directly related, and the main pathological marker is the abnormal aggregate of Lewy bodies of ⁇ -synuclein (a-synuclin).
  • olfactory disorder is a common symptom of early stages of neurodegenerative diseases such as AD and PD. More than 90% of Parkinson's patients are accompanied by olfactory disorder, which can be used as an indicator to distinguish PD from atypical par.
  • Kinson syndrome provides help for the early diagnosis and differential diagnosis of PD.
  • the mechanism of olfactory disturbance in neurodegenerative diseases is related to the accumulation of toxic proteins in the olfactory area. Under pathological conditions, the autophagy pathway in the olfactory-related area is damaged, resulting in the protein being unable to degrade normally, and accumulating and compressing neurons, inducing the progression of disease. In response to these abnormal protein aggregations, there are currently no clinically effective drugs.
  • the treatment of PD can only relieve symptoms by supplementing dopamine, and it can not play a therapeutic role in the disease. And as the disease worsens, dopamine therapy gradually fails and produces serious side effects. Therefore, there is an urgent need to develop new therapeutic drugs.
  • Autophagy inducers can enhance the occurrence of autophagic flow in neurons through various mechanisms, such as increasing the production of autophagosomes, promoting the fusion of autophagosomes with lysosomes, enhancing lysosomal function, and increasing the number of lysosomes Etc., to repair the damaged autophagy pathway, promote the degradation of toxic proteins, and reduce the nerve damage caused by their accumulation.
  • Some autophagy inducers can activate the autophagy pathway by binding to TFEB protein and are an effective autophagy inducer.
  • due to its strong hydrophobicity and small organic molecules it has poor drug-forming properties. After oral administration, it has low bioavailability, low brain enrichment, and short circulation time in the body.
  • nano preparations have been widely used in the fields of medicine and biology because of their advantages of protecting drugs from being destroyed, prolonging the effective drug maintenance time, controlling the release of drugs, and reducing the toxic and side effects of drugs.
  • nano drug particles encapsulated by liposomes, polymers and other carriers usually contain less than 10% of the drug, and the concentration of polymers and other carriers in the brain may bring potential toxic and side effects and become an obstacle to nano The particles are further applied to clinically difficult problems.
  • the blood-brain barrier prevents more than 98% of drugs from entering brain tissue, especially nano-drugs with a diameter of about 100 nm. It is more difficult to penetrate the blood-brain barrier to reach the nervous system. Therefore, there is an urgent need to develop a nano-delivery system with high drug loading, high brain targeting, safety and non-toxicity, which is applied to the treatment of neurological diseases of autophagy small molecule drugs.
  • the main purpose of the present invention is to provide a nano-autophagy inducer for nasal cavity and a preparation method thereof, in particular, a self-carrying carrier-free nasal nano-autophagy inducer and a preparation method for application to early neurodegeneration disease.
  • a nasal cavity autophagy inducer for prevention and treatment of early neurodegenerative diseases including a hydrophobic small molecule with autophagy induction effect and an amphipathic surfactant; first configure amphiphilic surfactant 1-10mg/mL and 0.5 -5mg/mL good solvent solution of autophagy-inducing drug, and then the good solvent solution is added dropwise to deionized water, the volume ratio of the good solvent solution to deionized water is (0.5-5): 50, dropwise addition At the same time, it is supplemented by air blowing to assist the volatilization of the good solvent; a self-supporting carrier-free nanoparticle suspension emulsion with a particle size of 50-200nm is prepared by reprecipitation method, and freeze-dried to prepare a freeze-dried powder; before use, freeze-dried The powder was resuspended in isotonic saline to obtain a carrier-free carrier-free nasal autophagy inducer.
  • the autophagy inducing agent of the present invention can eliminate abnormal protein aggregation by inducing autophagy.
  • the surface potential of the self-supporting carrier-free nanoparticles is -10 to -60 mV. More preferably, it is -10 to -30 mV.
  • the early neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.
  • the early neurodegenerative diseases are also accompanied by symptoms of olfactory dysfunction.
  • the nasal cavity autophagy inducer is a highly targeted enriched autophagy inducer at the olfactory bulb.
  • the nasal cavity autophagy inducer of the present invention has a significant clearance effect on abnormal protein aggregation in the olfactory area and other diseased areas.
  • the small hydrophobic molecule is a curcumin analog of the following structural formula, its cis-isomer or a mixture of the two in any ratio:
  • the small hydrophobic molecule is a mixture of the above-mentioned curcumin analogs and their cis-isomers, and the weight ratio of the cis-isomers in the mixture accounts for 25-35% of the total mixture.
  • the mixture whose cis-isomer has a weight ratio of 25-35% of the total mixture is prepared by the following method, and the curcumin analogue methanol solution is irradiated with ultraviolet light for 1.5-2.5h.
  • the concentration of the curcumin analogue in the methanol solution is 0.5-5 mg/ml, further preferably 0.5-1.5 mg/ml. If the time of ultraviolet irradiation is shorter than 1.5h, a sufficient amount of cis isomer cannot be generated, and if more than 2.5h, by-products will start to be produced, particularly preferably 2h of ultraviolet irradiation.
  • the nasal cavity autophagy inducer for prevention and treatment of early neurodegenerative diseases also includes oligosaccharides, chitosan oligosaccharide concentration in isotonic physiological saline solution is 0.01-0.2% (w/v) .
  • the amphiphilic surfactant is not limited, and may be any pharmaceutically acceptable, having a lipophilic group and a hydrophilic group and capable of forming a self-assembled nanoparticle structure with the autophagy-inducing drug molecule of the present invention Surfactant.
  • the amphiphilic surfactant of the present invention is a polyethylene glycol derivative, and more preferably, it is a carboxyl polyethylene glycol or polymaleic anhydride 18-carbonene-polyethylene glycol.
  • the gas is nitrogen or inert gas, preferably nitrogen.
  • Gas blowing assists the evaporation of the good solvent to ensure the formation of nanoparticles and at the same time to prevent hidden safety hazards caused by solvent residues.
  • the average particle diameter of the nasal cavity autophagy inducer is 50-200 nm, preferably 50-150 nm, and more preferably 50-120 nm.
  • the drug loading rate of the nasal nano-autophagy inducer is more than 25%.
  • the nasal autophagy inducer of the present invention further includes chitosan oligosaccharides, the concentration of chitosan oligosaccharides in isotonic saline solution is 0.01-0.2% (w/v), when used, the lyophilized powder contains Resuspend in oligosaccharide isotonic saline.
  • Chitosan oligosaccharides also known as chitosan oligosaccharides, oligomeric chitosan, molecular weight ⁇ 3200Da, with many unique features such as higher solubility than chitosan, fully soluble in water, easy to be absorbed and utilized by organisms.
  • Chitosan oligosaccharide is a non-toxic functional low molecular weight amino sugar, which is a polycation structure.
  • the present invention modifies it outside the nanoparticles to prevent the stimulation of the nasal environment by the drug; it is easy to be negatively charged with the mucosal cell surface
  • the role of the group can change the fluidity and permeability of the cell membrane and increase the absorption of nanoparticles.
  • chitooligosaccharide itself also has a certain immune regulation and neuroprotection, and its effect is 14 times that of chitosan.
  • the degree of polymerization of chitosan oligosaccharide used in the present invention is 2-20, or molecular weight ⁇ 3200Da.
  • the concentration of the oligosaccharide of the present invention in isotonic saline solution is 0.01-0.2% (w/v), if less than 0.01%, it is difficult to play a role in increasing absorption, preventing irritation, etc., if it is greater than 0.2 %, it is easy to cause negatively charged nanoparticles to aggregate.
  • nanoparticles are affected by the molecular structure of small hydrophobic molecules, and the non-covalent binding force between the molecules, due to different molecular configurations, can lead to differences in nanostructures.
  • the present invention adds amphiphilic surfactant before solvent exchange, mixes it with a hydrophobic small molecule in a certain proportion in an organic solvent, and then performs solvent exchange to obtain a carrier-free by reprecipitation method Wrapped nanoparticles.
  • the nasal cavity autophagy inducer of the present invention has no other carrier components, so its drug loading is high, low toxicity, good safety, its particle size is small and uniform, and it has high stability and long circulation time in the body.
  • the drug contained in the nasal nano-autophagy inducer is a hydrophobic organic small molecule with autophagy inducing effect, and after the improvement of its drug-forming property, it can be used for the prevention and treatment of neurodegenerative diseases.
  • Antioxidants can also be added in the present invention, and the antioxidants can be sodium metabisulfite, sodium bisulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, vitamin C, vitamin E, sulfur
  • the amount of one or more of urea is the conventional amount prescribed in pharmacy.
  • the invention can also be added with a preservative.
  • the preservative can be methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzalkonium bromide, benzalkonium
  • ammonium chloride chlorobutanol, phenethyl alcohol, thimerosal, phenylmercuric nitrate, sorbic acid, chlorhexidine, the amount of which is the conventional amount prescribed in pharmacy.
  • the osmotic pressure adjusting agent can also be added in the present invention.
  • the osmotic pressure adjusting agent may be one or more of sodium chloride, glucose, lactose, and mannitol, and the dosage is the conventional dosage prescribed in pharmacy.
  • the invention provides a self-carrying carrier-free nasal cavity autophagy inducer.
  • the nanoparticles are spherical or nearly spherical, and the surface potential is negatively charged.
  • the hydrophobic autophagy-inducing drug molecules are prepared into nanometers by reprecipitation method.
  • the particle suspension emulsion is freeze-dried to obtain nanoparticle freeze-dried powder.
  • the lyophilized powder is resuspended in isotonic saline to obtain the nasal cavity autophagy inducer, which is administered by nasal drip or nasal spray for the treatment of neurodegenerative diseases.
  • the nasal cavity autophagy inducer for preventing and treating early neurodegenerative diseases provided by the present invention
  • the delivery drug is a hydrophobic small molecule with autophagy induction effect
  • the delivery method is a self-carrying carrier-free nasal cavity nano delivery system, which uses nasal drops Or spray method, non-invasive, no carrier, no biodegradation problems and accumulation toxicity, drug loading is up to 25%, highly retains the binding ability of small molecules and targeted receptors, pH-responsive slow release in neurons
  • Molecular drugs specifically activate the autophagy pathway, thereby effectively eliminating the accumulation of toxic proteins in the brain of neurodegenerative diseases, especially have a special effect on the early olfactory disorder of the disease, and are of great significance to prevent the further deterioration of the disease.
  • the present invention provides a method for preparing a nasal cavity autophagy inducer as described in the first aspect, the method comprising the following steps:
  • the method for preparing the nasal nano-autophagy inducer of the present invention is carried out by the reprecipitation method.
  • the good solvent is converted into water (poor solvent)
  • the hydrophobic small molecules with autophagy induction effect are precipitated to form nanoparticles, and the parent is added Type surfactant, can further enhance its stability and water dispersibility.
  • This method is simple and easy to perform, does not require complicated operations and conditions, and can be carried out at room temperature.
  • the addition time of the amphiphilic surfactant should be dissolved in a good solvent together with small molecules before the nanoparticles are formed.
  • the aqueous phase is added dropwise to prepare the nanoparticles Particles.
  • gas preferably nitrogen blowing is used to remove the organic solvent.
  • the good solvent is miscible with water.
  • a good solvent refers to a solvent with a solute interaction parameter less than 0.5.
  • the good solvent is a mixture of one or more of acetone, methanol, ethanol and tetrahydrofuran, and more preferably tetrahydrofuran.
  • the water may be deionized water, distilled water or double-distilled water, etc., preferably deionized water.
  • the concentration of the hydrophobic drug molecule dissolved in the good solvent in the step (1) is 0.5-5 mg/mL, preferably 1 mg/mL.
  • the volume ratio of the good solvent to water in the step (1) is preferably (1-3): 50, for example, 1:50, 1.2:50, 1.5:50, 1.8:50, 1.9:50, 2.1:50, 2.5:50 or 2.8:50, preferably 2:50.
  • the reaction temperature in the step (1) is 20-30°C, more preferably 25°C.
  • the concentration of the amphiphilic surfactant added in the step (2) in a good solvent may be 1-10 mg/mL, such as 1 mg/mL, 2 mg/mL, 3 mg /mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL or 10 mg/mL, preferably 2 mg/mL.
  • the time of the ultrasonic dispersion in the step (2) is 3-30 min, such as 3 min, 4 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min or 28 min, more preferably 5 min.
  • Freeze-drying can significantly improve its stability.
  • the freezing temperature is lower than the low eutectic point of nanoparticles and water coexisting at 10-20°C and 10Pa pressure. 90h, preferably 48h.
  • cryoprotectants such as glucose, mannitol, lactose, NaCl, etc.
  • first add cryoprotectants such as glucose, mannitol, lactose, NaCl, etc.
  • cryoprotectants such as glucose, mannitol, lactose, NaCl, etc.
  • the nasal cavity autophagy inducer of the present invention can be administered by nasal route in the form of spray or nasal drops.
  • a nasal cavity autophagy inducer according to the present invention.
  • the drug can be absorbed through the mucosa of the olfactory area, and firstly reach the olfactory-related area of the brain and be enriched there. It is aimed at early neurodegeneration Symptoms common to sexual diseases have an olfactory dysfunction. It has a clear effect on abnormal protein aggregation in the olfactory area and other diseased areas, and is of great significance to prevent the further deterioration of neurodegenerative diseases such as early AD and PD.
  • the present invention relates to a high drug loading, high brain targeting, safe and non-toxic nasal cavity autophagy inducer delivery system, which is applied to a small hydrophobic organic molecule with autophagy induction effect, simple operation, Wide application range and strong universality.
  • a small molecule drug precursor it has the advantages of significantly improving water dispersibility and drug formation, enhancing bioavailability, reducing the frequency of administration, and reducing toxic and side effects.
  • the nano-system has no carrier, no biodegradation problems and accumulated toxicity, the drug loading rate is as high as 25% or more, and the target receptor binding ability of the original molecule is highly retained. It has pH-responsive drug release characteristics, can play a long-term, slow-release role, and has good application prospects in the treatment of nervous system diseases.
  • the nasal cavity autophagy inducer of the present invention through the nasal-brain direct pathway, efficiently delivers drugs into the brain by bypassing the blood-brain barrier along the olfactory nerve and other channels, which can avoid gastrointestinal tract degradation and liver first-pass effect, which is significant Enhance brain targeting, with high bioavailability, fast onset, good patient compliance, etc., and can reduce the accumulation of organs in the peripheral circulatory system and reduce the potential side effects of long-term use.
  • nasal preparations have no first-pass effect and reduce drug consumption.
  • nasal administration only requires nasal drip, spray, etc., which is convenient to use and non-invasive, and improves patient compliance.
  • FIG. 1 Scanning electron microscope (SEM) image of nasal nano-autophagy inducer.
  • a is the M1 nanoparticle prepared in Example 1;
  • b is the M1 nanoparticle prepared in Example 2 and loaded with the TPAAQ probe.
  • FIG. 1 Transmission electron microscopy (TEM) image of nasal nano-autophagy inducer.
  • a is the M1 nanoparticle prepared in Example 1;
  • b is the M1 nanoparticle prepared in Example 2 and loaded with the TPAAQ probe.
  • FIG. 3 Particle size distribution of nasal nano-autophagy inducer.
  • a is the M1 nanoparticle prepared in Example 1;
  • b is the M1 nanoparticle prepared in Example 2 and loaded with the TPAAQ probe.
  • FIG. 4 Nasal cavity autophagy inducer drug loading rate graph.
  • a is the M1 nanoparticle prepared in Example 1;
  • b is the M1 nanoparticle prepared in Example 2 and loaded with the TPAAQ probe.
  • Figure 10 Validation graph of the binding effect of M1 nanoparticles prepared in Example 1 to the target protein TFEB protein.
  • FIG. 13 M1 drug content in the brain and plasma of M1 nanoparticles of Example 3 after nasal brain administration.
  • FIG. Fluorescence biodistribution map of brain and organs of mice after nasal administration of M1 nanoparticles of TPAAQ fluorescent probe of Example 4.
  • Figure 15 Open field behavior diagram of Parkinson's model mice of Example 5.
  • Figure 17 Parkinson's model mice of Example 5 olfactory bulb and striatum, substantia nigra distribution map after nasal administration of M1 nanoparticles, where the arrows show M1 nanoparticles.
  • Figure 18 Test chart of expression levels of toxic proteins and autophagy pathway-related proteins in the brain olfactory bulb of the Parkinson's model mouse of Example 5.
  • Figure 19 Test graph of expression levels of toxic proteins and autophagy pathway-related proteins in the substantia nigra of the Parkinson's model mouse of Example 5.
  • Figure 22 is the nuclear magnetic spectrum of (e,e) compound with double trans configuration
  • Figure 23 is the nuclear magnetic spectrum of (e, z) cis-trans configuration compounds
  • Figure 24 is the HPLC-MS spectrum of the mixture
  • the mixed isomer is an isomer mixture of curcumin analogues of the following structural formula:
  • the curcumin analogue According to the hydrophobic properties of the curcumin analogue, it is dissolved in a good solvent and given different radiation conditions, different degrees of isomer transformation can occur, and different ratios of cis-trans isomer mixture of curcumin analogues can be obtained. Among them, the conversion rate of sunlight irradiation is the highest, but there are by-products generated; the conversion rate is followed by ultraviolet irradiation and radioactive iodine radiation, and the temperature has no effect on the structure of curcumin analogs under the condition of avoiding light.
  • the good solvent is preferably acetonitrile, methanol, ethanol, acetone, tetrahydrofuran.
  • the isomer product with a conversion rate of 30% can be obtained by UV irradiation for 2h, the preparation method is simple, and no by-product is generated.
  • a methanol solution containing 1 mg/mL of curcumin analogues seal with aluminum foil, and place them at 4°C, 25°C, and 50°C for 8 hours to obtain the reaction product 1-3; also configure the same concentration of turmeric
  • the methanol solution of the analogues was irradiated with sunlight for 2 hours, sunlight for 24 hours, ultraviolet radiation for 2 hours, and radioactive iodine 131 for 2 hours at room temperature to obtain the reaction products 4-7, to obtain various ratios of curcumin analogues. Constructor mixture.
  • the molecular weight of the product was determined by high performance liquid phase time-of-flight mass spectrometry. From Figure 20, the molecular weight of the curcumin analog and its conversion product are both 294.34. It is confirmed that the two are isomers, and further from the structure, it is Cis-trans isomer.
  • Example 1 it is preferable to prepare the product 6 with simple preparation method, easy control, and no by-product formation (UV irradiation for 2h, the isomer conversion rate of curcumin analogue is about 30%), as a representative mixed isomer M1, to conduct follow-up biological activity research.
  • the curcumin analog M1 used in this example is the product 6 prepared in Example 1.
  • Example 1 Using a scanning electron microscope (FEI Quanta200, Netherlands) according to the method in its specification, the M1 nanoparticles prepared in Example 1 were observed, and the scanning electron microscope diagram is shown in FIG. 1a. Using high-resolution transmission electron microscopy (FEI Technai F30, Netherlands) according to the method in its specification, observe the M1 nanoparticles prepared in Example 1, and its transmission electron microscopy diagram is shown in FIG. 2a.
  • the M1 nanoparticles prepared in Example 1 were subjected to dynamic light scattering measurement, and the average particle size of the M1 nanoparticles prepared in Example 1 was measured to be 62.73 nm, particle size distribution is shown in Figure 3a.
  • the M1 molecule prepared in Example 1 was dissolved in acetonitrile, and a series of concentrations of M1 in acetonitrile (6.25, 12.5, 25, 50, and 100ug/mL) were arranged in a gradient, and the absorbance was measured at 428nm using ultra high performance liquid chromatography (UPLC) To make a standard curve. Three batches of 100ug/mL M1 nanoparticles were taken, dissolved in acetonitrile, and ultrasonically measured for 5min. The same method was used to determine the M1 content in the nanoparticles using a standard curve. As shown in Figure 4a, the drug loading rate of M1 nanoparticles is (31.49 ⁇ 2.03)%.
  • the M1 small molecule prepared in Example 1 and the M1 nanoparticles prepared in Example 1 were dissolved in water and an organic solvent, as shown in FIG. 6, it can be seen that the M1 small molecule is difficult to dissolve in water and soluble in tetrahydrofuran.
  • the M1 nanoparticles can be dispersed in water and have the Tyndall effect under the laser.
  • the M1 nanoparticles prepared in Example 1 were divided into six equal parts, three parts were added to artificial nasal fluid, and three parts were added to 5% plasma. United States), then soaked in 200 ml of the same pH buffer, stirring continuously at 37 °C, at a certain time point to collect 2ml of solution from the solution. During the dialysis, 2ml of PBS was added after each sampling to keep the solution volume constant. The UV-VIS method was used to measure absorbance and calculate the amount of drug released. Each sample was tested three times, averaged, and statistically analyzed. The results are shown in Figure 7.
  • the M1 nanoparticles prepared in Example 2 have the property of slow release, and do not show the initial explosive drug release, but are released slowly and steadily, which is crucial for the application of M1 nanoparticles in vivo and can reduce the drug Toxicity, reduce drug leakage, etc.
  • M1 small molecule drugs As the concentration increases, free M1 can exhibit dose-dependent cytotoxicity, while the M1 nanoparticles in Example 2 at the same concentration have no toxic effect on N2a cells, probably due to M1 The sustained release of nanoparticles inhibits the accumulation toxicity of M1 small molecule drugs at higher concentrations.
  • the neuronal cell line PC12 cells were treated with MPP + neurotoxin, resulting in a neurotoxic cell model.
  • the M1 nanoparticles prepared in Example 1 were added to pre-treat the M1 nanoparticles group; the model control group without drug treatment, and the normal control group without MPP+neurotoxin. After incubation for 48 hours, the absorbance was measured according to literature methods. The results are shown in Figure 9.
  • the cell survival rate of the M1 nanoparticle group was significantly higher than that of the MPP+ model group.
  • the M1 nanoparticles prepared in Example 2 can be dose-dependently protected.
  • PC12 nerve cells reduce the cell damage induced by MPP+neurotoxin.
  • the target protein for the neuroprotective effect of free M1 molecule is the TFEB protein in the cytoplasm.
  • M1 promotes the dephosphorylation of the TFEB protein into the nucleus and up-regulates the expression of autophagy-related genes, thereby playing a neuroprotective role.
  • M1 nanoparticles prepared in Example 2 were added to MF7 cells overexpressing the fluorescently labeled TFEB protein, and the nucleus of TFEB was observed after 24 hours of treatment. The results are shown in FIG. 10.
  • the M1 nanoparticles prepared in Example 2 The particles can promote TFEB into the nucleus in a dose-dependent manner, confirming that the M1 nanoparticles retain the targeting properties of the original molecules.
  • GFP-LC3 protein is a green acid-responsive protein that can be degraded in acid lysosomes
  • RFP-LC3 protein is a red acid-stable protein that will not be degraded in lysosomes. Therefore, when autophagy When the pathway is activated and the flow of autophagy is smooth, the dots of red LC3 increase, indicating the induction of autophagic flow.
  • the M1 nanoparticles prepared in Example 1 were added for drug treatment, which was the M1 nanoparticle group.
  • the number of red LC3 spots was detected under a confocal microscope, as shown in FIG. 11 Compared with the control group, the number of red LC3 spots in the cells of the M1 nanoparticle group increased significantly, confirming that the M1 nanoparticles can induce the occurrence of autophagic flow. This is the chain effect of the activation of TFEB protein in the test (8).
  • Example 3 M1 nasal cavity nano-targeted delivery system with fluorescent probe TPAAQ
  • TPAAQ is a hydrophobic small molecule fluorescent probe excited at 473nm wavelength and emitted at 650nm wavelength, which can be used for in vivo fluorescence distribution monitoring of nanomaterials. Since it is also a hydrophobic small molecule, it is similar to the preparation process of the M1 nanoparticles in Example 1, and the M1 nasal nanoparticle preparation loaded with TPAAQ can be obtained by the same method.
  • the lyophilized powder is redispersed in isotonic saline, added dropwise to chitosan oligosaccharide (0.1w/v) saline solution, stirred for 0.5-2h, and reacted by physical adsorption for 1 hour, Centrifuge at a speed of 10000-150000*g for 5-30 min, discard the supernatant, remove the reactants, and purify the product to obtain a self-carrying carrier-free M1 nasal nano preparation modified with TPAAQ probes.
  • Nerve cells were cultured normally, the M1 nasal nano preparation containing the fluorescent probe TPAAQ prepared in Example 3 was added, and after 3 hours of culture, the cell uptake was observed at a specific wavelength with a laser confocal scanning microscope, as shown in Figure 12, from fluorescence The signal shows that the fluorescent probe TPAAQ-loaded M1 nasal nano preparation prepared in Example 3 can be taken up by cells in large amounts.
  • Example 4 Application of M1 nanoparticles transnasal brain targeted delivery system
  • Example 13 Six male C57BL/6J strains with a body weight of 25 g were selected and kept adaptively for 3 days.
  • the M1 nasal nano preparation prepared in Example 1 was dispersed in isotonic saline at a concentration of 5 mg/ml, and given to the nasal cavity of 15 ul of mice. After 24 hours, the brain tissue, cerebrospinal fluid and plasma were dissected out, and the brain tissue was divided into olfactory bulb and For the rest of the brain, after adding methanol to all samples to remove protein, triple quadrupole LC/MS was used to analyze the M1 drug content in the samples. The results are shown in Figure 13.
  • the M1 nasal cavity nano-targeted brain delivery system delivers M1 drugs into the olfactory bulb with extremely high targeting, and has a distribution in the cerebrospinal fluid that is three times higher than that in the plasma, and in other parts of the brain. Double the amount of drug distribution in plasma. It is confirmed that its absorption route is to reach the brain through the olfactory bulb and can be transmitted to other parts of the brain. Its transmission may be time-dependent, and it will continue to be transmitted backward through cerebrospinal fluid after 24h.
  • Example 5 Application of nasal brain targeted delivery system of M1 nanoparticles carrying TPAAQ fluorescent probe
  • mice Nine male C57BL/6J mice with a body weight of 25 g were selected and kept adaptively for 3 days. Disperse the M1 nasal cavity nano preparation with the fluorescent probe TPAAQ prepared in Example 2 in physiological saline at a concentration of 5 mg/ml, and give 15 ul of the nasal cavity of the mouse. After 24h and 48h, apply the small animal fluorescence imaging system to detect the mouse Brain in vivo fluorescence, and fluorescence signals in isolated organs such as brain, heart, liver, spleen, lung, kidney and blood, as shown in Figure 14, the brain signal is significantly stronger than other parts of the body and tissues.
  • the brain-targeted delivery system can successfully deliver the M1 nasal nanoformulation into the brain with high targeting, reducing the distribution of drugs in peripheral tissues.
  • Example 6 Therapeutic application of self-carrying carrier-free M1 nasal nano preparation in Parkinson's model mice
  • mice with a body weight of 25g were divided into three groups, the first group of wild-type group (WT group), the second group of model group (MPTP group), and the third group of model administration group (M1NPs) , 10 mice per group.
  • WT group wild-type group
  • MPTP group second group of model group
  • M1NPs model administration group
  • mice of the second and third groups were injected intraperitoneally with a 20 mg/kg dose of MPTP neurotoxin for five days to cause Parkinson's disease model.
  • the WT group and the MPTP group were given saline in the nasal cavity, and the M1NPs group was given the self-carrying carrier-free M1 nasal nano preparation, that is, the M1 nasal nano preparation prepared in Example 1 was dispersed in Infiltrated with normal saline, a fresh preparation was used, with a concentration of 1 mg/ml, and 15 ul was given to the mice nasally.
  • the medicine was administered four times a day, with a total of four doses. Behavioral observations were made two weeks after the model was made. Then mice were dissected, brain tissue was separated, and various pharmacological tests were performed.
  • the MPTP Parkinson mouse model explores symptoms such as dyskinesia and significant anxiety, which can be detected by the open field test. According to literature methods, the behavioral behavior of Parkinson's model mice in Example 5 was tested. The results are shown in Figure 15a. Compared with the wild-type mice in the control group, the trajectory of the model mice changed significantly, but after being treated with the M1 nasal nano preparation, the trajectory tended to be normal.
  • the clinical manifestations of Parkinson's disease mainly include resting tremor, bradykinesia, muscle stiffness, and posture and gait disorders.
  • the DigiGait imaging system is used on animals. By imaging animals under a transparent running belt, the software quantifies the characteristics of gait mechanics and posture index to detect the behavioral characteristics of Parkinson's model mice. The results are shown in Figure 16. Compared with wild-type mice, Parkinson's model mice have disordered gait signals, reduced coordination, and a marked reduction in the footprint of the foot. After treatment with the self-carrying carrier-free M1 nasal nano preparation, the above The pathological conditions were significantly improved, confirming that M1 nasal nano preparations can effectively improve the behavioral symptoms of Parkinson's disease.
  • mice of the treatment group were taken, dissected 24 hours after the last nasal administration of M1 nasal nano preparations, brain tissue was removed, the olfactory bulb, striatum and substantia nigra were separated, slices were fixed, and various brain regions of nanoparticles were observed under transmission electron microscope. distributed. As shown in Figure 17, it can be clearly seen that M1 nanoparticles are distributed in the brain olfactory bulb, striatum, and substantia nigra. It is confirmed that after the nasal cavity is administered with the M1 nasal nano preparation, nanoparticle prototypes can enter the brain tissue and the distribution of olfactory bulbs The largest amount suggests that its absorption pathway is olfactory nerve-mediated and spreads back to other areas.
  • Tyrosine hydroxylase is a key enzyme in the dopamine biosynthesis pathway
  • SNCA is an ⁇ -synuclein protein accumulated in the brain of Parkinson's disease
  • TFEB is a protein related to the autophagy pathway.
  • nasal administration of the M1 nano-formulation can clear the accumulation of neurotoxin in the olfactory bulb and alleviate the dopamine synthesis disorder caused by the toxin.
  • the drug effect may be related to the M1-induced TFEB protein-mediated autophagy pathway.
  • mice in the control group, model group and treatment group were sacrificed, brain tissue was taken, the olfactory bulb was separated, and the total protein was measured after homogenization.
  • the company's ⁇ -synuclein antibody detects the expression of monomer ⁇ -synuclein and aggregate ⁇ -synuclein in olfactory bulb homogenate. The results are shown in Figure 19a.
  • the present invention illustrates the detailed features and detailed methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the detailed features and detailed methods, that is, it does not mean that the present invention must rely on the detailed features and detailed methods Implementation.
  • Those skilled in the art should understand that any improvement to the present invention, equivalent replacement of optional components of the present invention, addition of auxiliary components, choice of specific modes, etc., fall within the scope of protection and disclosure of the present invention.
  • the invention discloses a nasal cavity autophagy inducer for preventing and treating early neurodegenerative diseases and a preparation method thereof, including a hydrophobic small molecule with autophagy induction effect and an amphiphilic surfactant; first, a good solvent solution is provided, Then, a self-carrying carrier-free nanoparticle suspension emulsion is prepared by a reprecipitation method, and freeze-dried to prepare a lyophilized powder; before use, the lyophilized powder is resuspended in isotonic saline to obtain.
  • the drug can be absorbed through the olfactory region mucosa under the route of nasal administration, and it first reaches the olfactory-related area of the brain and is enriched there. It is aimed at the symptoms common to early neurodegenerative diseases.
  • the disorder has a specific effect of relieving; it has a significant clearance effect on abnormal protein aggregation in the olfactory area and other disease areas, and is of great significance to prevent the further deterioration of neurodegenerative diseases such as early AD and PD.
  • the inducer of the present invention has no carrier, no biodegradation problem and accumulation toxicity, and the drug loading rate is as high as 25% or more, and has good industrial practicality.

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Abstract

一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法，包括具有自噬诱导作用的疏水性小分子以及双亲性表面活性剂；首先配置良溶剂溶液，然后通过再沉淀法制备成自携式无载体纳米颗粒悬乳液，冷冻干燥制备成冻干粉；临用前，将冻干粉在等渗生理盐水中复悬，即得。所述的鼻腔纳米自噬诱导剂，鼻腔给药途经下，药物可经嗅区黏膜吸收，首先到达并脑部的嗅觉相关区域并在此富集，针对于早期神经退行性疾病共有的症状嗅觉障碍具有特效缓解作用；对于嗅觉区和其它病变区域的异常蛋白聚集具有显著清除作用，对阻止早期AD、PD等神经退行性疾病的进一步恶化具有重要意义。所述诱导剂无载体、无生物降解问题和蓄积毒性，载药率高达25％以上。

Description

一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法 技术领域
本发明属于生物医药领域，涉及一种鼻腔纳米自噬诱导剂及其制备方法。具体来说是一种防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法。
背景技术
细胞自噬(Autophagy)，是通过吞噬自身细胞质蛋白或细胞器，并使其包被进入囊泡，与溶酶体融合进而降解其所包裹的内容物的过程，藉此实现细胞的代谢需要和细胞器的更新。神经元对自噬有严重依赖性。自噬途径的破坏，可导致神经元内泛素化的蛋白质聚集体蓄积，诱发神经元退化，继而导致神经退行性疾病的发生。
阿尔茨海默病(Alzheimer’s disease,AD)是最常见的神经退行性疾病，其两个主要特征，淀粉样β肽积累导致的老年斑块形成和Tau聚集导致的神经纤维缠结形成，可通过自噬通路激活而被有效降解。帕金森病(Parkinson’s disease,PD)在年龄60岁以上的人群中患病率为1％，主要表现为震颤等运动功能障碍，这是由多巴胺能神经元的退化引起的，其发病机制与自噬溶酶体途径的缺陷直接相关，主要病理标志物是α-突触核蛋白(a-synuclin)的异常聚集体Lewy小体。
根据多篇临床报道，嗅觉障碍是AD、PD等神经退行性疾病的早期阶段共有症状，超过百分之九十的帕金森患者都伴随有嗅觉障碍，可作为指标用于鉴别PD和非典型帕金森综合征，为PD的早期诊断及鉴别诊断提供帮助。神经退行性疾病的嗅觉障碍机制与毒性蛋白在嗅觉区域的蓄积相关。在病理情况下，嗅觉相关区域的自噬通路遭到损伤，导致蛋白无法正常降解，而蓄积压迫神经元，诱发疾病恶化进程。针对这些异常蛋白聚集，目前临床上还没有特效药物。如PD的治疗目前只能通过补充多巴胺来缓解症状，并不能对疾病起到治疗作用。且随着疾病恶化，多巴胺治疗逐渐失效，同时产生严重的副作用。因此，迫切需要开发新的治疗药物。
自噬诱导剂可通过多种机制增强神经元内自噬流的发生，如增多自噬小体生成、促进自噬小体与溶酶体的融合、增强溶酶体功能、增多溶酶体数量等，修复受损的自噬通路，促进有毒蛋白的降解，减少其蓄积而造成的神经损伤。某些自噬诱导剂 可通过结合TFEB蛋白而激活自噬通路，是一种有效的自噬诱导剂。然而，由于其是强疏水性的有机小分子，成药性差，经过口服途径给药，生物利用度低、脑内富集量少、体内循环时间短，种种问题限制了其在AD、PD等病理模型上的探索研究以及未来临床转化的应用。
随着纳米技术的快速发展，纳米制剂因具有保护药物不被破坏、延长有效药物维持时间、控制药物的释放、降低药物的毒副作用等优点，在医学和生物学领域得到了广泛的应用。报道较多的脂质体、聚合物等载体包载的纳米药物颗粒，通常载药量低于10％，且聚合物等载体在脑部的富集可能带来潜在的毒副作用，成为阻碍纳米颗粒进一步应用于临床的棘手问题。此外，血脑屏障作为一种重要的生理屏障，阻止了98％以上的药物进入脑组织，尤其是径约为100nm左右的纳米药物，更加难以穿透血脑屏障到达神经系统发挥作用。因此，亟需开发一种高载药量、高脑靶向性、安全无毒的纳米递送系统，应用于自噬小分子药物的神经系统疾病的治疗。
发明内容
本发明的主要目的在于提供一种用于鼻腔纳米自噬诱导剂及其制备方法，具体来说是一种自携式无载体鼻腔纳米自噬诱导剂及制备方法，以应用于早期神经退行性疾病。
本发明的技术方案如下：
一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂，包括具有自噬诱导作用的疏水性小分子以及双亲性表面活性剂；首先配置双亲性表面活性剂1-10mg/mL以及0.5-5mg/mL自噬诱导药物的良溶剂溶液，然后将所述良溶剂溶液向去离子水中滴加，所述良溶剂溶液与去离子水的体积比为(0.5-5)：50，滴加的同时辅以气吹，辅助良溶剂挥发；通过再沉淀法制备成粒径为50-200nm的自携式无载体纳米颗粒悬乳液，冷冻干燥制备成冻干粉；临用前，将冻干粉在等渗生理盐水中复悬，即得自携式无载体鼻腔纳米自噬诱导剂。
本发明的自噬诱导剂，能够通过诱导自噬，对异常蛋白聚集具有清除作用。
优选地，自携式无载体纳米颗粒的表面电势为-10～-60mV。更优选-10～-30mV。
优选地，所述的早期神经退行性疾病包括阿尔茨海默病和帕金森病。
所述的早期神经退行性疾病还伴随有嗅觉障碍症状，所述鼻腔纳米自噬诱导剂为嗅球部位高靶向性富集的自噬诱导剂。本发明的鼻腔纳米自噬诱导剂对于嗅觉区和其它病变区域的异常蛋白聚集具有显著清除作用。
优选地，所述疏水性小分子是以下结构式的姜黄素类似物、其顺式异构体或二 者的任意比例混合物：
Figure PCTCN2020070987-appb-000001
优选地，所述的疏水性小分子是上述姜黄素类似物及其顺式异构体的混合物，混合物中的顺式异构体的重量比占总混合物量的25-35％。
优选地，所述的顺式异构体的重量比占总混合物量的25-35％的混合物，由如下方法制得，将姜黄素类似物的甲醇溶液，加以紫外照射1.5-2.5h。
优选地，姜黄素类似物的甲醇溶液浓度为0.5-5mg/ml，进一步优选为0.5-1.5mg/ml。紫外照射的时间如果短于1.5h，则不能生成足够量的顺式异构体，如果多余2.5h，则会开始产生副产物，特别优选为紫外照射2h。
优选地，所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂，还包括寡聚糖，壳寡糖在等渗生理盐水溶液浓度为0.01-0.2％(w/v)。
本发明中，双亲性表面活性剂不受限制，可以是任何药学上可以接受、具有亲油性基团和亲水性基团且能与本发明的自噬诱导药物分子形成自组装纳米颗粒结构的表面活性剂。作为优选，本发明所述的双亲性表面活性剂为聚乙二醇衍生物，进一步优选，为羧基聚乙二醇或聚马来酸酐18碳烯-聚乙二醇。
作为优选，所述的气体为氮气或惰性气体，优选为氮气。气体吹，辅助良溶剂挥发，以保证纳米颗粒的形成，同时防止溶剂残留造成的安全性隐患。
优选地，所述鼻腔纳米自噬诱导剂的平均粒径为50-200nm、优选50-150nm、更优选50-120nm。
优选地，所述鼻腔纳米自噬诱导剂的载药率为25％以上。
优选地，本发明所述的鼻腔纳米自噬诱导剂还包括壳寡糖，壳寡糖在等渗生理盐水溶液浓度为0.01-0.2％(w/v)，使用时，冻干粉在含壳寡糖的等渗生理盐水中复悬。
壳寡糖，又叫壳聚寡糖、低聚壳聚糖，分子量≤3200Da，具有壳聚糖所没有的较高溶解度、可全溶于水、容易被生物体吸收利用等诸多独特功能。壳寡糖是一种无毒的功能性低分子量氨基糖，为多聚阳离子结构,本发明将其修饰在纳米颗粒外， 可防止药物对鼻腔内环境的刺激；易与粘膜细胞表面带负电荷的基团作用,可改变细胞膜的流动性和通透性，增加纳米颗粒的吸收，此外，壳寡糖自身还具有一定的免疫调节和神经保护作用，其效果是壳聚糖的14倍。
本发明所用的壳寡糖聚合度为2-20，或分子量≤3200Da。
优选地，本发明寡糖在等渗生理盐水溶液浓度为0.01-0.2％(w/v)，如果少于0.01％，则难以起到在增加吸收，防止刺激等方面中的作用，如果大于0.2％，则容易导致带负电的纳米颗粒聚集。
将冻干粉在等渗生理盐水中复悬，其冻干粉浓度可根据需要配置。优选为3-7mg/ml。
纳米颗粒的形成，受到疏水性小分子的分子结构影响，分子间为非共价结合力，因分子构型不同可导致纳米结构有差异。为增强纳米颗粒的稳定性，本发明在溶剂交换前，加入双亲性表面活性剂，与疏水性小分子以一定比例在有机溶剂中混匀后，再进行溶剂交换，通过再沉淀法得到无载体包裹的纳米颗粒。
本发明的鼻腔纳米自噬诱导剂无其它载体成分，因此其载药量高、低毒、安全性好，其粒径小且均匀，并且稳定性高、在体内循环时间长。
本发明中，鼻腔纳米自噬诱导剂所含药物为具有自噬诱导作用的疏水性有机小分子，在改善其成药性后，能够被用于神经退行性疾病的预防及治疗。
本发明还可以加入抗氧剂，所述的抗氧剂可以是焦亚硫酸氢钠、亚硫酸氢钠、亚硫酸钠、硫代硫酸钠、半胱氨酸盐酸盐、维生素C、维生素E、硫脲中的一种或多种，其用量为药剂学上所规定的常规用量。
本发明还可以加入防腐剂，所述的防腐剂可以是对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯、对羟基苯甲酸丁酯、苯扎溴铵、苯扎氯铵、三氯叔丁醇、苯乙醇、硫柳汞、硝酸苯汞、山梨酸、洗必泰中的一种或多种，其用量为药剂学上所规定的常规用量。
本发明还可以加入渗透压调节剂，所述的渗透压调节剂可以是氯化钠、葡萄糖、乳糖、甘露醇中的一种或多种，其用量为药剂学上所规定的常规用量。
本发明提供的一种自携式无载体鼻腔纳米自噬诱导剂，所述纳米颗粒呈球形或近似球形，表面电势为负电，是将疏水性的自噬诱导药物分子通过再沉淀法制备成 纳米颗粒悬乳液，冷冻干燥得到纳米颗粒冻干粉。临用前，将冻干粉复悬于等渗生理盐水中，即得鼻腔纳米自噬诱导剂，通过滴鼻或鼻喷雾的方式给药，用于神经退行性疾病的治疗。
本发明提供的用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂，递送药物为具有自噬诱导作用的疏水性小分子，递送方式为自携式无载体鼻腔纳米递送系统，采用滴鼻或喷雾方式，无创、无载体、无生物降解问题和蓄积毒性，载药量高达25％以上，高度保留小分子与靶向受体的结合能力，可pH响应性地在神经元内缓释小分子药物，特异性激活自噬通路，从而高效清除神经退行性疾病的脑内毒性蛋白累积，尤其对疾病早期的嗅觉障碍有特殊效果，对防止疾病的进一步恶化具有重要意义。
在第二方面，本发明提供一种如第一方面所述的鼻腔纳米自噬诱导剂的制备方法，所述方法包括如下步骤：
1)首先配置双亲性表面活性剂1-10mg/mL以及0.5-5mg/mL疏水性小分子药物的良溶剂溶液，然后将所述良溶剂溶液往去离子水中逐滴滴加：所述良溶剂溶液与去离子水的体积比为(0.5-5)：50，滴加的同时辅以气体吹，辅助良溶剂挥发，
2)通过再沉淀法制备成粒径为50-200nm的自携式无载体纳米颗粒悬乳液，冷冻干燥制备成冻干粉；
3)临用前，将冻干粉复悬于等渗生理盐水中，即得鼻腔纳米自噬诱导剂。
本发明制备鼻腔纳米自噬诱导剂的方法，通过再沉淀法进行，良溶剂转换进入水(不良溶剂)中的时候，将具有自噬诱导作用的疏水性小分子析出形成纳米颗粒，且加入双亲型表面活性剂，可进一步增强其稳定性和水分散性。本方法简单易行，不需要复杂的操作和条件，室温下即可进行。
需要强调的是，本发明中，双亲型表面活性剂的加入时间，应在纳米颗粒形成以前，即与小分子一同溶解于良溶剂中，混合均匀后，再逐滴滴加入水相，制备纳米颗粒。滴加时，辅以气体(优选为氮气)吹，用以除净有机溶剂。此方法不同于先形成纳米颗粒、后加两亲性表面活性剂作表面修饰的方法，产物亦不相同。
本发明制备鼻腔纳米自噬诱导剂的方法中，所述良溶剂与水互溶。根据Flory-Krigboum稀溶液理论，良溶剂是指与溶质相互作用参数小于0.5的溶剂。
优选地，所述良溶剂为丙酮、甲醇、乙醇和四氢呋喃中一种或多种的混合，更 优选为四氢呋喃。
本发明制备鼻腔纳米自噬诱导剂的方法中，所述水可以是去离子水、蒸馏水或双蒸水等，优选去离子水。
本发明制备鼻腔纳米自噬诱导剂的方法中，所述步骤(1)中疏水性药物分子溶于良溶剂中的浓度为0.5-5mg/mL,优选为1mg/mL。
本发明制备鼻腔纳米自噬诱导剂的方法中，所述步骤(1)中良溶剂与水的体积比优选为(1-3)：50，例如1：50、1.2：50、1.5：50、1.8：50、1.9：50、2.1：50、2.5：50或2.8：50，优选为2：50。
优选地，所述步骤(1)中反应温度为20-30℃，更优选为25℃。
本发明制备鼻腔纳米自噬诱导剂的方法中，所述步骤(2)中加入的双亲型表面活性剂在良溶剂的浓度可以为1-10mg/mL，例如1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL或10mg/mL，优选为2mg/mL。
优选地，所述步骤(2)中超声分散的时间为3-30min，例如3min、4min、5min、8min、10min、15min、20min、25min或28min，更优选为5min。
自组装纳米颗粒于水溶液中长期储存可能发生聚集，将其冷冻干燥，可明显提高其稳定性，冷冻温度低于纳米颗粒与水共存的低共熔点10-20℃、10Pa压力下冷冻干燥24-90h，优选为48h。
为避免冻干后纳米颗粒聚集和粒径变化，先加入冷冻保护剂，如葡萄糖、甘露醇、乳糖、NaCl等，在冷冻时促进大量微小冰晶形成，或使冻干品呈疏松状态，以利于纳米颗粒保持原形态并易于在水中再分散。
本发明所述的鼻腔纳米自噬诱导剂可采用喷雾或滴鼻形式通过鼻腔途径给药。
本发明的有益效果为：
(1)本发明涉及的一种鼻腔纳米自噬诱导剂，鼻腔给药途经下，药物可经嗅区黏膜吸收，首先到达并脑部的嗅觉相关区域并在此富集，针对于早期神经退行性疾病共有的症状嗅觉障碍具有特效缓解作用；对于嗅觉区和其它病变区域的异常蛋白聚集具有显著清除作用，对阻止早期AD、PD等神经退行性疾病的进一步恶化具有重要意义。
(2)本发明涉及的一种高载药量、高脑靶向性、安全无毒的鼻腔纳米自噬诱导 剂递送系统，应用于具有自噬诱导作用的疏水性有机小分子，操作简单、适用范围广且普适性强。相比于具有小分子药物前体，具有显著提升水分散性及成药性、增强生物利用度、降低给药频次、降低毒副作用等优势。相比于传统的脂质体或聚合物纳米载药系统，该纳米系统无载体、无生物降解问题和蓄积毒性，载药率高达25％以上，高度保留原分子的靶向受体结合能力，具有pH响应性的药物释放特性，可长效、缓释发挥作用，在神经系统疾病的治疗方面具有良好的应用前景。
(2)本发明涉及的鼻腔纳米自噬诱导剂，通过鼻脑直接通路，将药物沿嗅神经等途径绕过血脑屏障高效递送入脑，可避免胃肠道降解和肝脏首过效应，显著增强脑靶向性，具有生物利用度高、起效快、患者顺应性好等特点，并可降低外周循环系统脏器富集，降低长期服用的潜在副作用。与口服药相比，鼻腔制剂无首过效应，降低药物损耗。与静脉注射相比，鼻腔给药仅需滴鼻、喷雾等方式，使用方便无创、患者依从性提高。尤其是对于长期服药的神经退行性疾病患者，可以减轻病人的痛苦、患者顺应性好，便于自己用药，并降低长期用药带来的风险，具有良好的应用前景。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1.鼻腔纳米自噬诱导剂扫描电镜(SEM)图。其中a为实施例1制备的M1纳米颗粒；b为实施例2制备的载有TPAAQ探针的M1纳米颗粒。
图2.鼻腔纳米自噬诱导剂透射电镜(TEM)图。其中a为实施例1制备的M1纳米颗粒；b为实施例2制备的载有TPAAQ探针的M1纳米颗粒。
图3.鼻腔纳米自噬诱导剂粒径分布图。其中a为实施例1制备的M1纳米颗粒；b为实施例2制备的载有TPAAQ探针的M1纳米颗粒。
图4.鼻腔纳米自噬诱导剂药物装载率图。其中a为实施例1制备的M1纳米颗粒；b为实施例2制备的载有TPAAQ探针的M1纳米颗粒。
图5.实施例1制备的M1纳米颗粒表面电势图。
图6.实施例1制备的M1纳米颗粒丁达尔效应光学表征图。
图7.实施例1制备的M1纳米颗粒pH响应性药物释放曲线图。
图8.实施例1制备的M1纳米颗粒与小分子M1药物的细胞毒性图。
图9.实施例1制备的M1纳米颗粒的体外神经保护作用图。
图10.实施例1制备的M1纳米颗粒与靶蛋白TFEB蛋白结合效果验证图。
图11.实施例1制备的M1纳米颗粒诱导细胞自噬流荧光图。
图12.实施例2制备的载有TPAAQ荧光探针的M1纳米颗粒细胞摄取图。
图13.实施例3的M1纳米颗粒经鼻脑给药后小鼠脑和血浆中M1药物含量图。
图14.实施例4的TPAAQ荧光探针的M1纳米颗粒经鼻给药后的小鼠的脑及脏器荧光生物分布图。
图15.实施例5的帕金森模型小鼠旷场行为图。
图16.实施例5的帕金森模型小鼠步态行为图。
图17.实施例5的帕金森模型小鼠鼻腔给予M1纳米颗粒后的嗅球及纹状体、黑质分布图，其中箭头所示为M1纳米颗粒.
图18.实施例5的帕金森模型小鼠脑嗅球部位毒性蛋白及自噬通路相关蛋白表达含量测试图。
图19.实施例5的帕金森模型小鼠脑黑质部位毒性蛋白及自噬通路相关蛋白表达含量测试图。
图20实施例1中质谱图中分子量为294.34的化合物图谱
图21为实施例1不同条件下的姜黄素类似物转化率。
图22为(e,e)双反式构型化合物核磁谱；
图23为(e,z)顺反式构型化合物核磁谱；
图24为混合物HPLC-MS谱
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，一下实施例仅为发明的优选实施例，以便于更好地理解本发明，因而不应视为限定本发明的范围。对于本领域的技术人员来说，本发明可以有各种更改和变化，凡在本发明的精神和原则之内，所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。下述实施例中的实验方法，如无特殊说明，均为常规方法； 所用实验材料，如无特殊说明，均为自常规生化试剂厂商购买得到的。
实施例1混合异构体M1的制备
该混合异构体为以下结构式的姜黄素类似物的异构体混合物：
Figure PCTCN2020070987-appb-000002
根据申请人的计算机模拟结果，在混合物中，顺式同分异构体的比例越高，产物的生物活性越强。但实际中，产物越高，副产物的产率也越高。
在本实施例中，提供了一种可获得30％转化率的异构体产物，制备方法简单，且无副产物生成的方法。
根据姜黄素类似物的疏水性质，其溶解于良溶剂中，给予不同辐射条件，可发生不同程度的异构体转化，获得不同比例的姜黄素类似物顺反异构体混合物。其中，日光照射的转化率最高，但有副产物生成；转化率其次为紫外照射、放射性碘辐射，而避光条件下，温度对姜黄素类似物的结构无影响。
进一步地，良溶剂优选为乙腈、甲醇、乙醇、丙酮、四氢呋喃。
进一步地，UV照射2h可获得30％转化率的异构体产物，制备方法简单，无副产物生成。
姜黄素类似物的顺反异构体混合物M1(Mixture 1)制备方法
配置含有1mg/mL的姜黄素类似物的甲醇溶液，用铝箔纸密封后，分别于4℃、25℃及50℃的环境下放置8h，得到反应产物1-3；另配置同样同浓度的姜黄素类似物的甲醇溶液，室温下，分别加以日光照射2h、日光照射24h、紫外照射2h、放射性碘131辐射2h，得到反应产物4-7，以获得各种比例的姜黄素类似物顺反异构体混合物。
结果
(1)转化产物的分子量鉴定
应用高效液相联用飞行时间质谱仪测定产物的分子量，由图20可知，姜黄素类 似物及其转化产物的分子量均为294.34，确认二者为同分异构体，进一步由结构可知，为顺反异构体。
(2)顺反异构体转化率的测定：
应用高效液相色谱法(HPLC)，于384nm最大吸收波长下，测定样品溶液1-7中，姜黄素类似物的顺反异构体转化率。结果如图21所示，反应产物1-3中均无有新物质产生，说明未发生姜黄素类似物异构体转化(图21a-c)。反应产物4,日光照射2h后，姜黄素类似物含量有73.91％转化为其异构体(图21d)；而日光照射24h，反应产物5除姜黄素类似物异构体之外，还有许多复杂产物生成(图21e)。反应产物6，紫外照射2h，姜黄素类似物异构体转化率为29.59％(图21f)。反应产物7，放射性碘131辐射条件2h下，姜黄素类似物异构体转化率为27.91％(图21g)。
根据实施例1测试结果，优选制备方法简单、便于控制、无副产物生成的产物6(紫外照射2h，姜黄素类似物的异构体转化率约为30％)，作为代表性混合异构体M1，进行后续生物活性研究。
实施例2：姜黄素类似物M1鼻腔纳米制剂的制备
本实施例所用的姜黄素类似物M1，为实施例1中制备的产物6。
配置含有1mg/mL的M1及2mg/mL的羧基聚乙二醇的四氢呋喃溶液5mL，混匀，取200μL所述M1分子溶液，逐滴往5mL去离子水中滴加，滴加的同时辅以氮吹，以除净有机溶剂。25℃下搅拌10分钟后静置，获取M1自携式无载体纳米颗粒悬乳液，冷冻干燥形成冻干粉。临用前，将冻干粉重新分散于等渗的生理盐水中，将冻干粉重新分散于等渗的生理盐水中，逐滴加入至壳寡糖(0.1w/v)生理盐水溶液里，搅拌0.5-2h，即得自携式无载体M1鼻腔纳米制剂。
结果
(1)M1纳米颗粒形态、粒径及电位分布的测定
使用扫描电镜(FEI Quanta200，荷兰)按照其说明书中的方法，观察实施例1中制备的M1纳米颗粒，其扫描电镜图如图1a所示。使用高分辨透射电镜(FEI Technai F30，荷兰)按照其说明书中的方法，观察实施例1中制备的M1纳米颗粒， 其透射电镜图如图2a所示。使用激光粒度仪(马尔文，英国)按照其说明书中的方法，对实施例1中制备的M1纳米颗粒进行动态光散射测定，测得实施例1中制备的M1纳米颗粒的平均粒径为62.73nm，粒径分布如图3a所示。
(2)M1纳米颗粒载药率的测定
将实施例1中制备的M1分子溶解于乙腈中，梯度配置系列浓度的M1的乙腈溶液(6.25，12.5，25，50及100ug/mL)，应用超高效液相色谱(UPLC)于428nm测定吸光度，做出标准曲线，。取三批次100ug/mL的M1纳米颗粒，分别溶解于乙腈中，超声5min，同法测定，利用标准曲线计算纳米颗粒中的M1含量。如图4a所示，M1纳米颗粒的载药率为(31.49±2.03)％。
(3)M1颗粒的表面电势测定
使用激光粒度仪按照其说明书中的方法，对实施例1中制备的M1纳米颗粒进行
Zeta-电位分析，测得实施例1中制备的M1纳米颗粒平均电荷为-56.5mV，其
分布如图5所示。(4)M1颗粒的光学表征测定
将实施例1中制备的M1小分子及实施例1中制备的M1纳米颗粒分别溶解于水和有机溶剂中，如图6所示，可见M1小分子难以溶解于水，可溶于四氢呋喃中，而M1纳米颗粒可分散于水中，在激光下具有丁达尔效应。
(5)M1纳米颗粒药物释放曲线测定
将实施例1制备的M1纳米颗粒等分为六份，3份加入人工鼻液中，3份加入5％血浆，分别装入透析袋中，分散并稀释，分别加入透析袋(3500分子量，
Figure PCTCN2020070987-appb-000003
美国)中，接着浸泡在200毫升的相同pH的缓冲液里，37℃不断搅拌，于一定的时间点从溶液中收集2ml溶液。在透析过程中，每次取样后补充2ml PBS，使溶液体积保持恒定。采用UV-VIS法测定吸光度，计算药物释放量。每个样本进行3次测试，取平均值，统计分析，结果如图7所示。可见，实施例2制备的M1纳米颗粒具有缓慢释放的性质，没有显示初始爆发性药物释放，而是缓慢而稳定地释放，这对于M1纳米颗粒在体内的应用是至关重要的，可降低药物毒性，减少药物泄漏等。
(6)细胞毒性试验
按照文献(《细胞培养》，司徒镇强，世界图书出版公司，1996年)中的方法培养神经瘤母细胞N2a细胞，然后加入实施例1制备的M1纳米颗粒继续培养，加药24小时后按照文献(《细胞培养》，司徒镇强，世界图书出版公司，1996年)中的方 法(MTT法)检测细胞存活率，此为M1纳米颗粒组。用与M1纳米颗粒组含相同浓度的游离M1按相同方法处理N2a细胞的组为阳性对照组；不含疏水性药物的空白培养基培养的N2a细胞作为阴性对照组，其中以阴性对照组中细胞的存活率按100％计算。结果如图8所示，随着浓度的升高，游离M1可体现出剂量依赖性的细胞毒性，而实施例2中的M1纳米颗粒在相同浓度下，对N2a细胞无毒性作用，可能由于M1纳米颗粒的缓释作用，在较高浓度时抑制了M1小分子药物的蓄积毒性。
(7)M1纳米颗粒的神经保护作用测定
利用MPP+神经毒素处理神经细胞株PC12细胞，造成神经毒性细胞模型。在造模前6h，加入实施例1中制备的M1纳米颗粒预处理，为M1纳米颗粒组；不加药物处理的为模型对照组，未加MPP+神经毒素的为正常对照组。造模后继续培养48h，依照文献方法测吸光度，结果如图9所示，M1纳米颗粒组的细胞存活率比MPP+模型组显著升高，实施例2中制备的M1纳米颗粒可剂量依赖性保护PC12神经细胞，降低MPP+神经毒素对其诱导的细胞损伤。
(8)M1纳米颗粒与靶蛋白TFEB蛋白的结合效应测定
游离M1分子神经保护作用的靶蛋白是细胞浆内的TFEB蛋白，M1通过促进TFEB蛋白去磷酸化进入细胞核中，而上调自噬相关基因的表达，从而起到神经保护的作用。本实验中，在过表达荧光标记TFEB蛋白的MF7细胞中加入实施例2中制备的M1纳米颗粒，处理24h后观测TFEB入核情况，结果如图10所示，实施例2中制备的M1纳米颗粒可剂量依赖性地促进TFEB入核，证实M1纳米颗粒保留了原分子的靶向特性。
(9)M1纳米颗粒对细胞自噬流的诱导作用
当自噬被诱导时，其标志性蛋白LC3的表达量增多。通过构建表达GFP-RFP-LC3的慢病毒并感染N2a细胞，可在共聚焦荧光显微镜下检测药物对自噬流的作用。其中，GFP-LC3蛋白为绿色的酸响应性蛋白，可在酸性溶酶体内被降解，而RFP-LC3蛋白为红色的酸稳定蛋白，在溶酶体中不会被降解，因此，当自噬通路被激活，自噬流通畅时，红色LC3的点增多，显示自噬流的诱导作用。在感染了慢病毒的N2a细胞中，加入实施例1中制备的M1纳米颗粒进行药物处理，为M1纳米颗粒组，24h后，于共聚焦显微镜下检测红色LC3点的数量，如图11所示，与对照组相比，M1纳米颗粒组细胞内红色LC3点的数量显著增加，证实M1纳米颗粒能够诱导自噬流的 发生。这是测试(8)结果中TFEB蛋白被激活后的连锁效应。
实施例3：载荧光探针TPAAQ的M1鼻腔纳米制剂脑靶向性递送系统
TPAAQ是一种473nm波长激发、650nm波长发射的疏水性小分子荧光探针，可用于纳米材料的体内荧光分布监测。因其亦是疏水性小分子，故与实施例1的M1纳米颗粒制备过程相似，可同法获得载有TPAAQ的M1鼻腔纳米制剂。
配置含有1mg/mL的M1和2mg/mL的TPAAQ的四氢呋喃溶液5mL，混匀，取200μL所述M1分子溶液，逐滴加入到5mL去离子水中，同时辅以氮吹，以除净有机溶剂。25℃下磁力搅拌10分钟后静置，获取载荧光探针TPAAQ的M1自携式无载体纳米颗粒悬乳液，冷冻干燥形成冻干粉。临用前，将冻干粉重新分散于等渗的生理盐水中，逐滴加入至壳寡糖(0.1w/v)生理盐水溶液里，搅拌0.5-2h，利用物理吸附作用反应1小时，以10000-150000*g的转速离心5-30min，弃置上清液，除去反应物，将产物纯化即得壳寡糖修饰载有TPAAQ探针的自携式无载体M1鼻腔纳米制剂。
结果
(1)载荧光探针TPAAQ的M1纳米颗粒形态、粒径分布的测定
使用扫描电镜(FEI Quanta200，荷兰)按照其说明书中的方法，观察实施例1中制备的M1纳米颗粒，其扫描电镜图如图1b所示。使用高分辨透射电镜(FEI Technai F30，荷兰)按照其说明书中的方法，观察实施例1中制备的M1纳米颗粒，其透射电镜图如图2b所示。使用激光粒度仪(马尔文，英国)按照其说明书中的方法，对实施例1中制备的M1纳米颗粒进行动态光散射测定，测得实施例1中制备的M1纳米颗粒的平均粒径为178.2nm，粒径分布图如图3b所示。
(2)载荧光探针TPAAQ的M1纳米颗粒载药率的测定
利用实施例1的测试(2)所做M1乙腈溶液标准曲线，，取三批次100ug/mL的载荧光探针TPAAQ的M1纳米颗粒，分别溶解于乙腈中，超声5min，同法测定，利用标准曲线计算纳米颗粒中的M1含量。如图4b所示，载荧光探针TPAAQ的M1纳米颗粒的载药率为(26.95±1.50)％。
(3)细胞摄取实验
神经细胞正常培养，加入实施例3中制备的载荧光探针TPAAQ的M1鼻腔纳米制剂，培养3h后，用激光共聚焦扫描显微镜于特定波长下观测细胞摄取情况，如图 12所示，从荧光信号可见，实施例3中制备的载荧光探针TPAAQ的M1鼻腔纳米制剂可被细胞大量摄取。
实施例4：M1纳米颗粒经鼻脑靶向递送系统的应用
取体重为25g的雄性C57BL/6J品系小鼠6只，适应性饲养3天。将实施例1中制备的M1鼻腔纳米制剂分散于等渗生理盐水中，浓度5mg/ml，给予小鼠鼻腔15ul，24h后解剖取出脑组织、脑脊液和血浆，并将脑组织分为嗅球部分和大脑其余部分，所有样品分别加甲醇除蛋白后，应用三重四极杆液质联用色谱，分析样品内M1药物含量。结果如图13所示，M1鼻腔纳米制剂脑靶向递送系统极高靶向性地将M1药物递送入嗅球，并在脑脊液中有高于血浆内含量三倍的分布，且在大脑其它部位有血浆两倍量的药物分布。证实其吸收途径为经过嗅球而到达大脑，并可传递到大脑其它部位。其传递可能为时间依赖性，24h后会继续经由脑脊液向后传递。
实施例5：载有TPAAQ荧光探针的M1纳米颗粒经鼻脑靶向递送系统的应用
取体重为25g的雄性C57BL/6J品系小鼠9只，适应性饲养3天。将实施例2中制备的载荧光探针TPAAQ的M1鼻腔纳米制剂分散于生理盐水中，浓度5mg/ml，给予小鼠鼻腔15ul，分别于24h、48h后应用小动物荧光成像系统，检测小鼠脑部在体荧光，及离体脑、心、肝、脾、肺、肾等脏器和血液中的荧光信号，结果如图14所示，脑部信号显著强于身体其它部位和组织，提示实施例3脑靶向性递送系统可成功将M1鼻腔纳米制剂高靶向性递送入脑，降低药物在外周组织的分布。
实施例6：自携式无载体M1鼻腔纳米制剂在帕金森模型小鼠的治疗应用
取体重为25g的雄性C57BL/6J品系小鼠30只，分三组，第一组野生型组(WT组),第二组模型组(MPTP组)，第三组模型给药组(M1NPs)，每组10只小鼠。按照文献方法，将第二、三组小鼠持续腹腔注射20mg/kg剂量MPTP神经毒素五天，造成帕金森疾病模型。造模同期做给药处理，WT组、MPTP组小鼠鼻腔给予生理盐水，M1NPs组鼻腔给予自携式无载体M1鼻腔纳米制剂，即，将实施例1中制备的M1鼻腔纳米制剂分散于等渗生理盐水中，临用新制，浓度1mg/ml，鼻腔给予小鼠15ul。间隔一天给药，共给药四次，造模结束两周后进行行为学观测，后解剖小鼠，分离脑组织，做各项药理学检测。
结果
(1)旷场试验检测帕金森模型小鼠行为学表现
MPTP帕金森小鼠模型有探索运动障碍和显著焦虑等症状，可由旷场试验检测。根据文献方法，检测实施例5中帕金森模型小鼠的行为学表现。结果如图图15a所示，与对照组野生型小鼠相比，模型小鼠运动轨迹显著改变，而经M1鼻腔纳米制剂治疗后，轨迹趋于正常。统计数据显示，对比野生型小鼠，模型小鼠在矿场内的运动时间(图15b)、平均速度(图15c)和区域穿梭次数(图15d)等均显著减少，而经过自携式无载体M1鼻腔纳米制剂治疗后，上述病变情况均显著改善，证实M1鼻腔纳米制剂可有效缓解帕金森疾病模型的行为学症状。
(2)步态试验检测帕金森模型小鼠行为学表现
帕金森病的临床表现主要包括静止性震颤、运动迟缓、肌强直和姿势步态障碍等。在动物上采用DigiGait成像系统，通过在透明跑带下方对动物成像，软件量化步态力学和姿势指数等特征，可检测帕金森模型小鼠的行为学特征。结果如图16所示，对比野生型小鼠，帕金森模型小鼠的步态信号紊乱、协调性降低、脚掌着地面积显著减小，而经过自携式无载体M1鼻腔纳米制剂治疗后，上述病变情况均显著改善，证实M1鼻腔纳米制剂可有效改善帕金森疾病的行为学症状。
(3)组织电镜检测帕金森模型小鼠鼻腔给予M1纳米颗粒后的嗅球及纹状体、黑质分布
取三只治疗组小鼠，于最后一次鼻腔给予M1鼻腔纳米制剂24h后解剖，取出脑组织，分离嗅球、纹状体及黑质部位，固定切片，于透射电镜下观察纳米颗粒的脑各区域分布。如图17所示，可明显看到M1纳米颗粒在脑嗅球、纹状体及黑质部位内均有分布，证实鼻腔给予M1鼻腔纳米制剂后，可以纳米颗粒原型进入脑组织，而嗅球的分布量最多，提示其吸收途径为嗅神经介导，并向后扩散至其它区域。
(4)Western Blot法检测脑嗅球部位毒性蛋白及自噬通路相关蛋白表达含量
酪氨酸羟化酶(TH)是多巴胺生物合成途径的关键酶，SNCA是帕金森病脑内蓄积的α-突触核蛋白，TFEB是自噬通路相关蛋白。各组小鼠给药结束后，将对照组、模型组和治疗组的小鼠处死，取脑组织，分离嗅球部位，组织匀浆后测定蛋白总量，按照Western Blot的常规步骤，应用Santa Cruz公司的以上蛋白抗体，检测嗅球匀浆液中以上各蛋白的表达量。结果如图18a所示，统计表明，TH蛋白在模型组显著降低，而治疗组显著升高(图17b)，证实M1鼻腔纳米制剂可缓解毒素造成的脑 内多巴胺的合成障碍。与对照组和模型组相比，治疗组的脑嗅球中TFEB蛋白量显著升高(图18c)，证实M1鼻腔纳米制剂激活了嗅球内的TFEB蛋白，这是其药效的可能机制之一。此外，SNCA毒性蛋白含量在模型组中升高，而治疗组有降低趋势(图18d)，证明了M1鼻腔纳米制剂能够清除嗅球内的毒性蛋白。综上所述，鼻腔给予M1纳米制剂后，能够对嗅球内的神经毒素的蓄积进行清除，缓解毒素造成的多巴胺合成障碍，该药效可能与M1诱导TFEB蛋白介导的自噬通路相关。
(5)Western Blot法检测脑黑质部位毒性蛋白及自噬通路相关蛋白表达含量
各组小鼠给药结束后，将对照组、模型组和治疗组的小鼠处死，取脑组织，分离嗅球部位，组织匀浆后测定蛋白总量，按照Western Blot的常规步骤，应用Santa Cruz公司的α-突触核蛋白抗体，检测嗅球匀浆液中单体α-突触核蛋白以及聚集体α-突触核蛋白的表达量。结果如图19a所示，统计表明，单体及聚集体α-突触核蛋白在模型组均显著升高，而治疗组均有显著降低趋势(图19b及19c)，证实M1鼻腔纳米制剂可有效清除黑质病变部位的毒性蛋白蓄积,从而对疾病起到治疗作用。
申请人声明，本发明通过上述实施例来说明本发明的详细特征以及详细方法，但本发明并不局限于上述详细特征以及详细方法，即不意味着本发明必须依赖上述详细特征以及详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明选用组分的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
工业实用性
本发明公开了一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法，包括具有自噬诱导作用的疏水性小分子以及双亲性表面活性剂；首先配置良溶剂溶液，然后通过再沉淀法制备成自携式无载体纳米颗粒悬乳液，冷冻干燥制备成冻干粉；临用前，将冻干粉在等渗生理盐水中复悬，即得。本发明的鼻腔纳米自噬诱导剂，鼻腔给药途经下，药物可经嗅区黏膜吸收，首先到达并脑部的嗅觉相关区域并在此富集，针对于早期神经退行性疾病共有的症状嗅觉障碍具有特效缓解作用；对于嗅觉区和其它病变区域的异常蛋白聚集具有显著清除作用，对阻止早期 AD、PD等神经退行性疾病的进一步恶化具有重要意义。本发明诱导剂无载体、无生物降解问题和蓄积毒性，载药率高达25％以上，具有良好的工业实用性。

Claims (13)

  1. 一种自噬诱导剂，其特征在于，包括的疏水性小分子姜黄素类似物的顺式异构体或顺反异构体混合物，其中，所述的顺式异构体或顺反异构体混合物为以下结构式的姜黄素类似物通过在日光、紫外或放射性辐射照射产生：
    Figure PCTCN2020070987-appb-100001
  2. 如权利要求1所述的一种自噬诱导剂，其特征在于，混合物中的顺式异构体的重量比占总混合物量的25-35％。
  3. 如权利要求1所述的一种自噬诱导剂，其特征在于所述的自噬诱导剂表面装饰,药学上可以接受、具有亲油性基团和所述亲水性基团且能与所述的自噬诱导药物分子形成自组装纳米颗粒结构的表面活性剂。
  4. 如权利要求1或3所述的一种自噬诱导剂，其特征在于，用于早期神经退行性疾病治疗，包括阿尔茨海默病和帕金森病。
  5. 如权利要求4所述的一种自噬诱导剂，其特征在于，所述的早期神经退行性疾病还伴随有嗅觉障碍症状，所述鼻腔纳米自噬诱导剂为嗅球部位高靶向性富集的自噬诱导剂。
  6. 如权利要求1至5任一项所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂，剂型为鼻腔喷雾剂或滴鼻剂。
  7. 一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，其特征在 于：包括具有自噬诱导作用的疏水性小分子以及双亲性表面活性剂；首先配置双亲性表面活性剂1-10mg/mL以及0.5-5mg/mL自噬诱导药物的良溶剂溶液，然后将所述良溶剂溶液向去离子水中滴加，所述良溶剂溶液与去离子水的体积比为(0.5-5)：50，滴加的同时辅以气吹，辅助良溶剂挥发；通过再沉淀法制备成粒径为50-200nm的自携式无载体纳米颗粒悬乳液，冷冻干燥制备成冻干粉；临用前，将冻干粉在等渗生理盐水中复悬，即得自携式无载体鼻腔纳米自噬诱导剂。
  8. 根据权利要求6所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，其特征在于：所述具有自噬诱导作用的疏水性小分子是以下结构式的姜黄素类似物、其顺式异构体或二者的任意比例混合物，
    Figure PCTCN2020070987-appb-100002
  9. 根据权利要求7所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，其特征在于：所述的顺式异构体的重量比占总混合物量的25-35％的混合物，由如下方法制得，将姜黄素类似物姜黄素类似物的甲醇溶液，加以紫外照射1.5-2.5h。
  10. 根据权利要求6或8所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，其特征在于：还包括壳寡糖，壳寡糖在等渗生理盐水溶液浓度为0.01-0.2％(w/v)。
  11. 根据权利要求6或8所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，其特征在于：所述双亲性表面活性剂为聚乙二醇衍生物，所述的聚乙二醇衍生物为带负电的聚乙二醇衍生物。
  12. 根据权利要求10所述的一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，其特征在于：所述聚乙二醇衍生物为带有羧基的聚乙二醇衍生物或聚马来酸酐18碳烯-聚乙二醇。
  13. 一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂的制备方法，包括如下步骤：
    1)首先配置聚乙二醇衍生物1-10mg/mL以及0.5-5mg/mL疏水性小分子药物的良溶剂溶液，然后将所述良溶剂溶液往去离子水中滴加：所述良溶剂溶液与去离子水的体积比为(0.5-5)：50，滴加的同时辅以气体吹，辅助良溶剂挥发；
    2)通过再沉淀法制备成粒径为50-200nm的自携式无载体纳米颗粒悬乳液，冷冻干燥制备成冻干粉；
    临用前，将冻干粉复悬于等渗生理盐水中，即得鼻腔纳米自噬诱导剂。
PCT/CN2020/070987 2019-01-09 2020-01-08 一种用于防治早期神经退行性疾病的鼻腔纳米自噬诱导剂及其制备方法 WO2020143688A1 (zh)

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