WO2020151197A1 - 血脑屏障渗透性调控剂及其应用 - Google Patents

血脑屏障渗透性调控剂及其应用 Download PDF

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WO2020151197A1
WO2020151197A1 PCT/CN2019/095060 CN2019095060W WO2020151197A1 WO 2020151197 A1 WO2020151197 A1 WO 2020151197A1 CN 2019095060 W CN2019095060 W CN 2019095060W WO 2020151197 A1 WO2020151197 A1 WO 2020151197A1
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brain
drug delivery
delivery system
blood
brain barrier
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胡富强
温丽娟
袁弘
王凯
孟廷廷
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Zhejiang University
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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
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    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of pharmacy and relates to the application of SC79 or its analogues in the preparation of blood-brain barrier permeability regulators.
  • the invention also relates to the combined application of the blood-brain barrier permeability regulator and the brain-targeted drug delivery system and a kit containing the blood-brain barrier permeability regulator and the brain-targeted drug delivery system.
  • the blood brain barrier is a special system that protects and maintains the functions of the central nervous system. It is mainly composed of brain microvascular endothelial cells, tight junctions between endothelial cells, basement membranes, and glial cell foot processes around capillaries. Among them, endothelial cells and their tight junctions are the important morphological basis of the blood-brain barrier. Most macromolecular substances and more than 98% of small molecule drugs cannot leak through the blood-brain barrier. Only a few nutrients with a molecular weight of less than 500Da can passively diffuse or selectively cross the blood-brain barrier to maintain the physiology of the central nervous system. balance.
  • Brain capillary endothelial cells and the tight junctions expressed between cells constitute the hypotonic and high resistance characteristics of the blood-brain barrier.
  • Claudin-5, Occludin, etc. are key proteins that constitute tight junctions and play an important role in maintaining the integrity of the blood-brain barrier.
  • the blood-brain barrier expresses a large number of receptors, such as transferrin receptor, lipoprotein receptor, insulin receptor, acetylcholine receptor, diphtheria toxin receptor, folate receptor, etc., with corresponding ligands (such as transferrin, single Anti-OX26, CDX polypeptide, RVG29 polypeptide, TGN polypeptide, Angiopep-2 polypeptide, etc.) modify the surface of the nano-drug delivery system.
  • receptors such as transferrin receptor, lipoprotein receptor, insulin receptor, acetylcholine receptor, diphtheria toxin receptor, folate receptor, etc.
  • ligands such as transferrin, single Anti-OX26, CDX polypeptide, RVG29 polypeptide, TGN polypeptide, Angiopep-2 polypeptide, etc.
  • Angiopep-2 is a polypeptide derived from aprotinin, which can specifically target low-density lipoprotein receptor related protein 1.
  • Low-density lipoprotein receptor-related protein 1 is highly expressed in the blood-brain barrier and glioma cells. Therefore, modification of Angiopep-2 on the surface of reduction-responsive chitosan-stearylamine grafts can achieve blood brain The dual targeting effect of barrier and glioma.
  • central nervous system diseases can cause pathological destruction of the blood-brain barrier structure and increase the permeability of the blood-brain barrier.
  • the increased permeability of the blood-brain barrier can cause the internal environmental balance of the central nervous system to be disrupted; on the other hand, the pathological destruction of the blood-brain barrier can also significantly enhance the transport of brain-targeted drug delivery systems into the brain.
  • the brain-targeted drug delivery system can be transported into the brain through the receptor-mediated pathway and the pathological window of the blood-brain barrier at the same time, resulting in more significant therapeutic effects.
  • SC79 is a protein kinase B phosphorylation activator with a molecular formula of C 17 H 17 ClN 2 O 5 and a molecular weight of 364.78. It can activate the Claudin-5 and Occludin signaling pathways downstream of protein kinase B and down-regulate the tight junction proteins Claudin-5, The expression of Occludin.
  • SC79 analogs mainly refer to the mechanism of action similar to SC79, which can act on protein kinase B agonists, activate the downstream Claudin-5 and Occludin signaling pathways of protein kinase B and down-regulate the expression of tight junction proteins Claudin-5 and Occludin.
  • SC79 inhibits the activation of protein kinase B in the cytoplasm, and reproduces the primary cell function of protein kinase B signaling, thereby increasing the survival of neurons.
  • SC79 or its analogues to regulate the increase in permeability of the blood-brain barrier, promote the transfer efficiency of drug delivery systems into the brain, and improve the efficacy of brain-targeted drug delivery systems has not yet been reported.
  • SC79 or its analogues in combination with a brain-targeted drug delivery system to enhance the transfer efficiency of the brain-targeted drug delivery system into the brain and improve the anti-glioma efficacy of the brain-targeted drug delivery system has not been reported. .
  • the inventors of the present invention have discovered in research that the brain-targeted drug delivery system is accompanied by the recovery of the blood-brain barrier function during the treatment of brain tumors and other brain diseases, thereby reducing the efficiency of the brain-targeted drug delivery system to transport into the brain , Reduce the efficacy, which may be one of the main reasons for the failure of the treatment of brain diseases. Therefore, exploring the permeability regulation mechanism of the blood-brain barrier and maintaining the blood-brain barrier open during treatment has important research significance and scientific value for improving the efficacy of brain-targeted drug delivery systems.
  • the purpose of the present invention is to provide a blood-brain barrier permeability regulator, which can maintain the blood-brain barrier open during treatment using a brain-targeted drug delivery system, and improve the efficacy of the brain-targeted drug delivery system. And the combined application of the blood-brain barrier permeability regulator and the brain-targeted drug delivery system.
  • the inventors of the present invention have discovered through in-depth research that the protein kinase B (AKT) agonist SC79 and its analogs have a regulatory effect on the permeability of the blood-brain barrier, which can activate the Claudin-5 and Occludin signaling pathways downstream of protein kinase B.
  • AKT protein kinase B
  • Down-regulating the expression of tight junction proteins Claudin-5 and Occludin thereby increasing the permeability of the blood-brain barrier, enhancing the delivery efficiency of the brain-targeted drug delivery system into the brain, especially the Angiopep-2 modified glycolipid nano-drug
  • the transport efficiency of the system into the brain significantly improves the therapeutic effect of the brain-targeted drug delivery system.
  • the present invention is based on the above findings. Specifically, the present invention relates to the following invention contents.
  • the blood-brain barrier permeability regulator can increase the permeability of the blood-brain barrier and enhance the transport efficiency of the brain-targeted drug delivery system into the brain.
  • brain-targeted drug delivery system is an Angiopep-2 modified glycolipid nano drug delivery system.
  • Angiopep-2 modified glycolipid nano drug delivery system is an Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA.
  • siRNA having the following nucleotide sequence:
  • a brain-targeted drug delivery kit characterized in that it comprises a combination of a blood-brain barrier permeability regulator and a brain-targeted drug delivery system, the blood-brain barrier permeability regulator contains SC79 or its analogues As an effective ingredient, it can increase the permeability of the blood-brain barrier and enhance the transport efficiency of the brain-targeted drug delivery system into the brain.
  • the brain-targeted drug delivery system is an Angiopep-2 modified glycolipid nano drug delivery system.
  • kits according to 10 above, wherein the Angiopep-2 modified glycolipid nano drug delivery system is an Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA.
  • siRNA comprises an siRNA having the following nucleotide sequence:
  • the blood-brain barrier permeability modulator down-regulates the tight junction protein Claudin-5 by activating the Claudin-5 and Occludin signaling pathways downstream of protein kinase B , Occludin expression, thereby increasing the permeability of the blood-brain barrier.
  • kit according to any one of 9 to 12 above, wherein the kit is a kit for treating glioma.
  • brain-targeted drug delivery system is an Angiopep-2 modified glycolipid nano drug delivery system.
  • Angiopep-2 modified glycolipid nano drug delivery system is an Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA.
  • siRNA having the following nucleotide sequence:
  • the blood-brain barrier permeability modulator down-regulates the tight junction proteins Claudin-5, Claudin-5 and Occludin signaling pathways downstream of protein kinase B by activating the Claudin-5 and Occludin signaling pathways.
  • the expression of Occludin thereby increases the permeability of the blood-brain barrier.
  • brain-targeted drug delivery kit is a kit for treating glioma.
  • a blood-brain barrier permeability regulator with SC79 or its analogue as an effective ingredient which can increase the permeability of the blood-brain barrier and enhance the transport efficiency of the brain-targeted drug delivery system into the brain, especially It can enhance the transport efficiency of Angiopep-2 modified glycolipid nano drug delivery system into the brain.
  • the transfer efficiency of the brain-targeted drug delivery system into the brain can be enhanced, thereby improving the brain-targeted drug delivery system's resistance to brain diseases
  • the treatment effect has a significantly superior technical effect.
  • FIG. 3 In vitro Angiopep-2 modified glycolipid nano-drug delivery system reduces the down-regulated expression of tight junction protein Claudin-5/Occludin and promotes the recovery of pathological blood-brain barrier function.
  • the Transwell model of brain microvascular endothelial cells bEnd.3 simulates the physiological blood-brain barrier, and the Transwell model of brain microvascular endothelial cells bEnd.3 and the glioma cells U87 MG co-incubates the pathological blood-brain barrier.
  • Angiopep-2 modified glycolipid nano-drug delivery system was added to the Transwell chamber, the paclitaxel concentration was 0.5 ⁇ g/mL, and the siRNA concentration was 100 nM.
  • FIG 4 In the brain glioma model, Angiopep-2 modified glycolipid nano-drug delivery system promotes the recovery of pathological blood-brain barrier function and reduces the blood-brain barrier permeability.
  • Angiopep-2 modified glycolipid nano drug delivery system was injected into the tail vein at the dosage of paclitaxel 10mg/kg and siRNA 50nM/kg. After 48 hours, the Evans blue solution was injected into the tail vein at the dosage of 5mg/kg, and 4 hours later , The whole brain of nude mice was taken by cardiac perfusion.
  • Figure A shows the result of qualitative observation of the distribution intensity of Evans blue in the brain tissue with a small animal in vivo imaging instrument.
  • FIG. 5 In the brain glioma model, Angiopep-2 modified glycolipid nano-drug delivery system promotes the recovery of pathological blood-brain barrier function, and the amount of Evans blue penetrating into the brain is reduced.
  • FIG. 6 SC79 dose-dependently regulates the transmembrane resistance of the blood-brain barrier in vitro.
  • FIG. 7 SC79 in vitro affects the efficiency of FITC-dextran across the blood-brain barrier in a dose-dependent manner.
  • FIG. 8 In vitro SC79 was down-regulated in a dose-dependent manner to down-regulate the expression of Claudin-5/Occludin.
  • the Transwell model of brain microvascular endothelial cells bEnd.3 simulates the physiological blood-brain barrier. Different concentrations of SC79 (0, 5 ⁇ g/mL, 10 ⁇ g/mL) act on the Transwell chamber, and 24 hours later, the bEnd.3 on the Transwell chamber membrane is treated. Cells were stained with immunofluorescence. The figure shows the results of observation of Claudin-5/Occludin expression by laser confocal microscope.
  • FIG. 9 SC79 in vitro regulates the transmembrane resistance of the blood-brain barrier in a time-dependent manner.
  • the Transwell model of brain microvascular endothelial cells bEnd.3 simulates the physiological blood-brain barrier
  • the Transwell model of brain microvascular endothelial cells bEnd.3 and the glioma cells U87 MG co-incubates the pathological blood-brain barrier.
  • Figure 10 SC79 regulates the increase in permeability of the blood-brain barrier in vivo, and the amount of Evans blue that penetrates into the brain increases.
  • the SC79 doses of 5 mg/kg and 10 mg/kg were injected into the tumor-bearing animals through the tail vein. After 24 hours, the Evans blue solution was injected into the tail vein at the dose of 5 mg/kg. After 4 hours, the nude mice were taken by cardiac perfusion.
  • Figure A shows the result of qualitative observation of the distribution intensity of Evans blue in the brain tissue with a small animal live imaging instrument
  • Figure B shows the semi-quantitative result of Evans blue fluorescence signal.
  • FIG 11 SC79 in vivo regulates the increase in blood-brain barrier permeability in a time-dependent manner.
  • the SC79 doses of 5mg/kg and 10mg/kg were injected into the tumor-bearing animals through the tail vein.
  • the Evans blue solution was injected into the tail vein at the doses of 5mg/kg.
  • the whole brain of a nude mouse was taken by cardiac perfusion.
  • Figure A shows the result of qualitative observation of the distribution intensity of Evans blue in the brain tissue with a small animal in vivo imager
  • Figure B shows the semi-quantitative result of the fluorescence signal of Evans blue.
  • FIG. 12 In the glioma model, SC79 regulates the increase in permeability of the blood-brain barrier and enhances the distribution of Angiopep-2 modified glycolipid nano-drug delivery system in the brain.
  • the SC79 dose of 5mg/kg was injected into the tumor-bearing animal through the tail vein. After 24 hours, the Angiopep-2 modified glycolipid nano-administration system encapsulated the near-infrared fluorescent probe DiR was injected into the tail vein. After 4 hours, the heart was perfused. Take the whole brain of a nude mouse.
  • Figure A shows the qualitative observation of the distribution intensity of the Angiopep-2 modified glycolipid nano drug delivery system encapsulated with the near-infrared fluorescent probe DiR in the brain tissue with a small animal live imaging instrument.
  • Figure 13 The therapeutic effect of SC79 and Angiopep-2 modified glycolipid nano drug delivery system administered 24 hours in advance in the treatment of glioma.
  • the administration was started on the 4th day of tumor-bearing, and the Angiopep-2 modified glycolipid nano drug delivery system was injected into the tail vein at the dose of paclitaxel 10mg/kg and siRNA 50nM/kg.
  • the SC79 dose of 5mg/kg was injected into the tumor-bearing animals through the tail vein.
  • Angiopep-2 modified glycolipid nano-drug delivery system was injected into the tail vein again, and SC79 was injected into the tail vein three times, Angiopep-2 Modify the glycolipid nano drug delivery system four times.
  • Bioluminescence imaging technology monitors the growth of glioma in situ.
  • Figure A qualitatively reflects the size of the brain tumor on the 4th, 7th, 10th, 13th and 16th day of the tumor-bearing.
  • SC79 involved in the present invention is an agonist of protein kinase B (AKT), with a molecular formula of C 17 H 17 ClN 2 O 5 and a molecular weight of 364.78.
  • SC79 is a protein kinase B phosphorylation activator, which can activate the Claudin-5 and Occludin signaling pathways downstream of protein kinase B and down-regulate the expression of tight junction proteins Claudin-5 and Occludin.
  • Studies have found that in a mouse model of permanent focal cerebral ischemia, SC79 inhibits the activation of protein kinase B in the cytoplasm and reproduces the primary cell function of protein kinase B signaling, thereby increasing the survival of neurons.
  • SC79 analogs mainly refer to the mechanism of action similar to SC79, which can act on protein kinase B agonists, activate the downstream Claudin-5 and Occludin signaling pathways of protein kinase B and down-regulate the expression of tight junction proteins Claudin-5 and Occludin.
  • SC79 can activate the Claudin-5 and Occludin signaling pathways downstream of protein kinase B, down-regulate the expression of tight junction proteins Claudin-5 and Occludin, and increase the permeability of the blood-brain barrier. Therefore, SC79 can be used as an effective ingredient of the blood-brain barrier permeability regulator. Further studies have found that after the administration of SC79 0.5-24 hours, it can effectively increase the permeability of the blood-brain barrier, significantly enhance the transfer efficiency of the brain-targeted drug delivery system into the brain, and significantly improve the impact of the brain-targeted drug delivery system on the brain. The therapeutic effect of the disease.
  • brain diseases include but not limited to glioma, pituitary tumor, meningioma, brain metastasis and so on.
  • the occurrence of these brain diseases will cause the loss of the integrity of the blood-brain barrier tight junction structure and the abnormal barrier function, accompanied by the down-regulation of tight junction protein expression, the increase of blood-brain barrier permeability, and the destruction of the internal environment of the central nervous system.
  • a drug delivery system based on receptor-mediated transcytosis, for example, transferrin receptor, lipoprotein receptor, insulin receptor, acetylcholine receptor, diphtheria Corresponding ligands of toxin protein receptor and folate receptor, such as transferrin, monoclonal antibody OX26, CDX polypeptide, RVG29 polypeptide, TGN polypeptide, Angiopep-2 polypeptide and other brain-targeted drugs obtained by modifying the surface of nano-drug delivery system Delivery system.
  • receptor-mediated transcytosis for example, transferrin receptor, lipoprotein receptor, insulin receptor, acetylcholine receptor, diphtheria Corresponding ligands of toxin protein receptor and folate receptor, such as transferrin, monoclonal antibody OX26, CDX polypeptide, RVG29 polypeptide, TGN polypeptide, Angiopep-2 polypeptide and other brain-targeted drugs obtained by modifying the surface of nano-drug delivery system Delivery system.
  • Angiopep-2 is a polypeptide derived from aprotinin, which can specifically target low-density lipoprotein receptor-related protein 1.
  • Low-density lipoprotein receptor-related protein 1 is highly expressed in the blood-brain barrier and glioma cells. Therefore, modification of Angiopep-2 on the surface of reduction-responsive chitosan-stearylamine grafts can achieve blood brain The dual targeting effect of barrier and glioma.
  • the drug delivered by the Angiopep-2 modified glycolipid nano drug delivery system of the present invention is not particularly limited as long as it is a drug that can be used to treat brain diseases.
  • Examples of drugs that are commonly used in the art can be used to treat brain diseases.
  • paclitaxel, doxorubicin, etc., siRNA, plasmid DNA, etc. can also be used.
  • One of these drugs may be used, or two or more of them may be used in combination.
  • the Angiopep-2 modified glycolipid nano drug delivery system of the present invention is an Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA.
  • the Angiopep-2 modified glycolipid nano drug delivery system of the present invention is an Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA, which is used for the treatment of glioma.
  • the aforementioned siRNA preferably includes an siRNA having the following nucleotide sequence:
  • the Angiopep-2 modified glycolipid nano drug delivery system of the present invention has a significant curative effect on glioma by co-delivering paclitaxel and siRNA with the above-mentioned specific nucleotide sequence.
  • the present invention can enhance the transfer efficiency of the brain-targeted drug delivery system into the brain by using the above-mentioned blood-brain barrier permeability regulator and the brain-targeted drug delivery system in combination, thereby improving the brain-targeted drug delivery system
  • the treatment effect of the disease has a significantly superior technical effect.
  • the brain-targeted drug delivery system is administered 0.5 hours after the administration of the blood-brain barrier permeability modifier, more preferably 12 hours, and particularly preferably 24 hours.
  • the brain-targeted drug delivery kit of the present invention comprises a combination of the above-mentioned blood-brain barrier permeability regulator and a brain-targeted drug delivery system.
  • the aforementioned blood-brain barrier permeability regulator contains SC79 or its analogues as an effective ingredient, which can increase the permeability of the blood-brain barrier and enhance the transport efficiency of the brain-targeted drug delivery system into the brain.
  • the aforementioned brain-targeted drug delivery system is preferably an Angiopep-2 modified glycolipid nano drug delivery system, and more preferably an Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA.
  • the above-mentioned siRNA includes an siRNA having the following nucleotide sequence: 5'-GUCUAUCAGCGCAGCUACUTT-3' for the sense strand and 5'-AGUAGCUGCGCUGAUAGACTT-3' for the antisense strand.
  • the brain-targeted drug delivery kit of the invention is preferably a kit for the treatment of glioma, which has a significant curative effect on glioma.
  • the above-obtained Angiopep-2 targeting-modified reduction response chitosan-stearylamine grafted paclitaxel micelle solution was slowly mixed with 10 ⁇ M siRNA solution at an N/P ratio of 150 (w/w), and vortexed. Let stand for 30 minutes to obtain the Angiopep-2 modified glycolipid nano drug delivery system that co-delivers paclitaxel and siRNA.
  • the nucleotide sequence of the siRNA is: sense strand 5'-GUCUAUCAGCGCAGCUACUTT-3', antisense strand 5'-AGUAGCUGCGCUGAUAGACTT-3'.
  • the Angiopep-2 modified glycolipid nano drug delivery system was prepared according to the methods described in 1 and 2 above. At a cell density of 5 ⁇ 10 3 cells/well, 200 ⁇ L of well-growing U87 MG cell suspension was inoculated into a 96-well plate. Incubate at 37°C and 5% carbon dioxide until the cells adhere to the wall.
  • Angiopep-2 modified glycolipid nano drug delivery system was added, paclitaxel concentration was 0.5 ⁇ g/mL, siRNA concentration was 100 nM, untreated cells were used as blank control, and each group was repeated 3 times. After 48 hours of incubation, add 20 ⁇ L of 5 mg/mL Thiazole Blue to each well, continue incubating for 4 hours, discard the medium, add 200 ⁇ L of dimethyl sulfoxide to each well, and place the 96-well plate in a constant temperature shaking box for 15 minutes. The microplate reader measures the absorbance at 570nm. Calculate the cell inhibition rate according to the following formula:
  • Angiopep-2 modified glycolipid nano drug delivery system has a cell inhibition rate of 60.0 ⁇ 1.1%
  • siRNA and paclitaxel have a cell inhibition rate of 5.5 ⁇ 0.5% and 18.7 ⁇ 5.4% for U87 MG, respectively.
  • the Angiopep-2 modified glycolipid nano drug delivery system can significantly inhibit the growth of glioma, has a significant anti-glioma effect, and the effect is significantly better than siRNA and paclitaxel.
  • Angiopep-2 modified glycolipid nano drug delivery system down-regulates tumor cell VEGF expression
  • the Angiopep-2 modified glycolipid nano drug delivery system was prepared according to the methods described in 1 and 2 above. At a cell density of 4 ⁇ 10 5 cells/well, 2.0 mL of well-growing U87 MG cell suspension was inoculated into a 6-well plate , Incubate at 37°C and 5% carbon dioxide until the cells adhere to the wall. Add Angiopep-2 modified glycolipid nano drug delivery system, paclitaxel concentration is 0.5 ⁇ g/mL, siRNA concentration is 100nM, incubated for 48 hours, with untreated cells as a blank control, repeat 3 times for each group. qt-PCR detects tumor cell VEGF expression level, the results are shown in Figure 2.
  • Angiopep-2 modified glycolipid nano-drug delivery system can significantly down-regulate the expression level of VEGF mRNA in U87 MG tumor cells (21.1%), which is significantly better than siRNA (92.9%) and paclitaxel (78.5%) ).
  • Example 2 In vitro Angiopep-2 modified glycolipid nano drug delivery system promotes the recovery of pathological blood-brain barrier function
  • bEnd.3 brain microvascular endothelial cells bEnd.3 as the blood-brain barrier model cells
  • 0.5 mL of the well-growing bEnd.3 cell suspension was inoculated into the cell of a 12-well Transwell plate with a pore size of 0.4 ⁇ m at a cell density of 1 ⁇ 10 5 cells/well
  • the middle and outer compartments were supplemented with 1.5 mL of fresh culture medium.
  • the fresh culture medium was replaced every other day, and the incubation was continued for 15 days at 37°C and 5% carbon dioxide.
  • the cell transmembrane resistance is greater than 150 ohm ⁇ square centimeter ( ⁇ cm 2 ), it can be regarded as a physiological blood-brain barrier model.
  • U87 MG glioma cells were used as brain tumor model cells. At a cell density of 2 ⁇ 10 5 cells/well, 1.5 mL of well-growing U87 MG cell suspension was inoculated in a 12-well plate at 37°C, 5 Incubate under 5% carbon dioxide until the cells adhere to the wall. Then transfer the physiological blood-brain barrier constructed by the method described in 1 above, namely the Transwell chamber with bEnd.3 cells to a 12-well plate with U87 MG cells, and continue to co-culture for 24 hours to obtain pathological blood brain Barrier model.
  • the Angiopep-2 modified glycolipid nano drug delivery system was added to the Transwell chamber of the pathological blood-brain barrier model. After 48 hours of drug action, bEnd will grow. The 3-cell Transwell chamber was taken out, washed 3 times with PBS, fixed with 4% formaldehyde, blocked with 10% bovine serum albumin for 30 minutes, added Claudin-5/Occludin primary antibody and incubated overnight at 4°C, and finally labeled with fluorescent After the secondary antibody was incubated for 1 hour, the nucleus was counter-stained with DAPI. The expression of Claudin-5/Occludin was observed by laser confocal microscope, and the expression level of Claudin-5/Occludin was analyzed semi-quantitatively with Image J. The results are shown in Figure 3.
  • the effect of Angiopep-2 modified glycolipid nano-drug delivery system can reduce the down-regulated expression of tight junction protein Claudin-5/Occludin, and significantly reduce the degree of pathological blood-brain barrier damage, indicating that Angiopep-2 modified sugar
  • the lipid nano drug delivery system can promote the recovery of pathological blood-brain barrier function in vitro.
  • Example 3 Angiopep-2 modified glycolipid nano drug delivery system promotes the recovery of pathological blood-brain barrier function in glioma model animals
  • Example 4 SC79 activates the downstream signaling pathway of protein kinase B in a dose-dependent manner, promotes the down-regulation of tight junction proteins, and increases the permeability of the blood-brain barrier
  • SC79 activates the downstream signaling pathway of protein kinase B in a dose-dependent manner, promotes the down-regulation of tight junction protein Claudin-5/Occludin expression, and increases blood-brain barrier permeability.
  • Example 5 SC79 activates the downstream signaling pathway of protein kinase B in a time-dependent manner, promotes the down-regulation of tight junction proteins, and increases the permeability of the blood-brain barrier
  • Example 6 SC79 increases the permeability of the blood-brain barrier of glioma model animals and promotes the transportation of Angiopep-2 modified glycolipid nano-drug system into the brain
  • the Angiopep-2 modified glycolipid nano-administration encapsulated the near-infrared fluorescent probe DiR was injected into the tail vein.
  • the whole brain of nude mice was taken by cardiac perfusion, and the small animal in vivo imager qualitatively observed the distribution intensity of Angiopep-2 modified glycolipid nano drug delivery system in the brain tissue. The results are shown in Figure 12.
  • a blood-brain barrier permeability regulator with SC79 or its analogue as an effective ingredient which can increase the permeability of the blood-brain barrier and enhance the transport efficiency of the brain-targeted drug delivery system into the brain, especially It can enhance the transport efficiency of Angiopep-2 modified glycolipid nano drug delivery system into the brain.
  • the transfer efficiency of the brain-targeted drug delivery system into the brain can be enhanced, thereby improving the brain-targeted drug delivery system's resistance to brain diseases
  • the treatment effect has a significantly superior technical effect. Therefore, the present invention has great practicability in the medical field.

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Abstract

本发明涉及SC79或其类似物在制备血脑屏障渗透性调控剂中的应用。所述血脑屏障渗透性调控剂以SC79或其类似物作为有效成分，通过激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达，由此增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率，特别是能够增强Angiopep-2修饰的糖脂纳米给药系统入脑的转运效率。本发明还涉及一种试剂盒，其包含该血脑屏障渗透性调控剂和脑靶向药物递释系统。根据本发明，通过组合使用上述血脑屏障渗透性调控剂与脑靶向药物递释系统，能够增强脑靶向药物递释系统入脑的转运效率，提高脑靶向药物递释系统的疗效。

Description

血脑屏障渗透性调控剂及其应用 技术领域
本发明属于制药领域，涉及SC79或其类似物在制备血脑屏障渗透性调控剂中的应用。本发明还涉及该血脑屏障渗透性调控剂与脑靶向药物递释系统的组合应用以及包含该血脑屏障渗透性调控剂与脑靶向药物递释系统的试剂盒。
背景技术
血脑屏障(blood brain barrier,BBB)是保护、维持中枢神经系统功能的特殊系统，主要由脑微血管内皮细胞、内皮细胞之间的紧密连接、基膜以及毛细血管周围的胶质细胞足突构成，其中内皮细胞及其紧密连接是血脑屏障的重要形态学基础。大部分的大分子物质和超过98％的小分子药物都不能渗漏通过血脑屏障，只有少部分分子量小于500Da的营养物质可以被动扩散或者选择性跨过血脑屏障以维持中枢神经系统的生理平衡。
血脑屏障完整性缺失是脑部疾病发生、发展过程中的一个显著的病理学特性。脑毛细血管内皮细胞及细胞间表达的紧密连接构成血脑屏障的低渗和高电阻特性。其中，Claudin-5、Occludin等是构成紧密连接的关键蛋白，在维持血脑屏障完整性特性中扮演重要的角色。
另一方面，在对脑部疾病、特别是脑肿瘤的治疗中，通常需要在手术之外辅以药物治疗，然而，血脑屏障的存在严重限制了治疗药物的脑内转运。如何有效地提高治疗药物的入脑转运效率，是目前亟需解决的关键问题。
近年来，脑靶向药物递释系统的研究取得了一系列进展。其中，受体介导的转胞吞因其高亲和性、选择性和特异性，被视为是脑靶向递送最有效的途径之一。血脑屏障表达大量受体，如转铁蛋白受体、脂蛋白受体、胰岛素受体、乙酰胆碱受体、白喉毒素蛋白受体、叶酸 受体等，以相应配体(如转铁蛋白、单抗OX26、CDX多肽、RVG29多肽、TGN多肽、Angiopep-2多肽等)修饰纳米药物递释系统表面，静脉注射给药后经受体介导途径跨过BBB转运入脑，提高药物治疗效果。Angiopep-2是aprotinin衍生的多肽，它可以特异性地靶向低密度脂蛋白受体相关蛋白1。低密度脂蛋白受体相关蛋白1在血脑屏障和脑胶质瘤细胞都有高表达，因此，将Angiopep-2修饰于还原响应型壳聚糖-硬脂胺嫁接物表面，可实现血脑屏障和脑胶质瘤的双重靶向作用。
研究发现，许多中枢神经系统疾病可导致血脑屏障结构病理性破坏，血脑屏障渗透性增加。一方面，血脑屏障渗透性增加可造成中枢神经系统内环境平衡被破坏；而另一方面，血脑屏障病理性破坏也可以显著增强脑靶向药物递释系统的入脑转运，治疗药物的脑内递送增加，脑靶向药物递释系统可同时经受体介导途径和血脑屏障病理性开窗转运入脑，产生更显著的疗效。迄今为止，脑靶向药物递释系统研究大多侧重配体与其受体、靶细胞和靶组织间的相互作用，忽视了给药系统治疗疾病过程中，病理性血脑屏障功能的可能改变，所引起的给药系统渗透入脑效率降低问题，相关机制尚缺乏深入研究。
SC79是一种蛋白激酶B磷酸化激活剂，其分子式为C 17H 17ClN 2O 5，分子量为364.78，可激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达。SC79类似物主要是指作用机制与SC79类似，能作用于蛋白激酶B的激动剂，激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达。
近年来有研究发现，在永久性局部脑缺血小鼠模型中，SC79抑制细胞质中蛋白激酶B的活化，并重现蛋白激酶B信号的初级细胞功能，从而增加神经元的存活。但是，SC79或其类似物用于调控血脑屏障渗透性增加、促进药物递释系统入脑转运效率、提高脑靶向药物递释系统疗效的作用尚未报道。组合使用SC79或其类似物与脑靶向药物递释系统，增强脑靶向药物递释系统入脑的转运效率，提高脑靶向药物递释系统的抗脑胶质瘤疗效也未见任何报道。
发明内容
本发明的发明人在研究中发现，脑靶向药物递释系统治疗脑肿瘤等脑内疾病过程中会伴随着血脑屏障功能的恢复，从而降低脑靶向药物递释系统转运入脑的效率，降低疗效，这可能是脑部疾病治疗失败的主要原因之一。因此，探索血脑屏障渗透性调控机制，维持治疗过程中血脑屏障开放，对提高脑靶向药物递释系统的疗效具有重要的研究意义和科学价值。
为此，本发明的目的在于提供一种能够在利用脑靶向药物递释系统进行治疗过程中维持血脑屏障开放，提高脑靶向药物递释系统的疗效的血脑屏障渗透性调控剂、以及该血脑屏障渗透性调控剂与脑靶向药物递释系统的组合应用。
本发明的发明人经过深入的研究发现，蛋白激酶B(AKT)的激动剂SC79及其类似物对血脑屏障渗透性具有调控作用，其能够通过激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达，由此增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率，特别是能够增强Angiopep-2修饰的糖脂纳米给药系统入脑的转运效率，显著提高脑靶向药物递释系统的治疗效果。本发明正是基于上述发现而完成的发明。具体而言，本发明涉及以下发明内容。
1.SC79或其类似物在制备血脑屏障渗透性调控剂中的应用。
2.如上述1所述的应用，其特征在于，上述血脑屏障渗透性调控剂能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率。
3.如上述2所述的应用，其特征在于，上述脑靶向药物递释系统是Angiopep-2修饰的糖脂纳米给药系统。
4.如上述3所述的应用，其特征在于，上述Angiopep-2修饰的糖脂纳米给药系统是共递送紫杉醇和siRNA的Angiopep-2修饰糖脂纳米给药系统。
5.如上述4所述的应用，其特征在于，上述siRNA包含具有下述核苷酸序列的siRNA：
正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，
反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。
6.如上述1～5中任一项所述的应用，其特征在于，上述血脑屏障渗透性调控剂通过激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达，由此增加血脑屏障渗透性。
7.如上述1～5中任一项所述的应用，其特征在于，在施用上述血脑屏障渗透性调控剂0.5小时后施用脑靶向药物递释系统。
8.如上述1～5中任一项所述的应用，其特征在于，在施用上述血脑屏障渗透性调控剂24小时后施用脑靶向药物递释系统。
9.一种脑靶向给药试剂盒，其特征在于，其包含血脑屏障渗透性调控剂和脑靶向药物递释系统的组合，上述血脑屏障渗透性调控剂含有SC79或其类似物作为有效成分，能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率。
10.如上述9所述的试剂盒，其特征在于，上述脑靶向药物递释系统是Angiopep-2修饰的糖脂纳米给药系统。
11.如上述10所述的试剂盒，其特征在于，上述Angiopep-2修饰的糖脂纳米给药系统是共递送紫杉醇和siRNA的Angiopep-2修饰糖脂纳米给药系统。
12.如上述11所述的试剂盒，其特征在于，上述siRNA包含具有下述核苷酸序列的siRNA：
正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，
反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。
13.如上述9～12中任一项所述的试剂盒，其特征在于，上述血脑屏障渗透性调控剂通过激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达，由此增加血脑屏障渗透性。
14.如上述9～12中任一项所述的试剂盒，其特征在于，上述试剂盒是用于治疗脑胶质瘤的试剂盒。
15.血脑屏障渗透性调控剂与脑靶向药物递释系统的组合在脑 靶向给药试剂盒的制造中的应用，其特征在于，上述血脑屏障渗透性调控剂含有SC79或其类似物作为有效成分，能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率。
16.如上述15所述的应用，其特征在于，上述脑靶向药物递释系统是Angiopep-2修饰的糖脂纳米给药系统。
17.如上述16所述的应用，其特征在于，上述Angiopep-2修饰的糖脂纳米给药系统是共递送紫杉醇和siRNA的Angiopep-2修饰糖脂纳米给药系统。
18.如上述17所述的应用，其特征在于，上述siRNA包含具有下述核苷酸序列的siRNA：
正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，
反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。
19.如上述15～18中任一项所述的应用，其特征在于，上述血脑屏障渗透性调控剂通过激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达，由此增加血脑屏障渗透性。
20.如上述15～18中任一项所述的应用，其特征在于，上述脑靶向给药试剂盒是用于治疗脑胶质瘤的试剂盒。
发明的效果
根据本发明，能够提供一种以SC79或其类似物作为有效成分的血脑屏障渗透性调控剂，其能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率，特别是能够增强Angiopep-2修饰的糖脂纳米给药系统入脑的转运效率。通过组合使用该血脑屏障渗透性调控剂与脑靶向药物递释系统，能够增强脑靶向药物递释系统入脑的转运效率，进而提高脑靶向药物递释系统的对脑部疾病的治疗效果，具有显著优越的技术效果。
附图说明
图1：体外Angiopep-2修饰糖脂纳米给药系统对U87 MG脑胶质瘤细胞的抑制率。以5×10 3个/孔的密度，取200μL生长良好的U87  MG脑胶质瘤细胞悬液接种于96孔板中。细胞贴壁生长后，加入Angiopep-2修饰糖脂纳米给药系统，紫杉醇浓度为0.5μg/mL、siRNA浓度为100nM，以未处理组作为空白对照。四唑蓝比色法检测细胞生长抑制率(n＝3)。
图2：体外Angiopep-2修饰糖脂纳米给药系统下调U87 MG细胞VEGF mRNA表达。以4×10 5个/孔的密度，取2mL生长良好的U87 MG脑胶质瘤细胞悬液接种于6孔板中。细胞贴壁生长后，加入Angiopep-2修饰糖脂纳米给药系统，紫杉醇浓度为0.5μg/mL、siRNA浓度为100nM，孵育48小时，以未处理组作为空白对照。TRIzol提取胞内RNA，qt-PCR检测并根据△△Ct法计算肿瘤细胞VEGF mRNA的相对表达水平(n＝3)。
图3：体外Angiopep-2修饰糖脂纳米给药系统降低紧密连接蛋白Claudin-5/Occludin下调表达，促进病理性血脑屏障功能恢复。脑微血管内皮细胞bEnd.3的Transwell模型模拟生理性血脑屏障，脑微血管内皮细胞bEnd.3和脑胶质瘤细胞U87 MG共孵育的Transwell模型模拟病理性血脑屏障。Transwell小室中加入Angiopep-2修饰糖脂纳米给药系统，紫杉醇浓度为0.5μg/mL、siRNA浓度为100nM，孵育48小时后，对Transwell小室膜上的bEnd.3细胞进行免疫荧光染色，图A所示为激光共聚焦显微镜观察紧密连接蛋白Claudin-5/Occludin表达结果，图B是Image J对Claudin-5/Occludin表达的荧光半定量分析结果(n＝3)。
图4：脑胶质瘤模型中，Angiopep-2修饰糖脂纳米给药系统促进病理性血脑屏障功能恢复，血脑屏障渗透性降低。按照紫杉醇10mg/kg、siRNA 50nM/kg的给药剂量尾静脉注射Angiopep-2修饰糖脂纳米给药系统，48小时后，按照5mg/kg给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，图A所示为小动物活体成像仪定性观察脑组织中依文思蓝分布强度结果，图B所示为依文思蓝荧光信号半定量结果(n＝3)。
图5：脑胶质瘤模型中，Angiopep-2修饰糖脂纳米给药系统促进病理性血脑屏障功能恢复，依文思蓝渗透入脑含量减少。按照紫杉醇 10mg/kg、siRNA 50nM/kg的给药剂量尾静脉注射Angiopep-2修饰糖脂纳米给药系统，48小时后，按照5mg/kg给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，机溶剂N，N-二甲基甲酰胺萃取脑组织中依文思蓝，图中所示结果为紫外分光光度法定量检测的依文思蓝渗透至脑内量(n＝3)。
图6：体外SC79呈剂量依赖性调控血脑屏障跨膜电阻。脑微血管内皮细胞bEnd.3的Transwell模型模拟生理性血脑屏障，不同浓度的SC79(0、5μg/mL、10μg/mL)作用于Transwell小室，24小时后，细胞电阻仪检测细胞跨膜电阻值(n＝5)。
图7：体外SC79呈剂量依赖性影响FITC-右旋糖酐跨血脑屏障转运效率。脑微血管内皮细胞bEnd.3的Transwell模型模拟生理性血脑屏障，不同浓度的SC79(0、5μg/mL、10μg/mL)作用于Transwell小室，24小时后，PBS清洗Transwell小室和外室，将FITC-右旋糖酐的HBSS溶液加于Transwell小室，作用4小时，图中所示结果即为荧光分光光度计检测Transwell外室的FITC-右旋糖酐浓度(n＝3)。
图8：体外SC79呈剂量依赖性下调降低紧密连接蛋白Claudin-5/Occludin下调表达。脑微血管内皮细胞bEnd.3的Transwell模型模拟生理性血脑屏障，不同浓度的SC79(0、5μg/mL、10μg/mL)作用于Transwell小室，24小时后，对Transwell小室膜上的bEnd.3细胞进行免疫荧光染色，图中所示为激光共聚焦显微镜观察紧密连接蛋白Claudin-5/Occludin表达结果。
图9：体外SC79呈时间依赖性调控血脑屏障跨膜电阻。脑微血管内皮细胞bEnd.3的Transwell模型模拟生理性血脑屏障，脑微血管内皮细胞bEnd.3和脑胶质瘤细胞U87 MG共孵育的Transwell模型模拟病理性血脑屏障。SC79(5μg/mL)作用于Transwell小室，细胞电阻仪实时监测细胞跨膜电阻值(n＝5)。
图10：体内SC79调控血脑屏障渗透性增加，依文思蓝渗透入脑含量增加。分别按照5mg/kg、10mg/kg的SC79给药剂量尾静脉注射于荷瘤动物，24小时后，按照5mg/kg给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，图A所示为小动物活 体成像仪定性观察脑组织中依文思蓝分布强度结果，图B所示为依文思蓝荧光信号半定量结果。
图11：体内SC79呈时间依赖性调控血脑屏障渗透性增加。按照5mg/kg、10mg/kg的SC79给药剂量尾静脉注射于荷瘤动物，0小时、0.5小时、24小时后，按照5mg/kg给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，图A所示为小动物活体成像仪定性观察脑组织中依文思蓝分布强度结果，图B所示为依文思蓝荧光信号半定量结果。
图12：脑胶质瘤模型中，SC79调控血脑屏障渗透性增加，增强Angiopep-2修饰糖脂纳米给药系统脑内分布。按照5mg/kg的SC79给药剂量尾静脉注射于荷瘤动物，24小时后，尾静脉注射包封近红外荧光探针DiR的Angiopep-2修饰糖脂纳米给药系统，4小时后，心脏灌流取裸鼠完整大脑，图A所示为小动物活体成像仪定性观察脑组织中包封近红外荧光探针DiR的Angiopep-2修饰糖脂纳米给药系统分布强度结果，图B所示为包封近红外荧光探针DiR的Angiopep-2修饰糖脂纳米给药系统荧光信号半定量结果(n＝3)。
图13：体内提前24小时施用SC79，Angiopep-2修饰糖脂纳米给药系统给药治疗脑胶质瘤的疗效。荷瘤第4天开始给药，按照紫杉醇10mg/kg、siRNA 50nM/kg的给药剂量尾静脉注射Angiopep-2修饰糖脂纳米给药系统。48小时后，按照5mg/kg的SC79给药剂量尾静脉注射于荷瘤动物，24小时后，再次尾静脉注射Angiopep-2修饰糖脂纳米给药系统，累计尾静脉注射SC79三次、Angiopep-2修饰糖脂纳米给药系统四次。生物发光成像技术监测原位脑胶质瘤生长，图A定性反映荷瘤第4、7、10、13、16天脑肿瘤大小，图B是脑胶质瘤实时(第4、7、10、13、16天)生长的定量监测曲线(n＝5)。以生理盐水组为对照。
具体实施方式
本发明涉及的SC79是一种蛋白激酶B(AKT)的激动剂，其分子式为C 17H 17ClN 2O 5，分子量为364.78。SC79是一种蛋白激酶B磷酸化激活剂，可激活蛋白激酶B下游Claudin-5、Occludin信号通路 而下调紧密连接蛋白Claudin-5、Occludin的表达。有研究发现，在永久性局部脑缺血小鼠模型中，SC79抑制细胞质中蛋白激酶B的活化，并重现蛋白激酶B信号的初级细胞功能，从而增加神经元的存活。
SC79类似物主要是指作用机制与SC79类似，能作用于蛋白激酶B的激动剂，激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达。
本发明的发明人研究发现，SC79能够激活蛋白激酶B下游Claudin-5、Occludin信号通路，下调紧密连接蛋白Claudin-5、Occludin表达，增加血脑屏障渗透性。因此，SC79能够作为血脑屏障渗透性调控剂的有效成分使用。进一步的研究发现，在施用SC79 0.5～24小时后，可有效增加血脑屏障渗透性，显著增强脑靶向药物递释系统入脑的转运效率，显著提高脑靶向药物递释系统对脑内疾病的治疗效果。
作为脑内疾病，包括但不限于脑胶质瘤、垂体瘤、脑膜瘤、脑部转移瘤等。这些脑部疾病的发生都会引起血脑屏障紧密连接结构完整性缺失及屏障功能异常，伴随着紧密连接蛋白下调表达、血脑屏障渗透性增加、中枢神经系统内环境平衡被破坏。
作为脑靶向药物递释系统，可以例举基于受体介导的转胞吞的药物递释系统，例如，以转铁蛋白受体、脂蛋白受体、胰岛素受体、乙酰胆碱受体、白喉毒素蛋白受体、叶酸受体的相应配体，如转铁蛋白、单抗OX26、CDX多肽、RVG29多肽、TGN多肽、Angiopep-2多肽等修饰纳米药物递释系统表面而得到的脑靶向药物递释系统。这些脑靶向药物递释系统在静脉注射给药后，经受体介导途径跨过血脑屏障转运入脑。其中，Angiopep-2是aprotinin衍生的多肽，它可以特异性地靶向低密度脂蛋白受体相关蛋白1。低密度脂蛋白受体相关蛋白1在血脑屏障和脑胶质瘤细胞都有高表达，因此，将Angiopep-2修饰于还原响应型壳聚糖-硬脂胺嫁接物表面，可实现血脑屏障和脑胶质瘤的双重靶向作用。
作为本发明的Angiopep-2修饰的糖脂纳米给药系统所递送的药 物，只要是能够用于治疗脑部疾病的药物，就没有特别限定，可以列举本领域通常惯用的脑部疾病治疗药物，例如紫杉醇、阿霉素等，也可以使用siRNA、质粒DNA等。这些药物可以使用一种，也可以组合使用两种以上。
作为一个优选的例子，本发明的Angiopep-2修饰的糖脂纳米给药系统是共递送紫杉醇和siRNA的Angiopep-2修饰糖脂纳米给药系统。
作为一个更优选的例子，本发明的Angiopep-2修饰的糖脂纳米给药系统是共递送紫杉醇和siRNA的Angiopep-2修饰糖脂纳米给药系统，其用于治疗脑胶质瘤。
其中，上述siRNA优选包含具有下述核苷酸序列的siRNA：
正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，
反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。
本发明的Angiopep-2修饰的糖脂纳米给药系统通过共递送紫杉醇和具有上述特定核苷酸序列的siRNA，对脑胶质瘤具有显著疗效。
本发明通过组合使用上述血脑屏障渗透性调控剂与脑靶向药物递释系统，能够增强脑靶向药物递释系统入脑的转运效率，进而提高脑靶向药物递释系统的对脑部疾病的治疗效果，具有显著优越的技术效果。
优选在施用上述血脑屏障渗透性调控剂0.5小时之后、更优选12小时后、特别优选24小时后施用脑靶向药物递释系统。此外，优选在施用SC79 96小时之前、更优选72小时之前、特别优选48小时之前施用脑靶向药物递释系统。通过在上述时间范围内使用SC79，能够更加显著地提高脑靶向药物递释系统入脑的转运效率，进而提高脑靶向药物递释系统对脑内疾病的治疗效果。
本发明的脑靶向给药试剂盒包含上述血脑屏障渗透性调控剂和脑靶向药物递释系统的组合。上述血脑屏障渗透性调控剂含有SC79或其类似物作为有效成分，能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率。上述脑靶向药物递释系统优选为Angiopep-2修饰的糖脂纳米给药系统，更优选为共递送紫杉醇和 siRNA的Angiopep-2修饰糖脂纳米给药系统。
其中，优选上述siRNA包含具有下述核苷酸序列的siRNA：正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。发明的脑靶向给药试剂盒优选为用于治疗脑胶质瘤的试剂盒，其对脑胶质瘤具有显著疗效。
在下文中，结合实施例和附图对本发明进行详细说明。需要说明的是，以下的实施例仅为例示，下述实施例对本发明的保护范围并无限定作用。
实施例
实施例一 Angiopep-2修饰糖脂纳米给药系统的制备
1.Angiopep-2修饰的还原响应型壳聚糖-硬脂胺嫁接物载紫杉醇胶束 制备
精密称取10mg紫杉醇，溶解于无水乙醇中，制成2.0mg/mL的紫杉醇储备液备用。按照5％～50％的投药量，边搅拌边向浓度为2.0mg/mL的Angiopep-2修饰的还原响应型壳聚糖-硬脂胺嫁接物胶束溶液中加入紫杉醇储备液，室温下搅拌30分钟，反应结束后，对反应液进行探头超声20次(200W，工作2秒，间歇3秒)，随后将反应液转移至透析袋中，去离子水透析24小时，透析液定容，8000转/分钟离心10分钟，除去游离的紫杉醇，取上清液即为Angiopep-2靶向修饰的还原响应型壳聚糖-硬脂胺嫁接物载紫杉醇胶束。
2.Angiopep-2修饰糖脂纳米给药系统制备
将上述所得到的Angiopep-2靶向修饰的还原响应型壳聚糖-硬脂胺嫁接物载紫杉醇胶束溶液与10μM siRNA溶液按N/P比150(w/w)缓慢混合，涡旋后静置30分钟，得到共递送紫杉醇、siRNA的Angiopep-2修饰糖脂纳米给药系统。其中，siRNA的核苷酸序列为：正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。
3.Angiopep-2修饰糖脂纳米给药系统对U87 MG脑胶质瘤细胞的细 胞毒性
以U87 MG脑胶质瘤细胞为模型细胞，采用四唑蓝比色法评价 Angiopep-2修饰糖脂纳米给药系统对肿瘤细胞的生长抑制效果。根据上述1、2所述方法制备得到Angiopep-2修饰糖脂纳米给药系统，以5×10 3个/孔的细胞密度，取200μL生长良好的U87 MG细胞悬液接种于96孔板中，在37℃、5％二氧化碳条件下孵育至细胞贴壁。加入Angiopep-2修饰糖脂纳米给药系统，紫杉醇浓度为0.5μg/mL、siRNA浓度为100nM，以未处理细胞作为空白对照，每组重复3次。孵育48小时后，每孔加入5mg/mL噻唑蓝20μL，继续孵育4小时，弃去培养基，向每孔加入200μL二甲基亚砜，将96孔板置于恒温振荡箱中振荡15分钟。酶标仪测量570nm处吸光度值。根据以下公式计算细胞抑制率：
细胞抑制率(％)＝(1－实验组的吸光度/对照组的吸光度值)×100％
结果如图1所示。其中Angiopep-2修饰糖脂纳米给药系统对细胞抑制率为60.0±1.1％，siRNA和紫杉醇对U87 MG的细胞抑制率分别为5.5±0.5％、18.7±5.4％。
根据图1所示的结果可知，Angiopep-2修饰的糖脂纳米给药系统可显著抑制脑胶质瘤生长，具有显著的抗脑胶质瘤作用，且效果明显优于siRNA和紫杉醇。
4.Angiopep-2修饰糖脂纳米给药系统下调肿瘤细胞VEGF表达
根据上述1、2所述方法制备得到Angiopep-2修饰糖脂纳米给药系统，以4×10 5个/孔的细胞密度，取2.0mL生长良好的U87 MG细胞悬液接种于6孔板中，在37℃、5％二氧化碳条件下孵育至细胞贴壁。加入Angiopep-2修饰糖脂纳米给药系统，紫杉醇浓度为0.5μg/mL、siRNA浓度为100nM，孵育48小时，以未处理细胞作为空白对照，每组重复3次。qt-PCR检测肿瘤细胞VEGF表达水平，结果如图2所示。
根据图2所示的结果可知，Angiopep-2修饰糖脂纳米给药系统可显著下调U87 MG肿瘤细胞内VEGF mRNA表达水平(21.1％)，效果明显优于siRNA(92.9％)和紫杉醇(78.5％)。
实施例二 体外Angiopep-2修饰糖脂纳米给药系统促进病理性血脑屏障功能恢复
1.体外生理性血脑屏障模型构建
以脑微血管内皮细胞bEnd.3作为血脑屏障模型细胞，以1×10 5个/孔的细胞密度，取0.5mL生长良好的bEnd.3细胞悬液接种于孔径0.4μm的12孔Transwell板小室中，外室补加1.5mL新鲜培养液。隔天更换新鲜培养液，在37℃，5％二氧化碳条件下持续孵育15天。当细胞跨膜电阻值大于150欧姆·平方厘米(Ω·cm 2)时，即可视为生理性血脑屏障模型。
2.体外病理性血脑屏障模型构建
以U87 MG脑胶质瘤细胞作为脑肿瘤瘤模型细胞，以2×10 5个/孔的细胞密度，取1.5mL生长良好的U87 MG细胞悬液接种于12孔板中，在37℃，5％二氧化碳条件下孵育至细胞贴壁。然后将上述1所述方法构建的生理性血脑屏障即生长有bEnd.3细胞的Transwell小室转移至长有U87 MG细胞的12孔板中，继续共培养24小时，即可得到病理性血脑屏障模型。
3.体外Angiopep-2修饰糖脂纳米给药系统促进病理性血脑屏障功能 恢复
以上述2所述方法构建的病理性血脑屏障为模型，在病理性血脑屏障模型的Transwell小室中加入Angiopep-2修饰糖脂纳米给药系统，药物作用48小时后，将长有bEnd.3细胞的Transwell小室取出，用PBS清洗3次，4％甲醛固定，10％的牛血清白蛋白封闭30分钟后，加入Claudin-5/Occludin一抗并于4℃孵育过夜，最后用荧光标记的二抗孵育1小时，DAPI复染细胞核。激光共聚焦显微镜观察紧密连接蛋白Claudin-5/Occludin表达，Image J半定量分析Claudin-5/Occludin表达水平，结果如图3所示。
根据图3所示的结果可知，Angiopep-2修饰糖脂纳米给药系统作用，可减少紧密连接蛋白Claudin-5/Occludin下调表达，显著降低病理性血脑屏障破坏程度，说明Angiopep-2修饰糖脂纳米给药系统体外可促进病理性血脑屏障功能恢复。
实施例三 Angiopep-2修饰糖脂纳米给药系统促进脑胶质瘤模型动物病理性血脑屏障功能恢复
选择雄性BABL/c裸鼠(20±2g)为模型动物，借助脑立体定位仪，将表达虫荧光素酶的U87-luci脑胶质瘤细胞以5×10 5个细胞/5微升浓度接种于裸鼠纹状体(接种坐标于前囟前侧0.8毫米，右侧2毫米，深度为3毫米)区域，构建原位脑胶质瘤模型。按照紫杉醇10mg/kg、siRNA 50nM/kg的给药剂量尾静脉注射Angiopep-2修饰糖脂纳米给药系统，48小时后，按照5mg/kg的给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，小动物活体成像仪定性观察脑组织中依文思蓝分布强度，结果如图4所示。有机溶剂N，N-二甲基甲酰胺萃取脑组织中依文思蓝，紫外分光光度法定量检测依文思蓝渗透至脑内量，结果如图5所示。
根据图4、图5所示的结果可知，较荷瘤对照组，Angiopep-2修饰糖脂纳米给药系统给药作用后，依文思蓝渗透入脑含量减少，显示血脑屏障渗透性降低，表明Angiopep-2修饰糖脂纳米给药系统可降低血脑屏障渗透性，促进脑胶质瘤模型动物血脑屏障功能恢复。
实施例四 SC79呈剂量依赖性激活蛋白激酶B下游信号通路，促进紧密连接蛋白下调，增加血脑屏障渗透性
以实施例二的1所述方法构建的生理性血脑屏障为模型。
在生理性血脑屏障模型的Transwell小室中加入不同浓度(0、5μg/mL、10μg/mL)蛋白激酶B激动剂SC79，药物作用24小时后，将长有bEnd.3细胞的Transwell小室转移至另一12孔板中，PBS清洗3次，在小室中加入0.5mL HBSS营养液，外室补加1.5mL HBSS营养液，细胞于37℃平衡15分钟，跨膜电阻仪检测细胞跨膜电阻值，结果如图6所示。
在生理性血脑屏障模型的Transwell小室中加入不同浓度(0、5μg/mL、10μg/mL)蛋白激酶B激动剂SC79，药物作用24小时后，将长有bEnd.3细胞的Transwell小室转移至另一12孔板中，PBS清洗3次，在小室中加入含有FITC标记荧光标记的分子量为10kDa右旋糖酐HBSS溶液，外室补加1.5mL HBSS营养液，细胞于37℃避光孵育4小时后，荧光分光光度计检测经Transwell小室转移至外室的FITC-右旋糖酐的浓度，结果如图7所示。
在生理性血脑屏障模型的Transwell小室中加入不同浓度(0、5μg/mL、10μg/mL)蛋白激酶B激动剂SC79，药物作用24小时后，将长有bEnd.3细胞的Transwell小室取出，用PBS清洗3次，4％甲醛固定，10％的牛血清白蛋白封闭30分钟后，加入Claudin-5/Occludin一抗并于4℃孵育过夜，最后用荧光标记的二抗孵育1小时，DAPI复染细胞核。激光共聚焦显微镜观察紧密连接蛋白Claudin-5/Occludin表达情况，结果如图8所示。
根据图6、图7、图8所示的结果可知，SC79呈剂量依赖性激活蛋白激酶B下游信号通路，促进紧密连接蛋白Claudin-5/Occludin下调表达，增加血脑屏障渗透性。
实施例五 SC79呈时间依赖性激活蛋白激酶B下游信号通路，促进紧密连接蛋白下调，增加血脑屏障渗透性
以实施例二的1、2所述方法分别构建的生理性血脑屏障和病理性血脑屏障为模型，分别在生理性血脑屏障膜、病理性血脑屏障模型的Transwell小室中加入SC79(5μg/mL)刺激，实时监测细胞跨膜电阻值，结果如图9所示。
根据图9所示的结果可知，SC79刺激后，细胞跨膜电阻值呈时间依赖性下降，血脑屏障渗透性呈剂量依赖性增加，药物作用24小时后，SC79增加血脑屏障渗透性效果最明显。
实施例六 SC79增加脑胶质瘤模型动物血脑屏障渗透性，促进Angiopep-2修饰糖脂纳米给药系统入脑转运
选择雄性BABL/c裸鼠(20±2g)为模型动物，以0.2％注射级吐温80溶液稀释SC79，按照5mg/kg、10mg/kg的SC79给药剂量尾静脉注射，24小时后，按照5mg/kg的给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，小动物活体成像仪定性观察脑组织中依文思蓝分布强度，结果如图10所示。
根据图10所示的结果可知，较荷瘤对照组，SC79给药刺激后，依文思蓝渗透入脑含量增加，显示血脑屏障渗透性显著增加，表明SC79可增加血脑屏障渗透性。
选择雄性BABL/c裸鼠(20±2g)为模型动物，以0.2％注射级吐 温80溶液稀释SC79，按照5mg/kg、10mg/kg的SC79给药剂量尾静脉注射，0.5小时、24小时后，按照5mg/kg的给药剂量尾静脉注射依文思蓝溶液，4小时后，心脏灌流取裸鼠完整大脑，小动物活体成像仪定性观察脑组织中依文思蓝分布强度，结果如图11所示。
根据图11所示的结果可知，SC79给药刺激0.5小时后，依文思蓝渗透入脑含量增加；SC79给药刺激24小时，依文思蓝渗透入脑含量最高，显示SC79调控血脑屏障渗透性增加效果最明显。
选择雄性BABL/c裸鼠(20±2g)为模型动物，借助脑立体定位仪，将表达虫荧光素酶的U87-luci脑胶质瘤细胞以5×10 5个细胞/5微升浓度接种于裸鼠纹状体(接种坐标于前囟前侧0.8毫米，右侧2毫米，深度为3毫米)区域，构建原位脑胶质瘤模型。以0.2％注射级吐温80溶液稀释SC79，按照5mg/kg的SC79给药剂量尾静脉注射，24小时后，尾静脉注射包封近红外荧光探针DiR的Angiopep-2修饰糖脂纳米给药系统，4小时后，心脏灌流取裸鼠完整大脑，小动物活体成像仪定性观察脑组织中Angiopep-2修饰糖脂纳米给药系统分布强度，结果如图12所示。
根据图12所示的结果可知，较荷瘤对照组，SC79给药刺激后，Angiopep-2修饰糖脂纳米给药系统脑内分布增加，显示SC79可以提高Angiopep-2修饰糖脂纳米给药系统的脑内转运效率。
实施例七 SC79与Angiopep-2修饰糖脂纳米给药系统序贯治疗脑胶质瘤
选择雄性BABL/c裸鼠(20±2g)为模型动物，借助脑立体定位仪，将表达虫荧光素酶的U87-luci脑胶质瘤细胞以5×10 5个细胞/5微升浓度接种于裸鼠纹状体(接种坐标于前囟前侧0.8毫米，右侧2毫米，深度为3毫米)区域，构建原位脑胶质瘤模型。按照紫杉醇10mg/kg、siRNA 50nM/kg的给药剂量尾静脉注射Angiopep-2修饰糖脂纳米给药系统，48小时后，尾静脉注射SC79吐温溶液，24小时后待SC79调控血脑屏障渗透性至最大时，再次尾静脉注射Angiopep-2修饰糖脂纳米给药系统，累计尾静脉注射SC79三次、Angiopep-2修饰糖脂纳米给药系统四次。生物发光成像技术实时监测原位脑胶质瘤 生长。结果如图13所示。
根据图13所示的结果可知，较生理盐水对照组及单一的Angiopep-2修饰糖脂纳米给药系统治疗组，SC79和Angiopep-2修饰糖脂纳米给药系统序贯治疗可显著抑制脑胶质瘤的生长。
工业实用性
根据本发明，能够提供一种以SC79或其类似物作为有效成分的血脑屏障渗透性调控剂，其能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率，特别是能够增强Angiopep-2修饰的糖脂纳米给药系统入脑的转运效率。通过组合使用该血脑屏障渗透性调控剂与脑靶向药物递释系统，能够增强脑靶向药物递释系统入脑的转运效率，进而提高脑靶向药物递释系统的对脑部疾病的治疗效果，具有显著优越的技术效果。因此，本发明在医药领域中具有很大的实用性。

Claims (10)

  1. SC79或其类似物在制备血脑屏障渗透性调控剂中的应用。
  2. 如权利要求1所述的应用，其特征在于：
    所述血脑屏障渗透性调控剂能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率。
  3. 如权利要求2所述的应用，其特征在于：
    所述脑靶向药物递释系统是Angiopep-2修饰的糖脂纳米给药系统。
  4. 如权利要求3所述的应用，其特征在于：
    所述Angiopep-2修饰的糖脂纳米给药系统是共递送紫杉醇和siRNA的Angiopep-2修饰糖脂纳米给药系统。
  5. 如权利要求4所述的应用，其特征在于：
    所述siRNA包含具有下述核苷酸序列的siRNA，
    正义链5’-GUCUAUCAGCGCAGCUACUTT-3’，
    反义链5’-AGUAGCUGCGCUGAUAGACTT-3’。
  6. 如权利要求1～5中任一项所述的应用，其特征在于：
    所述血脑屏障渗透性调控剂通过激活蛋白激酶B下游Claudin-5、Occludin信号通路而下调紧密连接蛋白Claudin-5、Occludin的表达，由此增加血脑屏障渗透性。
  7. 如权利要求1～5中任一项所述的应用，其特征在于：
    在施用所述血脑屏障渗透性调控剂0.5小时后施用脑靶向药物递释系统。
  8. 如权利要求1～5中任一项所述的应用，其特征在于：
    在施用所述血脑屏障渗透性调控剂24小时后施用脑靶向药物递释系统。
  9. 一种脑靶向给药试剂盒，其特征在于：
    其包含血脑屏障渗透性调控剂和脑靶向药物递释系统的组合，
    所述血脑屏障渗透性调控剂含有SC79或其类似物作为有效成分，能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率，
    所述脑靶向药物递释系统是Angiopep-2修饰的糖脂纳米给药系统。
  10. 血脑屏障渗透性调控剂与脑靶向药物递释系统的组合在脑靶向给药试剂盒的制造中的应用，其特征在于：
    所述血脑屏障渗透性调控剂含有SC79或其类似物作为有效成分，能够增加血脑屏障渗透性，增强脑靶向药物递释系统入脑的转运效率，
    所述脑靶向药物递释系统是Angiopep-2修饰的糖脂纳米给药系统。
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