WO2019235746A1 - Administration intranasale efficace au cerveau - Google Patents

Administration intranasale efficace au cerveau Download PDF

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WO2019235746A1
WO2019235746A1 PCT/KR2019/005516 KR2019005516W WO2019235746A1 WO 2019235746 A1 WO2019235746 A1 WO 2019235746A1 KR 2019005516 W KR2019005516 W KR 2019005516W WO 2019235746 A1 WO2019235746 A1 WO 2019235746A1
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brain
ppa5
disease
tumor
nasal
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PCT/KR2019/005516
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Korean (ko)
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윤채옥
이수환
카살라다야난다
에스. 랭거로버트
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한양대학교 산학협력단
메사추세츠 인슈티튜트 오브 테크놀로지
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Publication of WO2019235746A1 publication Critical patent/WO2019235746A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—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
    • 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
    • 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
    • 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
    • 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 present invention effectively delivers a pH-sensitive bioreducible polymer that can be used as a drug carrier to the brain through nasal administration, thereby effectively transmitting brain by nasal administration used for the diagnosis, prevention or treatment of central nervous system disease, neurodegenerative disease or brain tumor. It's about technology.
  • Noninvasive tracking and imaging of diseased tissue can enable early detection with routinely performed diagnostic procedures that can lead to timely intervention of the disease and the overall improved prognosis of the patient.
  • Intra-nasal delivery is emerging as a new delivery system that can bypass the Blood-Brain Barrier, the largest barrier in the delivery of therapeutic agents for the treatment of central nervous system diseases.
  • the present invention provides a polymer represented by the following formula (1):
  • the present invention is also a polymer represented by the formula (1); And at least one drug selected from the group consisting of a therapeutic agent and a diagnostic agent.
  • the present invention also provides a method for treating any one of central nervous system diseases, neurodegenerative diseases or brain tumors, comprising nasal administration in a pharmaceutically effective amount to a subject in need thereof.
  • the pH-sensitive bioreducible polymer of the present invention can reduce side effects by bypassing the blood brain barrier through nasal administration and improving the bioavailability of the drug to lower the dose of the drug.
  • the nasal administered drug initially reaches the target disease site of the brain without passing through the bloodstream for systemic circulation, minimizing the negative side effects of overdose.
  • the present invention can efficiently deliver drugs to various parts of the brain tissue, which is suitable for the treatment of central nervous system diseases, neurodegenerative diseases or brain tumors.
  • the pH-sensitive bioreducible polymer of the present invention is labeled with a diagnostic label and delivered to brain tissue through nasal administration, which can be used for diagnosis of central nervous system disease, neurodegenerative disease or brain tumor.
  • FIG. 11 shows PTX concentrations in various regions of brain tissue after nasal or intravenous administration of PPA5-PTX-IR780 in brain tumor bearing mouse model.
  • FIG. 12 shows PTX concentrations in various regions of brain tissue after nasal or intravenous administration of PPA5-PTX-IR780 in normal mouse model.
  • FIG. 14 shows the siRNA distribution profiles of the brain after nasal administration of siRNA-Cy5.5 / PPA5-IR780 in a glioblastoma bearing mouse model.
  • FIG. 15 shows pDNA intensity-time profile after nasal or intravenous administration of pDNA-FITC / PPA5-IR780 in brain tumor bearing mouse model.
  • 16 shows the pDNA intensity-time profile after nasal or intravenous administration of pDNA-FITC / PPA5-IR780 in normal mouse model.
  • 17 shows bioavailability of pDNA in tumor bearing mouse models or brains of normal mice.
  • FIG. 18 shows the region-specific pDNA distribution profiles of the brain after nasal administration of the pDNA-FITC / polymer complex in a normal mouse model.
  • FIG. 23 shows H & E staining results showing potent antitumor efficacy of PPA5-PTX by nasal administration in U87MG / Fluc orthotopic brain tumor model.
  • the present invention relates to a polymer represented by the following formula (1).
  • the polymer of Formula 1 of the present invention is a compound having a high biodegradability in the intracellular pH range (about pH 6.0) and having a biodegradability and pH sensitivity targeting a low pH hypoxic tumor microenvironment, comprising: (i) immunity Consisting of escapable portions from immune reactions, (ii) chargeable portions, and (iii) bioreducible portions comprising disulfide bonds.
  • polymer of Formula 1 may be called 'PPA5' generated through the synthesis of mPEG-b-Pip-CBA (PPCBA) and polyethyleneimine-arginine (PEI-Arg).
  • polyethylene glycol acts as an immune response avoiding site
  • arginine is a charge site that imparts a positive charge to the polymer.
  • Arginine imparts a positive charge to the polymer, allowing it to form a complex by binding by ionic interaction with a negatively charged surface (eg, the negatively charged surface of an adenovirus).
  • N, N'-citamine bisacrylamide is a moiety that acts as a bioreducible site containing disulfide bonds, and the moiety that provides the pH sensitivity of the polymer is piperazine.
  • PPA5 For the synthesis of PPA5, mPEG-acrylate (5kDa), N, N'-citamine bisacrylamide and piperazine were reacted to synthesize mPEG-piperazine-N, N'-cystamine bisacrylamide (PPCBA), and then the PEI Add -Arg to synthesize PPCBA-PEI-Arg (PPA5).
  • the PPA5 polymer of the present invention is a pH sensitive type, and in particular, may target the microenvironment of tumor cells in hypoxic conditions and thus may be used as a drug carrier for delivering drugs to tumor cells.
  • the PPA5 polymer of the present invention may be used as a drug carrier for delivering a drug or the like in vivo by binding to a target component having a negatively charged surface due to the positive charge of the surface.
  • a drug carrier for delivering drugs to the target disease site.
  • the PPA5 polymer of the present invention is delivered to tumor cells in combination with a photosensitizer and can kill tumor cells through photothermal therapy during near-infrared irradiation in the 700-900 nm wavelength range.
  • PPA5 polymer of the present invention can be introduced into the brain tissue by bypassing the brain blood barrier through nasal administration can be used as a drug carrier.
  • the PPA5 polymer of the present invention may be combined with a diagnostic agent on the surface, bypass the cerebral blood barrier through nasal administration, and enter the brain tissue to be used for diagnosis of brain disease.
  • the present invention is a polymer represented by the formula (1); And at least one drug selected from the group consisting of a therapeutic agent and a diagnostic agent.
  • the drug carrier may preferably be a drug carrier for nasal administration.
  • the PPA5 polymer of the present invention may be introduced into the brain tissue by bypassing the brain blood barrier through nasal administration to effectively deliver drugs.
  • the drug carrier of the present invention may comprise a complex form of a PPA5 polymer and a drug.
  • the therapeutic agent may be one or more selected from the group consisting of a gene delivery system, a photosensitive agent, and a pharmaceutically active ingredient.
  • the gene delivery system may be in the form of (i) a naked recombinant DNA molecule, (ii) a plasmid, (iii) a viral vector, and (iv) a liposome or niosome that contains the Naked recombinant DNA molecule or plasmid. have.
  • All gene delivery systems used in conventional gene therapy can be applied, preferably plasmids; Adenovirus (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997)), Adeno-associated viruses: AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc.
  • Viral vectors such as vaccinia virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)), reovirus, poxvirus, Semiki forester virus, Measles virus; Liposomes containing the Naked recombinant DNA molecule or plasmid (Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002) or niosomes can be used.
  • vaccinia virus Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)
  • reovirus poxvirus
  • Semiki forester virus Measles virus
  • Liposomes containing the Naked recombinant DNA molecule or plasmid Methodhods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002
  • niosomes can be used.
  • Adenoviruses are widely used as gene transfer vectors because of their medium genome size, ease of manipulation, high titers, wide range of target cells and excellent infectivity. Both ends of the genome contain 100-200 bp of Inverted Terminal Repeat (ITR), which is an essential cis element for DNA replication and packaging.
  • ITR Inverted Terminal Repeat
  • the genome El region (E1A and E1B) encodes proteins that regulate transcription and transcription of host cell genes.
  • the E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.
  • the target nucleotide sequence to be delivered intracellularly is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably the E1B region) or E3 region, more preferably the deleted E1 region. do.
  • the term "deletion" as used in connection with a viral genome sequence has the meaning including not only that sequence in its entirety, but also partially deleted.
  • Adenoviruses have 42 different serotypes and subgroups of A-F. Among these, adenovirus types 2 and 5 belonging to subgroup C are the most preferred starting materials for obtaining the adenovirus vectors of the present invention. Biochemical and genetic information for adenovirus types 2 and 5 is well known.
  • Retroviruses are widely used as gene transfer vectors because they insert their genes into the host genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can infect them.
  • the target nucleotide sequence to be transported into the cell is inserted into the retroviral genome instead of the sequence of the retrovirus to produce a nonreplicating virus.
  • a packaging cell line is constructed that includes gag, pol and env genes but does not have long terminal repeat (LTR) and ⁇ sequences (Mann et al., Cell, 33: 153-159 (1983)).
  • RNA transcripts of the recombinant plasmids When a recombinant plasmid comprising the target nucleotide sequence, LTR, and ⁇ sequences to be transported is introduced into the cell line, the ⁇ sequences enable the production of RNA transcripts of the recombinant plasmids, which are packaged into a virus and the virus is Discharged to the medium (Nicolas and Rubinstein "Retroviral vectors," In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, 494-513 (1988)). The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.
  • MMLV Moroni murine rheumemia virus
  • EPO erythropoietin
  • the gene delivery system of the present invention can also be prepared according to this second generation retroviral vector construction strategy.
  • Adeno-associated virus is suitable as the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect various kinds of cells. Details of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368.
  • the AAV virus is a plasmid (McLaughlin et al., J. Virol., 62: 1963-) containing a gene sequence of interest (a target nucleotide sequence to be carried into a cell) with two AAV terminal repeats located next to it. 1973 (1988); and Samulski et al., J. Virol., 63: 3822-3828 (1989) and expression plasmids comprising wild type AAV coding sequences without terminal repeats (McCarty et al., J. Virol., 65: 2936-2945 (1991).
  • viral vectors can also be used in the gene delivery system of the present invention.
  • Vaccinia virus Panhlmann M. et al., Human Gene Therapy 10: 649-657 (1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses.Rodrigue and Denhardt, eds Stoneham: Butterworth, 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed.
  • viral vectors include Rio virus, poxvirus, Semiki Forester virus, and Measles virus.
  • Liposomes are automatically formed by phospholipids dispersed in the aqueous phase. Examples of successfully delivering foreign DNA molecules into liposomes into cells include Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982) and Nicolau et al., Methods Enzymol., 149: 157-176 (1987). Meanwhile, the most widely used reagent for transforming animal cells using liposomes is lipofectamine (Lipofectamine, Gibco BRL). Liposomes containing the target nucleotide sequence to be transported interact with the cell through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes to deliver the target nucleotide sequence into the cell.
  • lipofectamine Liposomes containing the target nucleotide sequence to be transported interact with the cell through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes to deliver the target nucleot
  • the gene delivery system may be a recombinant adenovirus.
  • recombinant adenovirus as a gene transfer vector
  • the use frequency of cancer gene therapy is steadily increasing.
  • the recombinant adenovirus has been in the spotlight as a gene carrier for cancer treatment because the immune response of the host induced by the virus used as a vector may not be a big problem or may act as an advantage.
  • the recombinant adenovirus may be a non-replicating adenovirus or a tumor killing adenovirus.
  • Non-replicating adenoviruses are recombinant by inserting therapeutic genes instead of E1 genes (all or part) which are essential for the replication of adenoviruses, which prevent them from replicating in the cells into which the adenoviruses are introduced.
  • Tumor-killing adenoviruses are adenoviruses that are partially deficient in the E1B 55kDa gene and are capable of propagation only in cells in which p53 is functionally inactivated. In cancer cells where p53 function is inhibited, virus proliferation occurs actively, whereas in normal cells, virus proliferation is inhibited. Therefore, tumor killing adenovirus does not affect normal cells and can selectively kill only cancer cells, which is particularly advantageous in the treatment of cancer.
  • the recombinant adenovirus has an inactivated E1B 19kDa gene, an E1B 55kDa gene or an E1B 19kDa / E1B 55kDa gene, and preferably has an inactivated E1B 19kDa and E1B 55kDa genes.
  • an inactivated E1B 19kDa gene is a gene in which a mutation (substitution, addition, partial deletion or total deletion) occurs in the gene that does not produce an active E1B 19 kDa protein.
  • a mutation substitution, addition, partial deletion or total deletion
  • the recombinant adenovirus of the invention may comprise an active E1A gene.
  • Recombinant adenoviruses comprising the E1A gene will have replicable properties.
  • the recombinant adenovirus of the present invention comprises an inactivated E1B 19kDa / E1B 55kDa gene and an active E1A gene.
  • the gene delivery system may be a replication-incompetent tumor-selective killing adenovirus with an E1 site deleted.
  • the vector preferably further comprises a selection marker.
  • selection marker in the present invention is intended to facilitate the selection of transformed cells by introducing a shRNA expression cassette.
  • the screening marker that can be used in the vector of the present invention is not particularly limited as long as it is a gene capable of easily detecting or measuring the introduction of the vector, but typically, drug resistance, nutritional requirements, resistance to cytotoxic agents or surfaces Markers that confer a selectable phenotype, such as expression of a protein, for example GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol Histidinol dehydrogenase gene (hiD) or guanine phosphosribosyltransferase (Gpt).
  • the photosensitizers are porphyrins (phorphyrins) compounds, chlorins (chlorins) compounds, bacteriochlorins (bacteriochlorins) compounds, phthalocyanines (phthalocyanines) compounds, naphthalocyanines (Naphthalocyanines) compounds and 5-aminolevulin ester It can be selected from the group consisting of 5-aminolevuline esters compounds.
  • it may be IR-780, Cu-64-DOTA, or the like, which exhibits a photothermal effect under near infrared rays in the 700-900 nm wavelength range.
  • the kind of the pharmaceutically active ingredient of the present invention is not particularly limited, and for example, anticancer agents, antibiotics, hormones, hormonal antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphokoxy, urokinase, streptokinase And one or more selected from the group consisting of tissue plasminogen activators, protease inhibitors, alkylphosphocholines, radioisotope labeling components, surfactants, cardiovascular drugs and nervous system drugs.
  • anticancer agents bevacizumab, temozolomide, nitrosourea, cisplatin, PCV (procarbazine + CCNU + vincristine), vinblastine, vinblastine (vinblastine) vincristine, vinflunine, vindesine, vinorelbine, cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, paclitaxel, Tecetaxel, ixabepilone, herceptin, erbitux, cyclophosphamide, doxorubicin, Etoposide, Topotecan, Topotecan, Carboplatin, procarbazine, mechlorethamine, ifosfamide, melphalan, chlorambucil, bisululfan, diactino Mycin (dactinomycin), daunorubicin, bleomycin (bleomycin), plicomycin, mitomycin, mi
  • the drug carrier of the present invention can be used for the prevention or treatment of brain tumors through the tumor targeting of PPA5 polymer, and the drug can be delivered to the target disease site through binding to other target components, thereby preventing the central nervous system disease or neurodegenerative disease. It can be used for prevention or treatment.
  • the central nervous system diseases include cognitive impairment, intellectual disability, cerebellar disease, epilepsy, neurodevelopmental disorder, dementia, autism spectrum disorder, Down syndrome, Rett syndrome, fragile X syndrome and the like.
  • the neurodegenerative diseases include ischemic stroke, traumatic brain injury, acute disseminated encephalomyelitis, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mild cognitive impairment, Alzheimer's disease, peak disease, senile dementia, progressive supranuclear palsy, subcortical dementia, Wilson's disease, multiple infarct disease, atherosclerotic dementia, AIDS-related dementia, cerebellar degeneration degeneration, spinocerebellar degeneration syndromes, Friedreichs ataxia, ataxia telangiectasia, epilepsy-related brain injury, spinal cord injury, restless legs syndrome , Huntington's disease, Parkinson's disease, striatonigral degeneration, cerebral vasculitis, mitochondria Mitochondrial encephalomyopathies, neuronal ceroid lipofuscinosis, spinal muscular atrophies, lysosomal storage disorders, leukodystrophies, Urea cycle defect disorder, hepatic ence
  • the brain tumor refers to tumors that occur in the brain and meninges.
  • the brain tumors are glioma, oligodendroglioma, glioblastoma, colloid cyst, epidermoid cyst meningioma, hemangioblastoma, lymphoma lymphoma, pituitary adenoma, metastatic tumor, or a combination thereof.
  • the glioma is glioblastoma multiforme (GBM).
  • GBM glioblastoma multiforme
  • the brain tumor may be a tumor metastasized from a primary brain tumor or other cancer.
  • the PPA5 polymer of the present invention may be labeled with a diagnostic agent (diagnostic marker), and the polymer labeled by the marker may be traced in vivo, thereby optically detecting and imaging a central nervous system disease, a neurodegenerative disease or a brain tumor. This is possible.
  • a diagnostic agent diagnostic marker
  • the diagnostic agent can be used without limitation as long as the substance can detect and recognize the target cell.
  • near-infrared fluorescent substance that can penetrate the living body such as cyanine, allophycocyanin, fluorescein, tetramethylrhodamine, BODIPY or Alexa ) Etc; Calcium-47, Carbon-11, Carbon-14, Chromium-51, Cobalt-57, Cobalt-58, Erbium-169, Fluorine-18, Gallium-67, Gallium-68, Hydrogen-3, Indium-111, Iodine- Radiopharmaceuticals such as 123, Iodine-131, Technetium-99m; Or MRI contrast agents.
  • the drug carrier of the present invention may be administered to tissues or cells isolated from a diagnosis subject and used by the PPA5 polymer and / or the diagnostic agent to detect a signal and obtain an image.
  • a magnetic resonance imaging apparatus or an optical imaging apparatus may be used.
  • the magnetic resonance imaging apparatus places a living body in a strong magnetic field and irradiates radio waves of a specific frequency to absorb energy into atomic nuclei such as hydrogen in biological tissues to make the energy high, and then stops propagating the atomic nuclei such as hydrogen. It is a device that allows energy to be emitted and converts this energy into a signal that is processed by a computer and imaged.
  • the magnetic resonance imaging apparatus is preferably a T2 spin-spin relaxation magnetic resonance imaging apparatus.
  • the drug carrier of the present invention may further comprise a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in drug carriers formulated in liquid solutions are sterile and biocompatible, which are saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol And one or more of these components can be mixed and used, and other conventional additives, such as antioxidant, a buffer solution, and bacteriostatic agent, can be added as needed.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Nasal administration formulations containing the drug delivery system of the present invention as an active ingredient may be formulated for injection or in the form of sprays such as aerosols that enable inhalation through the respiratory system.
  • the drug delivery agents of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials.
  • a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.
  • the drug carrier of the present invention may be injected into the nasal-brain administration route through the nasal-brain delivery drug delivery device.
  • the drug delivery device for nasal-brain delivery may use a known nebulizer form.
  • the drug carrier of the present invention may be used as a single therapy, but may also be used in combination with other conventional chemotherapy or radiation therapy, and when the combination therapy is performed, disease treatment may be more effectively performed.
  • the present invention treats any one of central nervous system diseases, neurodegenerative diseases or brain tumors, comprising nasal administration in a pharmaceutically effective amount to a subject in need thereof.
  • nasal administration in a pharmaceutically effective amount to a subject in need thereof.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment.
  • Suitable dosages of the drug delivery agents of the present invention vary depending on factors such as formulation method, mode of administration, age, weight, sex, degree of disease symptom, food, time of administration, rate of excretion and response to reaction, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment.
  • the drug delivery system of the present invention 1 ⁇ 10 5 - 1 ⁇ 10 15 pfu / ml of polymers or of polymer and includes a composite of the gene delivery system, a light feeling agent or a pharmaceutically active ingredient, a typically 1 ⁇ 10 10 pfu in two Inject once every two weeks.
  • the term "individual" of the present invention includes animals or humans, such as horses, sheep, pigs, goats, camels, antelopes, dogs, and the like, whose diseases may be ameliorated by the administration of the drug carrier according to the present invention.
  • the treatment method according to the present invention may be a method of treating animals except humans, but is not limited thereto. That is, in the case of humans, considering that the disease can be improved by administration of the drug delivery system according to the present invention, it can be sufficiently used in the treatment of humans.
  • Arginine was conjugated to polyethyleneimine according to the references (Journal of Colloid and Interface Science 348 (2010) 360-368, Biomaterials 31 (2010) 8759-8769).
  • Carboxylic acid groups of arginine amino acids 350 mg, 2.0 mmol
  • EDC / NHS EDC, 384 mg, 2.0 mmol
  • NHS 230 mg 2.0 mmol
  • arginine was activated by addition of polyethylenimine (PEI) (360 mg, 0.2 mmol) and
  • the product was dialyzed against secondary distilled water for 1 day (MWCO 1.0 kDa) to remove unreacted compound and lyophilized to give PEI-Arg as a white substance.
  • the chemical structure was 1 H NMR (600 MHz, D 2 O). Characteristic peak of PEI (2.0-3.0 ppm) and peak of arginine (1.66-(-HCCH 2 CH 2 CH 2 NH-); 1.86 (-HCCH 2 CH 2 CH 2 NH-); 3.24 ( -HCCH 2 CH 2 CH 2 NH-) and 3.86 (-HCCH 2 CH 2 CH 2 NH-) were identified.
  • U87MG-Fluc cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and 37 in DMEM (Gibco BRL, Grand Island, NY) medium containing 10% FBS (Gibco BRL) and HEPES (Gibco BRL). Incubated at 5 °C CO 2 conditions.
  • Non-replicating Ad (dE1 / GFP) and oncolytic Ad (DWP418, RdB / IL-12 / Decorin or HE5cT-Rd19-k35 / Decorin; oAd) expressing GFP (green fluorescent protein) under CMV promoter control at the E1 site
  • dE1 / GFP Non-replicating Ad
  • DWP418, RdB / IL-12 / Decorin or HE5cT-Rd19-k35 / Decorin; oAd expressing GFP (green fluorescent protein) under CMV promoter control at the E1 site
  • Ads were propagated in HEK293 cells, followed by CsCl (Sigma, St Louis, MI) gradient gradient purification.
  • the number of virus particles (VP) was calculated as absorbance 1 equal to 10 12 VP / mL in the OD 260 measurement.
  • Virus titers (Infectious titers, PFU / mL) were determined using a limiting dilution assay on HEK293 cells. Viral particle / PFU ratios of dE1 / GFP and DWP418 were 29: 1 and 81: 1, respectively.
  • MOI was calculated from viral titers.
  • Ad particles (2 ⁇ 10 10 VP / PBS, pH 7.4) were mixed with PPA5 polymer at various concentrations. As a result, the molar ratio of Ad particle per polymer became 1 * 10 ⁇ 5> and 1 * 10 ⁇ 6> . The solution was incubated for 30 minutes at room temperature before use.
  • the complex was injected by the nasal route and NIR imaging was performed 5 minutes, 6, 12, 18, 24, 48 h after administration.
  • Ad / PPA5-IR780 complex shows that the signal remains similar from 5 minutes to 18 h after injection, and then gradually decreases in a time dependent manner until 48 h.
  • the Ad / PPA5-IR780 complex entered the tumor tissue, many fluorescence signals were maintained in the tumor bearing brain region: the maximum fluorescence intensity in the tumor region was achieved 5 minutes after irradiation.
  • U87MG-Fluc a human glioblastoma cell line stably expressing the firefly luciferase gene
  • U87MG-Fluc a human glioblastoma cell line stably expressing the firefly luciferase gene
  • Bioluminescence imaging was performed at 6 day intervals to monitor tumor growth non-invasively.
  • Nasal injection of the Ad / PPA5-IR780 complex on day 21 was followed by laser irradiation on brain tumors 6 hours after administration.
  • the fluorescence intensity was 165 times lower in the Ad / PPA5-IR780-treated group (7.64 ⁇ 10 6 p / s) compared to the PBS-treated group (1.26 ⁇ 10 9 p / s). This result confirms the potent antitumor effect of the nasal administered Ad / PPA5-IR780 complex.
  • the U87MG-Fluc in situ glioblastoma model was used to compare the therapeutic efficacy and in vivo distribution profiles of various nasal administrations of nanocomposites with Ad and various types of IR780-bound polymers. All treatments were administered via the nasal route 6 days after cell injection. All tumors were laser irradiated 6 hours after nasal administration of treatment.
  • NIR imaging was performed every 24 hours after the first nasal administration of the therapeutic agent on day 6 after tumor cell injection.
  • Ad / PPA2-IR780 or Ad / PPA5-IR780 treated mice showed higher fluorescence intensity in brain tissues than Ad / PAMAM (G2) -IR780 treated mice; After administration of Ad / PAMAM (G2) -IR780, minimal or no fluorescence was detected in the brain region.
  • Ad / PAMAM-PEG Ad / PAMAM-PEG
  • Ad / PPSA Ad / APP
  • Ad / PPA5-IR780s were administered nasal to the mice three times apart.
  • One group of mice received Ad / PPA5-IR780 via the intravenous route (named Ad / PPA5-IR780 (I.V.) group). All tumors were irradiated with laser 6 h after treatment.
  • luciferase signals obtained from isotopic tumors of Ad / PPA5-IR780 (IN injection) treated mice were significantly lower than those of the other groups, and Ad / PAMAM-PEG , PPA5-IR780-mediated tumor fluorescence showing 30.5-, 29.5-, 9.66-, or 27.9-fold lower tumor fluorescence than Ad / PPSA, Ad / APP or Ad / PPA5-IR780 (IV injection) treated groups, respectively Nasal delivery is shown to induce much better therapeutic efficacy compared to intravenous delivery.
  • intraocular brain tumors expressing luciferase were injected intravenously (IV) with oAd / PPA5-IR780 or PBS, oAd or o Ad / PPA5-IR780 was injected nasal (IN). Twenty four hours after injection, tumors were irradiated with 808 nm laser for 3 minutes.
  • PTX-bound polymer variant (PPA5-PTX-IR780) was administered via either the intravenous or nasal route. At 0.5, 1, 2 and 6h post-injection, brain tissue was harvested and the amount of PTX in various anatomical regions of brain tissue was measured by LC-MS / MS.
  • the amount of PTX in the subregions of the brain rises irregularly within 0.5 h and reaches normal 2 h after nasal injection. After 2 h of nasal injection, intratumoral PTX concentration was the highest among the subregions of the brain.
  • PPA5-PTX-IR780 In normal mouse models, nasal injection of PPA5-PTX-IR780 induces efficient entry of PTX into brain tissue similar to that observed in tumor bearing mice in FIG. 11 (FIG. 12); Similar in vivo distribution patterns were observed in both mice, but the total amount of PTX was slightly lower in normal mice than in tumor bearing mice. These results suggest that the presence of tumors is not essential for efficient brain delivery of PPA5-PTX-IR780. Importantly, these results suggest that the PPA5-IR780 polymer backbone can be used as a drug carrier for the treatment of central nervous system disease, neurodegenerative diseases or brain tumors.
  • DTP (direct delivery to the nasal-brain) efficiency of PTX after nasal delivery of PPA5-PTX-IR780 has the values obtained in FIGS. 11 and 12, and is used to determine the nose-to-brain direct transport and the targeting targeting efficiency Calculated and analyzed.
  • DTP% represents the proportion of drugs delivered directly to the brain via the olfactory pathway.
  • PPA5-PTX-IR780 has the highest DTE% (1620.95 +/- 38.81 in tumor models and 1019.17 +/- 28.39 in normal models) and DTP% (93.83 +/- 0.97 in tumor models and 90.18 +/- 0.96 in normal models
  • Administration of PPA5-PTX-IR780 via the nasal route results in better delivery of the therapeutic to the brain than intravenous due to efficient delivery through the nasal region of the nasal cavity.
  • the amount of PTX delivered to the brain via the nasal passages was higher in the tumor model when compared to the tumor model and the normal model.
  • siRNA-Cy5.5 Cy5.5-labeled siRNA
  • FITC-labeled pDNA was complexed with PPA5-IR780 for nasal delivery.
  • the pDNA-FITC / PPA5-IR780 complex was administered via the intravenous and nasal route. At 0.5, 1, 2 and 6h after injection, the presence of pDNA was tested in brain tissue using a fluorescence reader.
  • pDNA-FITC was compared with P (DTT) PA5, P (DTT) PA5-IR780, PPA5, or PPA5-IR780 polymers.
  • the complex was formed and then administered by the nasal route.
  • fluorescence readers were used to test for the presence of pDNA in various regions of brain tissue.
  • FITC strength was treated by pDNA-FITC complexed with pH reactive polymer compared to pDNA-FITC treated groups (groups 2 and 3) complexed with non-pH reactive polymer. Higher in mice (groups 4 and 5).
  • these results indicated that the brain entry efficiency of the nasal-administered pDNA / polymer complex was not dependent on the photosensitive IR780 moiety, as Groups 2 and 4 showed similar accumulation as Groups 3 and 5 in various regions of brain tissue. .
  • pH sensitivity is important for effective polymer-mediated delivery of therapeutics via the nasal route.
  • the efficiency of delivery of the oAd / PPA5-Cu-64-DOTA-Herceptin complex to the brain via nasal, intraperitoneal and intravenous administration was measured via PET / CT imaging.
  • nasal delivery showed the strongest signal in the brain region compared to other delivery methods.
  • the binding of the therapeutic antibody and PPA5 could be effectively delivered to the brain in the nasal-administered experimental group rather than through the PBS or intravenous (IV).
  • the highest rate was detected as 20% of the initial dose 2 hours after administration.
  • PTX was successfully delivered to various regions of the brain by nasal delivery. In addition, up to 12% of the initial dose was detected in brain tissue.
  • nasal delivery of PPA5-PTX in the U87MG / Fluc orthotopic brain tumor model was determined by PTX I.V. Delivery and PTX I.N. It showed much better antitumor efficacy than delivery, and 1/25 doses of PTX concentration (I.N. delivery) showed much better efficacy than intravenous delivery.
  • the present invention can be used in the field of diagnosis and treatment of central nervous system diseases, neurodegenerative diseases or brain tumors.

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Abstract

La présente invention concerne une technique d'administration intranasale efficace au cerveau. Plus particulièrement, la présente invention concerne un effet d'application au diagnostic, à la prévention, ou au traitement d'une maladie cérébrale du système nerveux central, ou d'une tumeur cérébrale en administrant efficacement un médicament au cerveau par l'administration intranasale d'un polymère PPA5 bioréductible, sensible au pH, qui peut être utilisé comme support de médicament.
PCT/KR2019/005516 2018-05-08 2019-05-08 Administration intranasale efficace au cerveau WO2019235746A1 (fr)

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