Polymer nanocomposite for brain-targeted drug delivery and pharmaceutical composition comprising same

Abstract

The present invention relates to a polymer nanocomposite for brain-targeted drug delivery and a pharmaceutical composition comprising same. The polymer nanocomposite of the present invention has a structure in which (a) a cyclodextrin derivative bonded to a piperazine derivative, which is a brain-targeting ligand, and (b) a polymer chain bonded to a hydrophobic compound, which is a guest molecule, are self-assembled through host-guest interaction. The nanocomposite is loaded with a therapeutic drug and can efficiently penetrate the blood-brain barrier and deliver the drug to brain tissue through intranasal administration, which is a non-invasive route.

Description

Polymer nanocomposites for brain-targeted drug delivery and pharmaceutical compositions containing the same

[0001] The present invention relates to a drug delivery system (DDS) technology, and more specifically, to a non-invasive drug delivery platform capable of efficiently penetrating the blood-brain barrier (BBB) ​​to deliver drugs to the central nervous system (CNS). Specifically, the present invention relates to a nanocomposite formed based on hydrophobic interactions of hydrophobic molecules comprising a cyclodextrin derivative and a hydrophobic compound, and a pharmaceutical composition for treating brain diseases using the same.

[0002] The prevalence of central nervous system (CNS) diseases, such as degenerative brain diseases like Alzheimer's and Parkinson's, epilepsy, brain tumors, and stroke, is increasing worldwide, and the resulting socioeconomic burden is enormous. To effectively treat these diseases, therapeutic drugs must reach target sites within brain tissue at sufficient concentrations. However, the brain is protected by a highly selective barrier called the blood-brain barrier (BBB), which severely restricts the movement of most therapeutic drugs (especially macromolecular drugs and gene therapies) from the bloodstream to the brain parenchyma.

[0003] The blood-brain barrier is formed by the tight connection of endothelial cells in brain capillaries through tight junctions.

[0004] It acts as a physical barrier that strictly controls the influx of external substances. As a result, it is known that more than 98% of drugs administered into the systemic circulation fail to reach the brain. Various brain drug delivery technologies have been studied to overcome these limitations.

[0005] Traditional methods include invasive techniques involving opening the skull and directly injecting drugs into the brain; however, this places a significant burden on the patient and carries a high risk of infection and tissue damage, making it difficult to apply to repeated treatments. Another method involves temporarily opening the blood-brain barrier using substances such as mannitol, but this raises safety concerns as it can introduce unwanted substances into the brain. Additionally, drug delivery systems utilizing receptor-mediated transcytosis (RTA) based on specific receptors present in the blood-brain barrier (e.g., transferrin receptors, insulin receptors) are being developed, but there are limitations in delivery efficiency due to issues with receptor expression levels or saturation.

[0006] Recently, there have been active attempts to deliver drugs directly to the brain via the non-invasive route of intranasal administration. Intranasal administration suggests the possibility that drugs can bypass the blood-brain barrier and be delivered directly to the brain through the olfactory nerve pathway and the trigeminal nerve pathway. This route has the advantage of being easy to administer and enabling rapid drug delivery to the brain while minimizing systemic exposure. However, the actual efficiency of intranasal delivery to the brain remains low due to issues such as rapid mucociliary clearance, enzymatic degradation, and a limited absorption surface area.

[0007] Therefore, there is an urgent need to develop a new brain-targeted drug delivery platform that can dramatically improve therapeutic effects by maximizing the advantages of intranasal administration while increasing nasal mucosal permeability and extending the retention time in brain tissue.

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] Advanced Drug Delivery Reviews Volume 36, Issue 1, 1 March 1999, Pages 41-57

[0011] The present invention was devised to solve the problems of the prior art as described above, and aims to provide a new nanocomposite for brain-targeted drug delivery that can efficiently penetrate the blood-brain barrier through intranasal administration, a non-invasive route, and effectively deliver and retain drugs within brain tissue.

[0012] In addition, the present invention aims to provide a drug delivery system in which the brain-targeted drug delivery nanocomposite has low cytotoxicity and excellent biocompatibility, and can be universally applied to various types of therapeutic drugs such as viruses, nucleic acids, proteins, peptides, and compounds.

[0013] In addition, the present invention aims to provide a pharmaceutical composition effective for the prevention or treatment of various central nervous system diseases, such as epilepsy, brain tumors, and degenerative brain diseases, comprising the above-mentioned nanocomposite for brain-targeted drug delivery.

[0014] The composition of the polymer nanocomposite of the present invention is explained as follows based on the drawings.

[0015] To solve the above problem, the present invention, in one aspect, provides a polymer nanocomposite for brain-targeted drug delivery comprising: (a) a cyclodextrin derivative to which a piperazine derivative is bound; and (b) a polymer chain to which a hydrophobic compound is bound to one end, wherein the hydrophobic compound is included within the cyclodextrin derivative by hydrophobic interactions.

[0016] The composition of the polymer nanocomposite of the present invention is explained as follows based on the drawings.

[0017] 1. Polymer Nanocomplex

[0018] In the present invention, "polymer nanocomposite" refers to a composite structure formed by the self-assembly of two or more polymer components through non-covalent bonding, such as hydrophobic interaction, electrostatic attraction, or hydrogen bonding, as particles at the nanometer (nm) size level. In particular, the nanocomposite of the present invention forms a stable core-shell-like structure through host-guest interactions between the internal cavity of the cyclodextrin and the hydrophobic portion of the hydrophobic compound. Such a nanostructure can function as a carrier that encapsulates or binds drugs to its surface to enhance in vivo stability and facilitate delivery to target tissues.

[0019] 2. Cyclodextrin (CD) derivatives

[0020] In the present invention, "cyclodextrin derivative" refers to a modified form of cyclodextrin, which is a cyclic oligosaccharide in which D-glucopyranose units are connected by α-1,4-glycosidic bonds. Cyclodextrin has a three-dimensional structure in the form of a truncated cone, and is characterized by having a hydrophilic outer surface and forming a hydrophobic cavity in the interior. Due to these structural characteristics, it can form a stable inclusion complex by encapsulating another hydrophobic molecule (guest) of the appropriate size within the hydrophobic cavity.

[0021] In the present invention, to improve water solubility and biocompatibility, it is preferable to use a derivative in which some of the 2-, 3-, and -6 hydroxyl groups (-OH) of a natural cyclodextrin (α-CD, βCD, or γ-CD) are substituted with other functional groups. In one embodiment, the cyclodextrin derivative may be a natural cyclodextrin substituted with a functional group selected from methyl, ethyl, hydroxyethyl, sulfobutyl ether, carboxymethyl, sulfate, maleyl, and hydroxypropyl. Specifically, the cyclodextrin derivative may be 2-hydroxypropyl-gamma-cyclodextrin (2-hydroxypropyl-β-cyclodextrin, HP-β-CD) substituted with hydroxypropyl. HP-β-CD is a safe additive approved by the U.S. FDA and is widely used in drug delivery systems due to its high water solubility and low toxicity. In the present invention, HP-β-CD functions as a host that encapsulates hydrophobic compounds such as adamantan, forming the basic framework of the nanocomposite.

[0022] 3. Piperazine derivatives

[0023] In the present invention, "piperazine derivative" refers to a compound based on the structure of piperazine, a heterocyclic compound in which two nitrogen atoms are contained at the 1,4 positions of a six-membered ring. The piperazine structure is a key pharmacophore found in various central nervous system acting drugs and is known to interact particularly with serotonin (5-HT) receptors, dopamine receptors, etc.

[0024] In the present invention, such piperazine derivatives are utilized as brain-targeting ligands. Specifically, piperazine derivatives are bound to cyclodextrin derivatives so that when the nanocomposite is administered intranasally, it interacts with specific receptors in the olfactory or trigeminal neural pathways to improve permeability and retention into brain tissue. In one embodiment, the piperazine derivative may be ortho-methoxyphenyl piperazine (OPP). OPP is known to have an affinity for serotonin receptors (e.g., 5-HT1A, 5-HT2A), and experimental results of the present invention have demonstrated that the presence of OPP is a key factor in dramatically improving the brain delivery efficiency of the nanocomposite. In addition to OPP, other piperazine derivatives capable of interacting with receptors in the brain, such as N-methyl-N'-aminopropylpiperazine (NMAP), N-methylpiperazine (NMP), and N-isopropylpiperazine (NiPP), can also be used, but OPP demonstrated the best brain delivery efficiency.

[0025] Piperazine derivatives can be bonded to cyclodextrin derivatives, preferably hydroxypropyl-beta-cyclodextrin (2-hydroxypropyl-β-cyclodextrin, HP-β-CD), through covalent bonding. In one embodiment, piperazine derivatives can be bonded to the hydroxyl groups of HP-β-CD using glycidyl methacrylate (GMA) as a linker, as disclosed in FIG. 2, and compounds in which OPP is bonded to HP-β-CD may be interchangeably referred to herein as HP-β-CD-OPP or HP-β-CD-GMP-OPP.

[0026] 4. Hydrophobic Compounds and Polymer Chains

[0027] In the present invention, the term “hydrophobic compound” refers to an organic compound having low solubility in water and capable of forming an inclusion complex by non-covalently binding to the hydrophobic cavity of a cyclodextrin through hydrophobic interactions. Such hydrophobic compounds are selectively inserted into the non-polar cavity of the cyclodextrin to form a stable host-guest complex, thereby improving the structural stability and drug delivery efficiency of the nanocomposite. A representative example of a hydrophobic compound is adamantane.

[0028] Adamantane is a rigid hydrocarbon compound with a diamond-like structure in which three cyclohexane rings are fused. It exhibits high hydrophobicity and forms a very stable inclusion complex with β-cyclodextrin. Adamantane is used as an antiviral or a treatment for Parkinson's disease, but in the present invention, it functions as an object molecule that specifically binds to the cyclodextrin cavity.

[0029] In the nanocomposite of the present invention, the hydrophobic compound is generally bonded to the end of the polymer chain. The “polymer chain” is a hydrophilic polymer with excellent biocompatibility, which enhances the water solubility of the nanocomposite, provides a stealth effect that evades immune responses in vivo, and acts as a linker connecting drugs or functional molecules. In one embodiment, the polymer chain may be polyethylene glycol (PEG). PEG is a polymer composed of repeating ethylene oxide monomers, has low toxicity and high water solubility, and is widely used in drug delivery systems due to its anti-protein adsorption effect. The molecular weight of PEG may be in the range of 1 kDa to 10 kDa, 2 to 10 kDa, 3 to 10 kDa, 3 to 90 kDa, 3 to 8 kDa, 3 to 7 kDa, 3 to 6 kDa, or 3 to 5 kDa, and preferably may be about 3.4 kDa. Nanocomposites having such a structure form a structure in which a hydrophobic compound and PEG are combined (e.g., hydrophobic compound-PEG), and the hydrophobic compound portion is inserted into the cavity of a cyclodextrin derivative (e.g., hydroxypropyl-β-cyclodextrin-OPP, HP-β-CD-OPP) to form an inclusion complex, thereby forming a nanocomposite of the [HP-β-CD-OPP] / [hydrophobic compound-PEG] type overall. In this specification, this may be referred to as HCO / hydrophobic compound-PEG (HP-β-CD-OPP / Hydrophobic Compound-PEG).

[0030] The present invention is not limited to specific hydrophobic compounds (e.g., adamantane). Any compound having structural characteristics capable of being incorporated into the hydrophobic cavities of cyclodextrin may be used, and, for example, may include the following materials. By applying hydrophobic compounds selected from cyclic hydrocarbon compounds such as adamantan, adamantan derivatives (1-adamantanol, adamantan carboxylic acid), noradamantan, and tricyclodecane; lipophilic drugs such as paclitaxel, docetaxel, coumarin, retinol, and curcumin; aliphatic hydrophobic ligands such as alkyl chain-substituted phenylalkanes, cholesterol, and steroid backbone compounds; and hydrophobic aromatic compounds such as pyrene, anthracene, and phenylacetic acid derivatives, the binding affinity, stability, release characteristics, and drug loading capacity of the inclusion complex can be controlled, and a nanocomposite optimized for a specific drug or target can be designed.

[0031] 5. Combination with Therapeutic Agents

[0032] The polymer nanocomposite of the present invention can function as a carrier for delivering various "therapeutic drugs" to the brain. The therapeutic drug can be bound to another end of the polymer chain (e.g., PEG). Depending on the type of drug, various chemical bonding methods may be used.

[0033] In one embodiment, a reactive functional group may be introduced at the end of the polymer chain to facilitate binding with a therapeutic drug. For example, Ada-PEG-OPSS or Ada-PEG-SPDP can be synthesized by introducing an orthopyridyl disulfide (OPSS) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) functional group to the PEG end. The OPSS or SPDP functional group specifically reacts with molecules having a thiol (-SH) group to form a stable disulfide bond (-SS-). Therefore, viruses or proteins with thiol groups introduced to their surface, or small molecule drugs having thiol groups, can be easily bound.

[0034] The therapeutic drug that can be loaded onto the nanocomposite of the present invention is not limited thereto, but may be any one selected from the group consisting of compounds, peptides or proteins, viruses, nucleic acids, vaccine components, diagnostic or imaging contrast agents, radiotherapeutic agents, and photosensitizers. Definitions and examples for each category are described in detail below.

[0035] The term "compound" is a concept that includes organic or inorganic compounds and may include all pharmaceutically acceptable salts, solvates, hydrates, prodrugs, isomers, and isotope substituents. Examples include the anticancer agents paclitaxel, dosolubicin, and cisplatin; the anti-inflammatory agents dexamethasone and prednisolone; the immunomodulators tofacitinib and hydroxychloroquine; and the metabolic disease treatments metformin, atorvastatin, and amlodipine. In particular, examples of small molecule compounds for the treatment of brain-related diseases include the Alzheimer's disease treatments donepezil, galantamine, rivastigmine, and memantine; the Parkinson's disease treatments levodopa / carbidopa and selegiline; the epilepsy treatments lamotrigine, levetiracetam, and cenobamate; and the glioblastoma treatment temozolomide.

[0036] The term "peptide or protein" may include peptides, enzymes, hormones, antibodies, and modifications thereof that have physiological activity. Examples include insulin, GLP-1 analogs, interferon, erythropoietin, anti-VEGF antibodies, and anti-PD-1 antibodies. Examples for the treatment of brain-related diseases include neurotrophic factor (BDNF) mimic peptides, anti-IL-6 receptor antibodies, and transferrin receptor-targeting fusion proteins.

[0037] The term "virus" may refer to oncolytic viruses used for therapeutic purposes or viral vectors for gene delivery. Examples include oncolytic HSV-1, (oncolytic) adenovirus, reovirus, and adeno-associated virus (AAV) vectors. In particular, for the treatment of brain-related diseases, serotypes such as AAV-9, AAV-rh10, and AAV-PHP.eB, which have excellent neuroaffinity, or HSV-1 or adenovirus-based oncolytic viruses for the treatment of glioblastoma may be utilized.

[0038] Nucleic acids may include siRNA, shRNA, miRNA, antisense oligonucleotides (ASO), LNA-ASO, mRNA, plasmid DNA, CRISPR guide RNA, and ribonucleoprotein complexes. Examples include siRNA / ASOs targeting PCSK9 or TTR, mRNA vaccines encoding therapeutic proteins, CRISPR-Cas9-based gRNA, and plasmids. Examples for the treatment of brain-related diseases include SOD1-targeted ASOs (treatment of ALS), HTT-targeted ASOs (treatment of Huntington's disease), nucleic acid therapeutics targeting tau protein (MAPT) or α-synuclein (SNCA), and GBA or ARSA-coding mRNA for enzyme replacement.

[0039] Vaccine components refer to components including antigens and adjuvants, such as protein antigens, peptide antigens, mRNA vaccines, DNA vaccines, and virus-like particles. Examples of adjuvants include aluminum salts, monophosphoryl lipid A (MPLA), CpG ODN, and Poly(I:C). For brain-related diseases, β-amyloid or tau protein antigens for the treatment of Alzheimer's disease, α-synuclein antigen vaccines for the treatment of Parkinson's disease, etc. may be included.

[0040] Diagnostic or imaging contrast agents refer to contrast agents used in optical, MRI, CT, PET, SPECT, and ultrasound imaging. Examples include fluorescein, indocyanine green (ICG), cyanine (IR-780 / IRDye 800CW), Gd-DOTA, 18F-FDG, 68Ga-DOTATATE, 123I-ioflupane, and microbubbles. Examples for brain-related diagnostics include the amyloid marker 18F-florbetapyr, the tau marker 18F-flortaucipyr, and the dopamine transporter marker 123I-ioflupane, which are used for diagnosing Alzheimer's disease.

[0041] Radiotherapeutic agents refer to therapeutic agents in which a radioisotope is bound to a target ligand or antibody. Examples include 177Lu-DOTATATE, 90Y-labeled ligands, and 225Ac-PSMA ligands. For the treatment of brain tumors, 177Lu or 131I-labeled antibodies targeting EGFRvIII or somatostatin receptors may be used.

[0042] Photosensitizers refer to components that generate reactive oxygen species and induce cytotoxicity when irradiated with light of a specific wavelength. Examples include photoprin, tolaporfin, phortocyanin, and IR700. Fluorescent and photodynamic therapeutic agents based on 5-aminolevulinic acid (5-ALA)-derived protoporphyrin IX (PpIX) can be utilized for the treatment of brain-related diseases, particularly glioblastoma.

[0043] 6. Use for the prevention or treatment of central nervous system diseases

[0044] In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of central nervous system diseases, neurodegenerative diseases, or brain tumors comprising the polymer nanocomposite as an active ingredient. The composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0045] The aforementioned central nervous system disorders include cognitive impairment, intellectual disability, microcephaly, epilepsy, neurodevelopmental disorder, dementia, autism spectrum disorder, Down syndrome, Rett syndrome, and fragile X syndrome.

[0046] The aforementioned neurodegenerative diseases include ischemic stroke, traumatic brain injury, acute disseminated encephalomyelitis, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mild cognitive impairment, Alzheimer's disease, Pick's disease, senile dementia, progressive supranuclear palsy, subcortical dementia, Wilson's disease, multiple infarct disease, arteriosclerotic dementia, AIDS-related dementia, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedreich's ataxia, ataxia telangiectasia, epilepsy-related brain injury, spinal cord injury, and restless legs syndrome. Restless legs syndrome, Huntington's disease, Parkinson's disease, striatonigral degeneration, cerebral vasculitis, mitochondrial encephalomyopathies, neuronal ceroid lipofuscinosis, spinal muscular atrophies, lysosomal storage disorder, leukodystrophies, urea cycle defect disorder, hepatic encephalopathies, renal encephalopathies, metabolic encephalopathies,It may be porphyria, bacterial meningitis, viral meningitis, meningoencephalitis, prion diseases, poisonings with neurotoxic compounds, Guillain-Barre syndrome, chronic inflammatory neuropathies, polymyositis, dermatomyositis, or radiation-induced brain damage, etc.

[0047] The above brain tumor (or encephaloma) refers to a tumor that develops in the brain tissue and meninges. The above brain tumor may be a glioma, oligodendroglioma, glioblastoma, colloid cyst, epidermoid cyst, meningioma, hemangioblastoma, lymphoma, pituitary adenoma, metastatic tumor, or a combination thereof. For example, the glioma is glioblastoma multiforme (GBM). The above brain tumor may be a primary brain tumor or a tumor metastasized from another cancer.

[0048] In addition, the nanocomposite of the present invention can be labeled with a diagnostic agent (diagnostic marker), and the nanocomposite labeled by the marker can be tracked in vivo, thereby enabling optical detection and imaging of central nervous system diseases, neurodegenerative diseases, or brain tumors.

[0049] The above diagnostic agent may be used without limitation as long as it is a substance capable of detecting and recognizing target cells. For example, near-infrared fluorescent substances capable of penetrating the body, such as cyanin, allophycocyanin, fluorescein, tetramethylrhodamine, BODIPY, or Alexa; radiopharmaceuticals such as 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-123, Iodine-131, and Technetium-99m; or MRI contrast agents, etc.

[0050] One of the most significant features of the present invention is that it is optimized for intranasal administration. When administered intranasally, the nanocomposite of the present invention can enhance brain delivery efficiency through the following mechanism:

[0051] Enhanced mucosal permeability: The cyclodextrin structure itself can interact with the cell membranes of the nasal mucosa to temporarily increase permeability.

[0052] Brain Targeting and Improved Retention Rate: Piperazine derivatives (OPPs) bind to serotonin receptors present in olfactory and trigeminal neural pathways, promoting axonal transport along neural pathways and extending retention time within brain parenchyma.

[0053] Reduced systemic exposure: Since it is delivered directly to the brain without passing through the bloodstream, unnecessary drug accumulation in other organs such as the liver and lungs can be minimized, thereby reducing side effects.

[0054] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with the carrier. In the present invention, the term "pharmaceuticalally acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for drug delivery systems formulated as liquid solutions are those that are sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components; additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the product into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0055] As a nasal administration formulation containing the drug delivery system of the present invention as an active ingredient, it can be formulated in an injectable form or as a spray, such as an aerosol, that allows for inhalation through the respiratory tract. To formulate it as an injectable formulation, the drug delivery system of the present invention may be mixed in water with a stabilizer or a buffer to prepare a solution or suspension, and this may be formulated for unit administration in ampoules or vials. When formulating it as a spray, such as an aerosol, a propellant or the like may be combined with an additive to disperse the water-dispersed concentrate or wet powder.

[0056] The drug delivery system of the present invention can be injected via a nasal-brain administration route through a drug delivery device for nasal-brain delivery.

[0057] The above-mentioned nasal-brain drug delivery device may use a known nebulizer type.

[0058] The drug delivery system of the present invention can be used as a monotherapy, but it may also be used in combination with other conventional chemotherapy or radiation therapy, and when such combination therapy is performed, disease treatment is possible more effectively.

[0059] Accordingly, as another embodiment of the present invention, the present invention provides a method for delivering a drug to the brain via nasal administration, comprising the step of administering the pharmaceutical composition to an individual via the nose.

[0060] In another embodiment of the present invention, the present invention provides a method for treating any one of a central nervous system disease, a neurodegenerative disease, or a brain tumor, comprising the step of administering the pharmaceutical composition nasally in a pharmaceutically effective amount to an individual in need.

[0061] As another embodiment of the present invention, the present invention provides the use of a polymer nanocomposite or a pharmaceutical composition containing the same for use in the manufacture of a drug for preventing or treating any one of a central nervous system disease, a neurodegenerative disease, or a brain tumor.

[0062] The term "pharmaceuticalally effective amount" in this invention refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. Suitable dosages of the drug delivery system of this invention vary by factors such as the formulation method, mode of administration, patient's age, body weight, sex, severity of disease symptoms, diet, time of administration, rate of excretion, and responsiveness, and a physician of moderate skill can easily determine and prescribe a dosage effective for the intended treatment. Generally, the drug delivery system of this invention comprises a polymer or polymer-gene delivery system, a photosensitizer, or a complex of a pharmaceutically active ingredient at a concentration of 1 × 10⁵ to 1 × 10¹⁵ pfu / mL, typically 1 × 10¹⁶ 10 Inject PFU every other day for two weeks.

[0063] The term "individual" in the present invention includes animals such as horses, sheep, pigs, goats, camels, antelopes, and dogs, or humans, that have a disease whose symptoms can be improved by the administration of the drug delivery system according to the present invention. By administering the drug delivery system according to the present invention to an individual, the disease can be effectively prevented and treated. The treatment method according to the present invention may be a method for treating animals other than humans, but is not limited thereto. That is, considering that humans have a disease whose symptoms can be improved by the administration of the drug delivery system according to the present invention, it can be sufficiently used in the treatment of humans as well.

[0064] The polymer nanocomposite for brain-targeted drug delivery according to the present invention and the pharmaceutical composition containing the same exhibit the following excellent effects.

[0065] First, drugs can be delivered to the brain very efficiently through non-invasive intranasal administration. The nanocomposite of the present invention exhibits high brain delivery efficiency that was difficult to achieve with conventional drug delivery methods, through the synergistic effect of cyclodextrin and piperazine derivatives. In particular, it can increase the drug concentration in the brain by tens to hundreds of times compared to a control group without piperazine (OPP).

[0066] Second, it maximizes the therapeutic effect by inducing sustained retention of the drug within brain tissue. It has been confirmed that the nanocomposite of the present invention remains in brain tissue at a significant concentration for up to 7 days after administration. This enhances patient convenience by allowing for a sustained therapeutic effect while reducing the frequency of drug administration.

[0067] Third, it has excellent biocompatibility and low toxicity. HP-β PEG and other components of the nanocomposite are all known to be biocompatible substances, and it has been confirmed in actual cytotoxicity and animal studies that they do not cause significant liver or kidney toxicity or tissue damage.

[0068] Fourth, it significantly reduces drug accumulation in non-target organs such as the lungs, thereby minimizing the risk of systemic side effects. In particular, when delivering the gene therapy AAV, it reduces lung accumulation by more than 99% compared to the conventional method (Naked AAV) while increasing brain delivery efficiency by more than 150 times, enabling selective brain treatment.

[0069] Fifth, it is a universal platform technology applicable to various types of therapeutic drugs. The present invention has demonstrated that it can successfully deliver not only viral vectors such as AAV and adenovirus, but also various small molecule compounds such as paclitaxel, cenobamate, and donepezil to the brain. This suggests that the technology of the present invention is a platform technology applicable to the treatment of a wide range of central nervous system diseases, including brain tumors, epilepsy, and Alzheimer's disease.

[0070] FIG. 1 is a schematic diagram of the synthesis of an HCO / Ada-PEG-based drug delivery system according to one embodiment of the present invention.

[0071] FIG. 2 is a schematic diagram showing the synthesis process of HP-β-CD-Hum-OPP according to one embodiment of the present invention.

[0072] FIG. 3 is a schematic diagram showing the synthesis process of Ada-PEG-OPSS according to one embodiment of the present invention.

[0073] Figure 4 shows the results of the hydrogen nuclear magnetic resonance (¹H-NMR) spectra of HP-β-CD-GMA, OPP, and HP-β-CD-GMA-OPP.

[0074] Figure 5 shows the mass spectrometry (MALDI-TOF) results of HP-β-CD, HP-β-CD-GMA, and HP-β-CD-GMA-OPP and Ada-PEG 3.4 This is the result showing the ¹H-NMR spectrum of -OPSS.

[0075] Figure 6 is the result of a cytotoxicity evaluation comparing the cell viability of the HCO / Ada-PEG polymer and PEI polymer of the present invention over time at different concentrations.

[0076] Figure 7 shows the results of ex vivo near-infrared (NIR) fluorescence images of excised brain tissue 24 hours after intranasal administration of PBS (control group) and HCO / Ada-PEG-IR780, respectively.

[0077] Figure 8 is an ex vivo NIR fluorescence image result to compare brain delivery efficiency according to the type of piperazine derivative (OPP, NMAP, NMP, NiPP).

[0078] Figure 9 is an ex vivo NIR fluorescence image result showing changes in brain distribution over time (30 minutes to 7 days) after intranasal administration of HCO / Ada-PEG-IR780.

[0079] Figure 10 shows a schematic diagram of the formation of Naked AAV and HCO / Ada-PEG-AAV complexes, transmission electron microscope (TEM) images, and particle size and zeta potential measurement results.

[0080] Figure 11 shows the results of gene copy number (GC) distribution in the brain and lungs and pharmacokinetic profiles in blood and brain tissue after intranasal administration of Naked AAV and HCO / Ada-PEG-AAV.

[0081] Figure 12 shows the results of quantitative analysis of the number of gene copies in each region of the brain after intranasal administration of Naked AAV and AAV / polymer complexes at various ratios.

[0082] Figure 13 shows the results of histological examination of brain tissues from each treatment group (normal, untreated, Naked AAV, HCO / Ada-PEG-AAV) in an epilepsy-induced mouse model after H&E staining.

[0083] Figure 14 shows the results of monitoring the change in body weight over time for each treatment group in a DHPS- / - mouse model.

[0084] Figure 15 is a graph showing the results of the olfactory perception test (Sniffing number test) to evaluate cognitive function and search behavior in each treatment group.

[0085] Figure 16 shows the results of a toxicity evaluation analyzing blood liver function (ALT, AST) and kidney function (BUN, Creatinine) indicators for each treatment group (PBS, Naked AAV, HCO / Ada-PEG-AAV).

[0086] Figure 17 is an ex vivo NIR fluorescence image result to evaluate the synergistic effect on brain delivery efficiency of piperazine target group (OPP) and cyclodextrin (CD) structures.

[0087] Figure 18 shows the results of a quantitative analysis of the virus distribution by brain region over time after intranasal administration of a Naked Ad (adenovirus) and HCO-Ad complex.

[0088] Figure 19 shows the results of ex vivo NIR fluorescence images analyzing the distribution of various drugs (PTX, Tig, Gal, Don, Mem) in brain tissue after intranasal administration using the HCO platform.

[0089] Figure 20 shows the results of an analysis of neurobehavioral changes when cenobamate was administered via various routes and formulations in a PTZ-induced seizure model.

[0090] Figure 21 shows the results of measuring changes in tumor growth over time in each treatment group in a glioblastoma animal model using in vivo fluorescence signals.

[0091] Figure 22 shows ex vivo NIR fluorescence images of cerebral cortex extracted after intranasal administration of PBS, PTX-PEG-IR780, and HCO / PTX-PEG-IR780, respectively, in a large animal (pig) model.

[0092] Figure 23 is a graph showing a quantitative comparison of the average fluorescence signals in the cerebral cortex (Dorsal, Ventral) of the PTX-PEG-IR780 monotherapy group and the HCO / PTX-PEG-IR780 complex therapy group in a large animal (pig) model.

[0093] Figure 24 shows the results of comparing the number of gene copies in brain and lung tissues after intranasal administration of Naked AAV and HCO-AAV, demonstrating brain-selective delivery and reduced lung accumulation effects.

[0094] The present invention will be explained in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

[0095] [Preparation Example] Preparation of a polymer nanocomposite according to the present invention

[0096] Preparation Example 1: Synthesis of HP-β-CD-OPP

[0097] HP-β-CD-OPP (also referred to as 'CD-OPP' or 'HCO' in this specification), which acts as the host in the nanocomposite of the present invention, was synthesized in the following two steps.

[0098] 1-1. Synthesis of HP-β-CD-GMA

[0099] 2-hydroxypropyl-β-cyclodextrin (HP-β-CD, 1.5 g, 1.0 mM) and dimethylaminopyridine (DMAP, 0.1 g) were dissolved in dimethylformamide (DMF), after which glycidyl methacrylate (GMA, 1.42 g, 10 mM) was added. The reaction mixture was stirred at 50°C for 24 hours under a nitrogen atmosphere. After the reaction was complete, the solution was cooled to room temperature and precipitated by pouring it into an excess of hexane:ether (30:70) mixed solvent. The resulting precipitate was separated, redispersed in water, and freeze-dried to obtain HP-β-CD-GMA in the form of a white powder. (Fig. 2)

[0100] 1-2. Synthesis of HP-β-CD-OPP

[0101] The HP-β-CD-GMA (0.2 equivalents) and ortho-methoxyphenylpiperazine (OPP, 10.0 equivalents) synthesized above were dissolved in methanol, and then triethylamine was added. The reaction mixture was stirred at 45–50°C for 72 hours under a nitrogen atmosphere. After the reaction, the mixture was cooled to room temperature and dialyzed against ethanol using a dialysis membrane with a molecular weight cutoff (MWCO) of 1.0 kDa, followed by dialyzing against distilled water (DI water) for 24 hours to remove unreacted substances. The final solution was filtered and freeze-dried to obtain HP-β-CD-OPP (CD-OPP), a pale white solid. (Fig. 2)

[0102] Preparation Example 2: Synthesis of Ada-PEG-OPSS

[0103] Ada-PEG-OPSS, which acts as a guest and drug linker in the nanocomposite of the present invention, was synthesized in the following two steps. (Fig. 3)

[0104] 2-1. Synthesis of Ada-PEG-SH

[0105] Thiol-PEG-acetic acid (100 mg, 28.6 μM) was dissolved in chloroform. Dicyclohexylcarbodiimide (DCC, 12 mg, 57 μM) and DMAP (7 mg, 57 μM) were added to this, and the mixture was stirred at room temperature for 15 minutes. Then, a 1-adamantan methanol solution (10 mg, 60 μM, dissolved in chloroform) was slowly added, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction, the solution was concentrated and precipitated in a cold ether:hexane (1:1) mixed solvent. Finally, the precipitate was redissolved in water and freeze-dried to obtain Ada-PEG-SH.

[0106] 2-2. Synthesis of Ada-PEG-OPSS

[0107] The Ada-PEG-SH (36 mg, 10 μM) and 2,2'-dipyridyldisulfide (8.8 mg, 40 μM) synthesized above were placed in a round-bottom flask and dissolved in methanol under a nitrogen atmosphere. A catalytic amount of acetic acid was added, and the reaction mixture was stirred overnight at room temperature. After the reaction, the solution was concentrated under reduced pressure, and the polymer was precipitated in an excess amount of cold ether. Finally, the polymer was redispersed in water and freeze-dried to obtain the final product, Ada-PEG-OPSS. (Fig. 3)

[0108] Preparation Example 3: Formation of HCO / Ada-PEG-OPSS inclusion complex

[0109] The final nanocomposite, HCO / Ada-PEG, was prepared by forming an inclusion complex through hydrophobic interactions between HP-β-CD-OPP (HCO) obtained in Preparation Example 1 and Ada-PEG-OPSS obtained in Preparation Example 2. Specifically, HCO and Ada-PEG-OPSS were mixed in an aqueous solution at a specific mass ratio (e.g., 5:1) and gently stirred to allow the adamantan portion of Ada-PEG-OPSS to spontaneously be included within the cyclodextrin cavities of HCO. Through this process, a stable water-soluble nanocomposite is formed. (Fig. 1)

[0110] Preparation Example 4: Formation of AAV / Polymer Composite

[0111] To prepare an AAV / polymer composite, 5×10 7 The AAV solution from GC was mixed with an equal amount of HCO / Ada-PEG-OPSS. The mixture was gently stirred to ensure that the AAV was properly coated by the polymer. Through this process, a stable AAV / polymer composite was formed, and subsequently, the structural integrity and surface characteristics were confirmed through transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis.

[0112] [Example]

[0113] Example 1: Structural analysis of the synthesized polymer

[0114] Hydrogen nuclear magnetic resonance (¹H-NMR) analysis and MALDI-TOF mass spectrometry were performed to confirm the chemical structure and molecular weight of each step product (HP-β-CD-GMA, HP-β-CD-OPP, Ada-PEG-OPSS, etc.) synthesized in Preparation Examples 1 and 2. ¹H-NMR spectra were measured using a VARIAN 600 MHz instrument under D₂O or DMSO-d6 solvents. The molecular weight of each polymer was measured through MALDI-TOF analysis.

[0115] In the ¹H-NMR spectrum of HP-β-CD-GMA, in addition to the intrinsic peak of HP-β-CD (3.4–4.2 ppm), characteristic peaks corresponding to the vinyl group (-CH2=C) and methyl group (-CH3) of GMA (6.18, 5.76, and 1.9 ppm, respectively) were observed, confirming the successful incorporation of GMA. (Fig. 4) In the spectrum of the final product, HP-β-CD-OPP (CD-OPP), peaks corresponding to the aromatic protons (6.8–6.9 ppm) and piperazine protons (2.5–2.9 ppm) of OPP were clearly observed along with the HP-β-CD peak, proving the successful binding of OPP. (Fig. 3)

[0116] MALDI-TOF analysis results showed that the molecular weight of HP-β-CD was measured to be approximately 1.95 kDa and that of HP-β-CD-OPP was approximately 2.4 kDa, confirming the increase in molecular weight due to the binding of OPP. Additionally, as Ada-PEG-SH was converted to Ada-PEG-OPSS, the molecular weight increased from 3.63 kDa to 3.8 kDa, confirming the successful introduction of the OPSS functional group. (Fig. 5) These analysis results comprehensively support the successful synthesis of each target polymer component with the correct structure.

[0117] Example 2: Cytotoxicity Evaluation

[0118] To evaluate the biocompatibility of the synthesized HCO / Ada-PEG polymer, an MTT assay was performed on NIH3T3 fibroblasts. Cells were seeded into 96-well plates at a density of 1 × 10³ cells / well and cultured for 24 hours. Subsequently, HCO / Ada-PEG or the positive control polyethyleneimine (PEI) were dissolved in PBS at various concentrations (0.1–20 μg / mL) and treated. At 24, 48, 72, and 7 days after treatment, MTT reagents were added to each well, and the cells were incubated at 37°C for 4 hours. The supernatant was removed, and the precipitated formazan was dissolved in DMSO; the absorbance was then measured at 540 nm using a microplate reader. Relative cell viability was calculated by setting the viability of untreated cells to 100%.

[0119] At all time points and concentrations, the HCO / Ada-PEG polymer exhibited significantly lower cytotoxicity compared to PEI, a representative cytotoxic polymer (Fig. 6). For example, after 24 hours of treatment, at a concentration of 10 μg / mL, the cell viability of the HCO / Ada-PEG treatment group remained above 80%, whereas that of the PEI treatment group decreased sharply to less than 40%. In a 7-day long-term exposure experiment, HCO / Ada-PEG also maintained a high cell viability of over 90% even at a high concentration of 20 μg / mL. These results suggest that the HCO / Ada-PEG polymer possesses excellent biocompatibility and is a safe material for use as an in vivo drug delivery system.

[0120] Example 3: Evaluation of intranasal delivery efficiency (Ex Vivo NIR imaging)

[0121] To evaluate the brain delivery capability of the HCO / Ada-PEG polymer, HCO / Ada-PEG-IR780 was prepared by conjugating the polymer with the near-infrared fluorescent dye IR-780. This complex was dissolved in PBS at a concentration of 1 mg / ml and administered intranasally to anesthetized C57BL / 6 mice. Twenty-four hours after administration, the mice were sacrificed, and the head, whole brain, and dissected brain regions (olfactory bulb, cerebral cortex, etc.) were extracted for ex vivo NIR imaging. Fluorescence intensity was analyzed using Imaging Studio software.

[0122] In the control group administered PBS, almost no fluorescence signals were observed in brain tissue, whereas in the group administered HCO / Ada-PEG-IR780, strong fluorescence signals were clearly observed in several major regions of the brain (Fig. 7). In particular, high fluorescence intensity was observed in the olfactory bulb, cerebral cortex, and hypothalamus, which are known as major pathways for intranasal delivery. This demonstrates that the HCO / Ada-PEG polymer successfully passed through the nasal mucosal barrier and was efficiently delivered to important parts of the brain via olfactory and trigeminal neural pathways. These results prove that the nanocomposite of the present invention is highly useful for non-invasive brain targeting strategies.

[0123] Example 4: Evaluation of OPP part's brain transmission dependence

[0124] To determine whether the excellent brain delivery efficiency of HCO / Ada-PEG specifically depends on the piperazine derivative OPP, modified polymers were synthesized in which OPP was replaced with other piperazine derivatives (CD-NMAP, CD-NMP, CD-NiPP). After conjugating each polymer with IR-780, it was administered intranasally to mice in the same manner as in Example 3, and its distribution in the brain was analyzed via ex vivo NIR imaging after 24 hours.

[0125] Compared to the original HCO / Ada-PEG (CD-OPP) administration group containing OPP, the fluorescence signal in the brain was significantly reduced in the modified polymer administration groups in which OPP was replaced with other piperazine derivatives (Fig. 8). Among all modified polymers, the complex containing OPP exhibited the highest fluorescence intensity in all regions of the brain. This result clearly demonstrates that the OPP portion plays a crucial role in promoting brain influx and accumulation through interactions with specific receptors during the nasal delivery process, and is a key factor in the brain targeting performance of the platform of the present invention.

[0126] Example 5: Evaluation of intrabrain distribution and retention over time

[0127] To determine the kinetics of HCO / Ada-PEG polymers within the brain, a study was conducted on changes in distribution over time. After administering HCO / Ada-PEG-IR780 intranasally to mice, mice were sacrificed at various time points including 30 minutes, 2 hours, 6 hours, 1 day, 2 days, 3 days, and 7 days, brain tissues were extracted, and NIR imaging was performed.

[0128] During the initial period (30 minutes to 2 hours), fluorescent signals were primarily observed in the posterior regions of the brain associated with the trigeminal neural pathway. After 6 hours, the signals showed an intensifying pattern in the anterior regions associated with the olfactory pathway, specifically the olfactory bulb and the cerebral cortex (Fig. 9). This suggests that the polymer can be delivered to the brain through a dual-pathway mechanism involving rapid initial influx via the trigeminal neural pathway and subsequent redistribution via the olfactory neural pathway. Of particular note is that fluorescent signals were continuously observed in brain tissue for up to 7 days after administration. This implies that the HCO / Ada-PEG polymer can remain in the brain for a long period, demonstrating a highly advantageous characteristic for the treatment of chronic brain diseases requiring continuous drug release.

[0129] Example 6: AAV / Polymer Composite Formation and Characterization

[0130] To load the gene therapy adeno-associated virus (AAV) onto the platform of the present invention, the amine groups (-NH₂) on the surface of the AAV were thiolated into thiol groups (-SH) by treating them with 2-iminothiolane (2-IT). Subsequently, the thiolized AAV was mixed with an HCO / Ada-PEG complex containing Ada-PEG-OPSS, so that the AAV could be covalently bonded to the surface of the polymer nanocomposite through disulfide bonds between the OPSS functional groups and the thiol groups. The structural integrity and surface characteristics of the formed AAV / polymer complex (HCO / Ada-PEG-AAV) were analyzed using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurement.

[0131] TEM image analysis revealed that the AAV particles maintained their inherent spherical shape intact even after binding with the polymer, and no structural destruction was observed (Fig. 10). This implies that the polymer binding process did not negatively affect the physical stability of the AAV. DLS measurements showed that the average particle size of the Naked AAV was approximately 24 nm, while that of the HCO / Ada-PEG-AAV complex increased slightly to approximately 25.2 nm, suggesting that the polymer layer was successfully formed on the surface of the AAV. The zeta potential showed a tendency to decrease slightly in the complex (-1.43 mV) compared to the Naked AAV (-1.13 mV). These results comprehensively confirm that the AAV has stably bound to the polymer nanocomposite of the present invention to form a complex suitable for in vivo delivery.

[0132] Example 7: Evaluation of In Vivo Distribution and Pharmacokinetics of AAV / Polymer Complex

[0133] Naked AAV or AAV / polymer complexes were administered intranasally to C57BL / 6 mice three times. 24 hours after the final administration, brain and lung tissues were excised to extract DNA, and tissue distribution was evaluated by quantitatively analyzing the AAV genome via real-time polymerase chain reaction (real-time PCR). For pharmacokinetic studies, blood was collected at various time points (30 minutes to 48 hours) after the final administration, and brain tissue was harvested at 3, 6, 12, and 24 hours to quantitatively analyze the AAV genome in the same manner.

[0134] Tissue distribution analysis showed that the AAV / polymer complex administration group exhibited dramatically improved brain delivery efficiency compared to the Naked AAV administration group, while accumulation in the lungs was significantly reduced (Fig. 11). Specifically, the ratio of gene copy numbers in the brain to the lungs increased by 1,352 times compared to Naked AAV, demonstrating that the platform of the present invention highly specifically enhances the brain targeting ability of AAV. Pharmacokinetic analysis revealed that the AAV / polymer complex remained at high concentrations within brain tissue for an extended period. After 24 hours of administration, the residual amount of AAV genome in the brain of the complex administration group was 5.3 times higher than that of the Naked AAV administration group. This suggests that the platform can not only increase initial delivery efficiency but also extend the duration of action of the therapeutic agent in the brain, thereby enhancing the therapeutic efficacy for neurological diseases.

[0135] Example 8: Evaluation of Brain Tissue Distribution Efficiency of AAV / Polymer Complex

[0136] To evaluate the brain distribution efficiency according to the binding ratio of AAV and polymer, AAV / polymer complexes prepared at three different ratios (1:1E+5, 1:2E+5, 1:3E+5) were administered intranasally. After 24 hours of administration, the brain was dissected and the AAV gene copy number (GC) in each region (cerebral cortex, whole brain, etc.) was quantitatively analyzed.

[0137] All ratios of AAV / polymer complexes showed significantly improved brain delivery efficiency compared to Naked AAV (Fig. 12). In particular, in the cerebral cortex, the AAV / polymer (1:1E+5) complex showed a 13.8-fold increase in gene copy numbers compared to Naked AAV. In the whole brain, the AAV / polymer (1:2E+5) complex achieved a 6.9-fold higher gene copy number compared to Naked AAV. These results reaffirm that the polymer complex of the present invention can significantly improve the distribution efficiency of AAV throughout the brain.

[0138] Example 9: Evaluation of therapeutic efficacy in an epilepsy model (histological examination)

[0139] An AAV loaded with the epilepsy treatment gene DHPS (Deoxyhypusin synthase) was prepared in the form of an AAV / polymer complex and administered intranasally to genetically epileptic DHPS- / - mice. Specifically, Dhps fl / fl(Dhpstm1.1Mirm / J,stock#034895,JacksonLaboratory) mice showed no phenotypic differences compared to homozygous C57BL / 6 wild-type (WT) mice; accordingly, Dhpsfl / fl mice were used as controls for each CKO line. To induce epilepsy in 4-day-old Dhps- / - mice, intracranial injections were administered into the cerebral region using a Hamilton syringe. The injected solution consisted of 2 μl of AAV9-CMV-Cre and 1 μl of Trypan blue solution. Four days after the injection, the mice received three nasal administrations of either naked AAV or an AAV / polymer complex. Twenty-four hours after the last administration, the mice were euthanized by cervical dislocation. Subsequently, a necropsy was performed, and the brains were collected, fixed in 4% formalin for 24 to 48 hours. The tissues were paraffin-embedded after undergoing a series of alcohol and xylene treatments. Then, 10 μm thick serial sections were prepared and stained with 0.1% H&E. After deparaffinization, the slides were hydrated with distilled water after passing through alcohols of progressively decreasing concentration. All culture and staining procedures were performed at room temperature (RT).

[0140] H&E staining results showed that extensive neuronal necrosis and vacuolation were observed in the brain tissue of untreated epileptic mice, indicating severe brain damage (Fig. 13). In contrast, the brain tissue of the group treated with the AAV / polymer complex exhibited tissue morphology similar to that of normal mice, and the number of necrotic neurons was significantly reduced. This suggests that the AAV / polymer complex treatment of the present invention has therapeutic potential to effectively alleviate brain damage caused by epilepsy and protect tissues.

[0141] Example 10: Evaluation of therapeutic efficacy in an epilepsy model (weight change)

[0142] For epilepsy model mice identical to those in Example 9, systemic health status and treatment effects were evaluated by measuring body weight every other day during the treatment period. Body weight was measured every other day (every 2 days) using the ECL66111 Heavy Duty Animal Weighing Scale.

[0143] Untreated DHPS- / - mice showed a tendency for body weight to decrease starting from day 32, indicating disease exacerbation (Fig. 14). In contrast, mice treated with the AAV / polymer (1:1E+5) complex maintained a stable body weight or steadily increased, similar to normal control mice. This demonstrates that treatment with the AAV / polymer complex contributes to inhibiting disease progression and restoring the overall health of the mice.

[0144] Example 11. Evaluation of seizure severity in an epilepsy model

[0145] The seizure-suppressing effect of the AAV / polymer complex was evaluated using a DHPS- / - mouse model. Seizure severity was assessed using the time to hindlimb extension (THLE) as an indicator. The THLE of untreated DHPS- / - mice was 25.0 ± 0.00 seconds, and they did not survive the seizure. In contrast, mice treated with AAV / polymer (1:1E+4) showed a shortened THLE of 16 ± 2.00 seconds, and all individuals recovered. Upon treatment with AAV / polymer (1:1E+5), the THLE further decreased to 9.7 ± 2.08 seconds, and complete recovery was observed in all mice (Fig. 15). These results suggest that AAV / polymer treatment significantly reduces seizure severity and improves the recovery rate.

[0146] To evaluate neurobehavioral function, the context-object recognition memory test and the pole test were performed. The specific test methods are as follows.

[0147] Day 1. Habituation

[0148] Point the mouse toward the wall and place it in a context with no wall cues and no objects for 10 minutes.

[0149] Day 2. Training

[0150] First session: Place the mouse in context A for 10 minutes. In this environment, there are no wall cues, and two identical objects are placed at two corners facing each other.

[0151] Second session: Afterwards, place the mouse in the home cage for about 1 minute, then place it in context B for 10 minutes. This environment has striped visual cues on the walls, and two new identical objects are placed at two corners facing each other.

[0152] After the training ends, return the mouse to its home.

[0153] Day 3. Testing

[0154] Place the mouse back in Context B for 10 minutes. At this time, one of the two objects is the object used in Context B of Day 2, and the other is the object used in Context A.

[0155] Context-object recognition memory ability is evaluated by calculating the Discrimination Ratio (DR). The DR is calculated using the following formula:

[0156]

[0157] Here, t novel is the search time for a new object (an object used in context A), t familiar is the search time for a familiar object (an object used in context B).

[0158] In the object-context recognition memory test, the discrimination rate of untreated DHPS- / - mice was 38.77%, indicating impaired cognitive function. In contrast, mice treated with AAV / polymer (1:1E+5) showed a significantly improved discrimination rate of 56.09%, which was similar to that of normal mice (57.63%).

[0159] To evaluate motor coordination and agility, the Pole Test was performed as follows. A vertical rod 36 cm in length and 1.5 cm in diameter (with a rough surface wrapped in knitting yarn) was set up in the center of a square box. A mouse was placed at the top of the rod with its head facing downward and allowed to descend on its own. The test was conducted for a maximum of 120 seconds, and the time taken for the mouse to descend to the bottom was recorded. If the mouse fell off the rod, that attempt was excluded from the analysis.

[0160] As a result, in the Pole test, untreated DHPS- / - mice showed reduced motor coordination ability with a prolonged descent time of 56.5 seconds. In contrast, the AAV / polymer (1:1E+5) treatment group showed a significantly reduced descent time of 8.33 seconds, exhibiting motor function similar to that of the normal control group (7.42 seconds). These results suggest that AAV / polymer treatment restores both cognitive and motor functions in DHPS- / - mice.

[0161] In addition, exploratory behavior was evaluated using the Sniffing Number Test. Untreated DHPS- / - mice exhibited a low frequency of sniffing, indicating impaired cognitive function and reduced curiosity. In contrast, the AAV / polymer (1:1E+5) treatment group showed a frequency of odor detection similar to that of the normal control group, demonstrating a recovery in cognitive function and exploratory behavior.

[0162] A wire hang assessment was performed to evaluate motor coordination and muscle strength. Specifically, the forelimbs of the mice were placed on a wire 30-40 cm high, and the mice were instructed to grasp the wire (a safety pad was placed underneath). A stopwatch was started. The time until the mouse fell was recorded, or the mouse was lowered after a maximum of 120 seconds.

[0163] In the wire hanging test, short drop times were observed in untreated DHPS- / - mice, indicating impaired motor function and reduced muscle strength. On the other hand, the AAV / polymer (1:1E+5) treatment group showed drop times similar to normal control groups, suggesting that motor coordination and muscle strength improved after treatment.

[0164] The above results show that AAV / polymer treatment not only reduces the severity of seizures but also significantly improves both cognitive and motor functions in DHPS- / - mice.

[0165] Example 12: Evaluation of liver or kidney toxicity in an epilepsy model (neurobehavioral examination)

[0166] The toxicity of the AAV / polymer complex was evaluated by administering the AAV / polymer complex intranasally three times to C57 / BL6 mice (Dae Han Bio Link Co., Ltd., Seoul, South Korea) (dosage: 1 × 10¹ * GC / mouse). As a control, naked AAV was administered intranasally in the same manner.

[0167] 48 hours after administration, blood was collected from the submandibular vein of mice using a Lancet (Goldenrod Animal Lancet, Braintree Scientific, Massachusetts, USA). The collected blood was centrifuged at 4,000 rpm for 10 minutes to separate the serum, and then aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured as indicators of hepatotoxicity. The analysis was performed at the Neodin Medical Research Institute (Neodin Corporation, Seoul, South Korea).

[0168] The ALT, AST, creatinine, and BUN levels of the AAV / polymer complex administration group were all within the normal range (Fig. 16). Specifically, ALT was measured at 22.5 U / L, AST at 111.5 U / L, creatinine at <0.1 mg / dL, and BUN at 25.45 mg / dL, showing no significant difference from the control groups administered PBS or Naked AAV. These results confirm that the polymer nanocomposite of the present invention is a safe delivery vehicle that does not cause significant hepatotoxicity or renal toxicity in vivo.

[0169] Example 13: Evaluation of the synergistic effect between the CD structure and the piperazine target

[0170] We evaluated how the cyclodextrin (CD) structure and piperazine (OPP) target group, which are key components of the nanocomposite of the present invention, contribute to brain delivery efficiency and whether there is a synergistic effect between the two. To this end, four formulations were prepared: (i) PEG-IR780 (linker + fluorescent dye), (ii) Ada-PEG-IR780 (object + linker + fluorescent dye), (iii) CD / Ada-PEG-IR780 (subject + object complex), and (iv) HCO / Ada-PEG-IR780 (target group bound subject + object complex). Each formulation was administered intranasally to BALB / c nude mice at a final dose of 24 μL (2 mg / mL). The total amount was divided into six doses (4 μL / dose) and administered alternately to both nostrils at 4-minute intervals. Two hours after administration, mice were sacrificed and brains were extracted. The entire brain was placed on a black non-fluorescent tray for ex vivo near-infrared (NIR) fluorescence imaging. After acquiring whole brain images, the brain was dissected and compartmentalized into the olfactory bulb, cerebral cortex, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata, and ex vivo near-infrared (NIR) fluorescence imaging was performed once more.

[0171] As a result, the fluorescence intensity observed in the entire brain and head regions clearly increased in the order of PEG-IR780 < Ada-PEG-IR780 < CD / Ada-PEG-IR780 < HCO / Ada-PEG-IR780 (Fig. 17). In particular, the signal increase of CD / Ada-PEG-IR780 compared to Ada-PEG-IR780 suggests that the CD construct enhances nasal mucosal permeability through hydrophobic interactions. Furthermore, the superior signal increase of HCO / Ada-PEG-IR780 compared to CD / Ada-PEG-IR780 clearly demonstrates that the presence of piperazine (OPP) derivatives targeting serotonin receptors creates a synergistic effect that further maximizes brain delivery efficiency. In the regional analysis after dissection, the HCO / Ada-PEG-IR780 administration group also showed the strongest and most uniform distribution throughout the brain, including the olfactory bulb, cerebral cortex, and midbrain. This supports the optimization of brain delivery efficiency through the combination of (i) enhanced permeability via CD-based hydrophobic interactions and (ii) enhanced brain cell targeting and retention rates via OPP.

[0172] Example 14: Evaluation of intracerebral distribution of adenovirus-polymer complex

[0173] The platform of the present invention was applied to another viral vector, adenovirus (Ad), to evaluate its distribution within the brain. The adenovirus-polymer complex was prepared by combining thiolized adenovirus with HCO / Ada-PEG-SPDP in the same manner as in Example 8 to form the HCO / Ada-PEG-Ad complex. Specifically, for HCO / Ada-PEG-Ad, the HCO polymer was first mixed with Adamantane (Ada)-PEG-SPDP in a mass ratio of 5:1 to form HCO / Ada-PEG-SPDP through hydrophobic interaction. Then, to bind the adenovirus with HCO / Ada-PEG-SPDP, the amine groups on the surface of the virus were first thiolated for 30 minutes through a reaction with 2-IT (2-iminothiolane), and then free 2-IT was removed using a Zeba spin column. Subsequently, HCO / Ada-PEG-SPDP and thiolated Ad were reacted for 2 hours to form disulfide bonds between the OPSS-group of SPDP and the thiol group of thiolated Ad, thereby preparing the desired complex. Free HCO / Ada-PEG-SPDP was removed using a Zeba spin column.

[0174] Subsequently, experimental samples were prepared by adjusting the final viral concentration to 1E10 VP / 24μL through quantitative analysis using qPCR. Viruses from each experimental group (naked Ad, HCO-Ad) were administered intranasally (IN) at 4μL doses six times at 4-minute intervals (total 24μL). Brain tissues were collected from one animal from each group after 2, 12, and 24 hours. The collected brain tissues were dissected into the cerebral cortex, hypothalamus, midbrain, cerebellum, pons and medulla, and olfactory bulb, and genomic DNA was extracted from each tissue. Quantitative analysis targeting the Ad IX gene was then performed on the extracted DNA using TaqMan-based qPCR.

[0175] At all time points, the HCO / Ada-PEG-Ad administration group showed significantly higher viral detection levels throughout the brain compared to the Naked Ad administration group (Fig. 18). In particular, for the HCO / Ada-PEG-Ad administration group, the highest viral concentrations were identified in the olfactory bulb, pons and medulla oblongata, and cerebral cortex, which is consistent with the initial influx and diffusion pattern via the intranasal delivery route. Over time, viral detection levels remained high in the HCO / Ada-PEG-Ad administration group, whereas in the Naked Ad administration group, levels were low and decreased rapidly. These results demonstrate that the HCO platform can significantly improve the delivery efficiency, tissue penetration, and diffusion of adenoviruses within brain tissue, once again proving the versatility of the platform.

[0176] Example 15: Evaluation of Brain Distribution of Various Small Molecule Drugs

[0177] To further verify the versatility of the platform of the present invention, brain delivery efficiency was evaluated for various small molecule drugs for the treatment of central nervous system diseases. Specifically, the intrabrain distribution of HCO-based polymers was evaluated using BALB / c nude mice. The experimental groups were set as the control group (PBS), PC (HCO / PTX-PEG-IR780), HCO / Tig-PEG-IR780 (No. 1), HCO / Gal-PEG-IR780 (No. 2), HCO / Don-IR780 (No. 3), and HCO / Mem-PEG-IR780 (No. 4) for comparative analysis. A total of 24 μL (2 mg / mL) of polymer solution was administered intranasally (IN) to each experimental group, and the administration method used was 4 μL administered six times at 4-minute intervals. Two hours after administration, the mice were sacrificed and brain tissue was extracted, and ex vivo near-infrared (NIR) imaging was performed to analyze the fluorescence distribution within the brain tissue. In addition to whole-brain imaging, signal intensity by brain region, including the olfactory bulb, cerebral cortex, hypothalamus, midbrain, cerebellum, pons, and medulla oblongata, was compared and analyzed.

[0178] As a result, strong fluorescence signals were observed across the brain and head regions in all HCO-based drug conjugate administration groups compared to the control group (PBS). (Fig. 19) Although there were slight differences in brain distribution patterns depending on the type of drug, effective delivery to major brain regions such as the olfactory bulb, cerebral cortex, and midbrain was confirmed for all drugs. In particular, the highest fluorescence intensity was observed in the HCO / PTX-PEG-IR780 administration group loaded with paclitaxel. These results clearly demonstrate that this HCO platform is not limited to specific drugs and can function as a universal carrier for efficiently delivering small molecule drugs with various chemical structures to the brain.

[0179] Example 16: Evaluation of the anticonvulsant effect of cenobamate

[0180] The therapeutic effect of intranasal (IN) administration of the anticonvulsant cenobamate, prepared as an HCO3 / Drug complex, was evaluated. Specifically, a PTZ (pentylenetetrazole)-induced seizure model was utilized to assess whether intranasal (IN) delivery of cenobamate could improve motor function and induce anticonvulsant effects. Balb / c nude mice were used for the experiment, and the experimental groups were composed of a normal group, an untreated group, an intraperitoneal administration group (3 mg / kg and 30 mg / kg, IP), an intranasal administration group (3 mg / kg, IN), and an HCO3-based intranasal administration group (HCO3 / Drug IN 3 mg / kg). Cenobamate was administered as a single dose (24 μL), and seizures were induced 2 hours after administration by injecting PTZ (60 mg / kg, IP). Motor function and anticonvulsant effects were analyzed by performing various behavioral assessments within 30 minutes of PTZ injection.

[0181] To evaluate motor skills and mobility, average speed and total distance were measured, and mouse activity was analyzed using the mobility rate. Frozen events and total time frozen(s) were quantified through video analysis to assess changes in neuromotor function.

[0182] In addition, the Maximal Electroshock Seizure (MES) test was performed to analyze seizure duration and frequency. The MES test is a method that quantitatively evaluates seizure responses induced by forced electric shock and was used to compare the anticonvulsant effects of cenobamate.

[0183] In the evaluation of motor ability, the HCO / cenobamate intranasal administration group (HCO / Drug IN 3 mg / kg) showed the most significant improvement in motor ability compared to all other treatment groups (Fig. 20). Average speed and total distance traveled were maintained at the highest levels, and the frequency of rigidity reactions was also effectively reduced. In the MES test results, the HCO / cenobamate intranasal administration group most effectively shortened seizure duration, and all mice survived with a post-seizure mortality rate of 0%. In contrast, the group administered the same dose intranasally alone showed no therapeutic effect, with all mice dying. This implies that the HCO platform maximizes the intracerebral absorption of cenobamate, enabling a potent anticonvulsant effect even at low doses that could not be achieved with conventional administration methods.

[0184] Example 17: Evaluation of antitumor effects in a glioblastoma model

[0185] The efficacy of anticancer treatment was evaluated using an animal model of glioblastoma, a type of brain tumor. Specifically, the therapeutic effect on tumor growth was assessed using a glioblastoma orthotopic model (U87MG-FLUC, Balb / c nude mouse). After injecting U87MG-FLUC cells (3E+5 cells / mouse) into the intracerebral ventricle (ICV), drug administration was initiated on day 7, when tumor formation was expected. Drug administration was performed a total of four times at 3-day intervals, with each mouse receiving 24 μL of the drug. The experimental groups consisted of a PBS control group, an intravenous PTX group (PTX IV), an intranasal PTX group (PTX IN), and an intranasal HCO / PTX-PEG-IR780 group (HCO IN). The intravenous group was administered a solution containing 100 μg of PTX, while the intranasal group was administered a solution containing 10 μg of PTX. Intranasal (IN) administration was carried out by administering a total of 24 μL in 6 repetitions of 4 μL at 4-minute intervals.

[0186] IVIS imaging was performed to track changes in tumor growth after drug administration, and changes in tumor size were compared by quantitatively analyzing the fluorescence signal (total flux, p / s).

[0187] As a result, rapid tumor growth was observed in all PBS, PTX IV, and PTX IN administration groups. In particular, the PTX IV administration group showed minimal therapeutic effect, with death occurring on day 9 (Fig. 21). On the other hand, the HCO IN administration group maintained a very low fluorescence signal until day 9, effectively suppressing tumor growth. This demonstrates that even when using the same anticancer drug (PTX), intranasal administration via the HCO platform maximizes drug delivery to the brain tumor site, resulting in a superior antitumor effect compared to intravenous injection or intranasal administration of the drug alone.

[0188] Example 18: Evaluation of Brain Delivery Efficiency in Large Animals (Pigs)

[0189] To validate the results from the rodent model in large animals with anatomical structures more similar to humans, experiments were conducted on adult pigs weighing approximately 35 kg. Specifically, PTX-PEG was synthesized by covalently bonding PEG to Paclitaxel (PTX), and PTX-PEG-IR780 was prepared by labeling PTX-PEG with IR-780 (fluorescent dye). A hydrophobic complex (HCO / PTX-PEG-IR780) was formed by mixing PTX-PEG-IR780 with 2-hydroxypropyl-β-cyclodextrin (HCO), into which a serotonin receptor-targeting piperazine derivative was introduced. The final formulation was prepared with an effective concentration of PTX-PEG-IR780 at 2 mg / mL. PTX-PEG-IR780 alone and PBS were prepared as comparative formulations. A total of 10 mL was administered to each pig. 5 mL was injected into each nostril, and the pigs were incubated for 2 hours while maintaining respiratory anesthesia. Two hours after administration, the brain was extracted, the cortex was isolated, and ex vivo NIR imaging was performed.

[0190] As a result, the fluorescent signal observed in the cerebral cortex of pigs was almost non-existent in the PBS group, and showed only a limited increase in the PTX-PEG-IR780 monotherapy group (Fig. 22). On the other hand, in the HCO / PTX-PEG-IR780 complex administration group, a very strong fluorescent signal was observed throughout the cortex compared to the same conditions. Quantitative analysis of fluorescence intensity showed that the HCO complex administration group exhibited a signal approximately 4 times higher in the dorsal cortex and 6 times higher in the ventral cortex compared to the monotherapy group (Fig. 23). These results strongly support the fact that the HCO platform of the present invention can dramatically improve drug delivery efficiency to the brain (especially the cerebral cortex) through intranasal administration not only in rodents but also in large animal models similar to humans, suggesting a very high potential for clinical application.

[0191] Example 19: Evaluation of Brain-Selective Delivery and Reduction of Lung Accumulation of HCO-AAV

[0192] The effects of HCO-based AAV delivery on brain tissue distribution were evaluated using C57BL6 normal mice. The experimental groups consisted of a Naked AAV administration group and an HCO-based AAV administration group, and the final viral concentration was set at 1E10 GC (genome copy) / 24μL. Using the in-delivery method, 4μL doses were administered at 4-minute intervals for a total of 6 repeated doses. To bind the AAV surface to HCO, the viral amine groups were first thiolated via a 2-iminothiolane (2-IT) reaction. Subsequently, after removing the remaining 2-IT using a Zeba spin column, HCO / Ada-PEG-SPDP, HCO / PTX-PEG-SPDP, and HCO / IR780-PEG-SPDP were reacted with thiolated AAV for 2 hours each to form HCO-AAV complexes. The free HCO was then removed from the formed complexes using a Zeba spin column.

[0193] To quantitatively evaluate the number of viral genomes in each experimental group, viral genomes were quantitatively analyzed using TaqMan-based qPCR. Mice were sacrificed 2 hours after the last administration, and brain tissue was excised to extract genomic DNA, after which the viral distribution was quantitatively analyzed using qPCR with AAV-specific primers. In addition, qPCR analysis was performed on lung tissue in the same manner to compare the non-specific accumulation of viral transmission within lung tissue.

[0194] As a result, the number of viral genomes in brain tissue increased significantly by approximately 153 times in the HCO-AAV administration group (average 469.50 GC / mg) compared to the Naked AAV administration group (average 3.1 GC / mg) (Fig. 24). On the other hand, while the viral accumulation in lung tissue was very high in the Naked AAV administration group (6.15E+04 GC), it decreased dramatically by more than 99.7% to 1.79E+02 GC in the HCO-AAV administration group. These results clearly demonstrate that the HCO platform is a highly ideal brain-targeted delivery system that enhances the selective delivery of AAV to brain tissue by 153 times while almost completely blocking unwanted accumulation in non-target organs such as the lungs.

Claims

1. (a) a cyclodextrin derivative to which a piperazine derivative is covalently bonded; and (b) a polymer chain having a hydrophobic compound covalently bonded to one end; comprising, A polymer nanocomposite characterized in that the hydrophobic compound of (b) above is included within the cyclodextrin derivative of (a) above by host-guest interaction.

2. The polymer nanocomposite according to claim 1, wherein the cyclodextrin derivative is an α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin substituted with one or more functional groups selected from methyl, ethyl, hydroxyethyl, sulfobutyl ether, carboxymethyl, sulfate, maleyl, and hydroxypropyl.

3. A polymer nanocomposite according to claim 1, characterized in that the piperazine derivative is one or more selected from ortho-methoxyphenyl piperazine (OPP), N-methyl-N'-aminopropylpiperazine (NMAP), N-methylpiperazine (NMP), and N-isopropylpiperazine (NiPP).

4. A polymer nanocomposite according to claim 1, characterized in that the polymer chain is polyethylene glycol (PEG).

5. A polymer composite according to claim 1, characterized in that an orthopyridyl disulfide (OPSS) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) functional group is additionally bonded to the other end of the polymer chain that is not bonded to the hydrophobic compound.

6. A polymer nanocomposite of any one of paragraphs 1 to 5 and A brain-targeted drug delivery system comprising a therapeutic drug bound to a polymer nanocomposite.

7. A brain-targeted drug delivery system according to claim 6, characterized in that the therapeutic drug is bonded to the other end of the polymer chain of the polymer nanocomposite that is not bonded to a hydrophobic compound, and the therapeutic drug is bonded by forming a disulfide bond with the OPSS functional group through a thiol group.

8. A brain-targeted drug delivery system according to claim 6, characterized in that the therapeutic drug has a modality selected from the group consisting of compounds, peptides / proteins, viruses, nucleic acids, vaccine components, radiotherapeutic agents, and photosensitizers.

9. A pharmaceutical composition for the prevention or treatment of degenerative brain diseases, central nervous system diseases, or brain tumors, comprising the brain-targeted drug delivery system of claim 6 as an active ingredient.

10. A pharmaceutical composition according to claim 9, characterized in that the composition is a formulation for intranasal administration.

11. A pharmaceutical composition according to claim 9, characterized in that the central nervous system disease is any one selected from the group consisting of cognitive impairment, intellectual disability, microcephaly, epilepsy, neurodevelopmental disorder, dementia, autism spectrum disorder, Down syndrome, Rett syndrome, and fragile X syndrome.

12. In paragraph 9, neurodegenerative diseases include ischemic stroke, traumatic brain injury, acute disseminated encephalomyelitis, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mild cognitive impairment, Alzheimer's disease, Pick's disease, senile dementia, progressive supranuclear palsy, subcortical dementia, Wilson's disease, multiple infarct disease, arteriosclerotic dementia, AIDS-related dementia, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedreich's ataxia, ataxia telangiectasia, epilepsy-related brain injury, and spinal cord Injury, restless legs syndrome, Huntington's disease, Parkinson's disease, striatonigral degeneration, cerebral vasculitis, mitochondrial encephalomyopathies, neuronal ceroid lipofuscinosis, spinal muscular atrophies, lysosomal storage disorder associated with the central nervous system, leukodystrophies, urea cycle defect disorder, hepatic encephalopathies, renal encephalopathies, metabolic encephalopathies,A pharmaceutical composition characterized by being any one selected from the group consisting of porphyria, bacterial meningitis, viral meningitis, meningoencephalitis, prion diseases, poisonings with neurotoxic compounds, Guillain-Barre syndrome, chronic inflammatory neuropathies, polymyositis, dermatomyositis, and radiation-induced brain damage.

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