Drug delivery system for penetrating blood-brain barrier, and composition for treating or diagnosing brain diseases comprising same

Abstract

The present invention relates to a drug delivery system capable of efficiently penetrating the blood-brain barrier (BBB). More specifically, the present invention relates to a drug delivery platform which fuses transthyretin (TTR) or a functional variant thereof with a peptide sequence comprising a certain amino acid sequence, and thus can be applied to a therapeutic agent for brain diseases and a diagnostic composition.

Description

Drug delivery system for permeating the blood-brain barrier and composition for treating or diagnosing brain diseases containing the same

[0001] The present invention relates to a drug delivery system capable of efficiently passing through the blood-brain barrier (BBB). More specifically, the invention relates to a drug delivery platform applicable to compositions for treating and diagnosing brain diseases by fusing transthyretin (TTR) or a functional variant thereof with a peptide sequence containing a specific amino acid sequence.

[0002]

[0003] The blood-brain barrier (BBB) ​​is a cellular barrier composed of tight junctions with a highly high electrical resistance of 0.1 Ω·m or more between the vascular endothelial cells in contact with the associated pericytes and astrocytes, and is a highly selectively permeable barrier that separates circulating blood from brain extracellular fluid in the central nervous system (CNS), serving as a gateway that regulates the entry and exit of substances.

[0004] The blood-brain barrier blocks bacteria, pathogens, and potential hazardous substances in the blood that can be transported through the blood from reaching the brain, but it also blocks most central nervous system drugs from reaching the brain, resulting in low drug efficiency. To compensate for this, these drugs are administered at high doses, which can cause serious side effects in surrounding organs. Therefore, there is a need to discover an efficient drug delivery system capable of penetrating the blood-brain barrier to prevent negative systemic effects while ensuring the therapeutic effect of the drugs.

[0005] In this regard, the Swiss pharmaceutical company Roche has developed an antibody-based 'brain shuttle.' The brain shuttle binds to transferrin receptors expressed in the epithelial tissue of cerebral blood vessels, is transported into cerebral blood vessel cells, and then moves into the brain parenchyma. While conducting Phase 3 clinical trials with gantenerumab, a monoclonal antibody targeting amyloid beta plaques—toxic proteins in brain neurons identified as a cause of dementia—Roche is conducting Phase 1 clinical trials with a redesigned dual antibody that attaches the brain shuttle to gantenerumab.

[0006] However, antibody-based drug delivery systems are not suitable for application to various types of drugs. Since antibodies are high-molecular-weight substances that are significantly larger than low-molecular-weight substances such as small molecule compounds and peptides, attaching a single drug to a single antibody actually reduces drug delivery efficiency. Consequently, the drugs attached to antibodies are substances that are highly effective but difficult to use due to toxicity issues. To address these problems, research is being conducted on various drug delivery systems utilizing small molecule compounds, peptides, and aptamers, which have molecular weights smaller than antibodies.

[0007]

[0008] To ensure the therapeutic effect of drugs, it is necessary to discover an efficient drug delivery system capable of penetrating the blood-brain barrier.

[0009]

[0010] One objective of the present invention is to provide a drug delivery system for permeating the blood-brain barrier.

[0011] Another objective of the present invention is to provide a composition for the prevention or treatment of brain diseases comprising the above-mentioned drug delivery system and drug.

[0012] Another objective of the present invention is to provide a composition for diagnosing brain diseases comprising the above-mentioned drug delivery system and a substance for diagnosing brain diseases.

[0013] Another objective of the present invention is to provide a brain disease diagnostic kit comprising the above-mentioned brain disease diagnostic composition.

[0014] Another objective of the present invention is to provide a method for manufacturing the above-mentioned drug delivery system for permeating the blood-brain barrier.

[0015] Another object of the present invention is to provide a drug carrier-drug conjugate in which the drug carrier and temozolomide or a derivative thereof are chemically combined.

[0016] Another objective of the present invention is to provide a method for treating a brain disease comprising the drug delivery system and the drug.

[0017] Another objective of the present invention is to provide a use for the drug delivery system to pass through the blood-brain barrier (BBB).

[0018]

[0019] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0020]

[0021] The present invention relates to a drug delivery system capable of permeating the blood-brain barrier and a composition for treating or diagnosing brain diseases comprising the same. The drug delivery system of the present invention has the ability to selectively pass through the blood-brain barrier (BBB) ​​and can significantly improve the delivery efficiency of drugs or diagnostic substances into the brain. This solves the problem of low BBB permeability of existing drugs and can maximize the efficacy of brain disease treatments and diagnostic compositions.

[0022]

[0023] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0024]

[0025] Figure 1 confirms the expression of the TTR-CPP protein according to one embodiment of the present invention.

[0026] Figure 2 shows the process of producing a TTR-CPP-TMZ polymer through the polymerization reaction of TTR-CPP protein and Temozolomide.

[0027] Figure 3 shows the results of confirming the absorption rate of U87 cells treated with temozolomide (TMZ), TMZ-TTR, and TMZ-CPP-TTR.

[0028] Figure 4 shows the drug distribution in brain slices after (a) 4 hours and (b) 24 hours of TTR-CPP-TMZ, confirming the efficiency of BBB penetration.

[0029] Figure 5 shows the survival rates according to treatment with TMZ, TTR-TMZ, and TTR-CPP-TMZ in a brain tumor mouse model.

[0030] Figure 6 illustrates the process of producing TTR-CPP-immobilized exosomes and loading drugs.

[0031] Figure 7 shows the survival rates of brain tumor mouse models treated with TMZ, TTR-TMZ, and TTR-CPP-TMZ in exosome-based experiments.

[0032] Figure 8 shows the LRP1 binding sites of TTR and TTR-CPP.

[0033]

[0034] One embodiment of the present invention is a drug delivery system for penetrating the blood-brain barrier.

[0035] In one embodiment, the drug delivery system is characterized by comprising transthyretin or a functional variant thereof; and a peptide comprising an amino acid sequence represented by the following general formula 1:

[0036] [General Formula 1]

[0037] X1X2X3X4X5X6R n

[0038] Here,

[0039] X1 to X6 are histidine, lysine, or arginine, and

[0040] R is arginine, and

[0041] n is 0 to 7.

[0042] In a specific embodiment according to any one of the preceding embodiments, the peptide is characterized by comprising transthyretin; and an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 48.

[0043] In a specific embodiment according to any one of the preceding embodiments, the peptide is characterized by comprising transthyretin; and an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 6.

[0044] In a specific embodiment according to any one of the preceding embodiments, the peptide is characterized by comprising a peptide composed of the amino acid sequence of SEQ ID NO. 1.

[0045] In a specific embodiment according to any one of the preceding embodiments, the amino acid sequence is characterized as a functional equivalent or variant having (i) 95% or more identity with SEQ ID NO. 1 and (ii) a peptide that maintains 80% or more of blood-brain barrier permeability activity.

[0046] In an embodiment according to any one of the preceding embodiments, the drug delivery system is characterized by passing through the blood-brain barrier (BBB).

[0047] In a specific embodiment according to any one of the preceding embodiments, the drug delivery system is characterized by passing through the blood-brain barrier (BBB) ​​by binding to LRP1 (Low-density lipoprotein receptor-related protein 1).

[0048] In a specific embodiment according to any one of the preceding embodiments, the transtyretin and the peptide containing the amino acid sequence are connected by a covalent bond or a peptide bond.

[0049] Another embodiment of the present invention is a pharmaceutical composition for the prevention or treatment of brain disease comprising the drug delivery system and the drug.

[0050] In one embodiment, the drug is characterized as being a physiologically active substance having a preventive or therapeutic effect on brain diseases.

[0051] In an embodiment according to any one of the preceding embodiments, the drug is characterized by being one or more selected from the group consisting of temozolomide, donepezil, rivastigmine, galantamine, paclitaxel, doxorubicin, and osimertinib.

[0052] In a specific embodiment according to any one of the preceding embodiments, the brain disease is characterized by being one or more selected from the group consisting of brain tumor, Alzheimer's disease, stroke, paralysis, dementia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, thrombosis, embolism, transient ischemic attack, lacune, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disorder, cerebral functional coma, and brain cancer.

[0053] In a specific embodiment according to any one of the preceding embodiments, the drug is characterized by being chemically bonded to an amino group (NH₂) or a carboxyl group (COOH) of the drug delivery system.

[0054] In a specific embodiment according to any one of the preceding embodiments, the drug delivery system is characterized by being bound to the surface of an exosome.

[0055] Another embodiment of the present invention is a composition for diagnosing brain diseases comprising a substance for diagnosing brain diseases and said drug delivery system.

[0056] In one embodiment, the drug delivery system is characterized by comprising transthyretin; and a peptide comprising an amino acid sequence represented by the following general formula 1:

[0057] [General Formula 1]

[0058] X1X2X3X4X5X6R n Here,

[0059] X1 to X6 are histidine, lysine, or arginine, and

[0060] R is arginine, and

[0061] n is 0 to 7.

[0062] In a specific embodiment according to any one of the preceding embodiments, the peptide is characterized by comprising transthyretin; and an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 6.

[0063] In a specific embodiment according to any one of the preceding embodiments, the peptide is characterized by comprising a peptide composed of the amino acid sequence of SEQ ID NO. 1.

[0064] In a specific embodiment according to any one of the preceding embodiments, the brain disease diagnostic material is characterized by being one or more selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

[0065] In an embodiment according to any one of the preceding embodiments, the composition for diagnosing brain diseases is characterized by further comprising a labeling substance including a fluorescent substance, a radioisotope, or a magnetic resonance imaging (MRI) contrast agent.

[0066] Another embodiment of the present invention is a brain disease diagnostic kit comprising the brain disease diagnostic composition and further comprising a labeling reagent, a buffer, and an indicator.

[0067] Another embodiment of the present invention is a method for manufacturing a drug delivery system for permeating the blood-brain barrier.

[0068] In one embodiment, the method is characterized by comprising a drug delivery system comprising transthyretin and a peptide comprising an amino acid sequence represented by the following general formula 1, and by introducing and expressing a recombinant expression vector, which is formed by linking a transthyretin-coding nucleic acid sequence and a nucleic acid sequence encoding said amino acid sequence, into a host cell:

[0069] [General Formula 1]

[0070] X1X2X3X4X5X6R n Here,

[0071] X1 to X6 are histidine, lysine, or arginine, and

[0072] R is arginine, and

[0073] n is 0 to 7.

[0074] Another embodiment of the present invention is a drug delivery system-drug conjugate for penetrating the blood-brain barrier.

[0075] In one embodiment, the drug delivery-drug conjugate is characterized by temozolomide or a derivative thereof being chemically bound to a drug delivery agent comprising transthyretin and a peptide having an amino acid sequence represented by the following general formula 1:

[0076] [General Formula 1]

[0077] X1X2X3X4X5X6R n Here,

[0078] X1 to X6 are histidine, lysine, or arginine, and

[0079] R is arginine, and

[0080] n is 0 to 7.

[0081] Another embodiment of the present invention is a method for treating brain diseases.

[0082] In one embodiment, the treatment method is characterized by preventing or treating a brain disease by administering a drug delivery vehicle comprising transthyretin and an amino acid sequence represented by the following general formula 1, and a drug, into the body of a mammal:

[0083] [General Formula 1]

[0084] X1X2X3X4X5X6R n Here,

[0085] X1 to X6 are histidine, lysine, or arginine, and

[0086] R is arginine, and

[0087] n is 0 to 7.

[0088] Another embodiment of the present invention is for use in passing a drug delivery system across the blood-brain barrier (BBB).

[0089] In one embodiment, the use is characterized by comprising transthyretin and an amino acid sequence represented by the following general formula 1:

[0090] [General Formula 1]

[0091] X1X2X3X4X5X6R n Here,

[0092] X1 to X6 are histidine, lysine, or arginine, and

[0093] R is arginine, and

[0094] n is 0 to 7.

[0095]

[0096] The present invention will be described in more detail below.

[0097] Meanwhile, each description and embodiment disclosed herein may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of the invention. Furthermore, the scope of the invention is not to be limited by the specific descriptions provided below.

[0098] In addition, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the invention described in this application using only ordinary experiments. In addition, such equivalents are intended to be included in the invention.

[0099]

[0100] One embodiment of the present invention relates to a drug delivery system for permeating the blood-brain barrier.

[0101] Specifically, one embodiment of the present invention may be a drug delivery system comprising, but is not limited to: transthyretin or a functional variant thereof; and a peptide comprising an amino acid sequence represented by the following general formula 1:

[0102] [General Formula 1]

[0103] X1X2X3X4X5X6R n Here,

[0104] X1 to X6 are histidine, lysine, or arginine, and

[0105] R is arginine, and

[0106] n is 0 to 7.

[0107] Specifically, it may include an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 48, and may also include an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 6, and more specifically, may include an amino acid sequence consisting of SEQ ID NO. 1.

[0108] The term “transthyretin (TTR)” in the present invention refers to a homototetrameric protein composed of 508 amino acids and having a molecular weight of approximately 55 kDa. The TTR protein is composed of four identical monomers having a β-planar structure and is known to be independently regulated, synthesized, and expressed in blood and cerebrospinal fluid. The TTR may include any polypeptide sequence known as a TTR protein in the ordinary art.

[0109] The term “functional variant of transthyretin” of the present invention means a polypeptide having (i) 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of transthyretin, and (ii) substantially the same biological activity as transthyretin. Such functional variants may include naturally occurring isoforms, mutants containing conservative amino acid substitutions, variants in which some amino acids at the N-terminus or C-terminus are deleted or added, or recombinant proteins that retain the β-structure and LRP1 binding ability of transthyretin. For example, the functional variants of the present invention also include TTR variants with enhanced tetrameric stability, such as the conserved variant between human TTR (Homo sapiens, UniProt P02766) and mouse TTR (Mus musculus, P07309), or the T119M (Threonine 119 → Methionine) variant. The variants may include variations within a range that bind to the TTR receptor LRP1 (Low-density lipoprotein receptor-related protein 1) or maintain interaction with thyroxine or retinol-binding protein (RBP).

[0110] In addition, the present invention may include functional equivalents and variants of the peptide.

[0111] A functional equivalent of the above-mentioned peptide includes a peptide that does not alter the overall activity of the peptide, and a peptide capable of performing the same functional action is included within the scope of the present invention. The above-mentioned variant refers, for example, to a sequence in which one or several amino acids are deleted, substituted, or added, or to a sequence having 95% or more, preferably 98% or more, and more preferably 99% or more identity with the above-mentioned amino acid sequence. Here, "identity" refers to the ratio (%) of the number of identical amino acid residues in one amino acid sequence to the total number of amino acid residues in one amino acid sequence, including the number of gaps, when two amino acid sequences are aligned to achieve the highest degree of agreement without introducing or not introducing gaps. In addition, "several" refers to an integer from 2 to 10, for example, an integer from 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Specific examples of natural variants include variants based on polymorphisms such as SNPs (single nucleotide polymorphisms) or splice variants. It is preferable that the above substitution be a conservative amino acid substitution. This is because if it is a conservative amino acid substitution, it can have a structure or properties substantially equivalent to that of a peptide having the above amino acid sequence.

[0112] Conservative amino acids are known to include mutually nonpolar amino acids (glycine, alanine, phenylalanine, valine, leucine, isoleucine, methionine, proline, tryptophan) and polar amino acids (amino acids other than nonpolar amino acids), charged amino acids (acidic amino acids (aspartic acid, glutamic acid) and basic amino acids (arginine, histidine, lysine)) and uncharged amino acids (amino acids other than charged amino acids), aromatic amino acids (phenylalanine, tryptophan, tyrosine), branched amino acids (leucine, isoleucine, valine) and aliphatic amino acids (glycine, alanine, leucine, isoleucine, valine), etc. Additionally, they may include peptides in which structural stability against heat, pH, etc., is increased or peptide activity is increased by mutation or modification in the amino acid sequence.

[0113]

[0114] The peptides composed of the amino acid sequences of SEQ ID NOs. 1 to 6 above may be chemically synthesized. When prepared by chemical synthesis, they may be prepared by chemical synthesis methods widely known in the art (Creighton, Proteins; Structures and Molecular Principles, WH Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid-phase synthesis, fragment condensation, F-MOC, or T-BOC chemical methods (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton, Florida, 1997; A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, 1989).

[0115] In addition, the above peptide may be prepared by genetic engineering methods, but is not limited to the following methods. First, a DNA sequence encoding the above peptide is prepared according to conventional methods. The DNA sequence may be prepared by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., one sold by Biosearch or Applied iosystems). The prepared DNA sequence is inserted into a vector containing one or more expression regulatory sequences (e.g., promoter, enhancer, etc.) operably linked to the DNA sequence to regulate the expression of the DNA sequence, and host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformed cells are cultured under a medium and conditions suitable for expressing the DNA sequence, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. This recovery may be performed using methods known in the art (e.g., chromatography). In the foregoing, the term "substantially pure peptide" means that the peptide according to the present invention substantially does not contain any other protein derived from the host. For genetic engineering methods for synthesizing the peptide of the present invention, reference may be made to the following literature: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Editions; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; Hitzeman et al., J. Biol. Chem.,255:12073-12080, 1990.

[0116] The drug delivery system of the present invention is fused with Transthyretin (TTR) and a functional variant thereof; and a peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 6, and it was confirmed that it can pass through the blood-brain barrier (BBB) ​​by binding to LRP1, known as a blood-brain barrier receptor, through a receptor-mediated endocytosis mechanism (Example 5).

[0117] The term “LRP1 (Low-density lipoprotein receptor-related protein 1)” of the present invention is a multi-ligand receptor belonging to the LDL receptor family, which repeatedly contains a complement-type repeat (CR) domain and can bind to various protein ligands. The drug delivery system of the present invention is useful as a drug delivery platform capable of selectively transporting therapeutic drugs or diagnostic substances to the brain, and thus can be effectively utilized for the treatment or diagnosis of brain diseases. The drug delivery system of the present invention is characterized as a fusion protein formed by directly connecting transthyretin (TTR) or a functional variant thereof and a peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 48 by a covalent bond or a peptide bond.

[0118] Specifically, the peptide may be bound to the N-terminus or C-terminus of transthyretin, and such binding forms may be realized through recombinant fusion methods widely used in the art or chemical coupling (e.g., EDC / NHS binding reaction, maleimide-thiol coupling, amide coupling, etc.). The fusion protein maintains the stable homotomer structure of transthyretin and is conferred a binding affinity with the LRP1 receptor (Low-density lipoprotein receptor-related protein 1) by the peptide sequences of SEQ ID NOs. 1 to 48, thereby exhibiting the ability to penetrate the blood-brain barrier (BBB) ​​via a receptor-mediated mechanism. Due to these structural characteristics, the fusion protein of the present invention is highly useful as a platform protein for the intrabrain delivery of therapeutic drugs or diagnostic substances.

[0119] Another embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of brain diseases.

[0120] The pharmaceutical composition of the present invention comprises the above-mentioned drug delivery system and drug.

[0121] The drug delivery system is as described above.

[0122] The above drug may be bound to the terminal carboxyl group (COOH) or amino group (NH2) of the drug delivery system.

[0123] The above drug may be bonded to the terminal carboxyl group (COOH) or amino group (NH₂) of the drug delivery system by covalent bonds or peptide bonds, and may also be connected via a linker if necessary.

[0124] The above-mentioned drug is a bioactive compound having a preventive or therapeutic effect on brain diseases, and can be used without limitation as long as a carboxyl group or an amino group is present at the terminal end of the molecular structure. The drug usable in the present invention may be an anticancer agent or a treatment for central nervous system (CNS) diseases, and may be, for example, temozolomide, donepezil, rivastigmine, galantamine, paclitaxel, doxorubicin, or osimertinib, but is not limited thereto.

[0125] The above brain disease may be a disease or condition that occurs in or has a cause in the central nervous system, and may be, for example, a brain tumor, Alzheimer's disease, stroke, paralysis, dementia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, thrombosis, embolism, transient ischemic attack, lacune, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disorder, cerebral coma, or brain cancer, but is not limited thereto.

[0126] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier that is commonly used in formulations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0127] The pharmaceutical composition of the present invention may additionally include, in addition to the above components, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). Suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the method of formulation, mode of administration, age, body weight, sex, severity of disease symptoms, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the intended treatment. Meanwhile, the dosage of the pharmaceutical composition of the present invention is not limited thereto and may be 0.01 to 2000 mg / kg (body weight) per day.

[0128] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0129] In the present invention, the term "prevention" refers to any act of completely or partially suppressing or delaying the onset of brain disease by administering a drug delivery system or composition comprising Transthyretin or a peptide having an amino acid sequence represented by General Formula 1, a functional variant thereof, and "treatment" refers to any act of completely or partially improving or benefiting the symptoms of brain disease, including reduction / improvement of symptoms, relief of pain from symptoms, reduction in the incidence of brain disease, or other changes in the patient that increase the therapeutic effect.

[0130] In the present invention, the term "administration" means introducing a specific substance to a patient by any appropriate method. The route of administration of the composition is not specifically limited thereto, but may be administered through any general route in which the composition can reach a target in vivo. For example, it may be administered orally, or by parenteral administration routes including, but not limited to, the skin, intravenously, intramuscularly, intra-arterially, intramedullaryly, intrathecally, intraventricularly, pulmonaryly, transdermally, subcutaneously, intra-abdominally, intranasally, gastrointestinally, topically, sublingually, vaginally, or rectally. Meanwhile, since peptides are digested upon oral administration, it is desirable for the oral composition to be formulated to coat the active agent or protect it from degradation in the stomach. Specifically, the composition in the present invention may be administered in the form of an injection. Additionally, the composition in the present invention may be administered by any device capable of delivering the active ingredient to target cells.

[0131] In the present invention, "individual" refers to an individual suspected of having a brain disease, and refers to an individual that has developed or may develop the said disease. In one embodiment, the individual may be a mammal including humans, mice, livestock, etc., but any individual capable of being treated with a composition comprising the transthyretin of the present invention or a functional variant thereof and a peptide comprising an amino acid sequence represented by General Formula 1 is included without limitation. Additionally, humans may be excluded from the individual of the present invention, but are not limited thereto.

[0132]

[0133] Another embodiment of the present invention relates to a composition for diagnosing brain diseases.

[0134] The composition of the present invention comprises a substance for diagnosing brain diseases and a drug delivery system, wherein the drug delivery system comprises Transthyretin or a functional variant thereof; and a peptide comprising an amino acid sequence represented by General Formula 1.

[0135] Transthyretin or a functional variant thereof; and a peptide comprising an amino acid sequence represented by General Formula 1, as described above.

[0136] Since the above drug delivery system can penetrate the blood-brain barrier (BBB) ​​through an LRP1 (Low-density lipoprotein receptor-related protein 1) receptor-mediated mechanism, it can effectively deliver the diagnostic substance of the diagnostic composition into brain tissue.

[0137] The above-mentioned material for diagnosing brain diseases may be, for example, an antibody, aptamer, DNA, RNA, protein, or polypeptide, and may be a biorecognition molecule capable of specifically binding to a biomarker of a specific brain disease (e.g., amyloid beta, tau protein, alpha-synuclein, GFAP, or tumor marker EGFRvIII, etc.).

[0138] In the diagnostic composition of the present invention, the drug delivery vehicle and the diagnostic substance may be coupled by a covalent bond, peptide linkage, or non-covalent interaction. For example, they can be stably coupled using a maleimide-thiol, EDC / NHS coupling, or biotin-streptavidin coupling system.

[0139] Additionally, the diagnostic composition of the present invention may further include a label capable of generating a detectable signal. The label may be, for example, a fluorescent label, a radioisotope, an enzyme label, a magnetic resonance imaging (MRI) contrast agent, or a positron emission tomography (PET) contrast agent, but is not limited thereto.

[0140] The above labeling substance may be combined by a direct labeling or indirect labeling method, and the detection method may include fluorescence imaging, radioimaging, MRI, or PET image analysis.

[0141] From the above characteristics, the brain disease diagnostic composition of the present invention can be effectively used for the early diagnosis or imaging of lesion sites of brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, or cerebrovascular disease by penetrating the blood-brain barrier.

[0142] Another embodiment of the present invention relates to a kit for diagnosing brain diseases.

[0143] The diagnostic kit of the present invention comprises the above-mentioned composition for diagnosing brain diseases, and the composition comprises a drug delivery system comprising Transthyretin or a functional variant thereof and a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 48, and a substance for diagnosing brain diseases.

[0144] Since the above drug delivery system can cross the blood-brain barrier (BBB) ​​through an LRP1 (Low-density lipoprotein receptor-related protein 1) receptor-mediated mechanism, it can significantly improve the delivery efficiency of diagnostic substances that target biomarkers in the brain.

[0145] The diagnostic kit of the present invention may also further include a labeling reagent, a buffer, and an indicator.

[0146]

[0147] Another aspect of the present invention relates to a method for preparing a drug delivery system comprising transthyretin or a functional variant thereof and a peptide comprising an amino acid sequence represented by the following general formula 1.

[0148] The above method includes the step of introducing and expressing a recombinant expression vector, in which a transthyretin coding sequence and the amino acid sequence are linked, into a host cell.

[0149] The above-described recombinant expression vector may be constructed by linking a full-length or partial fragment sequence of the transthyretin (TTR) gene with a peptide sequence selected from the group consisting of SEQ ID NOs 1 to 6. Linking may be performed using commonly known DNA linkage techniques. Host cells may include, but are not limited to, Escherichia coli (E. coli), yeast, CHO (Chinese hamster ovary), HEK293, or Sf9 insect cells. In particular, to ensure stable protein expression and folding, a vector for non-secretory cells or a vector for secretory cells may be used. After recovery from the host cells, the expressed protein may be purified using a nickel column (Ni-NTA affinity), protein A / G affinity, or size-exclusion chromatography. If necessary, steps for refolding and endotoxin removal may be performed after purification. The transthyretin-peptide fusion protein produced by the above method can be utilized as a drug delivery vehicle capable of effectively passing through the blood-brain barrier (BBB).

[0150]

[0151] Another aspect of the present invention relates to a drug carrier-drug conjugate in which temozolomide or a derivative thereof is chemically bound to a drug carrier comprising transthyretin or a functional variant thereof and a peptide having an amino acid sequence represented by the following general formula 1.

[0152] The above bonding can be carried out through covalent reactions such as amino group-carboxyl group reactions, maleimide-thiol bonding, or carbodiimide (EDC / NHS)-mediated bonding, depending on the functional group of the drug.

[0153] In addition to temozolomide, the above structure can be equally applied to other anticancer drug derivatives such as paclitaxel, doxorubicin, and osimertinib.

[0154]

[0155] The above conjugate functions to deliver the drug to tumor cells within brain tissue after the transthyretin-peptide carrier penetrates the BBB via the LRP1 receptor. This increases the accumulation concentration of the drug in the brain and reduces exposure to non-target tissues, thereby improving the anticancer effect and safety profile.

[0156]

[0157] Another aspect of the present invention relates to a drug delivery system comprising transthyretin or a functional variant thereof and a peptide comprising an amino acid sequence represented by the following general formula 1, and a method for preventing or treating brain disease by administering the drug into the body of a mammal.

[0158] In the above method, the drug delivery system selectively crosses the blood-brain barrier through the LRP1 receptor to deliver the drug into the brain tissue targeted for treatment.

[0159] The above-mentioned drugs are anticancer agents, neuroprotective agents, anti-inflammatory agents, or anti-protein agglutinants, and may include, but are not limited to, temozolomide, donepezil, rivastigmine, paclitaxel, or osimertinib.

[0160] The above method may be performed via intravenous, subcutaneous, intramuscular, subdural, or intrathecal administration, and may be administered once or repeatedly depending on the therapeutic purpose.

[0161] The treatment method of the present invention is particularly useful for the treatment of brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), stroke, etc., and can effectively solve the problem where the therapeutic effect was limited due to the low penetration of existing drugs into the blood-brain barrier (BBB).

[0162]

[0163] Another aspect of the present invention relates to the use of a drug delivery system comprising transthyretin or a functional variant thereof and a peptide comprising an amino acid sequence represented by the following general formula 1 for crossing the blood-brain barrier (BBB).

[0164] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0165]

[0166] Example 1. Production of TTR-CPP Protein

[0167] The TTR gene was synthesized by isolating RNA from hepatocytes (HepG2), synthesizing cDNA using reverse transcriptase, and obtaining the gene via RT-PCR using TTR-specific primers with the synthesized template. The CPP sequence (HKRRRR, SEQ ID NO. 1) was added to the primer sequences used to finally secure the TTR-CPP gene. The synthesized TTR-CPP conjugate gene was homologously recombinated using an infusion enzyme into the pBAD vector, a vector system optimized for the production of soluble proteins, utilizing the XhoⅠ / HindⅢ enzyme site, and cloned into TOP10 E. coli. Cloning was confirmed by culturing a single colony of TOP10 E. coli and treating it with XhoⅠ / HindⅢ enzymes to select clones that matched the size of the TTR / CPP gene. The TTR-CPP conjugate gene was finally verified through gene sequencing analysis of the selected clones.

[0168] To isolate the TTR-CPP fusion protein, the cloned TTR-CPPE.coli was cultured at 37°C under shaking (225–250 rpm) overnight until the absorbance O.D600 was 1–2. The following day, the culture medium was added to each test tube along with LB medium and cultured until the O.D600 reached approximately 0.5. To induce TTR-CPP protein expression in the pBAD / His vector, L-arabinose was added to achieve a final concentration of 0.00002–0.2%, and the culture was continued for an additional 4 hours. A portion of the culture medium was centrifuged to remove the supernatant, SDS sample buffer was added, and after heating for 5 minutes, SDS-PAGE was performed. TTR / CPP protein expression was confirmed via Coomassie blue staining following protein electrophoresis (Fig. 1, top).

[0169] Since TTR / CPP protein expression was most highly induced under 0.02% L-arabinose conditions, TTR-CPPE.coli was cultured in large quantities under the same conditions to obtain a 0.9 g cell pellet. Cells were extracted by adding DNase I and lysozyme to a non-ionic detergent buffer and centrifuged. The supernatant was filtered to separate water-soluble proteins, and His-tag TTR / CPP proteins were isolated using a resin capable of isolating His-tag proteins. High-purity TTR-CPP proteins were produced by washing and extracting the TTR / CPP protein-bound resin. Samples recovered from each purification step were subjected to protein electrophoresis, and the TTR / CPP protein was confirmed to be 21.18 kDa in size using an anti-His antibody (Fig. 1, bottom). The production yield of TTR-CPP protein was calculated to be 5.25 mg / L culture.

[0170]

[0171] Example 2. Preparation of TTR-CPP-temozolomide polymer

[0172] A TTR-CPP-Tz polymer was prepared by combining temozolomide, a brain tumor treatment agent having an amine group on the -COOH of TTR-CPP (Fig. 2).

[0173] TTR-CPP protein and EDC were mixed at a molar concentration of 1:5 and slowly stirred with a stirrer at room temperature for 30 minutes. Temozolomide was added at the same molar concentration as TTR-CPP protein and stirred for an additional hour at room temperature.

[0174] Unreacted monomers were removed by filtering with a molecular weight 3,000 microfilter (centricon).

[0175]

[0176] Example 3. Results of cell and animal experiments

[0177] 3-1. Effect of drug uptake on brain tumor cell line U-37 cells

[0178] A 12 mm cover slip was placed in each well of a 24-well plate and coated with Poly-L-Lysine solution (Sigma). Then, U-87 MG cells were seeded onto the cover slip at a density of 1 x 10⁵ cells / well and cultured for 24 hours at 37°C under 5% CO₂ conditions.

[0179] Alexa 647 fluorescence (Alexa Fluor® Antibody Labeling Kits) labeled drugs (temozolomide (TMZ), TMZ-TTR, TMZ-CPP-TTR) were treated and reacted for 1, 6, and 12 hours. Cells were treated with 4% paraformaldehyde in PBS and fixed for 20 minutes.

[0180] The slides were washed three times with 1X PBS at 10-minute intervals. A mounting solution containing DAPI (Invitrogen) was placed on a slide glass, and the cover slip surface where the cells had grown was placed in contact with the mounting solution. After staining at room temperature for 24 hours, the slides were stored at 4°C. Subsequently, images were captured at 10X magnification using a confocal microscope. A Carl Zeiss LSM800 confocal microscope was used.

[0181] As a result, as shown in Figure 3, the intracellular absorption rate of temozolomide bound to the TTR-CPP protein increased rapidly after 12 hours of reaction. Compared to temozolomide bound only to the TTR protein, the absorption rate of the drug using the TTR / CPP protein was significantly improved, so it was analyzed that binding CPP is advantageous for increasing the absorption rate of temozolomide in brain tumor cells.

[0182]

[0183] 3-2. BBB Crossing Effect of TTR / CPP-Temozolomide Polymer

[0184] The experiment was conducted with the experimental groups in Table 1 below.

[0185] Group Observation TimeControl24hEmozolomide (TMZ)-Alexa 6474h / 24hTTR-CPP-Alexa 6474h / 24hTTR-CPP-Emozolomide (TMZ)-Alexa 6474h / 24h

[0186]

[0187] TTR-CPP-TMZ was labeled with Alexa Fluor 647 (ThermoFisher) according to the manufacturer's manual. The amounts of temozolomide and TTR-CPP used as control substances were prepared at the same molar concentration as the amount of TTR-CPP-TMZ (9.5 μM).

[0188] Perfusion was performed 4 or 24 hours after the drug was IV injected (Lectin 488 injection).

[0189] Subsequently, after extracting the brain, it was treated overnight with 4% PFA at 4°C for post-fixation. The brain was sliced ​​into 1 mm sections using a matrix and treated with Binaree Starting Solution at 4°C until settling. Clearing was performed by treating with Tissue Clearing Solution B (Binaree Inc., Korea) at 37°C for 2 hours. After washing with DW, the tissue was treated overnight with Mounting & Storage Solution (Binaree Inc.) at 37°C. Afterward, cleared 1 mm tissues were imaged using confocal microscopy (Nikon, 4 or 20x objective lens).

[0190] As a result of observing the drug distribution, it appeared as shown in Fig. 4 (a) after 4 hours and Fig. 4 (b) after 24 hours.

[0191] As shown in Figure 4, the drug with TTR / CPP bound to temozolomide showed a higher absorption rate across the BBB compared to when temozolomide was treated alone. Consequently, it was observed that the intracerebral distribution of TTR / CPP-Temozolomide, in which TTR / CPP is bound to temozolomide, increased rapidly.

[0192] Based on the results of this experiment, it was determined that TTR / CPP could serve as a BBB-crossing drug delivery system that enhances the brain absorption of temozolomide.

[0193]

[0194] 3-3. Cancer removal effect in brain tumor model mice

[0195] Temozolomide, TTR-Temozolomide (TTR-TMZ), and TTR-CPP-Temozolomide (TTR-CPP-TMZ) were prepared at the same molar concentration relative to temozolomide (48 μM).

[0196] Ten BALB / c-nude mice (4 weeks old) were assigned to each test group, and the following substances were administered to each test group.

[0197] - G1: Control group

[0198] - G2: TMZ

[0199] - G3: TTR-TMZ (TTR-Temozolomide)

[0200] - G4: TTR-CPP-TMZ (TTR-CPP-Temozolomide)

[0201] 1×10⁶ U87 cells using a stereotactic system 5 The number of cells was determined and injected into each mouse. Starting 3 days after cell injection, 100 μL of PBS, TMZ, TTR-TMZ, and TTR-CPP-TMZ were injected into the mouse brains via the tail vein at 2-week intervals for 4 weeks (final concentration 10 mg / kg).

[0202] While checking the mouse lethality rate daily, a Kaplan-Meier graph was constructed using the lethality rates of each test group after rearing for 120 days (Fig. 5).

[0203] The 120-day survival rates of the control group, TMZ, TTR-TMZ, and TTR-CPP-TMZ groups were 10%, 10%, 40%, and 50%, respectively, showing the highest survival rate when using TTR / CPP as a carrier.

[0204] Although TMZ showed a slightly higher survival rate up to 63 days compared to the PBS control group, it showed the same survival rate as the control group upon long-term administration, indicating that its anticancer effect as a standalone substance is limited due to the characteristics of the substance.

[0205] On the other hand, for the BBB-crossing inducing substances, TTR-TMZ and TTR-CPP-TMZ, which are test substances formed by binding TTR and TTR-CPP to TMZ, the 50% survival time (median survival time) was found to be 36.1% and 44.6% higher, respectively, compared to the control group.

[0206] These results appear to be correlated with the very high BBB penetration efficiency of TTR-CPP-Temozolomide in the brain tumor absorption rate of Figure 3 and the drug distribution test of Figure 4.

[0207]

[0208] Example 4. Production and Efficacy of Exosome-Conjugated BBB Shuttle

[0209] 4.1 Exosome Separation

[0210] Exosomes were isolated from HEK293 cells. HEK293 cells were placed in a 100 mm culture plate at a density of 5 x 10⁶ 5 Cells were added and cultured for 72 hours in DMEM containing 10% exosome-depleted FBS. At harvest, the culture medium was centrifuged at 1500 x g for 30 minutes to remove cells and cellular byproducts. The supernatant was filtered through a 0.2 µm syringe filter to remove large vesicles or lipid particles. Subsequently, the filtrate (60 mL) was placed in the Centricon ®Exosomes were concentrated by placing the sample in a Plus-70 filter and centrifuging at 3500 x g for 40 minutes at 4°C. To recover the concentrate, the filter was placed in reverse into a filtrate collection cup and centrifuged again at 1000 x g for 5 minutes at 4°C. Subsequently, the concentrated medium was transferred to a TLA120.1 rotor ultracentrifuge tube and centrifuged at 100,000 x g for 70 minutes at 4°C using an Optima MAX-TL ultracentrifuge (Beckman Coulter Inc., Carlsbad, CA, USA). The supernatant was discarded, the exosome pellet was washed with 1 mL of phosphate-buffered saline (PBS), and the ultracentrifugation process was repeated. Finally, the supernatant was removed, and the exosome pellet was stored at -80°C until further use. The pellet was resuspended in 100 µl of PBS for further analysis.

[0211]

[0212] 4.2. Fabrication of TTR-CPP Immobilized Exosomes and Drug Loading

[0213] To improve BBB penetration efficiency, Exo-TTR-CPP-TMZ was prepared by conjugating TTR-CPP using HEK293-derived exosomes. Specifically, DSPE-PEG (2000) Biotin (Merck, USA) was immobilized on the surface of exosomes, and the recombinant TTR-CPP protein prepared in Example 2 was conjugated with streptavidin protein using a dedicated kit (Streptavidin conjugation kit, Abcam, USA) according to the test method provided by the manufacturer. The exosomes immobilized with biotin were reacted with streptavidin-TTR-CPP to ensure that TTR-CPP was immobilized on the surface of the exosomes through biotin-streptavidin binding. To load temozolomide, exosomes and temozolomide were mixed in a 1:1 weight ratio. The process of treating with ultrasound (40 kHZ, 400 W) for 30 seconds followed by a 10-second rest was repeated 6 times, after which the samples were left on ice for 2 minutes and then left at 37°C for an additional 1 hour. Any drug not loaded onto the exosomes was removed using an ultrafine filter paper (3 kDa). To calculate the amount of loaded temozolomide, a 330 nm absorbance standard curve was constructed, and the concentration was determined.

[0214]

[0215] 4.3. Animal Studies on the Drug Efficacy of Exo-TTR-CPP_TMZ

[0216] Twelve BALB / c-nude mice (4 weeks old) were assigned to each test group, and the following substances were administered to each test group.

[0217] - G1: Normal control group (Normal)

[0218] - G2: Negative control group (Control)

[0219] - G3: TMZ

[0220] - G4: Exo-TTR-TMZ

[0221] - G5: Exo-TTR-CPP-TMZ

[0222] 1×10⁶ U87 cells using a stereotactic system 5 The number of cells was determined and injected into each mouse, and starting 3 days after cell injection, 100 μL of PBS, TMZ, TTR-TMZ, and TTR-CPP-TMZ were injected into the mouse brain via the tail vein at weekly intervals for 4 weeks (final concentration 10 mg / kg).

[0223] While checking the mouse lethality rate daily, a Kaplan-Meier graph was constructed using the lethality rates of each test group after rearing for 120 days (Fig. 7).

[0224] The 120-day survival rates of the negative control, TMZ, TTR-TMZ, and TTR-CPP-TMZ groups were 17%, 25%, 25%, and 30%, respectively, showing the highest survival rate when using TTR / CPP as a carrier.

[0225] In terms of 50% median survival time, TMZ, Exo-TTR-TMZ, and Exo-TTR-CPP-TMZ increased by 2%, 3%, and 8%, respectively, compared to the negative control group, indicating that when TMZ is loaded onto exosomes conjugated with TTR-CPP, the efficiency increases slightly compared to the negative control group.

[0226]

[0227] Example 5. In silico docking analysis

[0228] The binding potential of TTR and TTR-CPP with LRP1 (Low-density lipoprotein receptor-related protein 1), known as the blood-brain barrier (BBB) ​​receptor, was evaluated through in silico docking simulations.

[0229] Specifically, docking simulations were performed using the HDOCK web-based platform. This platform can predict the binding of protein-protein and protein-peptide complexes and evaluates binding stability through docking scores and confidence scores. The subjects of analysis were the TTR protein, TTR-CPP (HKRRRR (Sequence No. 1)), and modified CPPs with extended arginine repeat lengths (TTR-hk9r, hk11r, ​​hk16r, hk20r). Since the binding sites within LRP1 are expected to be complement-type repeat (CR) domain clusters, binding affinity for these sites was predicted.

[0230] The result is as follows.

[0231] TTR alone: ​​docking score -258.84, confidence score 0.8981

[0232] TTR-HKRRRR: docking score -271.14, confidence score 0.9185

[0233] Since a lower docking score is interpreted as indicating higher complex stability, it was confirmed that TTR-CPP with attached CPP can bind to LRP1 more stably than TTR alone.

[0234]

[0235] In addition, as shown in Table 2 below, in the analysis of binding strength according to Arginine repeat length, the Arg 3~6 (R3-R6) range was found to be the most suitable for interaction with the LRP1 CR domain pocket.

[0236] As the number of Arg repeats increases (as the number of + charges increases), the interaction strength with - charges increases; however, due to the spatial constraint of the acidic pocket of the CDR, it was predicted that excessively long Arg repeats (e.g., R16, R20) would cause steric hindrance within the pocket, distorting the binding directionality and increasing the possibility of non-specific binding.

[0237] Rank1212Docking inputDocking ScoreConfidence Scorecontrol-258.84-243.90.89810.8674LRP1_M +TTR+4r_HKRRRR-271.14-262.50.91850.9047LRP1_M +TTR+9R-275.69-266.90.92510.912LRP1_M +TTR+11R-276.41-273.060.92610.9214LRP1_M +TTR+KHRRRR-272.26-267.280.92020.9126LRP1_M +TTR+RRHKRR-276.43-252.460.92610.8859LRP1_M +TTR+RRRKHRR-276.52-262.860.92620.9053LRP1_M +TTR+RRRRHK-257.87-253.490.89640.8879LRP1_M +TTR+RRRRKH-265.7-260.350.910.9009LRP1_M +HKRRRR-252.09-235.120.88510.8458LRP1_M +KHRRRR-267.02-248.290.91220.8772LRP1_M +RRHKRR-245.86-243.550.87180.8666LRP1_M +RRKHRR-247.7-226.890.87590.8231LRP1_M +RRRRHK-249.91-239.940.88060.858

[0238] In the table above, H represents histidine, K represents lysine, and R represents arginine.

[0239]

[0240] SEQ ID NOs 1 to 48 are combinations of histidine, lysine, and arginine with a balanced positive charge distribution, allowing them to be stably inserted into the acidic pocket of LRP1. That is, if the Arg length is too short, electrostatic attraction is insufficient, and if it is too long, binding becomes unstable due to steric constraints, whereas SEQ ID NOs 1 to 6 are predicted to appear as structures with optimized charge-spatial balance.

[0241] Even when CPP modified sequences (HKHKHK, HKRRHK, etc.) were applied, the docking score tended to decrease as the number of Arg repeats increased, but stability deteriorated beyond a certain length.

[0242] In the case of single cationic peptides without TTR (e.g., HKHKHK alone), the docking score was high, confirming the lack of a stabilization effect by the TTR domain. This suggests that the stabilizing scaffold effect of the TTR domain is essential for maintaining the bond.

[0243] In addition, the TTR-CPP (SEQ Nos. 1 to 48) complex was found to bind to an LRP1 binding site different from that of TTR alone (Fig. 8), suggesting that the binding site and binding pattern are structurally readjusted by the attachment of CPP.

[0244] In the case of TMZ drug conjugates (TTR-HKRRRR-TMZ, TTR-HK20R-TMZ), the docking score generally tended to increase (weakening of binding) upon TMZ binding. This suggests that the increase in spatial volume and changes in charge distribution due to TMZ conjugation may partially interfere with the interaction with LRP1.

[0245]

[0246] From the above results, it was confirmed at the molecular level that TTR-CPP has the potential to act as a drug delivery vehicle capable of crossing the BBB through strong electrical interactions with the acidic pocket of the LRP1 CR domain. In particular, it was found that the structure most stably fits the acidic pocket structure of LRP1 when the Arg repeat length is approximately 3 to 6, and it was confirmed that SEQ ID NOs 1 to 6 are the best combinations in terms of electrical binding strength and spatial complementarity.

[0247] Therefore, the TTR-CPP (sequence numbers 1 to 6) fusion proposed in the present invention is considered to be an optimal CPP combination capable of selectively passing through the BBB to deliver drugs or diagnostic substances.

[0248]

[0249] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. Transthyretin or a functional variant thereof; and a drug delivery system comprising a peptide having an amino acid sequence represented by the following general formula 1: [General Formula 1] X1X2X3X4X5X6R n Here, X1 to X6 are histidine, lysine, or arginine, and R is arginine, and n is 0 to 7.

2. A drug delivery system according to claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 48.

3. A drug delivery system according to claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 6.

4. A drug delivery system according to claim 1, wherein the peptide comprises a peptide having the amino acid sequence of SEQ ID NO.

1.

5. A drug delivery system according to any one of claims 1 to 4, wherein the drug delivery system passes through the blood-brain barrier (BBB) ​​by binding to LRP1 (Low-density lipoprotein receptor-related protein 1).

6. In Paragraph 1, A drug delivery system in which the above transtyretin and the peptide comprising the above amino acid sequence are connected by a covalent bond or a peptide bond.

7. A pharmaceutical composition for the prevention or treatment of brain disease comprising the drug delivery system and the drug of claim 1.

8. In claim 7, the above drug is a physiologically active substance having a therapeutic effect on brain diseases, a pharmaceutical composition for the prevention or treatment of brain diseases.

9. A pharmaceutical composition for the prevention or treatment of brain disease according to claim 7, wherein the drug is one or more selected from the group consisting of temozolomide, donepezil, rivastigmine, galantamine, paclitaxel, doxorubicin, and osimertinib.

10. A pharmaceutical composition for the prevention or treatment of brain disease according to claim 7, wherein the brain disease is one or more selected from the group consisting of brain tumor, Alzheimer's disease, stroke, paralysis, dementia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, thrombosis, embolism, transient ischemic attack, lacune, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disorder, cerebral functional coma, and brain cancer.

11. In Paragraph 7, A pharmaceutical composition for the prevention or treatment of brain diseases, wherein the above drug is chemically bonded to the amino group (NH₂) or carboxyl group (COOH) of the above drug delivery system.

12. In any one of paragraphs 7 through 11, The above drug delivery system is a pharmaceutical composition for the prevention or treatment of brain diseases, which is bound to the surface of an exosome.

13. A composition for diagnosing brain diseases comprising a substance for diagnosing brain diseases and a drug delivery system of claim 1.

14. A composition for diagnosing brain diseases according to claim 13, wherein the brain disease diagnosing material is one or more selected from the group consisting of antibodies, aptamers, DNA, RNA, proteins, and polypeptides.

15. In Paragraph 13, The above-mentioned composition for diagnosing brain diseases further comprises a labeling substance including a fluorescent, radioisotope, or magnetic resonance imaging (MRI) contrast agent.

16. A brain disease diagnostic kit comprising a composition for diagnosing a brain disease according to any one of claims 13 to 15, and further comprising a labeling reagent, a buffer, and a indicator.

17. A method for preparing a drug delivery system comprising: transtyretin or a functional variant thereof; and a peptide comprising an amino acid sequence represented by the following general formula 1, wherein A method comprising the step of introducing and expressing a recombinant expression vector, in which a transthyretin-coding nucleic acid sequence and a nucleic acid sequence coding for the said amino acid sequence are linked, into a host cell: [General Formula 1] X1X2X3X4X5X6R n Here, X1 to X6 are histidine, lysine, or arginine, and R is arginine, and n is 0 to 7.

18. A drug carrier-drug conjugate comprising a trans-styretin or a functional variant thereof; and a peptide comprising an amino acid sequence represented by the following general formula 1, wherein temozolomide or a derivative thereof is chemically bound to a drug carrier: [General Formula 1] X1X2X3X4X5X6R n Here, X1 to X6 are histidine, lysine, or arginine, and R is arginine, and n is 0 to 7.

19. Transtyretin or a functional variant thereof; and a drug delivery system comprising an amino acid sequence represented by the following general formula 1, and a method for treating brain disease by administering the drug into the body of a mammal to prevent or treat brain disease: [General Formula 1] X1X2X3X4X5X6R n Here, X1 to X6 are histidine, lysine, or arginine, and R is arginine, and n is 0 to 7.

20. Transtyretin or a functional variant thereof; and a drug delivery system comprising an amino acid sequence represented by the following general formula 1 for use in crossing the blood-brain barrier (BBB): [General Formula 1] X1X2X3X4X5X6R n Here, X1 to X6 are histidine, lysine, or arginine, and R is arginine, and n is 0 to 7.

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