Drug carrier for penetrating Blood-brain barrier using sialyllactose

Abstract

A blood-brain barrier permeable drug delivery system is provided by conjugating sialyllactose, which is capable of permeating the blood-brain barrier, to a drug. Since the drug delivery system can deliver not only small molecule drugs with small molecular weights but also large molecular weight antibodies to the brain, it is applicable to various types of treatments or diagnostic materials for brain diseases.

Description

Technology Field

[0001] The present invention relates to a drug delivery system that penetrates the blood-brain barrier using sialyllactose. Background Technology

[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 vascular endothelial cells in contact with associated pericytes and astrocytes, and is a highly selective 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 macromolecules and are significantly larger than small molecules such as small molecule compounds and peptides, attaching a single drug to a single antibody actually reduces drug delivery efficiency. Therefore, 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] Accordingly, the inventors confirmed through experiments that a fluorescent substance with a small molecular weight sialyllactose attached can pass through the blood-brain barrier, and further confirmed that if sialyllactose is attached to different substances, it can pass through the blood-brain barrier, thereby completing the present invention. The problem to be solved

[0008] The problem that the present invention aims to solve is to provide a drug delivery system capable of passing through the blood-brain barrier using sialyllactose with a low molecular weight. means of solving the problem

[0010] In order to achieve the above technical problem, one embodiment of the present invention provides a blood-brain barrier permeable drug delivery system characterized by being represented by the following structural formula 1, according to one aspect of the present invention.

[0011] [Structural Formula 1]

[0012] SL-LX

[0013] Here,

[0014] SL is sialylcactose or its salt, and

[0015] X is the target physiologically active substance for delivery, and

[0016] L may be a bond or chemical linker connecting the above SL and X.

[0017] In one embodiment, the sialyllactose may be 2,3-sialyllactose or 2,6-sialyllactose.

[0018] In one embodiment, the target physiologically active substance for delivery may be selected from the group consisting of a physiologically active substance having a therapeutic effect on brain diseases or a small molecule drug for diagnosis, peptide, antibody, protein, natural or modified ssDNA, dsDNA, RNA, siRNA, and ASO.

[0019] In one embodiment, the chemical linker may be used without limitation as long as it has a structure that is structurally connected to both SL and X. For example, the chemical linker may be selected from the group consisting of SL and -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH3)- and may be selected from the group consisting of X and -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH3)-. The two connections can be selected from the group having structures -(CH2)a-(NHCO)b-(CH2)c-, -(CH2)a-(CONH)b-(CH2)c-, -(CH2)a-(CO)b-(CH2)c-, -(CH2)a-(NH)b-(CH2)c-, and -(CH2)a-(O)b-(CH2)c-. In this case, a may be an integer from 0 to 10, b may be an integer from 0 to 1, and c may be an integer from 0 to 10.

[0020] In one embodiment, the chemical linker may be a cleavable linker that promotes release after delivering a target physiologically active substance to the brain.

[0021] In one embodiment, the chemical linker can be bonded to the hydroxyl group (-OH) of the lactose portion of sialyllactose.

[0022] In one embodiment of the present invention, according to another aspect of the present invention, a pharmaceutical composition for treating or diagnosing brain diseases comprising the aforementioned blood-brain barrier permeable drug delivery system is provided. Effects of the invention

[0024] A drug delivery system according to one embodiment of the present invention can be applied to drugs with low brain penetration efficiency to effectively deliver the drug to the brain, and thus can be used for the treatment and diagnosis of various brain diseases without toxicity issues caused by high-dose drug administration.

[0025] 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 of the invention or the claims. Brief explanation of the drawing

[0027] Figure 1 shows the experimental results comparing the brain penetration of Cy5.5 combined with lactose and sialyllactose. Figure 2 shows the result of quantifying the fluorescence signal intensity of the brain part of Figure 1 and the result of measuring the fluorescence signal intensity after extracting the brain in the experiment of Figure 1. Figure 3 shows the experimental results comparing the brain penetration of Cy5.5 combined with sialic acid and sialyllactose. FIG. 4 is a structural formula of dopamine combined with sialic lactose according to an embodiment of the present invention. Figure 5 shows the experimental results confirming the brain penetration of dopamine combined with 2,6-siallyllactose in animals. Figure 6 shows the results of measuring the fluorescence signal of the brain part in the experiment of Figure 5 and the results of measuring the fluorescence signal of the extracted brain. Figure 7 shows the results of measuring fluorescence signals in cross-sections of brains extracted from animals administered dopamine conjugated with 2,6-siallyllactose. Figure 8 shows the experimental results confirming the brain penetration of dopamine combined with 2,3-siallyllactose in animals. Figure 9 shows the results of measuring the fluorescence signal of the brain part in the experiment of Figure 8 and confirming the fluorescence signal of the extracted brain. FIG. 10 is the structural formula of methotrexate combined with sialyllactose according to one embodiment of the present invention. Figure 11 shows the experimental results confirming the brain penetration of sialyllactose-conjugated methotrexate in animals. Figure 12 shows the results of measuring the fluorescence signal of the brain part in Figure 11 and the results of measuring the fluorescence signal of the extracted brain. Figure 13 shows the results of confirming the anticancer effect of sialyllactose-conjugated methotrexate in an animal model of brain cancer. FIG. 14 is the structural formula of gemcitabine combined with sialyllactose according to one embodiment of the present invention. Figure 15 is the experimental result confirming the brain penetration of gemcitabine combined with sialyllactose according to one embodiment of the present invention in animals. Figure 16 is the experimental result confirming the anticancer activity of gemcitabine conjugated with sialyllactose according to one embodiment of the present invention in a cell line. Figure 17 is the experimental result confirming the brain penetration of an antibody bound to sialylcose according to one embodiment of the invention in animals. Figure 18 is the result of measuring the fluorescence signal of the brain part in Figure 17. Specific details for implementing the invention

[0028] The present invention will be described in detail below.

[0029] The following description should be understood as an example to aid in understanding the present invention, and the spirit or scope of the present invention is not limited by the following description.

[0031] According to one aspect of the present invention, a blood-brain barrier permeable drug delivery system can be provided, characterized by being represented by the following structural formula 1.

[0032] [Structural Formula 1]

[0033] SL-LX

[0034] Here,

[0035] SL is sialyllactose or its salt, X is a drug to be delivered, and L may be a bond or chemical linker connecting SL and X.

[0036] The above sialyllactose is a type of human milk oligosaccharide (HMO) most abundant in colostrum, and is a form in which sialic acid is attached to lactose. Depending on the position where sialic acid binds to lactose, two types may exist: 2,3-sialyllactose represented by structural formula 2 below and 2,6-sialyllactose represented by structural formula 3 below.

[0037] [Structural Formula 2]

[0038]

[0039] [Structural Formula 3]

[0040]

[0041] The above sialyllactose can be extracted from breast milk, chemically synthesized, or synthesized using an enzymatic reaction. Preferably, it can be synthesized using an enzymatic reaction that is environmentally friendly and safe, as it enables mass production and does not use toxic catalysts or organic solvents.

[0042] The salt of the above sialyllactose may be Na, but is not limited thereto.

[0043] The above-mentioned drug to be delivered may be a physiologically active substance having a therapeutic effect on brain diseases or a substance for diagnosis, and may be selected from the group consisting of small molecule drugs, peptides, antibodies, proteins, natural or modified ssDNA, dsDNA, RNA, siRNA, and ASO.

[0044] For example, the above small molecule drugs are anticancer agents such as Cisplatin, Carboplatin, Melphalan, Chlorambucil, Dacarbazine, Capecitabine, Cytarabine, Vinblastine, Vincristine, Paclitaxel, Docetaxel, Etoposide, Topotecan, Irinotecan, Dactinomycin, Doxorubicin, Daunorubicin, Mitomycin, Bleomycin, Temsirolimus, and Everolimus, It may be lenalidomide or a pharmaceutically acceptable salt thereof, memantine, donepezil, galantamine or a pharmaceutically acceptable salt thereof which are treatments for Alzheimer's disease, pramipexole, ropinirol, amantadine or a pharmaceutically acceptable salt thereof which are treatments for Parkinson's disease, and dopamine, acetylcholine, citicoline, choline alfoscerate (Alpha-GPC) or a pharmaceutically acceptable salt thereof which are neurotransmitters and brain function enhancers.

[0045] The above antibodies may be, for example, anticancer drugs such as Trastuzumab, Cetuximab, Bevacizumab, Pertuzumab, Pembrolizumab, Nivolumab, Ipilimumab, Atezolizumab, and Durvalumab, and may be Alzheimer's disease treatment drug Aducanumab.

[0046] The above protein or peptide may be, for example, leuprolide or octreotide, nerve growth factor (NGF; brain-derived neurotrophic factor, BDNF; neurotrophin-3, NT-3), and transforming growth factor-β (TGF-β).

[0047] The above-mentioned brain disease may be a disease or condition that originates in or has a pathogenesis in the central nervous system. For example, it may be a brain tumor, cerebellar atrophy, amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's disease, dementia, and Parkinson's disease, but is not limited thereto.

[0048] The above SL and X can be directly bonded to each other. For example, the functional group of SL and the functional group of X can be directly bonded, and if the direct bonding does not inhibit the blood-brain barrier permeability of SL and the physiological activity of X, or if the bond breaks down after penetrating the blood-brain barrier so that SL and X are separated, they can be directly bonded without a linker. Preferably, the lactose portion hydroxyl group (-OH) of SL and a functional group that does not affect the physiological activity of X can be bonded.

[0049] In addition, the above SL and X may be connected by a chemical linker. For example, the chemical linker may bind to the functional group of SL and the functional group of X, respectively, without inhibiting the blood-brain barrier permeability of SL and the physiological activity of X. Preferably, the chemical linker may bind to the lactose portion hydroxyl group (-OH) of SL and to the functional group that does not affect the physiological activity of X, respectively.

[0050] The chemical linker above may be used to have a structure that is structurally connected to both SL and X. The chemical linker above may be selected from the group consisting of SL and -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH3)- and may be selected from the group consisting of X and -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH3)-. The link between the two links may be selected from the group having structures of -(CH2)a-(NHCO)b-(CH2)c-, -(CH2)a-(CONH)b-(CH2)c-, -(CH2)a-(CO)b-(CH2)c-, -(CH2)a-(NH)b-(CH2)c-, and -(CH2)a-(O)b-(CH2)c-. At this time, a may be an integer from 0 to 10, b may be an integer from 0 to 1, and c may be an integer from 0 to 10.

[0051] The chemical linker may be a cleavable linker that promotes release after delivering the target drug to the brain. For example, pH-sensitive linkers, peptidase-sensitive linkers, and photo-unstable linkers may be used. Preferably, a peptidase-sensitive linker that is easily cleavable by peptidases present in the cell may be used. For example, it may be selected from those that are easily cleavable by peptidases such as cathepsin B, C, and D, which are peptidases preferentially expressed in tumor tissue.

[0053] The above blood-brain barrier permeable drug delivery system may be included in a pharmaceutical composition for the treatment or diagnosis of brain diseases, and the pharmaceutical composition may be provided as a pharmaceutical composition comprising the blood-brain barrier permeable drug delivery system alone or comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0054] The term “pharmaceuticalally acceptable” above means exhibiting properties that are not toxic to cells or humans exposed to the composition.

[0055] Furthermore, the above pharmaceutical composition may be provided by mixing it with a conventionally known agent for treating or diagnosing brain diseases. That is, the above pharmaceutical composition may be administered in combination with a known compound having a therapeutic or diagnostic effect for brain diseases.

[0056] The term “administration” above refers to the introduction of a specific substance into an individual by an appropriate method, and the term “individual” refers to all target animals, including humans, rats, mice, and livestock, intended for the treatment or diagnosis of brain diseases. Specific examples may include mammals, including humans.

[0057] The routes of administration of the above pharmaceutical composition are not limited to these but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal.

[0058] The above pharmaceutical composition may be administered orally or parenterally. When administering parenterally, it is preferable to select a method of external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but is not limited thereto. Oral administration may be preferred from the perspective of selecting a more effective route of absorption.

[0059] When formulating the above pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, humectants, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin, with the above extract. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0061] The present invention will be explained in more detail below through examples and test examples. However, the following examples and test examples are intended to illustrate the present invention and do not limit the scope of the present invention.

[0063] <Example>

[0064] 1. Experiment on the penetration of sialyllactose into the blood-brain barrier

[0065] To confirm the potential of sialyllactose as a drug delivery system that penetrates the blood-brain barrier, brain penetration experiments were conducted with sialic acid, lactose, and sialyllactose.

[0066] First, the fluorescent substance Cy5.5 (Genechem, Cat no. GCCD0064) was attached to lactose, 2,3-siallyllactose (GCB100), and 2,6-siallyllactose (GCB200), and samples were prepared by dissolving each substance in 10% DMSO and then diluting them (the final DMSO concentration was approximately 2% of the total volume). Each sample was administered intravenously to BALB / c nude mice at a concentration of 1 μmol / kg, after which the fluorescence signals in the brains of the nude mice were observed at time intervals (1, 2, 4, 6, 8, and 24 hours) using Perkin Elmer IVIS spectrum CT. After 24 hours of administration, the nude mice were perfused with physiological saline, the brains were extracted, and the fluorescence present in the brains was measured (Figs. 1 and 2). As a result, Cy5.5 (control) was found to barely penetrate the blood-brain barrier, and all Cy5.5s with different sialyllactose attached compared to lactose were found to be well delivered to the brain.

[0067] Next, the fluorescent substance Cy5.5 (Genechem, Cat no. GCCD0064) was attached to sialic acid, 2,3-siallyllactose (GCB100), and 2,6-siallyllactose (GCB200), and samples were prepared by dissolving each substance in 10% DMSO and then diluting them (the final DMSO concentration was approximately 2% of the total volume). Each sample was administered intravenously to BALB / c nude mice at a concentration of 1 μmol / kg, and the fluorescence signals in the brains of the nude mice were measured at time intervals (10 min, 30 min, 1, 2, 4, 6, 8, and 24 hours) using a Perkin Elmer IVIS spectrum CT. After 24 hours of administration, the nude mice were perfused with physiological saline, the brains were extracted, and the fluorescence present in the brains was measured. As a result, as shown in Figure 3, Cy5.5 (control) was found to barely penetrate the blood-brain barrier, and while brain penetration was similar even with sialic acid attached, Cy5.5 with sialyllactose attached was well delivered to the brain, and in particular, the effect of 2,6-sialyllactose was found to be higher than that of 2,3-sialyllactose.

[0068] In summary, attaching sialyllactose improves the passage of Cy5.5, which has low brain permeability, across the blood-brain barrier, and it was confirmed that Cy5.5 attached with sialyllactose has significantly higher brain permeability compared to sialic acid or lactose, which are parts of sialyllactose.

[0070] 2. Sialyllactose-bound dopamine in vivo experiment

[0071] Parkinson's disease is caused by the loss of dopamine neurons. Currently, L-dopa, a dopamine precursor capable of penetrating the brain, is used as a treatment instead of dopamine, which has low brain permeability; however, this leads to side effects such as levodopa-induced dyskinesia. Therefore, we conducted an experiment to determine whether L-dopa could be replaced by combining dopamine with sialyllactose to increase brain permeability.

[0072] 2-1) Preparation of Sialyllactose-Conjugated Dopamine

[0073] To prepare GCB007 (2,3-siallyllactose-dopamine conjugate) and GCB008 (2,6-siallyllactose-dopamine conjugate), 5 g of dopamine was dissolved in 50 ml of dimethylformamide and 11.6 ml of triethylamine. Then, 8.95 g of MMTr-Cl was slowly added while stirring at 0°C for 2 hours. Afterward, the mixture was concentrated and purified using silica gel to obtain 4.5 g of the substance. To 1.3 g of the purified substance, 0.06 g of 4-dimethylaminopyridine was dissolved in 100 ml of methylene chloride, 0.6 g of 4-nitrophenyl chloroformate was added, and the mixture was stirred for 4 hours. After the reaction was complete, the mixture was fractionated with 100 ml of 0.5 M aqueous citric acid solution to obtain the methylene chloride layer. This layer was treated with sodium sulfate, filtered through a glass filter, and concentrated to obtain 2 g of the substance. 16 ml of dimethylformamide and 2 ml of triethylamine were added to 2 g of the above substance and stirred. 0.54 g each of Aminohexyl-2,3-siallyllactose and 2,6-siallyllactose were added and stirred for 24 hours, after which the mixture was concentrated and purified with silica gel to obtain 0.3 g of substance. 3 ml of methanol was added to the above substance and stirred, followed by the addition of 6 ml of acetic acid and stirring for about 8 hours. After the reaction was complete, the mixture was concentrated and purified with silica gel to obtain GCB007 (2,3-siallyllactose-dopamine conjugate) and GCB008 (2,6-siallyllactose-dopamine conjugate).

[0074] To prepare GCB009 (2,3-siallyllactose-dopamine conjugate) and GCB010 (2,6-siallyllactose-dopamine conjugate), 24 ml of dimethylformamide and 1 ml of pyridine were added to 1.2 g each of carboxyl-2,3-siallyllactose and carboxyl-2,6-siallyllactose and stirred, after which 0.61 g of N-hydroxysuccinimide was added and stirred at 60°C for 90 minutes to concentrate, yielding 1 g of the substance. Then, 0.67 g of the substance obtained through the above process was mixed with 3.2 ml of dimethylformamide and 1.1 ml of triethylamine, stirred, and then 0.15 g of dopamine was added and stirred for 24 hours, concentrated, and purified with silica gel to prepare 0.06 g each of GCB009 (2,3-siallyllactose-dopamine conjugate) and GCB010 (2,6-siallyllactose-dopamine conjugate).

[0075] The structures of GCB007 (2,3-siallyllactose-dopamine conjugate), GCB008 (2,6-siallyllactose-dopamine conjugate), GCB009 (2,3-siallyllactose-dopamine conjugate) and GCB010 (2,6-siallyllactose-dopamine conjugate) are shown in Fig. 4.

[0076] 2-2) Experiment on Blood-Brain Barrier Permeation of Sialyllactose-Conjugated Dopamine

[0077] The fluorescent substance Cy5.5 was attached to dopamine, GCB007, and GCB008, and a blood-brain barrier penetration experiment was conducted. The experiment was conducted in the same manner as the sialyllactose blood-brain barrier penetration experiment described above.

[0078] First, the in vivo fluorescence distribution and fluorescence signals in the brain of dopamine with Cy5.5 attached and GCB008 were examined (Fig. 5), and the fluorescence distribution and fluorescence signals in the brain were measured after extracting the brain at different times (Fig. 6). As a result, it was confirmed that dopamine with sialyllactose attached had a higher penetration rate into the brain compared to dopamine without sialyllactose attached.

[0079] Additionally, to confirm whether the fluorescent substance is distributed not only throughout the entire area but also in the brain cross-section, GCB008 with Cy5.5 attached was administered using PerCP / Cy5.5 (Abcam, Cat no. ab102911) as the control substance, and after 24 hours, the brain was extracted and the cross-section was cut to check the fluorescence distribution. As a result, as shown in Figure 7, it was confirmed that dopamine bound to sialyllactose was detected in the brain cross-section as well.

[0080] Next, to confirm that the brain penetration phenomenon was not a transmission effect caused by the fluorescent substance Cy5.5, a blood-brain barrier penetration experiment was conducted on GCB007 attached with Cy5.5, using Cy5.5 as a control.

[0081] The in vivo fluorescence distribution and fluorescence signal of Cy5.5 and GCB007 attached to Cy5.5 were examined over time (Fig. 8), and the fluorescence distribution and fluorescence signal of the brain were examined after extracting the brain 24 hours later (Fig. 9). As a result, Cy5.5 was hardly detected in the brain, whereas GCB007-Cy5.5 was clearly detected, confirming that the brain penetration of dopamine bound to sialyllactose was induced by sialyllactose rather than Cy5.5.

[0083] 3. Sialyllactose-conjugated methotrexate in vivo experiment

[0084] 3-1) Preparation of methotrexate combined with sialyllactose

[0085] 10 ml of pyridine and 0.337 ml of triethylamine were added to 1 g of methotrexite and stirred, then 0.027 g of 4-dimethylaminopyridine and 0.887 g of 4-nitrophenyl chloroformate were slowly added and stirred for 4 hours. After the reaction, the mixture was concentrated to obtain 1 g of the substance. To the 1 g of the above substance, 15 ml of dimethylformamide and 2.42 ml of triethylamine were added and stirred, then 0.63 g each of Aminohexyl-2,3-sialyllactose and Aminohexyl-2,6-sialyllactose were added and the mixture was stirred for 12 hours to allow the reaction to proceed. After the reaction, the mixture was concentrated and purified with silica gel to obtain GCB016 (2,3-sialyllactose-methotrexate conjugate) and GCB017 (2,6-sialyllactose-methotrexate conjugate). The structures of GCB016 (2,3-siallyllactose-methotrexate conjugate) and GCB017 (2,6-siallyllactose-methotrexate conjugate) are shown in Fig. 10.

[0086] 3-2) Blood-brain barrier penetration test of sialyllactose-conjugated methotrexate

[0087] Cy5.5 was attached to methotrexate and used as a control substance, and Cy5.5 was attached to GCB017 to conduct a blood-brain barrier penetration experiment.

[0088] The time-dependent in vivo fluorescence distribution and fluorescence signals in the brain of methotrexate and GCB017 conjugated with Cy5.5 were examined (Figs. 11 and 12A), and the fluorescence distribution images and fluorescence signals of the brain extracted 24 hours after administration were examined (Fig. 12B). As a result, when methotrexate was administered, almost no fluorescence signal appeared in the brain, whereas when methotrexate conjugated with sialyllactose was administered, a strong fluorescence signal appeared, confirming that sialyllactose can induce delivery to the brain when bound to methotrexate.

[0089] 3-3) Experiment to Confirm Brain Tissue Penetration and Material Dissociation of Sialyllactose-Conjugated Methotrexate

[0090] To determine the tissue distribution and dissociation of sialyllactose-conjugated methotrexate, GCB017 was administered, and the presence of GCB017 and its dissociated products (linker-6'SL (L-6'SL) and linker) was checked in the brain and pancreas at different time points. Specifically, GCB017 was dissolved in 10% DMSO and then diluted to prepare samples (the final DMSO concentration was approximately 2% of the total volume). The samples were administered intravenously (IV) at a concentration of 80 mg / kg or orally (PO) at a concentration of 80 mg / kg. Subsequently, at different time points (0, 0.5, 2, and 4 hours), anesthesia and cardiac blood collection were performed, followed by perfusion with physiological saline and extraction of the brain and pancreas. Each excised tissue was homogenized in 50 mM Tris-HCl buffer (pH 7.5) at a 1:5 ratio and centrifuged at 3,000 rpm at 4 ℃ for 20 minutes to obtain the supernatant, which was then mass spectrometerized (SCIEX Triple Quad 3500 LC-MS / MS system).

[0091] (ng / ml) brain GCB017 L-6'SL linker Vehicle G1 101 N / D N / D N / D 102 N / D N / D N / D GCB017 (80 mg / kg, IV) 0.5h G2 201 557.75 N / D N / D 202 246.04 N / D N / D 203 82.46 N / D N / D 2h G3 301 52.73 N / D N / D 302 66.80 N / D N / D 303 41.10 N / D N / D 4h G4 401 39.94 N / D N / D 402 57.44 N / D N / D 403 161.80 N / D N / D GCB017 (80 mg / kg, PO) 0.5h G5 501 34.78 N / D N / D 502 169.99 N / D N / D 2h G6 601 N / D N / D N / D 602 N / D N / D N / D 4h G7 701 30.34 N / D N / D 702 N / D N / D N / D

[0092] As shown in Table 1, the substance and its dissociates were detected in brain tissue not only when GCB017 was administered intravenously but also when administered orally, which means that methotrexate, which is rarely delivered to the brain, can be delivered across the blood-brain barrier as a conjugate with sialyllactose and subsequently dissociated in the brain.

[0093] 3-4) Evaluation of the efficacy of sialyllactose-conjugated methotrexate in a brain cancer animal model

[0094] To confirm whether GCB017 is delivered to the brain and exerts the anticancer effect of methotrexate, an anticancer efficacy evaluation was performed using a brain cancer model constructed in mice. The brain cancer model was constructed by transplanting U-87MG, a brain cancer cell line expressing luciferase. For brain transplantation, each individual was induced with injectable anesthesia using isoflurane and secured to a stereotaxic arm. After making a longitudinal incision of approximately 1 cm in the scalp and exposing the injection site with a microdrill, the cell line was transferred into a 25 μl Hamilton syringe fitted with a 31-gauge needle. The cell line was then transplanted at a rate of 1 μl / min to a site 1 mm anterior, 2 mm lateral, and 3 mm deep relative to the bregma. After transplantation, the cells were kept in place for about 10 minutes to prevent backflow and ensure proper absorption before the syringe was removed. The incision was sutured and disinfected, and normal rearing was performed after confirming that the anesthesia had worn off. Methotrexate and GCB017 were administered intravenously twice a week for four weeks, for a total of eight times, starting one week after the model was created, and the size of the brain tumors was confirmed through optical imaging (Fig. 13). In the case of the negative control group (Vehicle), tumor growth was prominently observed on day 20, and an increasing pattern was confirmed as time progressed to days 24 and 27. Similarly, in the positive control group administered methotrexate, it was confirmed that the tumor size gradually increased starting from day 20. On the other hand, compared to the administration of the negative and positive control groups, it was confirmed that in the experimental group administered 22 μmol / kg of GCB017, tumor formation was delayed and the tumor size was controlled. Based on the results of the preceding Examples 3-2 and 3-3, it is determined that the methotrexate conjugate linked to sialyllactose according to the present invention has improved BBB permeability than methotrexate, thereby inhibiting the formation and growth of tumors.

[0095] 4. Blood-brain barrier penetration test of sialyllactose-conjugated gemcitabine

[0096] 4-1) Preparation of gemcitabine combined with sialyllactose

[0097] - Preparation of 2,3-Sialyllactose-Gemcitabine Conjugate: 25 ml of dimethylformamide and 0.89 ml of triethylamine were added to 1.4 g of gemcitabine and stirred, then 0.94 g of imidazole and 1.44 g of TBDMS-Cl were added and stirred for 4 hours. After concentrating and purifying with silica gel, 1.9 g of the substance was obtained. 12.5 ml of pyridine and 0.6 g of 4-dimethylaminopyridine were added to this substance and stirred, followed by the slow addition of 1.39 ml of ditert-butyl dicarbonate. After stirring and concentrating, the substance was fractionated with a 0.5 M aqueous citric acid solution and methylene chloride. After treatment with sodium sulfate and filtration with a glass filter, the resulting 1.2 g of substance was concentrated and purified, and then dissolved in 8 ml of pyridine. 0.03 g of 4-dimethylaminopyridine and 0.327 g of succinic anhydride were added to this, stirred, and then concentrated to fractionate into a 0.5 M aqueous citric acid solution and methylene chloride. 0.6 g of the substance obtained by treating with sodium sulfate, filtering with a glass filter, concentrating, and purifying, and 0.013 g of 4-dimethylaminopyridine were dissolved in 6 ml of pyridine and 0.42 ml of triethylamine. 0.248 g of 4-nitrophenyl chloroformate was added to this and stirred for 4 hours; after the reaction was complete, it was fractionated into a 0.5 M aqueous citric acid solution and 100 ml of methylene chloride. From this, a methylene chloride layer was collected, treated with sodium sulfate, filtered with a glass filter, and concentrated to obtain 0.6 g of material. To this, 6 ml of dimethylformamide and 0.62 ml of triethylamine were added and stirred, then 0.5 g of Aminohexyl-3'SL was added and stirred. To the 0.25 g of material obtained by concentrating and purifying with silica gel, 5 ml of distilled water and 0.025 ml of acetic acid were added and stirred at 60°C for 24 hours, then concentrated and purified with silica gel to obtain Gemcitabine-succinate-aminohexyl-3'SL (GCB033).

[0098] - Preparation of 2,6-Sialyllactose-Gemcitabine Conjugate: 2.28 g of gemcitabine was mixed with 41 ml of dimethylformamide and 1.45 ml of triethylamine, and after stirring, 1.53 g of imidazole and 2.35 g of TBDMS-Cl were added. The mixture was stirred for 4 hours, concentrated, and purified with silica gel to obtain 1.9 g of the substance. To 3.75 g of the substance obtained by this method, 50 ml of pyridine and 1.2 g of 4-dimethylaminopyridine were added, and after stirring, 2.7 ml of ditert-butyl dicarbonate was slowly added. After stirring and concentration, the mixture was fractionated with a 0.5 M aqueous citric acid solution and methylene chloride. 3.2 g of the substance obtained by treating with sodium sulfate, filtering with a glass filter, concentrating, and purifying was dissolved in 21 ml of pyridine, 0.082 g of 4-dimethylaminopyridine and 0.87 g of succinic anhydride were added, and after stirring and concentration, the mixture was fractionated into a 0.5 M aqueous citric acid solution and methylene chloride. 2.9 g of the substance obtained by treating with sodium sulfate, filtering with a glass filter, concentrating, and purifying was dissolved along with 0.061 g of 4-dimethylaminopyridine in 29 ml of pyridine and 2.5 ml of triethylamine, then 1.21 g of 4-nitrophenyl chloroformate was added and stirred for 4 hours. After the reaction was complete, the mixture was fractionated into a 0.5 M aqueous citric acid solution and 100 ml of methylene chloride. To the 2.9 g of material obtained by collecting the methylene chloride layer, treating it with sodium sulfate, filtering it through a glass filter, and concentrating it, 29 ml of dimethylformamide and 3 ml of triethylamine were added and stirred. Then, 2.4 g of Aminohexyl-6'SL was added and stirred, and to the 1.2 g of material obtained by concentrating and purifying it with silica gel, 24 ml of distilled water and 0.12 ml of acetic acid were added and stirred at 60 degrees for 24 hours. After concentrating and purifying it with silica gel, Gemcitabine-succinate-aminohexyl-6'SL (GCB034) was obtained.

[0099] The structures of GCB033 (conjugate of 2,3-siallyllactose-gemcitabine) and GCB034 (conjugate of 2,6-siallyllactose-gemcitabine) prepared as above are as shown in Fig. 14.

[0100] 4-2) Blood-brain barrier penetration test of sialyllactose-conjugated gemcitabine

[0101] To confirm the blood-brain barrier penetration of gemcitabine conjugates, blood-brain barrier penetration experiments were performed by attaching Cy5.5 to GCB033 and GCB034, respectively. Cy5.5 alone and gemcitabine with only Cy5.5 attached were used as controls. First, samples were prepared by dissolving each substance in 10% DMSO and diluting it (the final DMSO concentration was approximately 2% of the total volume). Each sample was administered intravenously to BALB / c nude mice at a concentration of 1 μmol / kg. Fluorescence signals in the brains of the nude mice were measured at time intervals (10 min, 30 min, 1, 2, 4, 6, 8, and 24 hours) using a Perkin Elmer IVIS spectrum CT. After 24 hours, the nude mice were perfused with physiological saline, the brains were extracted, and the fluorescence present in the brains was measured.

[0102] As shown in Figure 15, the results showed a stronger fluorescence signal when Cy5.5 attached to gemcitabine combined with sialyllactose was administered compared to gemcitabine alone, confirming that the brain penetration rate of gemcitabine was enhanced by sialyllactose.

[0103] 4-3) Experiment to Confirm Brain Tissue Penetration and Material Dissociation of Sialyllactose-Conjugated Gemcitabine

[0104] To confirm the tissue distribution and dissociation of sialyllactose-conjugated gemcitabine, GCB034 was administered, and the presence of GCB034 and its dissociated product, linker-6'SL (L-6'SL), and linker was checked in the brain and pancreas at time intervals. Specifically, GCB034 was dissolved in 10% DMSO and diluted to prepare samples (the final DMSO concentration was approximately 2% of the total volume). The samples were administered intravenously (IV) at a concentration of 82 mg / kg or orally (PO) at a concentration of 246 mg / kg. Subsequently, at time intervals (0, 0.5, 2, and 4 hours), anesthesia and cardiac blood collection were performed, followed by perfusion with normal saline and extraction of the brain and pancreas. Each excised tissue was homogenized in 50 mM Tris-HCl buffer (pH 7.5) at a 1:5 ratio and centrifuged at 3,000 rpm at 4 ℃ for 20 minutes to obtain the supernatant, which was then mass spectrometerized (SCIEX Triple Quad 3500 LC-MS / MS system).

[0105] (ng / ml) brain GCB034 L-6'SL linker Vehicle G1 101 N / D N / D N / D 102 N / D N / D N / D 103 N / D N / D N / D GCB034 (82 mg / kg, IV) 0.5h G2 201 N / D N / D N / D 202 137.90 5.42 N / D 203 N / D N / D N / D 2h G3 301 N / D N / D N / D 302 62.21 N / D N / D 303 66.96 5.49 N / D 4h G4 401 N / D N / D N / D 402 N / D N / D N / D 403 N / D N / D N / D

[0106] As shown in Table 2, the results showed that when GCB034 was administered intravenously, it crossed the BBB and the presence of the substance and some dissociated products were detected in brain and pancreatic tissues, confirming that gemcitabine combined with sialyllactose can be delivered to the brain and subsequently dissociated in the brain.

[0107] 4-4) Experiment to Confirm the Anticancer Activity of Sialyllactose-Conjugated Gemcitabine

[0108] To investigate the effect of binding with sialyllactose on the anticancer activity of gemcitabine, which is used as an anticancer agent, cancer cell lines (BxPC-3) were treated with GCB034 and gemcitabine at various concentrations (5, 10, 20, 40 nM). After 72 hours, cell viability was checked, and the apoptosis markers PARP and Caspase-3 were examined. As a result, it was confirmed that GCB034 could induce cancer cell apoptosis at all treated concentrations and exhibited a higher anticancer effect compared to gemcitabine without sialyllactose binding (Fig. 16A). Corresponding to these results, it was confirmed that the decrease in PARP and the increase in Caspase-3 cleavage of the pro form, which occur as apoptosis progresses, were significantly concentration-dependent in the gemcitabine conjugate (Fig. 16B).

[0110] 5. Blood-brain barrier penetration experiment of sialyllactose-conjugated antibodies

[0111] GCB027 (sialyllactose + Anti-NCAM) was prepared by linking an Anti-NCAM antibody (14-0567-82, eBioscience) to sialyllactose using an SH linker (3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester, sigma-aldrich, P3415). Cy5.5 was attached to the Anti-NCAM antibody to serve as a control, and blood-brain barrier penetration experiments were performed with GCB027 attached with Cy5.5. As a result of measuring the in vivo fluorescence distribution and fluorescence signals in the brain of Anti-NCAM attached with Cy5.5 and GCB027 over time (Figures 17 and 18), it was confirmed that attaching sialyllactose enables even large molecular weight antibodies to be delivered to the brain more effectively.

[0113] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0114] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A pharmaceutical composition for treating or diagnosing brain diseases comprising a blood-brain barrier permeable drug delivery system characterized by being represented by the following structural formula 1: [Structural Formula 1] SL-LX, wherein SL is sialyllactose or its salt, X is methotrexate or gemcitabine, L is connected to SL by a group selected from -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS- and -N(CH3)-, X is connected to a group selected from -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS- and -N(CH3)-, and the connection between the two links is -(CH2)a-(NHCO)b-(CH2)c-, -(CH2)a-(CONH)b-(CH2)c-, -(CH2)a-(CO)b-(CH2)c-, A chemical linker selected from the group consisting of -(CH2)a-(NH)b-(CH2)c- and -(CH2)a-(O)b-(CH2)c-, wherein a is an integer from 0 to 10; b is an integer from 0 to 1; and c is an integer from 0 to 10. Claim 2 A pharmaceutical composition for treating or diagnosing brain diseases comprising a blood-brain barrier permeable drug delivery system, characterized in that, in claim 1, the sialyllactose is 2,3-sialyllactose or 2,6-sialyllactose. Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 A pharmaceutical composition for treating or diagnosing brain diseases comprising a blood-brain barrier permeable drug delivery system, wherein, in claim 1, the chemical linker is bonded to the hydroxyl group (-OH) of the lactose portion of sialyllactose. Claim 7 delete

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