Sialyl lactose-based drug delivery system for blood-brain barrier penetration

Abstract

The present invention provides a drug delivery system for penetrating the blood-brain barrier, which is obtained by binding sialyllactose, which is capable of penetrating the blood-brain barrier, to a drug. The drug delivery system can deliver not only small molecular weight drugs, but also antibodies, which have a large molecular weight, to the brain, and is therefore applicable to various brain disease treatments and diagnostic substances.

Description

[Technical Field]

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

[0002] The blood-brain barrier (BBB) ​​is a cellular barrier composed of tight junctions with a very high electrical resistance (over 0.1 Ω·m) between vascular endothelial cells and associated pericytes and astrocytes. It is a highly selective permeable barrier that separates circulating blood from brain extracellular fluid in the central nervous system (CNS), and acts as a barrier regulating the entry and exit of substances.

[0003] The blood-brain barrier blocks the delivery of bacteria, pathogens, and potentially harmful substances carried through the blood to the brain, but it also blocks the delivery of many central nervous system drugs to the brain, resulting in low efficacy of these drugs. To compensate for this, these drugs are administered in high doses, which can cause serious side effects in peripheral organs. Therefore, there is a need to develop an efficient drug delivery system that can penetrate the blood-brain barrier to prevent negative systemic effects and ensure the therapeutic effects of the drugs.

[0004] With this in mind, 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, transports it into cerebrovascular cells, and then moves into the brain parenchyma. Roche is currently conducting Phase III clinical trials for gantenerumab, a monoclonal antibody that targets amyloid beta plaques, a toxic protein in brain nerve cells that has been identified as a cause of dementia, and is currently conducting Phase I clinical trials for a redesigned double antibody that attaches a brain shuttle to gantenerumab.

[0005] However, antibody-based drug delivery systems are not suitable for application to a variety of drugs. Antibodies are high-molecular-weight substances and are significantly larger than low-molecular-weight substances such as small-molecular-weight compounds and peptides. Therefore, attaching a single drug to a single antibody actually reduces the drug delivery efficiency. Therefore, drugs attached to antibodies are substances that have excellent efficacy but are difficult to use due to toxicity issues. To address these issues, research is underway into various drug delivery systems using small-molecular-weight compounds, peptides, aptamers, and other substances with molecular weights smaller than antibodies.

[0006] Therefore, the inventors confirmed through experiments that fluorescent substances with small molecular weight sialyllactose attached can pass through the blood-brain barrier, and further confirmed that when sialyllactose is bound to different substances, they can also pass through the blood-brain barrier, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to provide a drug delivery system that uses low molecular weight sialyllactose and can pass through the blood-brain barrier.

[0008] The technical problems that the present invention aims to achieve are not limited to the above-mentioned technical problems, and other technical problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0009] In order to achieve the above technical objectives, one embodiment of the present invention provides a drug delivery system for penetrating the blood-brain barrier, which is represented by the following structural formula 1:

[0010] [ka] where: SL is sialyllactose or a salt thereof; X is a physiologically active substance to be delivered; L may be a bond or a chemical linker connecting said SL and X.

[0011] In one embodiment, the sialyllactose may be 2,3-sialyllactose or 2,6-sialyllactose.

[0012] In one embodiment, the physiologically active substance to be delivered is a physiologically active substance having a therapeutic effect on a brain disease, or may be selected from the group consisting of a small molecule drug for diagnosis, a peptide, an antibody, a protein, natural or modified ssDNA, dsDNA, RNA, siRNA, and ASO.

[0013] In one embodiment, the chemical linker can be used without limitation as long as it has a structure that is structurally linked to both SL and X. For example, the chemical linker can be linked to SL by a group selected from the group consisting of -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH)-, and can be linked to X by a group selected from the group consisting of -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH)-. The linkage between both links can be selected from the group having the structures -(CH)a-(NHCO)b-(CH)c-, -(CH)a-(CONH)b-(CH)c-, -(CH)a-(CO)b-(CH)c-, -(CH)a-(NH)b-(CH)c-, and -(CH)a-(O)b-(CH)c-. In this case, a may be an integer of 0 to 10, b may be an integer of 0 to 1, and c may be an integer of 0 to 10.

[0014] In one embodiment, the chemical linker may be a cleavable linker that facilitates the release of the biologically active substance to be delivered to the brain after delivery.

[0015] In one embodiment, the chemical linker can be bonded to the hydroxy group (-OH group) of the lactose moiety of sialyllactose.

[0016] In one embodiment of the present invention, according to another aspect of the present invention, there is provided a pharmaceutical composition for treating or diagnosing brain diseases, comprising the above-mentioned drug delivery system for penetrating the blood-brain barrier. [Effects of the Invention]

[0017] The drug delivery system according to one embodiment of the present invention can be applied to drugs that have low brain penetration efficiency and can efficiently deliver drugs to the brain, and therefore can be used in the treatment and diagnosis of various brain diseases without the toxicity problems associated with the administration of high doses of drugs.

[0018] The effects of the present invention are not limited to the effects described above, but should be understood to include all effects that can be inferred from the configuration of the invention described in the description of the present invention or the claims. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of an experiment comparing the brain penetration of lactose- and sialyllactose-conjugated Cy5.5. [Figure 2] FIG. 2 shows the results of quantifying the fluorescent signal intensity of the brain portion of FIG. 1 and the results of measuring the fluorescent signal intensity of the brain excised in the experiment of FIG. [Figure 3] FIG. 3 shows the results of an experiment comparing the brain penetration of Cy5.5 conjugated with sialic acid and sialyllactose. [Figure 4] FIG. 4 shows the structural formula of dopamine bound to sialyllactose according to one embodiment of the present invention. [Figure 5] FIG. 5 shows the results of an experiment in which the brain penetration of 2,6-sialyllactose-conjugated dopamine was confirmed in animals. [Figure 6] FIG. 6 shows the results of measuring the fluorescent signal in the brain portion and the excised brain in the experiment of FIG. [Figure 7] FIG. 7 shows the results of measuring the fluorescent signal in a cross section of the brain extracted from an animal to which 2,6-sialyllactose-bound dopamine had been administered. [Figure 8] FIG. 8 shows the results of an experiment in which the brain penetration of 2,3-sialyllactose-conjugated dopamine was confirmed in animals. [Figure 9] FIG. 9 shows the results of measuring the fluorescent signals in the brain portion in the experiment of FIG. 8 and the results of checking the fluorescent signals in the excised brain. [Figure 10] FIG. 10 is a diagram showing the structural formula of methotrexate conjugated with sialyllactose according to one embodiment of the present invention. [Figure 11]FIG. 11 shows the results of an experiment in which brain penetration of sialyllactose-bound methotrexate was confirmed in animals. [Figure 12] FIG. 12 shows the results of measuring the fluorescent signal in the brain portion of FIG. 11 and the results of measuring the fluorescent signal in the excised brain. [Figure 13] FIG. 13 shows the results of confirming the anti-cancer effect of sialyllactose-conjugated methotrexate in an animal brain tumor model. [Figure 14] FIG. 14 shows the structural formula of gemcitabine conjugated with sialyllactose according to one embodiment of the present invention. [Figure 15] FIG. 15 shows the experimental results of confirming the brain penetration of gemcitabine conjugated with sialyllactose according to one embodiment of the present invention in animals. [Figure 16] FIG. 16 is a diagram showing the experimental results of confirming the antitumor activity of gemcitabine conjugated with sialyllactose according to one embodiment of the present invention in a cell line. [Figure 17] FIG. 17 shows the results of an experiment in which the brain penetration of an antibody conjugated with sialyllactose according to one embodiment of the present invention was confirmed in animals. [Figure 18] FIG. 18 shows the results of measuring the fluorescent signal in the brain region of FIG. BEST MODE FOR CARRYING OUT THE INVENTION

[0020] The present invention will be described in detail below.

[0021] The following description should be understood as an example to help understand the present invention, and the spirit or scope of the present invention is not limited by the following description.

[0022] According to one aspect of the present invention, there is provided a drug delivery system for penetrating the blood-brain barrier, which is represented by the following structural formula 1:

[0023] [ka] where: SL may be sialyllactose or a salt thereof, X may be a drug to be delivered, and L may be a bond or a chemical linker connecting the SL and X.

[0024] Sialyl lactose is a type of human milk oligosaccharide (HMO) that is most abundant in breast milk colostrum, and is a form in which sialic acid is attached to lactose (milk sugar). Depending on the position where sialic acid is bonded to lactose, there are two types: 2,3-sialyllactose represented by the following structural formula 2 and 2,6-sialyllactose represented by the following structural formula 3.

[0025] [ka]

[0026] [ka]

[0027] The 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 because it can be mass-produced and does not use toxic catalysts or organic solvents.

[0028] The salt of sialyllactose may be, but is not limited to, Na.

[0029] The drug to be delivered may be a physiologically active substance having a therapeutic effect on brain diseases or a diagnostic substance, which may be selected from the group consisting of small molecule drugs, peptides, antibodies, proteins, natural or modified ssDNA, dsDNA, RNA, siRNA, and ASO.

[0030] For example, small molecule drugs include anticancer drugs such as cisplatin, carboplatin, melphalan, chlorambucil, dacarbazine, capecitabine, cytarabine, vinblastine, vincristine, paclitaxel, docetaxel, etoposide, topotecan, irinotecan, dactinomycin, doxorubicin, daunorubicin, mitomycin, bleomycin, tetanus, and tetanus. The drug may be musirolimus, everolimus, lenalidomide, or a pharmaceutically acceptable salt thereof; memantine, donepezil, galantamine, or a pharmaceutically acceptable salt thereof, which are therapeutic agents for Alzheimer's disease; pramipexole, ropinirole, amantadine, or a pharmaceutically acceptable salt thereof, which are anti-Parkinson's disease drugs; or dopamine, acetylcholine, citicoline, choline alfocerate (Alpha-GPC), which are neurotransmitters and brain function improvers, or a pharmaceutically acceptable salt thereof.

[0031] The antibody may be, for example, anticancer drugs such as trastuzumab, cetuximab, bevacizumab, pertuzumab, pembrolizumab, nivolumab, ipilimumab, atezolizumab, and durvalumab, or may be aducanumab, an agent for treating Alzheimer's disease.

[0032] The 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).

[0033] The brain disease can be a disorder or disease that occurs in or has an etiology in the central nervous system, such as, but not limited to, brain tumor, cerebellar atrophy, Lou Gehrig's disease, stroke, Alzheimer's disease, dementia, and Parkinson's disease.

[0034] The SL and X can be directly bonded to each other. For example, a functional group of SL2 can be directly bonded to a functional group of X, and the direct bond can be broken down after permeating the blood-brain barrier, separating SL and X without inhibiting the blood-brain barrier permeability of SL or the physiological activity of X. In this case, the direct bond can be formed without a linker. Preferably, the hydroxyl group (-OH group) of the lactose moiety of SL can be bonded to a functional group that does not affect the physiological activity of X.

[0035] Alternatively, the SL and X can be linked via a chemical linker. For example, the chemical linker can bind to a functional group of the SL and a functional group of the X, respectively, without interfering with the blood-brain barrier permeability of the SL or the physiological activity of the X. Preferably, the chemical linker can bind to the hydroxyl group (—OH group) of the lactose moiety of the SL and to a functional group of the X that does not affect the physiological activity of the X.

[0036] The chemical linker can have a structure structurally linked to both SL and X. The chemical linker can be linked to SL by a group selected from the group consisting of -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH)-, and can be linked to X by a group selected from the group consisting of -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS-, and -N(CH)-. The link between both links can be selected from the group having the structures -(CH)a-(NHCO)b-(CH)c-, -(CH)a-(CONH)b-(CH)c-, -(CH)a-(CO)b-(CH)c-, -(CH)a-(NH)b-(CH)c-, and -(CH)a-(O)b-(CH)c-. In this case, a may be an integer of 0 to 10, b may be an integer of 0 to 1, and c may be an integer of 0 to 10.

[0037] The chemical linker may be a cleavable linker that promotes release of the drug after delivery to the brain. For example, a pH-sensitive linker, a peptidase-sensitive linker, or a photolabile linker may be used. Preferably, a peptidase-sensitive linker that can be easily cleaved by peptidases present in cells may be used. For example, a linker that is easily cleaved by peptidases such as cathepsin B, C, and D, which are peptidases preferentially expressed in tumor tissues, may be selected.

[0038] The drug delivery system for penetrating the blood-brain barrier may be included in a pharmaceutical composition for treating or diagnosing a brain disease, and the pharmaceutical composition may contain the drug delivery system for penetrating the blood-brain barrier alone or may be provided as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0039] The term "pharmaceutical acceptable" means that the composition exhibits the property of being non-toxic to cells or humans exposed to the composition.

[0040] Furthermore, the pharmaceutical composition may be provided in combination with a conventionally known therapeutic or diagnostic agent for brain diseases, i.e., the pharmaceutical composition may be administered in combination with a known compound having a therapeutic or diagnostic effect on brain diseases.

[0041] The term "administration" as used herein means introducing a predetermined substance into an individual by an appropriate method, and the term "individual" refers to all animals, including humans, such as mice, rats, and livestock, for the purpose of diagnosing or treating brain diseases. Specific examples include mammals, including humans.

[0042] Routes of administration of the pharmaceutical compositions include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0043] The pharmaceutical composition can be administered orally or parenterally. For parenteral administration, it is preferable to select topical application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but is not limited to these. From the viewpoint of selecting a more efficient absorption route, oral administration can be preferably selected.

[0044] The pharmaceutical compositions are formulated using commonly used diluents or excipients, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. These solid formulations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid formulations include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, may be used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention will be described in more detail below with reference to examples and test examples. However, the following examples and test examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. [Example]

[0046] 1. Blood-brain barrier penetration experiment of sialyllactose To confirm the feasibility of sialyllactose as a drug delivery vehicle for blood-brain barrier penetration, brain penetration experiments were carried out for sialic acid, lactose, and sialyllactose.

[0047] First, the fluorescent dye Cy5.5 (Genechem, Cat. No. GCCD0064) was attached to lactose, 2,3-sialyllactose (GCB100), and 2,6-sialyllactose (GCB200). Each sample was dissolved in 10% DMSO and diluted to prepare samples (final DMSO concentration: approximately 2% of the total volume). Each sample was intravenously administered to BALB / c nude mice at 1 μmol / kg. Fluorescence signals in the nude mouse brain were monitored at various times (1, 2, 4, 6, 8, and 24 hours) using a Perkin Elmer IVIS spectrum CT. At 24 hours after administration, the nude mice were perfused with saline, and the brains were removed and measured for brain fluorescence (Figures 1 and 2). The results showed that Cy5.5 (control group) barely penetrated the blood-brain barrier. Compared to lactose, all Cy5.5 with different sialyllactose groups were well delivered to the brain.

[0048] Next, the fluorescent dye Cy5.5 (Genechem, Cat. No. GCCD0064) was attached to sialic acid, 2,3-sialyllactose (GCB) 100, and 2,6-sialyllactose (GCB) 200. Each compound was dissolved in 10% DMSO and diluted to prepare samples (final DMSO concentration: approximately 2% of the total volume). Each sample was intravenously administered to BALB / c nude mice at 1 μmol / kg, and the fluorescence signal in the nude mouse brain was measured at intervals (10 min, 30 min, 1, 2, 4, 6, 8, and 24 h) using a Perkin Elmer IVIS spectrum CT. 24 h after administration, the nude mice were perfused with saline, and the brains were removed and the fluorescence in the brain was measured. As a result, as shown in Figure 3, Cy5.5 (control group) was shown to hardly penetrate the blood-brain barrier, and brain penetration was at a similar level even when sialic acid was attached, whereas Cy5.5 attached with sialyllactose was well transmitted to the brain, and in particular, the effect of 2,6-sialyllactose was shown to be greater than that of 2,3-sialyllactose.

[0049] In summary, when sialyllactose was attached, Cy5.5, which has low brain permeability, was able to pass through the blood-brain barrier more easily, and it was confirmed that Cy5.5 with sialyllactose attached had a much higher brain permeability than sialic acid or lactose, which are part of sialyllactose.

[0050] 2. In vivo experiments of sialyllactose-bound dopamine Parkinson's disease is caused by the loss of dopamine neurons. Currently, L-dopa, a dopamine precursor that can penetrate the brain, is used as a treatment instead of dopamine, which has poor brain penetration. However, side effects such as movement disorders (levodopa-induced dyskinesia) occur. Therefore, an experiment was conducted to investigate whether sialyllactose could be attached to dopamine to increase brain penetration and replace L-dopa.

[0051] 2-1) Preparation of dopamine bound to sialyllactose To prepare GCB007 (2,3-sialyllactose-dopamine conjugate) and GCB008 (2,6-sialyllactose-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 with stirring at 0°C and stirred for 2 hours. The solution was then concentrated and purified on silica gel to yield 4.5 g of material. 1.3 g of the purified material was dissolved in 100 ml of methylene chloride and 0.06 g of 4-dimethylaminopyridine. 0.6 g of 4-nitrophenyl chloroformate was added and stirred for 4 hours. After the reaction was complete, the mixture was fractionated with 100 ml of 0.5 M aqueous citric acid. The methylene chloride layer was treated with sodium sulfate, filtered through a glass filter, and concentrated to yield 2 g of material. 16 ml of dimethylformamide and 2 ml of triethylamine were added to the 2 g of material and stirred. 0.54 g each of aminohexyl-2,3-sialyllactose and 2,6-sialyllactose was added, stirred for 24 hours, concentrated, and then purified with silica gel to obtain 0.3 g of substance. 3 ml of methanol was added to the substance and stirred, and then 6 ml of acetic acid was added and stirred for about 8 hours. After the reaction was complete, the mixture was concentrated and purified with silica gel to obtain GCB007 (2,3-sialyllactose-dopamine complex) and GCB008 (2,6-sialyllactose-dopamine complex).

[0052] To prepare GCB009 (2,3-sialyllactose-dopamine conjugate) and GCB010 (2,6-sialyllactose-dopamine conjugate), 1.2 g of carboxyl-2,3-sialyllactose and 1.2 g of carboxyl-2,6-sialyllactose were each added with 24 ml of dimethylformamide and 1 ml of pyridine and stirred. 0.61 g of N-hydroxysuccinimide was then added and stirred at 60 ° C for 90 minutes. After concentrating, 1 g of material was obtained. 0.67 g of the material obtained through the above process was added with 3.2 ml of dimethylformamide and 1.1 ml of triethylamine and stirred. 0.15 g of dopamine was added and stirred for 24 hours. The mixture was then concentrated and purified with silica gel to prepare 0.06 g of GCB009 (2,3-sialyllactose-dopamine conjugate) and GCB010 (2,6-sialyllactose-dopamine conjugate), respectively.

[0053] The structures of GCB007 (2,3-sialyllactose-dopamine conjugate), GCB008 (2,6-sialyllactose-dopamine conjugate), GCB009 (2,3-sialyllactose-dopamine conjugate), and GCB010 (2,6-sialyllactose-dopamine conjugate) are shown in Figure 4.

[0054] 2-2) Blood-brain barrier penetration experiment of sialyllactose-bound dopamine Dopamine, GCB007, and GCB008 were attached with the fluorescent substance Cy5.5 and subjected to a blood-brain barrier penetration experiment in the same manner as the blood-brain barrier penetration experiment for sialyllactose.

[0055] First, we confirmed the time-dependent fluorescence distribution in the body and the fluorescence signal in the brain of Cy5.5-conjugated dopamine and GCB008 (Figure 5). After the brains were removed at different times, the fluorescence distribution and fluorescence signal in the brain were measured (Figure 6). As a result, we confirmed that dopamine conjugated with sialyllactose had a higher brain penetration rate than dopamine without sialyllactose.

[0056] Furthermore, to confirm whether the fluorescent substance was distributed not only over the entire area but also in the brain cross-sections, GCB008 with Cy5.5 attached was administered using PerCP / Cy5.5 (Abcam, Cat no. ab102911) as a control substance, and the brain was removed and sectioned 24 hours later, after which the fluorescence distribution was confirmed. As shown in Figure 7, it was confirmed that dopamine bound to sialyllactose was also detected in the brain cross-sections.

[0057] Next, to confirm that the brain penetration phenomenon was not due to the effect of the fluorescent substance Cy5.5, a blood-brain barrier penetration experiment was carried out using Cy5.5 as a control group and GCB007 with Cy5.5 attached.

[0058] The time-dependent fluorescence distribution and fluorescence signal of Cy5.5 and Cy5.5-conjugated GCB007 were examined (Figure 8), and the brain was then removed 24 hours later and the fluorescence distribution and fluorescence signal in the brain were examined (Figure 9). As a result, Cy5.5 was barely detected in the brain, while GCB007-Cy5.5 was clearly detected, confirming that the brain penetration of sialyllactose-conjugated dopamine was induced by sialyllactose, not Cy5.5.

[0059] 3. In vivo experiments of methotrexate conjugated with sialyllactose 3-1) Preparation of methotrexate conjugated with sialyllactose To 1 g of methotrexate, 10 ml of pyridine and 0.337 ml of triethylamine were added 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 a substance. To 1 g of the above material, 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 stirred for 12 hours. 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-sialyllactose-methotrexate conjugate) and GCB017 (2,6-sialyllactose-methotrexate conjugate) are shown in FIG.

[0060] 3-2) Blood-brain barrier penetration experiment of methotrexate bound to sialyllactose Cy5.5 was attached to methotrexate and used as a control substance, and Cy5.5 was attached to GCB017 for blood-brain barrier penetration experiments.

[0061] The time-dependent fluorescence distribution in the body and the fluorescence signal in the brain of Cy5.5-conjugated methotrexate and GCB017 were examined (Figures 11 and 12A), and the fluorescence distribution image and fluorescence signal of the brain excised 24 hours after administration were examined (Figure 12B). As a result, when methotrexate was administered, almost no fluorescence signal was observed in the brain, but when methotrexate conjugated with sialyllactose was administered, a strong fluorescence signal appeared, confirming that when sialyllactose binds to methotrexate, it can induce delivery to the brain.

[0062] 3-3) Brain tissue penetration and substance dissociation confirmation experiment of methotrexate bound to sialyllactose To confirm the tissue distribution and dissociation of methotrexate bound to sialyllactose, GCB017 was administered, and the presence of GCB017 and its dissociated products (linker-6'SL (L-6'SL) and linker) was confirmed in the brain and pancreas at different times. More specifically, GCB017 was dissolved in 10% DMSO and diluted to prepare samples (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 100 mg / kg. After cardiac blood collection, the brain and pancreas were isolated by perfusion with saline. Each tissue was homogenized in 50 mM Tris-HCl buffer (pH 7.5) at a ratio of 1:5 and centrifuged at 3,000 rpm for 20 minutes at 4°C. The supernatant was analyzed by mass spectrometry (SCIEX Triple Quad 3500 LC-MS / MS system).

[0063] [Table 1]

[0064] As a result, as shown in Table 1, the substance and its dissociated products were confirmed in brain tissue not only when GCB017 was administered intravenously but also when it was administered orally. This means that methotrexate, which is hardly transported to the brain, can be transported across the blood-brain barrier as a complex with sialyllactose and then dissociated from the brain.

[0065] 3-4) Efficacy evaluation of sialyllactose-conjugated methotrexate in brain tumor animal models To determine whether GCB017 is delivered to the brain and exhibits the anticancer effects of methotrexate, we established a mouse brain tumor model and evaluated its anticancer efficacy. The brain tumor model was created by implanting the luciferase-expressing brain tumor cell line, U-87MG. For brain implantation, each mouse was anesthetized with isoflurane injection and secured in a stereotaxic arm. A 1cm longitudinal incision was made in the scalp, and the injection site was exposed with a microdrill. The cell line was transferred into a 25μl Hamilton syringe equipped with a 31-gauge needle and placed at a depth of 3mm, 1mm anterior, 2mm lateral, and 3mm below the bregma line, at a rate of 1ml / min. After implantation, the cell line was left in place for approximately 10 minutes to prevent backflow and ensure good absorption. The syringe was then removed, the incision was sutured, disinfected, and the mouse was allowed to recover from anesthesia and fed normally. Starting one week after model creation, methotrexate and GCB017 were administered intravenously twice weekly for four weeks, a total of eight times. Brain tumor size was monitored via optical imaging (Figure 13). In the negative control (vehicle) group, tumor growth was evident on the 20th, and continued over time on the 24th and 27th days. The methotrexate-administered positive control group also showed gradual tumor growth from the 20th day. Meanwhile, tumor formation was delayed and tumor size was suppressed in the experimental group administered 22 μmol / kg of GCB017, compared with the negative and positive control groups. Considering the results of Examples 3-2 and 3-3, this is believed to be due to the fact that the methotrexate conjugate linked to sialyllactose according to the present invention inhibits tumor formation and growth with improved BBB permeability compared to methotrexate.

[0066] 4. Blood-brain barrier penetration experiment of gemcitabine conjugated with sialyllactose 4-1) Preparation of gemcitabine conjugated with sialyllactose Preparation of 2,3-sialyllactose-gemcitabine conjugate: 1.4 g of gemcitabine was added to 25 ml of dimethylformamide and 0.89 ml of triethylamine and stirred. Then, 0.94 g of imidazole and 1.44 g of TBDMS-Cl were added and stirred for 4 hours. This mixture was concentrated and purified on silica gel to yield 1.9 g of product. To this was added 12.5 ml of pyridine and 0.6 g of 4-dimethylaminopyridine and stirred. 1.39 ml of di-tert-butyl dicarbonate was slowly added. The mixture was stirred, concentrated, and fractionated into 0.5 M aqueous citric acid and methylene chloride. After treatment with sodium sulfate, the mixture was filtered through a glass filter, concentrated, and purified. 1.2 g of the resulting product was dissolved in 8 ml of pyridine. To this mixture was added 0.03 g of 4-dimethylaminopyridine and 0.327 g of succinic anhydride and stirred. The mixture was then concentrated and fractionated into 0.5 M aqueous citric acid and methylene chloride. This was treated with sodium sulfate, filtered through a glass filter, concentrated, and purified to obtain 0.6 g of material. 0.013 g of 4-dimethylaminopyridine was 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, the mixture was fractionated into 100 ml of 0.5 M aqueous citric acid solution and methylene chloride. The methylene chloride layer was treated with sodium sulfate, filtered through a glass filter, and concentrated to obtain 0.6 g of material. 6 ml of dimethylformamide and 0.62 ml of triethylamine were added and stirred, followed by 0.5 g of aminohexyl-3'SL. This was concentrated and purified with silica gel to obtain 0.25 g of material. 5 ml of distilled water and 0.025 ml of acetic acid were added and stirred at 60°C for 24 hours. The mixture was then concentrated and purified with silica gel to obtain gemcitabine-aminohexyl succinate-3'SL (GCB033).

[0067] Preparation of 2,6-sialyllactose-gemcitabine conjugate: 2.28 g of gemcitabine was added to 41 ml of dimethylformamide and 1.45 ml of triethylamine and stirred, followed by the addition of 1.53 g of imidazole and 2.35 g of TBDMS-Cl. After stirring for 4 hours and concentrating, the mixture was purified on silica gel to yield 1.9 g of the product. 3.75 g of the resulting product was added to 50 ml of pyridine and 1.2 g of 4-dimethylaminopyridine and stirred, followed by the slow addition of 2.7 ml of di-tert-butyl dicarbonate. After stirring, the mixture was concentrated and fractionated into 0.5 M aqueous citric acid solution and methylene chloride. This was treated with sodium sulfate, filtered through a glass filter, and concentrated. The resulting 3.2 g of material was dissolved in 21 mL of pyridine, and 0.082 g of 4-dimethylaminopyridine and 0.87 g of succinic anhydride were added. The mixture was stirred and concentrated, and then fractionated into 0.5 M aqueous citric acid and methylene chloride. This mixture was treated with sodium sulfate, filtered through a glass filter, concentrated, and purified to yield 2.9 g of material. 0.061 g of 4-dimethylaminopyridine was dissolved in 29 mL of pyridine and 2.5 mL of triethylamine, and 1.21 g of 4-nitrophenyl chloroformate was added and stirred for 4 hours. After completion of the reaction, the mixture was partitioned between 0.5 M aqueous citric acid and 100 mL of methylene chloride. The methylene chloride layer was treated with sodium sulfate and filtered through a glass filter. 2.9 g of the resulting material was concentrated and stirred with 29 mL of dimethylformamide and 3 mL of triethylamine. 2.4 g of aminohexyl-6'SL was added to this and stirred, then concentrated. After purifying on silica gel, 1.2 g of the resulting substance was added to 24 ml of distilled water and 0.12 ml of acetic acid, stirred at 60°C for 24 hours, concentrated, and purified on silica gel to obtain gemcitabine-succinate-aminohexyl-6'SL (GCB034).

[0068] The structures of GCB033 (2,3-sialyllactose-gemcitabine conjugate) and GCB034 (2,6-sialyllactose-gemcitabine conjugate) prepared as described above are shown in FIG.

[0069] 4-2) Blood-brain barrier penetration experiment of gemcitabine bound to sialyllactose To confirm the blood-brain barrier permeability of gemcitabine conjugates, we conjugated Cy5.5 to GCB033 and GCB034, respectively, and performed blood-brain barrier permeation experiments. Cy5.5 alone and gemcitabine conjugated with Cy5.5 alone served as controls. Each compound was dissolved in 10% DMSO and diluted to prepare samples (final DMSO concentration: approximately 2% of the total volume). Each sample was intravenously administered at 1 μmol / kg to BALB / c nude mice. Fluorescence signals in the nude mouse brains were measured at intervals (10 min, 30 min, 1, 2, 4, 6, 8, and 24 h) using a Perkin Elmer IVIS spectrum CT. After 24 h, the nude mice were perfused with saline, and the brains were removed and the fluorescence signals in the brains were measured.

[0070] As shown in Figure 15, the results showed that when Cy5.5 attached to gemcitabine bound to sialyllactose was administered compared to gemcitabine, a stronger fluorescent signal was observed, confirming that sialyllactose improves the brain permeability of gemcitabine.

[0071] 4-3) Brain tissue penetration and substance dissociation confirmation experiment of gemcitabine bound to sialyllactose To confirm the tissue distribution and dissociation of sialyllactose-conjugated gemcitabine, we administered GCB034 and examined the presence of GCB034 and its dissociated product, linker-6'SL (L-6'SL), and the linker in the brain and pancreas at various times. Specifically, GCB034 was dissolved in 10% DMSO and diluted to prepare samples (final DMSO concentration: approximately 2% of the total volume). The samples were administered intravenously (IV) at 82 mg / kg or orally (PO) at 246 mg / kg. After anesthesia at various times (0, 0.5, 2, and 4 hours), cardiac blood was collected, perfused with saline, and the brain and pancreas were removed. Each excised tissue was homogenized in 50 mM Tris-HCl buffer (pH 7.5) at a ratio of 1:5, centrifuged at 3,000 rpm at 4°C for 20 minutes, and the supernatant was analyzed by mass spectrometry (SCIEX Triple Quad 3500 LC-MS / MS system).

[0072] [Table 2]

[0073] As shown in Table 2, the results showed that when GCB034 was administered intravenously, it passed through the BBB and the presence of the substance and some of its dissociated products were detected in the brain and pancreatic tissue, confirming that gemcitabine bound to sialyllactose was delivered to the brain and then dissociated from the brain.

[0074] 4-4) Antitumor activity confirmation experiment of gemcitabine bound to sialyllactose To investigate the effect of sialyllactose conjugation on the antitumor activity of gemcitabine, a cancer cell line (BxPC-3), we treated the cancer cell line with GCB034 and gemcitabine at various concentrations (5 nM, 10 nM, 20 nM, and 40 nM) and then monitored cell viability and cell death markers PARP and caspase-3 after 72 hours. We confirmed that GCB034 not only induced cancer cell death at all concentrations, but also demonstrated superior anticancer efficacy compared to gemcitabine without sialyllactose (Figure 16A). Consistent with these results, we also observed a significant decrease in pro-PARP and an increase in caspase-3 cleavage, which occur as cell death progresses, in a concentration-dependent manner with the gemcitabine conjugate (Figure 16B).

[0075] 5. Blood-brain barrier penetration experiment of sialyllactose-conjugated antibodies GCB027 (sialyllactose + anti-NCAM) was prepared by linking 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, and a blood-brain barrier penetration experiment was performed with GCB027 attached to Cy5.5 as a control substance. The time-dependent distribution of fluorescence in the body and the fluorescence signal in the brain of Cy5.5-attached anti-NCAM and GCB027 were measured (Figures 17 and 18). The results confirmed that the attachment of sialyllactose improved delivery of large molecular weight antibodies to the brain.

[0076] The above disclosure of the present invention is for illustrative purposes only, and those skilled in the art will appreciate that the present invention may be easily modified into other specific forms without departing from the spirit or scope of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. For example, components described as a single unit may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0077] The scope of the present invention is defined by the claims that follow, and all modifications and variations that fall within the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims

1. A pharmaceutical composition for treating or diagnosing brain diseases, comprising a drug carrier for penetrating the blood-brain barrier, characterized by being represented by the following structural formula 1: 【Chemistry 1】 where: SL is 2,6-sialyllactose or a salt thereof; X is the drug to be delivered; L is a bond or chemical linker connecting SL and X.

2. 2. The pharmaceutical composition for the treatment or diagnosis of brain diseases according to claim 1, wherein the drug to be delivered is a physiologically active substance having a therapeutic effect on brain diseases, or is selected from the group consisting of small molecule drugs, peptides, antibodies, proteins, natural or modified ssDNA, dsDNA, RNA, siRNA, and ASO for diagnosis.

3. The chemical linker may be SL and -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS- and -N(CH ３ )--, and linked together; X and -CONH-, -C(=O)-, -NH-, -O-, =N-, -SS- and -N(CH ３ )--, and linked together; Between both links, -(CH ２ )a-(NHCO)b-(CH ２ ) c-, -(CH ２ )a-(CONH)b-(CH ２ ) c-, -(CH ２ )a-(CO)b-(CH ２ ) c-, -(CH ２ )a-(NH)b-(CH ２ )c- and -(CH ２ )a-(O)b-(CH ２ )c-, In this case, a is an integer from 0 to 10. b is an integer from 0 to 1; 2. The pharmaceutical composition for the treatment or diagnosis of brain diseases according to claim 1, wherein c is an integer of 0 to 10.

4. 2. The pharmaceutical composition for the treatment or diagnosis of brain diseases according to claim 1, wherein the chemical linker is a cleavable linker that promotes the release of the target physiologically active substance after delivery to the brain.

5. 2. The pharmaceutical composition for the treatment or diagnosis of brain diseases according to claim 1, wherein the chemical linker is bonded to a hydroxy group (-OH group) of the lactose moiety of 2,6-sialyllactose.

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