Inhibitors of nervous system vascular barrier breakdown, and therapeutic agents for nervous system diseases.

Abstract

The present invention addresses the problem of providing a suppressant for nervous system vascular barrier breakdown. Provided is a suppressant for nervous system vascular barrier breakdown, the suppressant comprising (a) or (b) as an active ingredient: (a) an enzyme capable of specifically detaching or decomposing a sugar chain of low-glycosylated basigin; and (b) an antibody, an aptamer, or a lectin that specifically binds to low-glycosylated basigin and suppresses the nervous system vascular barrier breakdown caused by basigin.　Preferably, the sugar chain of the low-glycosylated basigin is high-mannose sugar chain.

Description

Background Art

[0001] The present invention relates to an inhibitor of disruption of the nervous system vascular barrier and a therapeutic agent for nervous system diseases.

Technical Field

[0002] In nervous system tissues such as the adult brain, spinal cord, and retina, nervous system vascular barriers such as the blood-brain barrier and blood-retinal barrier are formed between the blood and the nervous system tissues, maintaining an optimal tissue microenvironment in which nerve cells can function normally. The nervous system vascular barrier is basically maintained in the adult after induction during ontogeny, that is, in a state where the "nervous system vascular barrier is closed", but is under dynamic regulation that changes in response to environmental changes or some stimuli. The involvement of the nervous system vascular barrier in the pathogenesis and treatment of intractable nervous system diseases can be classified into the following two categories. 1) Due to the presence of the nervous system vascular barrier ("the state where the nervous system vascular barrier is closed"), systemically administered therapeutic drugs cannot reach the nerve tissue parenchyma, and as a result, it hinders the treatment of brain tumors, neurological infections, etc. In this case, artificially "opening" the nervous system vascular barrier becomes a novel treatment for overcoming intractable nervous system diseases. 2) There are many nervous system diseases in which the "opened" state of the nervous system vascular barrier is central to the deterioration of the disease state (such as ischemic brain diseases, diabetic retinopathy, etc.). In this case, artificially "closing" the nervous system vascular barrier becomes a novel treatment for overcoming intractable nervous system diseases. Therefore, if the artificial control of the nervous system vascular barrier can control the "closed state" and the "opened state", improvement or complete cure of intractable nervous system diseases for which there is no useful treatment method in current medicine is expected.

[0003] For over half a century, many researchers both in Japan and abroad have conducted research to clarify "how the nervous system vascular barrier is regulated," but translational research has yet to yield results that can be expected to be applied to clinical practice. Development of new therapeutic drugs is underway for many intractable neurological diseases for which there are still no effective treatments, but one of the biggest obstacles in the drug discovery process is the existence of the nervous system vascular barrier. Many attempts have been made to pass drugs through the nervous system vascular barrier and reach the nerve tissue parenchyma. Among these, regarding methods to change the nervous system vascular barrier from a 'closed state' to an 'open state', currently in clinical practice there is a method to artificially change the nervous system vascular barrier to an 'open state' by intravascular administration of hypertonic mannitol solution, but this method deforms endothelial cells through dehydration, causing physical and irreversible dissociation of cells. In addition to this method, many other studies being conducted by researchers also involve strategies that lead to the disruption of the structure of the nervous system vascular barrier itself, namely the tight connections between vascular endothelial cells, and tissue damage is a major problem, as is the case with intravascular administration of hypertonic mannitol. Furthermore, regarding the nervous system vascular barrier, methods for opening it using proteinases such as matrix metalloproteinase (see Non-Patent Literature 1) have also been disclosed, but these methods destroy the molecules that form the nervous system vascular barrier, making temporary and reversible opening of the nervous system vascular barrier difficult.

[0004] Furthermore, there have been no reports of promising research results regarding methods for changing the nervous system vascular barrier from an 'open state' to a 'closed state'. In this situation, the inventors have conducted research for approximately a quarter of a century and have recently obtained interesting findings that have a high potential to lead to the establishment and clinical application of an artificial control system for the nervous system vascular barrier. Specifically, they have identified a disintegrin and metalloproteinase (ADAM)12, ADAM17, and basigin, which are expressed and localized on the cell membrane of nervous system vascular endothelial cells, as molecules responsible for regulating the nervous system vascular barrier (see Patent Documents 1 and 2, and Non-Patent Documents 2 and 3). In particular, they have found that suppressing the expression of basigin causes the barrier to change from an 'open state' to a 'closed state'. On the other hand, it is known that there are highly glycosylated basigin and low glycosylated basigin. Furthermore, highly glycosylated basidines have been identified as basidines involved in the induction of matrix metalloproteinase (MMP) expression, the adhesion of leukocytes to vascular endothelial cells and their migration outside the blood vessels, and diabetes-related blood-brain barrier disruption (see Non-Patent Literature 4). However, low-glycosylated basidines have not received much attention, and therefore research on them is still incomplete, with many aspects of their function remaining unclear. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2014 / 174834 brochure [Patent Document 2] International Publication No. 2017 / 073232 brochure [Non-patent literature]

[0006] [Non-Patent Document 1] Pengyu Pan et al., Neuroscience Letters Volume 649, 10 Pages 7-13, 2017 [Non-Patent Document 2] Cui, D et al., Sci Rep 5, 12796; doi: 10.1038 / srep12796, 2015. [Non-Patent Document 3] Mitsuru Arima et al., Sci Rep 6, 38445; doi: 10.1038 / srep38445, 2016. [Non-Patent Document 4] Yanan Xie et al. Journal of Neuroinflammation (2019) 16:72 https: / / doi.org / 10.1186 / s12974-019-1460-1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide an agent that inhibits the breakdown of the nervous system vascular barrier. [Means for solving the problem]

[0008] Basigin is known to have several important physiological functions, including involvement in the nervous system vascular barrier and neuronal function. Therefore, there are concerns that nonspecific suppression or stimulation of physiological functions by basigin may lead to serious side effects. Accordingly, the inventors conducted a detailed analysis of the molecular structure of basigin from the perspective of the nervous system vascular barrier. Basigin is a molecule with glycosylation, and the existence of two different N-linked glycosylated molecules is known: highly glycosylated basigin (hereinafter also referred to as "Basigin-HG") and lowly glycosylated basigin (hereinafter also referred to as "Basigin-LG"). The inventors found that Basigin-LG is an essential molecule for regulating the nervous system vascular barrier, that Basigin-LG is a glycoprotein modified with high mannose-type glycans, and that Basigin-LG is useful as a specific target for artificial control of the nervous system vascular barrier, thus completing the present invention. Furthermore, because basigin-HG possesses diverse biological activities, targeting and suppressing its biological activity raises concerns about various side effects. In contrast, targeting basigin-LG as the primary target is thought to reduce these side effects. Moreover, by using molecules that primarily target basigin-LG, it may be possible to provide groundbreaking inhibitors of neurovascular barrier disruption and therapeutic agents for neurological diseases.

[0009] In other words, the present invention is as follows: [1] (a) Enzymes that specifically release or degrade the glycans of low-glycozyme basidines; (b) Antibodies, aptamers, or lectins that specifically bind to low-glycozyme basidines and suppress basidine-induced disruption of the nervous system vascular barrier; An inhibitor of nervous system vascular barrier disruption, comprising (a) or (b) as the active ingredient. [2] The inhibitor of neurovascular barrier disruption described in [1] above, characterized in that the glycans of the low-glycozyme-modified basidine are high-mannose type glycans. [3] An inhibitor of neurovascular barrier disruption according to [1] or [2] above, characterized in that the enzyme that specifically releases or degrades the glycans of low-glycozyme basidine does not substantially release or degrade the glycans of high-glycozyme basidine. [4] The inhibitor of nervous system vascular barrier disruption described in [3] above, characterized in that the molecular weight of the low-glycozyme-modified basidine is 35,000 or less, and the molecular weight of the high-glycozyme-modified basidine is 40,000 to 60,000. [5] An inhibitor of nervous system vascular barrier disruption according to any one of [1] to [4] above, characterized in that the enzyme that specifically releases or degrades the glycans of low-glycosylation basidine is endoglycosidase F1 or endoglycosidase H. [6] (a) Enzymes that specifically release or degrade the glycans of low-glycozyme basidines; (b) Antibodies, aptamers, or lectins that specifically bind to low-glycozyme basidines and suppress basidine-induced disruption of the nervous system vascular barrier; A therapeutic agent for neurological diseases caused by disruption of the nervous system vascular barrier, comprising (a) or (b) as the active ingredient. [7] The neurological disease treatment agent according to [6] above, characterized in that the neurological disease is a neurocranial disease or a retinal neurological disease. [8] The neurological disease treatment agent described in [7] above, characterized in that the neurological disease is cerebral edema. [9] The neurological disease treatment agent described in [7] above, characterized in that the retinal neurological disease is retinal edema.

[0010] Furthermore, another embodiment of the neurological disease treatment agent of the present invention is: [i] A method for treating neurological diseases caused by disruption of the neurovascular barrier function, characterized by administering either (a) or (b) of (a) an enzyme that specifically releases or degrades the glycans of low-glycozyme basidine; or (b) an antibody, aptamer, or lectin that specifically binds to low-glycozyme basidine and suppresses the disruption of the neurovascular barrier by basidine. [(ii)] For use as a therapeutic agent for nervous system diseases caused by the breakdown of the nervous system vascular barrier function, an enzyme that specifically releases or degrades the sugar chain of (a) low-glycosylation-modified basigin or (b) an antibody, aptamer, or lectin that specifically binds to low-glycosylation-modified basigin and suppresses the breakdown of the nervous system vascular barrier by basigin, [(iii)] The use of (a) an enzyme that specifically releases or degrades the sugar chain of low-glycosylation-modified basigin; (b) an antibody, aptamer, or lectin that specifically binds to low-glycosylation-modified basigin and suppresses the breakdown of the nervous system vascular barrier by basigin; in the preparation of a therapeutic agent for nervous system diseases caused by the breakdown of the nervous system vascular barrier function, can include [i] to [iii]. [Advantages of the Invention]

[0011] According to the present invention, it becomes possible to suppress the breakdown of the nervous system vascular barrier and to treat nervous system diseases caused by the breakdown of the nervous system vascular barrier. [Brief Description of the Drawings]

[0012] [Figure 1] It is a figure showing an image of basigin having three N-linked glycosylation modification sites in the extracellular domain. [Figure 2] In Example 1, it is a figure showing the result of Western blot analysis. [Figure 3A] In Example 2, it is a figure showing the result of Western blot analysis 36 hours after culturing mouse cerebral vascular endothelial cell line (bEnd.3 cells) and introducing Basigin siRNA#1, Basigin siRNA#2, and Non-silence siRNA (NC siRNA). [Figure 3B]In Example 2, after culturing bEnd.3 cells and introducing Basigin siRNA#1, Basigin siRNA#2, and Non-silence siRNA, 36 hours later, they were placed under 1% hypoxia for 45 minutes (under Hypoxia) or left as they were (under 20% normal oxygen: Normoxia), and this is a figure showing the results of observing the change in the expression of claudin-5 by fluorescence immunostaining. [Figure 3C] In Example 2, after culturing bEnd.3 cells and introducing Basigin siRNA#1, Basigin siRNA#2, and Non-silence siRNA, 36 hours later, they were placed under 1% hypoxia for 45 minutes (under Hypoxia) or left as they were (under 20% normal oxygen: Normoxia), and this is a figure showing the results of measuring the trans-epithelial electrical resistance (TEER). [Figure 4] In Example 3, this is a figure showing the results of performing Western blot analysis by treating Basigin expressed in bEnd.3 cells with Endoglycosidase H (Endo H) or Peptide-N-Glycosidase F (PNGase F). [Figure 5A] In Example 4, bEnd.3 cells were grown as a monolayer, and this is a figure showing the results of observing the Claudin-5 expression level after treatment with Endo H by fluorescence immunostaining. [Figure 5B] In Example 4, bEnd.3 cells were grown as a monolayer, and this is a graph showing the quantification of the Claudin-5 expression level after treatment with Endo H. [Figure 5C] In Example 4, bEnd.3 cells were grown as a monolayer, and this is a figure showing the results of measuring the TEER after treatment with Endo H. [Figure 6] In Example 5, after administering a tracer to mice raised under normal oxygen conditions (Normoxia) and mice raised under 7 - 8% hypoxia (Hypoxia), this is a figure showing the results of observing the leakage of the tracer by confocal microscopy. [Figure 7]This figure shows the results of observing tracer leakage using a confocal microscope after administering the tracer to diabetic mice and non-diabetic mice, respectively, with EndoH-administered and non-EndoH-administered groups. [Modes for carrying out the invention]

[0013] One aspect of the inhibitor of nervous system vascular barrier disruption of the present invention is not particularly limited as long as it contains either (a) or (b) of (a) an enzyme that specifically releases or degrades the glycans of low-glycozyme basidine; or (b) an antibody, aptamer, or lectin that specifically binds to low-glycozyme basidine and suppresses nervous system vascular barrier disruption caused by basidine; and is hereinafter also referred to as "the present inhibitor of nervous system vascular barrier disruption." Another aspect of the present invention is a therapeutic agent for nervous system diseases caused by nervous system vascular barrier disruption, containing either (a) or (b) of (a) an enzyme that specifically releases or degrades the glycans of low-glycozyme basidine; or (b) an antibody, aptamer, or lectin that specifically binds to low-glycozyme basidine and suppresses nervous system vascular barrier disruption caused by basidine; and is hereinafter also referred to as "the present therapeutic agent for nervous system diseases."

[0014] In this specification, the term "neurovascular barrier" refers to a mechanism that restricts the exchange of substances between the blood and the tissue fluid of the nervous system. Examples of suitable examples include the blood-brain barrier, which restricts the exchange of substances between the blood and the tissue fluid of the brain, and the blood-retinal barrier, which restricts the exchange of substances between the blood and the tissue fluid of the retina.

[0015] In this specification, suppression of nervous system vascular barrier breakdown means suppressing the failure of mechanisms that restrict the exchange of substances between the blood and the tissue fluid of the nervous system, thereby preventing the entry of neurotoxic molecules into tissues and the leakage of plasma components.

[0016] In this specification, basidine is a glycoprotein belonging to the immunoglobulin superfamily localized to the cell membrane, and is also known as EMMPRIN (extracellular matrix metalloproteinase inducer), CD147 (cluster of differentiation 147), M6, GP42, HT7, OX-47, MC31, CE9, or forth22. Basidine is known to be a receptor for cyclophyllin A (CypA), a member of the cyclophyllin family that possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity. Human basidine is preferred as the basidine used, and the amino acid sequence of human basidine can be cited as SEQ ID NO: 1, which is published by the National Center for Biotechnology Information (NCBI) as accession number NP_940991.1.

[0017] Basidine is a glycoprotein with glycosylation modifications, possessing N-linked glycans in which N-acetylglucosamine (GlcNAc) is glycosidically bonded to the nitrogen (N) of the amide group in the side chain of the asparagine (Asn) residues at positions 44, 152, and / or 186 in the amino acid sequence shown in SEQ ID NO: 1. The molecular weight of basidine varies depending on the type and number of sugars added during the extension of these N-acetylglucosamine residues.

[0018] In this specification, low-glycozyme basigins (Basigin-LG) refer to basigins whose N-linked glycans are low-glycozyme and have a molecular weight of 35,000 or less, while high-glycozyme basigins (Basigin-HG) refer to basigins whose N-linked glycans are high-glycozyme and have a molecular weight of 40,000 to 60,000. The molecular weight of the core protein in low-glycozyme basigins or high-glycozyme basigins is approximately 27,000.

[0019] In this specification, a high-mannose type glycan refers to a glycan in which only mannose is bonded to a trimannosyl core structure consisting of two molecules of N-acetyl-D-glucosamine and three molecules of mannose, which are N-linked glycans. A complex type glycan refers to a hybrid type glycan in which mannose and N-acetylglucosamine are bonded to the above trimannosyl core structure.

[0020] The enzyme used herein to specifically release or degrade the glycans of low-glycozyme basidine is not particularly limited as long as it specifically acts on the glycans of low-glycozyme basidine and releases or degrades them. Preferably, such an enzyme specifically acts on the high-mannose type glycans of low-glycozyme basidine and releases or degrades the high-mannose type glycans of low-glycozyme basidine. Here, "specifically releasing or degrading the glycans of low-glycan-modified basidines" refers to enzymes that release or degrade the glycans of low-glycan-modified basidines, such as the high-mannose type glycans of low-glycan-modified basidines, but substantially do not release or degrade the glycans of high-glycan-modified basidines, such as the complex type glycans of high-glycan-modified basidines, or enzymes whose activity in releasing or degrading the glycans of low-glycan-modified basidines, such as the high-mannose type glycans of low-glycan-modified basidines, is 3 times or more, preferably 5 times or more, and more preferably 10 times or more, compared to the activity in releasing or degrading the glycans of high-glycan-modified basidines, such as the complex type glycans of high-glycan-modified basidines. Enzymes that specifically release or degrade the glycans of such low-glycozyme basidines include glycosidases that specifically act on the glycans of low-glycozyme basidines to release them, and α-mannosidases that specifically act on the glycans of low-glycozyme basidines to degrade them. Preferably, these are endoglycosidase H or endoglycosidase F1, but are not limited to these.

[0021] As a method for measuring the activity of releasing or degrading the glycans of low-glycozyme basidines or high-glycozyme basidines, for example, one method involves reacting the glycans of low-glycozyme basidines or high-glycozyme basidines with the enzyme to be measured, and detecting the amount of the reaction product as a change in molecular weight by SDS-PAGE, a change in retention time by HPLC, or a change in size by TLC.

[0022] In this specification, "specifically binding to low-glycozyme basidine" means binding to the glycan portion and / or protein portion (core protein portion) of low-glycozyme basidine, and substantially not binding to the glycan portion of high-glycozyme basidine.

[0023] In the antibodies, aptamers, or lectins described herein that specifically bind to the glycans of low-glycosylated basidines and suppress basidine-induced disruption of the nervous system vascular barrier, whether or not basidine-induced disruption of the nervous system vascular barrier can be determined, for example, by measuring the Claudin-5 expression level and TEER of a brain vascular endothelial cell line monolayer, and / or by evaluating tracer leakage after administering the tracer to mice, as shown in the examples described below.

[0024] The antibodies described herein that specifically bind to low-glycotype basidines and suppress basidine-induced neurovascular barrier disruption are not particularly limited as long as they bind to the glycan portion and / or protein portion of low-glycotype basidines, and do not bind to or substantially do not bind to the glycan portion of high-glycotype basidines, and have the effect of suppressing basidine-induced neurovascular barrier disruption when bound to low-glycotype basidines. Furthermore, the phrase "do not bind to or substantially do not bind to the glycan portion of high-glycotype basidines" also includes cases where binding to or affinity for the glycan portion of high-glycotype basidines does not affect the action of high-glycotype basidines.

[0025] The type of antibody may be any type, including human antibodies, chimeric antibodies, or humanized antibodies, as well as antibody fragments such as F(ab')2, Fab, diabody, Fv, ScFv, or Sc(Fv)2. Furthermore, the antibody may be polyclonal or monoclonal. Chimeric antibodies or humanized antibodies can be produced genetically engineered according to conventional methods. Antibody fragments can be produced by methods such as digesting full-length antibodies with pepsin or papain. As described in the examples below, since the glycans of low-glycozyme basidine are high-mannose glycans, antibodies that bind to low-glycozyme basidine can also be said to be antibodies that use the high-mannose glycans and / or protein portion of low-glycozyme basidine as epitopes.

[0026] Antibodies that bind to the low-glycozyme basidine described above can be produced using living cells such as cerebral vascular endothelial cell lines, such as bEnd.3 cells, or cerebral vascular endothelial cells expressing low-glycozyme basidine by genetic engineering, or purified low-glycozyme basidine as an immunosource, and known antibody production methods, such as hybridoma or phage display. In particular, by using living cells that highly express the low-glycozyme basidine, antibodies that maintain a three-dimensional structure closer to that in vivo and are specific to low-glycozyme basidine can be obtained. Antibodies useful for the treatment of neurological diseases, especially antibodies that specifically bind to low-glycozyme basidine, can be selected based on their property of preferentially binding to low-glycozyme basidine in screening methods such as ELISA using purified highly-glycozyme basidine and low-glycozyme basidine, or cells expressing highly-glycozyme basidine and low-glycozyme basidine.

[0027] In this specification, lectins that bind to low-glycotropic basidine and not to high-glycotropic basidine are not particularly limited as long as they have the characteristics of binding to low-glycotropic basidine and not binding to or substantially not binding to high-glycotropic basidine, and when bound to low-glycotropic basidine, they have the effect of suppressing the breakdown of the nervous system vascular barrier by basidine. The term "lectin" above refers to proteins other than antibodies or enzymes that bind to glycans. Such lectins can be selected from lectins that recognize α-mannosyl residues. Examples of lectins that recognize α-mannosyl residues include concanavalin A (ConA) from Canavalia gladiola, pea lectin (PSA) from Pisum sativum, lentil lectin (LCA) from Lens culinaris, snowdrop lectin (GNL) from Galanthus nivalis, NPL from Narcissus pseudonarcissus, banana lectin (BanLec) from Musa paradisiaca (banana), West African bean lectin (BMA) from Bowringia midbraedii, and towayblade (LOA) from Listera ovata. From these lectins, one should select a lectin that specifically binds to the glycans of low-glycosylated basidines, preferably to high-mannose glycans. Furthermore, the phrase "substantially does not bind to the glycans of high-glycosylated basidines" also includes cases where binding to or affinity for the glycans of high-glycosylated basidines does not affect the action of high-glycosylated basidines.

[0028] Examples of lectins that specifically bind to the glycans of low-glycozyme basidines include lectins whose interaction strength with high-mannose type glycans, as measured by frontal affinity chromatography, is 5 times or more, preferably 10 times, and more preferably 100 times or more, than that with non-high-mannose type glycans.

[0029] Whether the above-mentioned antibody or lectin has the characteristic of specifically binding to the glycans of low-glycozyme basidine can be confirmed, for example, by separating basidine into low-glycozyme basidine and high-glycozyme basidine based on molecular weight and examining whether the antibody, aptamer, or lectin binds to them. Methods for separating low-glycozyme basidine and high-glycozyme basidine include SDS-PAGE and gel filtration chromatography. Furthermore, whether the antibody, aptamer, or lectin binds to the separated low-glycozyme basidine or high-glycozyme basidine can be confirmed by binding the antibody, aptamer, or lectin labeled with a fluorescent dye, fluorescent protein, biotin, etc., to the separated low-glycozyme basidine or high-glycozyme basidine and detecting the label. Additionally, lectins that bind to high-mannose glycans can be obtained, for example, by the method described in Japanese Patent Publication No. 4051030 using affinity chromatography, and can be further purified and selected as needed.

[0030] In this specification, neurological diseases resulting from a breakdown of the nervous system's vascular barrier function can preferably include diseases caused by a breakdown of the vascular barrier function leading to disruption of the tissue microenvironment, more preferably include neurological diseases such as cerebral edema, cerebral infarction, degenerative dementia such as Alzheimer's disease, vascular dementia, and ischemic brain disease, or retinal neurological diseases such as retinal edema, and particularly preferably include cerebral edema or retinal edema. Note that "nerve system blood vessels" refers to blood vessels in nervous system tissue and does not include blood vessels in tissues other than nervous system tissue.

[0031] The method of administering the neurological disease treatment agent of the present invention is not particularly limited as long as the desired neurological disease treatment effect is obtained, and can include intravenous administration, oral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, transpulmonary administration, etc. In particular for retinal neurological diseases, intravitreous administration can be mentioned. Furthermore, the dosage of the neurological disease treatment agent of the present invention is not particularly limited and can be appropriately adjusted according to the physical condition, disease state, weight, age, sex, etc. of the subject or animal. As for the dosage, for example, it can be 0.01 μg to 100 g / kg body weight per day as the active ingredient enzyme, antibody, aptamer, or lectin, more preferably 0.1 μg to 10 g / kg body weight, and even more preferably 1 μg to 1 g / kg body weight, and the neurological disease treatment agent of the present invention may be used in combination with other neurological disease treatment agents. The number of administrations and the duration of administration of the neurological disease treatment agent of the present invention are also not particularly limited, and the daily dose can be administered once a day or divided into several doses. Furthermore, there are no particular restrictions on the origin or type of organism of the cells or tissues to be administered, but they are preferably mammals, such as humans, monkeys, cattle, horses, sheep, pigs, dogs, cats, rats, mice, and hamsters, and humans are particularly preferred.

[0032] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0033] [Example 1] (Western blot analysis) Basigins are glycoproteins with three N-linked glycosylation sites in their extracellular domain (Figure 1), and both highly glycosylated basigins (Basigin-HG) and less glycosylated basigins (Basigin-LG) are known to exist. Therefore, the glycosylation of each was analyzed by Western blotting.

[0034] Mouse vascular endothelial cell line (bEnd.3 cells: obtained from the American Type Culture Collection) was cultured in a monolayer in Dulbecco's modified Eagle medium (Sigma-Aldrich) containing 4500 mg / L glucose supplemented with 10% FBS (fetal bovine serum) at 37°C under 5% CO2. Six days after reaching confluence, tunicamycin, an N-linked glycan inhibitor, was added to the medium at a concentration of 2.5 μg / ml, and the cells were cultured for another 24 hours. On the seventh day after reaching confluence, bEnd.3 cells were lysed in tissue lysis buffer (100 μl PBS containing 0.5% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 1% Protease / Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific)), and the supernatant was obtained by centrifugation at 15,000 rpm for 15 minutes. Next, the samples were denatured in 2x Laemmli Sample Buffer at 99°C for 5 minutes, then electrophoresed on a 12% SDS polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked by incubation in a blocking solution (5% skim milk) for 1 hour. Then, rabbit anti-basigin antibody (1 μg / ml: Scrum) was incubated as the primary antibody at 4°C overnight. After washing with TBS-T (0.05% Tween-20, TBS), the antibody was incubated in a secondary antibody (HRP-labeled anti-rabbitk IgG, Dako) at room temperature for 1 hour. After washing with TBS-T, chemiluminescence was detected using an Amersham Imager (GE Healthcare).

[0035] The results of the Western blot analysis described above are shown in Figure 2. The right lane (+) represents the case where tunicamycin was added on day 6 after the start of culture and reaching confluence, while the left lane (-) represents the case where the culture was maintained until day 7 without the control tunicamycin. In the absence of the glycosylation inhibitor tunicamycin, two relatively wide bands of different sizes were detected. On the other hand, in the presence of tunicamycin, both relatively wide bands disappeared and converged into a single band that was judged to be the core protein. Therefore, it was shown that the respective bands in the left lane (-) correspond to basigin-HG and basigin-LG, and the expression of both was confirmed.

[0036] [Example 2] (Relationship between Basigin-HG or Basigin-LG expression and vascular barrier function) Example 1 confirmed that Basigin expresses both Basigin-HG and Basigin-LG. Therefore, we used a Basigin expression suppression system in an in vitro experimental system by introducing Basigin-specific siRNAs (two types, #1 and #2) to analyze the involvement of Basigin-HG and Basigin-LG in the regulation of the nervous system vascular barrier.

[0037] First, as siRNAs targeting basigin (basigin-specific siRNAs), we used two types shown in Table 1 below: Basigin siRNA #1 (sense strand sequence: SEQ ID NO: 2, antisense strand sequence: SEQ ID NO: 3) and Basigin siRNA #2 (sense strand sequence: SEQ ID NO: 4, antisense strand sequence: SEQ ID NO: 5), as well as Non-silence siRNA (Silencer® Select Negative Control #1 siRNA (NC siRNA) catalog number 4390843: Thermo Fisher Scientific). In SEQ ID NOs. 2-5, the base sequences represented by lowercase letters represent overhang sequences.

[0038] [Table 1]

[0039] In vitro, two dishes of mouse brain vascular endothelial cell line bEnd.3 (obtained from ATCC) were prepared and grown as a monolayer in Dulbecco's modified Eagle medium (Sigma-Aldrich) containing 4500 mg / L glucose supplemented with 10% FBS (fetal bovine serum) at 37°C under 5% CO2. 5.5 days after the start of culture and reaching confluence, each of the above siRNAs was introduced to a final concentration of 10 nM. 36 hours after basigin siRNA introduction, one dish was subjected to 1% hypoxia for about 45 minutes, while the other was left untreated (20% normal oxygen).

[0040] Subsequently, we performed Western blot analysis on bEnd.3 cells, observed changes in claudin-5 expression by immunofluorescence staining, and measured the TEER of the bEnd.3 cell monolayer, an indicator of vascular barrier function, according to the method of Koto T et al. (Am. J. Pathol. (2007) 170:1389-1397).

[0041] Western blot analysis was performed as follows: 36 hours after introduction of Basigin siRNA (7 days after confluence of culture: under Normoxia), bEnd.3 cells treated with Basigin siRNA, NC siRNA, or control without siRNA were lysed in tissue lysis buffer (100 μl PBS containing 0.5% Triton X-100, 1% SDS, and 1% Protease / Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific)) and centrifuged at 15,000 rpm for 15 minutes to obtain the supernatant. Subsequently, SDS-PAGE, transfer to PVDF, antibody treatment, and chemiluminescence detection were performed in the same manner as in Example 1.

[0042] Immunofluorescence staining was performed using the following method. First, cells 7 days after reaching confluence were cultured under either Hypoxia or Normoxia for approximately 45 minutes each. Each bEnd.3 cell group was then fixed with 100% methanol at room temperature for 5 minutes and incubated in 10% Non-Immune Goat Serum (Invitrogen) for 30 minutes to block nonspecific antibody binding. Next, the cells were reacted with rabbit polyclonal antibody against claudin-5 (1 / 25 dilution, Invitrogen) overnight at 4°C. Subsequently, the cells were washed with phosphate-buffered saline (PBS) and incubated with Alexa Fluor 488 goat anti-rabbit IgG (1 / 200 dilution, Eugene) at room temperature for 1 hour under protection from light. Finally, the stained cells were mounted on Fluoromount (Diagnostic BioSystems) and observed under a Zeiss LSM5 Pascal laser confocal microscope (Carl Zeiss).

[0043] Figure 3A shows the results of the Western blot analysis. As shown in Figure 3A, 36 hours after siRNA introduction, Basigin-LG expression was almost completely eliminated, while no significant decrease in Basigin-HG expression levels was observed. From these results, it can be said that the bEnd.3 cells used for immunofluorescence staining contained only Basigin-HG and not Basigin-LG. Furthermore, Figure 3B shows the results of immunofluorescence staining, and Figure 3C shows the results of measuring the TEER of the bEnd.3 cell monolayer. As shown in Figure 3B, in the case of a control without Basigin siRNA or when non-silence siRNA was introduced, the disappearance of claudin-5 from the cell membrane of bEnd.3 cells was observed under 1% hypoxia. On the other hand, in a situation where Basigin-LG expression was almost completely eliminated and Basigin-HG expression remained, no significant decrease in Claudin-5 from the cell membrane was detected even under 1% hypoxia. Furthermore, as shown in Figure 3C, Basigin-LG expression was almost completely eliminated, and it was shown that neurovascular barrier formation ability was not lost under conditions where Basigin-HG expression remained. Therefore, it was revealed that Basigin-LG is involved in the decrease of Claudin-5 from the cell membrane and the breakdown of the neurovascular barrier under 1% hypoxic conditions (Hypoxia). In addition, when bEnd.3 cells treated with Basigin siRNA, NC siRNA, or control cells without siRNA were lysed with tissue lysis buffer 72 hours after siRNA introduction in the same manner as above, and the supernatant was obtained and Western blot analysis was performed, it was confirmed that the expression level of Basigin-HG was lower compared to 36 hours after siRNA introduction (not shown). Therefore, in Figure 3A, the fact that Basigin-HG expression level was not lowered despite the almost complete elimination of Basigin-LG expression after treatment with Basigin siRNA is thought to be due to the difference in the half-lives of each protein.

[0044] [Example 3] (Glycan analysis of basidine) Based on the results of Example 2 described above, we hypothesized that Basigin-LG is an essential molecule for regulating the nervous system vascular barrier. If Basigin-LG is an essential molecule, it will be possible to establish an artificial control method for the nervous system vascular barrier that specifically targets only Basigin-LG. As a result, it is thought that the side effects that are a concern can be reduced by suppressing the expression of Basigin-HG, which is responsible for many of the physiological functions of basigin.

[0045] Therefore, we investigated the glycan structures of Basigin-HG and Basigin-LG. The glycan structures of Basigin expressed in bEnd.3 cells were analyzed using Endoglycosidase H (Endo H), an enzyme that specifically degrades high-mannose type glycans among N-linked glycans, and Peptide-N-Glycosidase F (PNGase F), an enzyme that degrades all N-linked glycans.

[0046] Mouse brain vascular endothelial cell line (bEnd.3 cells: obtained from ATCC) was cultured. Seven days after reaching confluence, bEnd.3 cells were lysed in tissue lysis buffer (100 μl PBS containing 0.5% Triton X-100, 1% SDS, and 1% Protease / Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific)) and centrifuged at 15,000 rpm for approximately 15 minutes to obtain the supernatant. Next, to perform an enzymatic reaction with Endo H or PNGase F (New England Biolabs), 55 μl of the supernatant was mixed with Glycoprotein Denaturing Buffer (10x) (New England Biolabs) included with Endo H and PNGase F, and the mixture was reacted at 100°C for 10 minutes to obtain the reaction product. Subsequently, the obtained reaction product was mixed with the provided GlycoBuffer (10x) (New England Biolabs) and 1,000 units of Endo H or PNGase F, and reacted at 37°C for 60 minutes. As a control, the reaction was carried out with no buffer or with the above buffer only (without enzyme). Next, as in Example 1, each reaction solution was subjected to SDS-PAGE, transfer to PVDF, antibody treatment, and detection of chemiluminescence.

[0047] The results of the Western blot analysis are shown in Figure 4. It was shown that the glycans of Basigin-HG are degraded by PNGase F but not by Endo H, while the glycans of Basigin-LG are degraded by both PNGase F and Endo H. In other words, it was revealed that both the glycans of Basigin-HG and Basigin-LG are N-linked glycans, and that the glycan of Basigin-LG is a high-mannose type glycan.

[0048] [Example 4] (Relationship between basigin-LG and the regulation of the nervous system's vascular barrier - in vitro) We investigated in vitro whether Basigin-LG is a glycosylated basigin essential for regulating the nervous system's vascular barrier.

[0049] bEnd.3 cells were cultured in a monolayer using the same method as in Example 1. On the 7th day after the start of culture and reaching confluence, Endo H was treated with 1,000 units for 60 minutes, as in Example 3. As a control, the cells were reacted with either no buffer or the above buffer only (without enzyme). Furthermore, one group was subjected to 1% hypoxia (hypoxia), while the other group was left untreated (20% normal oxygen:normoxia). Changes in claudin-5 expression were observed by immunofluorescence staining, and the TEER of the bEnd.3 cell monolayer, an indicator of vascular barrier function, was measured. The immunofluorescence staining results were obtained according to the method of Cui D et al. (Sci. Rep. 5:12796(2015) doi:10.1038 / srep12796). Three fields of view were randomly selected from each sample and photographed. Three lines were drawn on each photograph, and the fluorescence intensity of claudin-5 at the intersections with the cell membrane was measured. The average value was calculated and quantified. Similarly, measurements were performed on three samples per group, and the average values ​​were compared. The immunofluorescence staining results are shown in Figure 5A, the graph quantifying the immunofluorescence staining results is shown in Figure 5B, and the results of measuring the TEER of the bEnd.3 cell monolayer are shown in Figure 5C.

[0050] Figures 5A and 5B show that no significant decrease in cell membrane Claudin-5 was detected in the bEnd.3 cell layer pretreated with Endo H, even under hypoxic stimulation. Figure 5C shows that the bEnd.3 cell layer pretreated with Endo H retained its ability to form a nervous system vascular barrier even under hypoxic stimulation. These results clearly indicate that basigin-LG is an essential molecule for regulating the nervous system vascular barrier.

[0051] [Example 5] (Relationship between basigin-LG and the regulation of the nervous system's vascular barrier - in vivo) We analyzed whether the results of the in vitro experimental system in Example 4, which showed that "Basigin-LG is an essential molecule for regulating the nervous system vascular barrier," could be reproduced in an in vivo experimental system using the mouse retina. The retina is a tissue that is formed during individual development as the central nervous system budding, and like the brain, it is part of the central nervous system. Since the vascular system of the retina can be observed and evaluated two-dimensionally along its entire length in the longitudinal direction, the retina was used as the analytical material in this example as a representative of the central nervous system.

[0052] 3,000 units of Endo H were administered intravenously to mice via the tail vein, and the mice were reared for 40 hours under either 20% normal oxygen conditions (Normoxia) or 7-8% hypoxia conditions (Hypoxia) alongside control mice (without Endo H administration). Subsequently, fluorescent dyes were administered intracardiacly as tracers, and retinal stretch specimens were prepared. The permeability of retinal vessels (an indicator of blood-retinal barrier function) was evaluated by observing the leakage of the injected tracer using a confocal microscope. Two fluorescent dyes were used as tracers: Tetramethylrhodamine-conjugated lysine fixable dextran (10 kDa; Thermo Fisher Scientific) and Hoechst® stain H33252 (534 Da; Thermo Fisher Scientific).

[0053] As shown in Figure 6, in mice not administered Endo H, pigment leakage from blood vessels was detected in the hypoxic retina, indicating that the neurovascular barrier was 'open'. In contrast, in mice administered Endo H, no significant pigment leakage from blood vessels was detected even in the hypoxic retina, indicating that the neurovascular barrier was 'closed'. Therefore, in vivo experiments also demonstrated that basigin-LG is an essential molecule for regulating the neurovascular barrier.

[0054] [Example 6] (Restoration of the vascular barrier by intravenous administration of Endo H to diabetic mice) We analyzed whether the results of the in vitro experimental system in Example 4, which showed that "Basigin-LG is an essential molecule for regulating the nervous system's vascular barrier," could be reproduced in an in vivo experimental system using the retinas of diabetic mice.

[0055] Seven-week-old C57B6 / N mice were fasted for four hours, and their blood glucose and body weight were measured. Next, 150 mg / kg of streptozotocin (dissolved in citrate buffer) was injected intraperitoneally. Four days after the streptozotocin injection, the mice were fasted for another four hours, and their blood glucose and body weight were measured again. Diabetic mice with blood glucose levels of 250 mg / dl or higher were designated as blood-retinal barrier disruption model mice.

[0056] The diabetic mice and non-diabetic mice (C57B6 / N mice injected intraperitoneally with citrate buffer) obtained above were each divided into two groups: one group administered 3,000 units of Endo H via the tail vein (+), and the other group not administered Endo H (-). Both groups were reared for 40 hours. Subsequently, a fluorescent dye was administered intracardiacly as a tracer, and retinal stretch specimens were prepared. The permeability of retinal vessels (an indicator of blood-retinal barrier function) was evaluated by observing the leakage of the injected tracer using a confocal microscope. The same fluorescent dye as in Example 5 was used as the tracer. The results are shown in Figure 7.

[0057] In diabetic mice not administered Endo H, pigment leakage from blood vessels in the retina was detected, indicating an 'open' neurovascular barrier. In contrast, Endo H administration eliminated significant pigment leakage from blood vessels in the retina; that is, Endo H administration closed the neurovascular barrier. Therefore, in an in vivo experimental system using diabetic mice, basigin-LG was demonstrated to be an essential molecule for regulating the neurovascular barrier.

[0058] In summary, the above examples demonstrate that Basigin-LG is an essential molecule for regulating the nervous system vascular barrier, that Basigin-LG is a protein modified with high-mannose type glycans, and that Basigin-LG is useful as a specific target for establishing artificial control methods for the nervous system vascular barrier. Furthermore, since other physiological functions besides nervous system vascular barrier regulation are reported to be carried out by Basigin-HG, it is expected that specifically targeting Basigin-LG will enable the establishment of artificial control methods for the nervous system vascular barrier with minimal side effects. [Industrial applicability]

[0059] The 'open state' of the nervous system-vascular barrier is being utilized in the treatment of neurological diseases, where a central role in the worsening of the condition.

Claims

1. An inhibitor of nervous system vascular barrier disruption, comprising endoglycosidase F1 or endoglycosidase H as an active ingredient, which releases or degrades the high-mannose type glycans of low-glycosylation basidine.

2. The inhibitor of nervous system vascular barrier disruption according to Claim 1, characterized in that endoglycosidase F1 or endoglycosidase H does not substantially release or degrade the complex-type glycans of highly glycosylated basidine.

3. An inhibitor of nervous system vascular barrier disruption according to claim 1 or 2, characterized in that the molecular weight of the low-glycozyme-modified basidine is 35,000 or less, and the molecular weight of the high-glycozyme-modified basidine is 40,000 to 60,000.

4. A therapeutic agent for neurological diseases caused by disruption of the nervous system vascular barrier, comprising endoglycosidase F1 or endoglycosidase H as an active ingredient, which releases or degrades the high-mannose type glycans of low-glycosylation basidine.

5. The neurological disease treatment agent according to claim 4, characterized in that the neurological disease is a neurocranial disease or a retinal neurological disease.

6. The neurological disorder treatment agent according to claim 5, characterized in that the neurological disorder is cerebral edema.

7. The neurological disease treatment agent according to claim 5, characterized in that the retinal neurological disease is retinal edema.

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