Reversible opener of the nervous system vascular barrier

Abstract

The present invention addresses the problem of providing a reversible opening agent for a nervous system vascular barrier.　To solve the problem, a reversible opening agent for a nervous system vascular barrier is prepared, the reversible opening agent comprising, as an active ingredient, a ligand having an agonistic action on basigin. It is preferable that: the ligand contains, as an active ingredient, a polypeptide, etc., comprising an amino acid sequence represented in SEQ ID NO: 1 or a salt thereof; and the ligand is a cyclophilin A that has lost peptidyl-prolyl cis-trans isomerase activity thereof.

Description

[Technical Field]

[0001] The present invention relates to agents that reversibly open the neurovascular barrier. [Background technology]

[0002] In multicellular organisms, including humans, adult nervous system tissues such as the brain and retina are separated from other tissues by barriers formed by tissue-specifically differentiated blood vessels, such as the blood-brain barrier (BBB) ​​and blood-retinal barrier (BRB). These barriers severely restrict the transfer of substances between the blood and the nervous tissue parenchyma. These nervous system vascular barriers are induced during ontogeny and are essentially closed in adults. However, by skillfully regulating their barrier function, they maintain an optimal tissue microenvironment for normal neuronal function. While the nervous system vascular barrier is crucial for the function of nervous tissues, it also prevents systemic therapeutic agents from reaching the affected nervous tissue parenchyma, making the disease intractable.

[0003] The development of novel therapeutic agents (drug discovery) for many intractable neurological disorders for which no effective treatments are yet available is currently underway. However, one of the most significant obstacles in this drug discovery process is the existence of the neurovascular barrier. Many attempts have been made to penetrate the neurovascular barrier and deliver drugs to neural tissue parenchyma. One such approach involves intravascular administration of a hypertonic solution of mannitol, which dehydrates cerebral vascular endothelial cells, deforming them and physically dissociating them, followed by administration of the desired therapeutic agent. However, this approach is not effective due to the cell damage it causes. Furthermore, a method for opening the neurovascular barrier using proteinases such as matrix metalloproteinases (see Non-Patent Document 1) has been disclosed, but this method destroys the molecules that form the neurovascular barrier, making temporary and reversible opening of the neurovascular barrier difficult.

[0004] Despite extensive research to date, no effective method has been established for crossing the neurovascular barrier and delivering drugs to neural tissue parenchyma. Therefore, the development of drugs or methods for artificially opening the closed neurovascular barrier with minimal side effects would remove a major barrier to drug discovery for neurological diseases, leading to improved patient prognosis and even the establishment of useful treatments aimed at complete cure. Furthermore, drugs that open the neurovascular barrier would be applicable to many intractable neurological diseases, making their contribution to clinical medicine extremely broad.

[0005] The present inventors began their analysis of the regulatory mechanisms of the neurovascular barrier, focusing first on the mechanism by which the neurovascular barrier opens in response to hypoxic stimulation. They reported a cascade in which hypoxic stimulation causes claudin-5 to disappear from the plasma membrane of vascular endothelial cells, resulting in neurovascular barrier opening (see Non-Patent Document 2). Further analysis identified a disintegrin and metalloproteinase (ADAM) 12, ADAM17, and basigin, which are plasma membrane molecules expressed in vascular endothelial cells that form the neurovascular barrier, as molecules involved in the hypoxic stimulation-induced disappearance of claudin-5 from the plasma membrane of vascular endothelial cells. Furthermore, they reported that while ADAM12 and ADAM17 function relatively specifically in the process by which the neurovascular barrier opens in response to hypoxic stimulation, basigin functions commonly in the process by which the neurovascular barrier opens in response to various stimuli, including not only hypoxic stimulation but also inflammation (see Patent Document 1, Non-Patent Documents 3 and 4).

[0006] Cyclophilin A (CypA) was identified as an intracellular molecule that binds to cyclosporin A, a drug that inhibits T lymphocyte activation. Subsequent studies have shown that CypA is expressed in cell types other than T lymphocytes, that it is also a molecule that functions when secreted extracellularly, and that it binds to basigin when secreted extracellularly. Furthermore, it has been shown that extracellular CypA is involved in several biological phenomena, such as the modulation of inflammatory processes.

[0007] It has been reported that cyclophilin A is conjugated with a compound that promotes transport across the blood-brain barrier, such as a transferrin receptor-binding antibody, and used as a neuroprotective agent (see Patent Document 2), that the cyclophilin A / MMP9 pathway is involved in the degradation of blood-brain barrier molecules by apoE4 (see Non-Patent Document 5), and that cyclophilin A and basigin (CD147) are involved in apoptosis during subarachnoid hemorrhage (see Non-Patent Document 6). However, it was not previously known that cyclophilin A reversibly opens the neurovascular barrier. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2017 / 073232 Brochure [Patent Document 2] Special Publication No. 2008-530029 [Non-patent literature]

[0009] [Non-Patent Document 1] Neuroscience Letters Volume 649, 10 Pages 7-13, 2017 [Non-patent document 2] Am. J. Pathol. Volume 170, Issue 4, Pages 1389-1397, 2007 [Non-patent document 3] Scientific Report 5, 12796; doi: 10.1038 / srep12796, 2015 [Non-patent document 4] Scientific Report 6, 38445; doi: 10.1038 / srep38445, 2016 [Non-Patent Document 5] JAMA Neurol. September 1; 70(9): 1198-1200; doi:10.1001 / jamaneurol.2013.3841 [Non-patent document 6] Critical Care Medicine, September 2015, Volume 43; Number 9 e369-381 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide an agent for reversibly opening the vascular barrier to the nervous system. [Means for solving the problem]

[0011] The present inventors have conducted extensive research to solve the above-mentioned problems. First, they found in vitro that stimulation of brain microvascular endothelial cells with cyclophilin A (CypA), a known basigin agonist, leads to the loss of neurovascular barrier properties in brain microvascular endothelial cells, and that this loss of neurovascular barrier properties is temporary and reversible. Furthermore, in vivo analysis revealed that injection of CypA into mice temporarily and reversibly opens the neurovascular barrier in vivo, and that pre-injection of CypA actually enables the delivery of systemically administered doxorubicin, a drug with low brain penetration, to mouse neural tissues. Based on these findings, the present invention has been completed.

[0012] That is, the present invention is as follows. [1] A reversible agent for opening the nervous system vascular barrier, containing as an active ingredient a ligand that has agonistic activity against basigin. [2] The reversible opening agent for the nervous system vascular barrier according to [1] above, wherein the ligand is a polypeptide ligand. [3] The polypeptide ligand (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are added, substituted, deleted, and / or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having the ability to reversibly open the nervous system vascular barrier; and (3) A polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1 and having the ability to reversibly open the nervous system vascular barrier; The reversible opening agent for the nervous system vascular barrier according to [2] above, characterized in that it contains as an active ingredient one or more polypeptides or salts thereof selected from the group consisting of: [4] The reversible opening agent for the nervous system vascular barrier according to [2] or [3] above, wherein the polypeptide ligand is cyclophilin A that has lost peptidyl-prolyl cis-trans isomerase activity. [5] The reversible opening agent for the vascular barrier of the nervous system according to any one of [1] to [4] above, for use in simultaneous or sequential administration with at least one therapeutic agent selected from therapeutic agents for neurodegenerative diseases, therapeutic agents for retinal diseases, therapeutic agents for psychiatric disorders, therapeutic agents for central nervous system tumors, and therapeutic agents for epilepsy.

[0013] Further, other aspects of the present invention are as follows. <1> A method for delivering at least one therapeutic agent selected from a therapeutic agent for neurodegenerative diseases, a therapeutic agent for retinal diseases, a therapeutic agent for psychiatric disorders, a therapeutic agent for central nervous system tumors, and a therapeutic agent for epilepsy to the brain of a subject, the method comprising the step of administering to the subject a reversible agent for opening the vascular barrier of the nervous system, which comprises, as an active ingredient, a ligand having an agonistic effect on basigin, and at least one therapeutic agent selected from a therapeutic agent for neurodegenerative diseases, a therapeutic agent for retinal diseases, a therapeutic agent for psychiatric disorders, a therapeutic agent for central nervous system tumors, and a therapeutic agent for epilepsy, in simultaneous or sequential combination. (2) Use of a ligand having agonistic activity against basigin to produce a reversible agent for opening the nervous system vascular barrier. [Effects of the Invention]

[0014] By using the reversible opening agent for the vascular barrier of the present invention, it becomes possible to reversibly open the vascular barrier of the nervous system. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the results of treatment with 200, 300, or 400 ng / ml of CypA in Example 1. Figure 1a shows the results of fluorescent immunostaining, Figure 1b shows the results of quantification of claudin-5 signals on the cell membrane, and Figure 1c shows the results of measuring the transdermal electrical resistance (TEER) of the cell monolayer. [Figure 2] 2A and 2B show the results of treatment with 300 ng / ml of CypA with PPIase activity (CypA ​​in the figure) or without PPIase activity (CypA / PPIase- in the figure) in Example 2. Figure 2A shows the results of fluorescent immunostaining, Figure 2B shows the results of quantification of claudin-5 signals on the cell membrane, and Figure 2C shows the results of measuring the TEER of the cell monolayer. [Figure 3] 3A and 3B show the results of time-dependent treatment with 300 ng / ml of CypA in Example 3. Fig. 3A shows the results of fluorescent immunostaining, Fig. 3B shows the results of quantification of claudin-5 signals on the cell membrane, and Fig. 3C shows the results of measuring the TEER of the cell monolayer. [Figure 4] Figure 4 shows the results of examining changes in barrier function due to CypA administration using mouse retinal tissue in Example 4. Figure 4a shows the results of fluorescent immunostaining, and Figure 4b shows the results of leakage of intravenously injected tracer dye. [Figure 5] 1 shows the results of observing the uptake of doxorubicin into the cerebrum, liver, and kidney using a laser confocal microscope after intravenous administration of doxorubicin to mice with or without CypA pre-administration in Example 5. The upper panel shows mice without CypA pre-administration, and the lower panel shows mice with CypA pre-administration. a and e show HE staining findings of the cerebrum, and b, c, d, f, g, and h show doxorubicin fluorescence findings of the cerebrum, liver, and kidney. [Figure 6] FIG. 1 shows the results of quantifying the uptake of doxorubicin into the cerebrum, liver, and kidney after intravenous administration of doxorubicin to mice with or without prior administration of CypA in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0016] Basigin, as used herein, is a glycoprotein belonging to the immunoglobulin superfamily that is localized in the cell membrane and is also known as EMMPRIN (extracellular matrix metalloproteinase inducer), CD147 (cluster of differentiation 147), HT7, OX-47, or fourth 22. Basigin is known to be a receptor for cyclophilin A (CypA), a member of the cyclophilin family that possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity. Human basigin is preferred, and examples of human basigin include SEQ ID NOS: 2-4, which are published at the National Center for Biotechnology Information (NCBI) under accession numbers NP_001719.2, NP_940991.1, and NP_940992.1, respectively.

[0017] The ligand having agonistic activity against basigin in this specification is not particularly limited as long as it is a compound that binds to basigin and reversibly opens the neurovascular barrier. Such a ligand may be any of a polypeptide, an antibody, a protein, a nucleic acid, and a low molecular weight compound, but is preferably a polypeptide.

[0018] More preferred examples of such polypeptide ligands include the following polypeptides or salts thereof: (1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in which one or several amino acids have been added, substituted, deleted, and / or inserted, and which has the ability to reversibly open the nervous system vascular barrier; (3) one or more peptides selected from the group consisting of peptides having an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 1 and having the ability to reversibly open the nervous system vascular barrier;

[0019] Furthermore, the polypeptide ligand can also include cyclophilin A (CypA), a member of the cyclophilin family. Cyclophilin A (CypA) is a substance identified as an intracellular molecule that binds to cyclosporin A, a drug that suppresses the activation of T lymphocytes. Cyclophilin A The amino acid sequence of this compound is available from the NCBI website and is published under the accession number NP_066953.1 (SEQ ID NO: 1). Cyclophilin A (CypA) may be either a CypA with peptidyl-prolyl cis-trans isomerase (PPIase) activity or a CypA without PPIase activity, but from the viewpoint of further reducing side effects, a CypA without PPIase activity is preferred.

[0020] The above-mentioned "amino acid sequence in which one or several amino acids have been added, substituted, deleted, and / or inserted" refers to an amino acid sequence in which any number of amino acids have been added, substituted, deleted, and / or inserted, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 3, even more preferably 1 to 2, and most preferably 1. The above-mentioned "amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1" means that the sequence identity with the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more.

[0021] The polypeptide may be a natural or modified polypeptide. Examples of modified polypeptides include D- or L-peptides; α-, β-, or γ-peptides; N-methylpeptides; azapeptides; polypeptides having one or more amide (i.e., peptide) bonds substituted with one or more urea, thiourea, carbamate, or sulfonylurea bonds; and polypeptides modified by the addition of a biochemical functional group. The C-terminus of the polypeptide may be a carboxyl group (-COOH), carboxylate (-COO-), amide (-CONH), or ester (-COOR). Examples of R in the ester include C alkyl groups such as methyl, ethyl, n-propyl, isopropyl, or n-butyl; C cycloalkyl groups such as cyclopentyl and cyclohexyl; C aryl groups such as phenyl and α-naphthyl; and phenyl-C alkyl groups such as benzyl and phenethyl.

[0022] The salt in the "polypeptide or a salt thereof" herein is not particularly limited as long as it is a pharmacologically acceptable salt. Specific examples include inorganic salts such as hydrochloride, sulfate, and phosphate; organic acid salts such as acetate and citrate; alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as magnesium salt and calcium salt.

[0023] The CypA may be commercially available, may be prepared by known genetic engineering techniques based on the nucleotide sequence encoding CypA, or may be prepared by known amino acid synthesis techniques based on the amino acid sequence of CypA. The method for preparing the polypeptide is not particularly limited, and the polypeptide can be prepared according to known peptide synthesis methods based on the amino acid sequence of the polypeptide. Peptide synthesis methods may be, for example, solid-phase synthesis or liquid-phase synthesis. When preparing the polypeptide using solid-phase synthesis, for example, a peptide or amino acid that can constitute the polypeptide can be condensed with the remaining portion using solid-phase synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method), followed by removing the protecting group (if the product has a protecting group) to prepare the desired polypeptide. For solid-phase synthesis, commercially available peptide synthesizers such as APEX396 (Advanced Chemtech), 433A (Applied Biosystems), PS3 (Protein Technologies), 9050 (Perceptive), and PSSM-8 (Shimadzu) can be used. Resins used in solid-phase synthesis are not particularly limited, and examples include Rink amide AM Resin, Fmoc-AA-Wang Resin, and AA-2-Cl-Trt Resin. Furthermore, when preparing the above polypeptides using liquid-phase synthesis, the polypeptides can be prepared by stepwise condensation of N-protected amino acid derivatives one residue at a time. Depending on the presence or absence of protecting groups, methods such as the dicyclohexylcarbodiimide (DCC) method, the active ester method, and the mixed acid anhydride method can be used. The polypeptides can also be prepared using condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent, and diisopropylcarbodiimide (DIPCDI). Furthermore, commercially available products can also be used.

[0024] As used herein, the term "neurovascular barrier" refers to a mechanism that restricts the exchange of substances between blood and tissue fluid of the nervous system, and suitable examples include the blood-brain barrier, which restricts the exchange of substances between blood and tissue fluid of the brain, and the blood-retina barrier, which restricts the exchange of substances between blood and tissue fluid of the retina. Note that the term "nervous blood vessels" refers to blood vessels in nervous system tissues and does not include blood vessels in tissues other than nervous system tissues.

[0025] As used herein, "reversible opening of the vascular barrier to the nervous system" means that the mechanism that limits the exchange of substances between blood and nervous tissue fluid is rendered inoperative or impaired, thereby temporarily and reversibly enabling exchange of substances between blood and nervous tissue fluid. Here, reversible opening means that the vascular barrier to the nervous system does not remain open, but rather opens in such a way that, after a predetermined period of time, the exchange of substances between blood and nervous tissue fluid can be restored to the restricted state.

[0026] As used herein, "temporary" refers to the time that has elapsed since the administration of the reversible opening agent of the nervous system vascular barrier of the present invention to a subject, and can be 0.2 to 24 hours, with the lower limit being 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, and the upper limit being 20 hours, 18 hours, 16 hours, 12 hours, 10 hours, or 9 hours.

[0027] The reversible nervous system vascular barrier opening agent of the present invention may be administered simultaneously or sequentially with at least one therapeutic agent selected from therapeutic agents for neurodegenerative diseases, retinal diseases, psychiatric disorders, central nervous system tumors, and epilepsy. Here, the term "administered simultaneously" means that two or more agents are administered simultaneously to the same subject. Additionally, the term "administered sequentially" means that two or more agents are administered sequentially, i.e., sequentially at a certain interval, or separately to the same subject.

[0028] In one embodiment according to the invention, the reversible opening of the nervous system vascular barrier by the reversible opening agent lasts for 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, or 5 minutes.

[0029] When the doses are administered sequentially or individually at regular intervals, the "regular intervals" can be 0.5 minutes to 10 hours, with lower limits of, for example, 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours, and upper limits of, for example, 8 hours, 6 hours, 5 hours, 4 hours, and 3 hours.

[0030] The agent for opening the nervous system vascular barrier of the present invention may contain as an active ingredient a ligand having agonistic activity against basigin, and may further contain, as necessary, pharmaceutically acceptable conventional ingredients such as carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonicity agents, additives, coating agents, solubilizers, lubricants, glidants, solubilizers, flavorings, sweeteners, solvents, gelling agents, nutrients, etc. Specific examples of such ingredients include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0031] The method of administration of the agent for opening the vascular barrier of the present invention is not particularly limited as long as it achieves the desired effect of opening the vascular barrier of the present invention, and examples thereof include intravenous administration, oral administration, intravitreal administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, and pulmonary administration. Furthermore, the dosage of the agent for opening the vascular barrier of the present invention is not particularly limited and can be adjusted appropriately depending on the physical condition, condition, weight, age, sex, etc. of the subject or test animal. The dosage may be, for example, 0.01 μg to 100 g / kg body weight, more preferably 0.1 μg to 10 g / kg body weight, and even more preferably 1 μg to 1 g / kg body weight per day, and may be administered in a single dose or multiple doses (e.g., 2 to 4 times) per day.

[0032] The therapeutic agent is not particularly limited as long as it is at least one therapeutic agent selected from the group consisting of therapeutic agents for neurodegenerative diseases, therapeutic agents for retinal diseases, therapeutic agents for psychiatric diseases, therapeutic agents for central nervous system tumors, and therapeutic agents for epilepsy. Examples of therapeutic agents for neurodegenerative diseases include therapeutic agents for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, multiple sclerosis, myasthenia gravis, cerebral infarction, and vascular dementia. Examples of therapeutic agents for retinal diseases include therapeutic agents for retinal diseases such as diabetic retinopathy, age-related macular degeneration, retinal edema, retinal detachment, proliferative vitreoretinopathy, uveitis, ocular infection, retinopathy of prematurity, neovascular maculopathy, and chorioretinitis. Examples of therapeutic agents for psychiatric diseases include therapeutic agents for depression, schizophrenia, and panic disorder. Examples of therapeutic agents for central nervous system tumors include therapeutic agents for central nervous system tumors such as glioma, central nervous system malignant lymphoma, glioblastoma multiforme, gliosarcoma, etc. Examples of such therapeutic agents include low molecular weight compounds, antisense oligonucleotides, ribozymes, proteins, polypeptides, and peptides.

[0033] The subjects to which the agent for opening the nervous system vascular barrier of the present invention is administered are not particularly limited, and examples thereof include mammals such as humans, monkeys, cows, horses, sheep, pigs, dogs, cats, rats, mice, and hamsters.

[0034] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Example]

[0035] [Example 1] (In vitro analysis using monolayer culture of mouse cerebrovascular endothelial cell line (1)) As mentioned above, the present inventors have previously reported that the loss of claudin-5 from the plasma membrane of vascular endothelial cells leads to vascular barrier opening. Therefore, we investigated the effect of CypA administration on the expression level of claudin-5.

[0036] In vitro, mouse brain microvascular endothelial cell line bEnd.3 cells (obtained from the American Type Culture Collection) were grown as monolayers in Dulbecco's modified Eagle's medium containing 4500 mg / L glucose (Sigma-Aldrich) supplemented with 10% FBS (fetal bovine serum) at 37°C under 5% CO2. CypA (200, 300, or 400 ng / ml; BioVendor Research and Diagnostic Products) was added to the culture supernatant of the monolayer-cultured bEnd.3 cells and incubated for 3 hours. The expression of claudin-5 in bEnd.3 cells was then monitored by immunofluorescence staining, and the claudin-5 signal on the cell membrane was quantified. Furthermore, the TEER of the bEnd.3 cell monolayer, an indicator of vascular barrier function, was measured.

[0037] Fluorescent immunostaining was performed as follows. First, cultured bEnd.3 cells were fixed with 100% methanol for 5 minutes at room temperature and then incubated with 10% Non-Immune Goat Serum (Invitrogen) for 30 minutes to block nonspecific antibody binding. Next, the cells were incubated overnight at 4°C with a rabbit polyclonal antibody against claudin-5 (1 / 25 dilution, Invitrogen). After washing with phosphate-buffered saline (PBS), the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1 / 200 dilution, Eugene) for 1 hour at room temperature, protected from light. Stained cells were then mounted in Fluoromount (Diagnostic BioSystems) and observed under a Zeiss LSM5 Pascal laser confocal microscope (Carl Zeiss). For quantitative analysis, the fluorescence intensity of claudin-5 on the plasma membrane was measured using the operation menu installed in LSM5 Pascal 6, 7.3. Random photographs were taken of the culture dish field, and five lines were drawn on each photograph. The fluorescence intensity at points on the cell membrane where the lines intersected was then quantified. The average fluorescence intensity at approximately 80 points was calculated as the expression level of claudin-5 on the cell membrane of each monolayer. All experiments were performed in triplicate.

[0038] Figure 1a shows the results of immunofluorescence staining, Figure 1b shows the quantification of claudin-5 signal on the cell membrane, and Figure 1c shows the TEER measurement results of bEnd.3 cell monolayers. Figures 1a and 1b show that the claudin-5 level on the cell membrane significantly decreased 3 hours after CypA administration. Furthermore, Figure 1c shows that CypA administration correlated with the claudin-5 level on the cell membrane, and that a significant decrease in barrier function occurred 3 hours after CypA administration. These results confirmed that a single administration of CypA opens the barrier of bEnd.3 cell monolayers.

[0039] [Example 2] (In vitro analysis using monolayer culture of mouse cerebrovascular endothelial cell line (2)) CypA is known to have PPIase activity, so we investigated whether PPIase activity is involved in the barrier opening action of CypA by using a CypA lacking PPIase activity.

[0040] Fluorescent immunostaining, quantification of claudin-5 signals on the cell membrane, and measurement of TEER of bEnd.3 cell monolayers were performed in the same manner as in Example 1, except that CypA lacking PPIase activity (300 ng / ml: BioVendor Research and Diagnostic Products) was used. The results are shown in Figures 2a to 2c, respectively.

[0041] As shown in Figures 2a-c, the use of CypA lacking PPIase activity resulted in a significant decrease in claudin-5 signaling on the cell membrane, as well as a significant decrease in barrier function, indicating that CypA-mediated opening of the neurovascular barrier is independent of PPIase activity.

[0042] [Example 3] (In vitro analysis using monolayer culture of mouse cerebrovascular endothelial cell line (3)) To evaluate the time course of CypA-induced opening of the neurovascular barrier, we performed the same experiment as in Example 1, varying the treatment time with CypA. Specifically, the CypA concentration was 300 ng / ml, and the cells were incubated for 1, 3, 6, 9, and 12 hours after administration. After this, we performed immunofluorescence staining, quantification of claudin-5 signals on the cell membrane, and measurement of TEER of bEnd.3 cell monolayers.

[0043] Figure 3a shows the results of immunofluorescence staining, Figure 3b shows the quantification of claudin-5 signal on the cell membrane, and Figure 3c shows the time course of TEER measurements of bEnd.3 cell monolayers. Figures 3a and 3b show that the level of claudin-5 on the cell membrane significantly decreased 3 hours after CypA administration, but recovered to pre-CypA levels by 6 hours. Furthermore, Figure 3c shows that CypA administration correlated inversely with the level of claudin-5 on the cell membrane, and a significant decrease in barrier function occurred 3 hours after CypA administration, which returned to pre-CypA levels by 6 hours. These results confirm that a single dose of CypA temporarily and reversibly opened the barrier of bEnd.3 cell monolayers. Furthermore, the opened barrier function recovered after several hours in a self-limiting manner.

[0044] [Example 4] (In vivo analysis using mouse retinal tissue) Based on the results of Example 1, changes in barrier function in vivo due to CypA administration were investigated using mouse retinal tissue. The retina is a tissue formed by the budding of the central nervous system during ontogeny, and is a part of the central nervous system, just like the brain. Because the retinal vasculature can be observed and evaluated two-dimensionally along its entire length in the longitudinal direction, in this example, the retina was used as an analytical material as a representative of the central nervous system.

[0045] CypA (200 μg / kg) was administered intravenously to 7-week-old male C57B6 / N mice (Japan SLC). Three, six, or 24 hours after the vitreous injection, changes in the expression level of claudin-5 localized in the endothelial cell membrane of the retinal vasculature and retinal vasculature permeability were measured. Changes in claudin-5 expression were measured by fluorescent immunostaining using a rabbit polyclonal antibody against claudin-5, as in Example 1. The retinal vasculature permeability was measured as follows.

[0046] First, 500 μL of PBS containing 100 μg / mL Hoechst dye H33258 (molecular weight 534 Da, Sigma-Aldrich) and 1 mg / mL tetramethylrhodamine-conjugated lysine-fixable dextran (molecular weight 10,000 Da, Thermo Fisher Scientific) was injected into the left ventricle. After injection of the Hoechst dye, the eyes were enucleated and immediately fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature, protected from light, to prepare retinal flat mounts. They were mounted in fluorescent mounting medium and examined under a Zeiss LSM510 META laser confocal microscope (Carl Zeiss) to measure retinal vasculature permeability (an indicator of blood-retinal barrier function) by observing leakage of the injected tracer. Experiments were performed at least three times independently.

[0047] As shown in Figure 4a, claudin-5 expression levels on the plasma membrane of peripheral microvascular endothelial cells were found to decrease 3 h after intravenous injection of CypA, but they returned to physiological levels by 24 h. Furthermore, as shown in Figure 4b, leakage of the intravenously injected tracer dye increased 3 h after injection of CypA, decreased by 6 h compared with 3 h, and returned to undetectable physiological levels by 24 h. These data demonstrate that a single intravenous injection of CypA results in a transient and reversible opening of the retinal vasculature's blood-vascular barrier.

[0048] [Example 5] (Analysis of drug uptake into neural tissue parenchyma) Based on the results of Examples 1 to 4 above, we conducted the following experiment, suspecting that administering CypA in combination with a therapeutic agent might reversibly open the vascular barrier of the nervous system, allowing the therapeutic agent to reach the substance of the nervous tissue.

[0049] Doxorubicin was administered intravenously to mice with and without CypA pretreatment, and the uptake of doxorubicin into the cerebrum, liver, and kidney was monitored 3 hours later. The fluorescence emitted by doxorubicin was used to assess the uptake of doxorubicin into the cerebrum, liver, and kidney.

[0050] Doxorubicin hydrochloride (6.25 mg / kg; FUJIFILM Wako Pure Chemical Corporation), an anticancer drug with low brain penetration, was injected into the tail vein of mice with or without preinjection of CypA (200 μg / kg). Preinjection of CypA was performed intravenously 3 h before the doxorubicin injection. Mice were sacrificed 3 h after doxorubicin injection, and the cerebrum, liver, and kidney were collected and cryo-embedded in OCT compound. Next, 30 μm-thick cryosections were prepared and observed under an LSM710 laser confocal microscope (Carl Zeiss). Doxorubicin fluorescence was excited with a 488 nm argon laser, and emission was observed through a 530 nm long-pass filter.

[0051] The upper panels (a, b, c, d) of Figure 5 show the cerebrum, liver, and kidney of mice without CypA pre-administration, with a showing HE staining of the cerebrum, and b, c, and d showing doxorubicin fluorescence of the cerebrum, liver, and kidney, respectively. The lower panels (e, f, g, and h) show the cerebrum, liver, and kidney of mice with CypA pre-administration, with e showing HE staining of the cerebrum, and f, g, and h showing doxorubicin fluorescence of the cerebrum, liver, and kidney, respectively. Furthermore, Figure 6 is a graph quantifying the doxorubicin uptake in the cerebrum, liver, and kidney shown in Figure 5. As is clear from Figure 5, no doxorubicin fluorescence signal was detected in the cerebrum of mice without CypA pre-administration (upper panel). In contrast, a significant doxorubicin fluorescence signal was detected in the cerebrum of mice with CypA pre-administration (lower panel). These results indicate that pre-administration of CypA enabled intravenously administered doxorubicin to penetrate the vascular barrier and reach the neural tissue parenchyma. Therefore, CypA can be used to temporarily and reversibly open the vascular barrier in neural tissue with minimal damage to endothelial cells, allowing drugs to reach the neural tissue and potentially treat neurological disorders. Furthermore, in the liver and kidney, which have vasculature lacking barrier function, strong fluorescent doxorubicin signals were detected in the parenchyma of mice, regardless of whether or not CypA was pre-administered, and the fluorescent signal intensity was not significantly affected by CypA pre-administration. [Industrial Applicability]

[0052] Among the therapeutic strategies for intractable neurological diseases, the present invention makes it possible to deliver drugs to nervous tissues that cannot be delivered using current medical and pharmaceutical technologies. This means that the options for drugs that can be used to treat neurological diseases will be greatly expanded, and by removing a major obstacle that has always stood in the way of drug discovery, namely the inability to pass through the nervous system's vascular barrier, drugs will be usable in the medical industry.

Claims

1. It has an agonist effect on basigin, (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (2) A polypeptide consisting of an amino acid sequence in which 1 to 3 amino acids are added, substituted, deleted, and / or inserted in the amino acid sequence shown in SEQ ID NO: 1, and having a reversible opening action of the nervous system vascular barrier; and (3) A polypeptide consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 and having the ability to reversibly open the nervous system vascular barrier; A reversible agent for opening the nervous system vascular barrier, comprising as an active ingredient one or more polypeptides or salts thereof selected from the group consisting of:

2. A reversible opening agent for the nervous system vascular barrier according to claim 1, characterized in that the polypeptide is cyclophilin A that has lost peptidyl-prolyl cis-trans isomerase activity.

3. A reversible opening agent for the nervous system vascular barrier according to claim 1 or 2, for use in simultaneous or sequential administration with at least one therapeutic agent selected from therapeutic agents for neurodegenerative diseases, therapeutic agents for retinal diseases, therapeutic agents for psychiatric disorders, therapeutic agents for central nervous system tumors, and therapeutic agents for epilepsy.

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