Pharmaceutical composition for preventing or treating cerebral small vessel disease

Abstract

This pharmaceutical composition for preventing or treating cerebral small vessel disease comprises, as an active ingredient, a fibronectin accumulation inhibitor or a tissue metalloprotease inhibitor-3 accumulation inhibitor. In the pharmaceutical composition, the fibronectin accumulation inhibitor may be an expression inhibitor of fibronectin or Adamtsl2. In the pharmaceutical composition, the fibronectin accumulation inhibitor may be expression inhibitors of fibronectin and Adamtsl2. In the pharmaceutical composition, the fibronectin accumulation inhibitor may be an angiotensin II 1-type receptor antagonist. In the pharmaceutical composition, the fibronectin accumulation inhibitor may be candesartan.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cerebral small vessel disease. This application claims priority based on Japanese Patent Application No. 2020-084683 filed in Japan on May 13, 2020, the content of which is incorporated herein by reference.

Background Art

[0002] Cerebral small vessel disease, which develops due to cerebrovascular degeneration with hypertension and diabetes as risk factors, causes dementia and gait disorders. The pathogenesis of cerebral small vessel disease is unknown, and no effective treatment has been developed for this disease. One of the major reasons for this is the lack of an appropriate animal model that functionally and pathologically reproduces the disease state. Vascular disease research with similar risk factors has remained at the level of peripheral large vessel research. The main pathological condition of these blood vessels is atherosclerotic, which is different from the arteriolosclerotic type presented by cerebral small vessel disease. Therefore, it is necessary to clarify the molecular pathological mechanism of cerebral small vessel disease and develop a treatment method in light of the pathological condition of this disease.

[0003] Cerebral small vessel diseases include not only sporadic cases but also hereditary cerebral small vessel diseases caused by several genetic backgrounds. Among them, Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), which is caused by mutations leading to the loss of expression or enzymatic activity of high-temperature requirement serine protease A1 (HTRA1), a type of serine protease, is known to present a disease pattern similar to sporadic cerebral small vessel diseases, such as arteriolosclerosis of cerebral small vessels and extensive leukoencephalopathy (see, for example, Non-Patent Document 1). HTRA1 cleaves a wide range of substrates, such as precursor transforming growth factor β1 (pro-TGFβ1), fibronectin, and latent TGFβ-binding protein 1 (LTBP1). Furthermore, the accumulation of these substrates has been confirmed in the brains of CARASIL patients (see, for example, Non-Patent Document 2) and in the brains obtained from HTRA1-deficient (HTRA1- / -) mice (see, for example, Non-Patent Document 3). However, the site of the cerebral artery where these substrates first accumulate and which substrate contributes to arterial damage have not yet been clarified.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] The present invention has been made in view of the above circumstances, and provides a novel pharmaceutical composition effective for the prevention or treatment of cerebral small vessel disease. [Means for Solving the Problems]

[0006] As a result of intensive studies to achieve the above object, the inventors found that in HTRA1-deficient mice, fibronectin and fibronectin-binding proteins, as well as tissue metalloprotease inhibitor 3 (TIMP3), accumulated in the intima of leptomeningeal arteries and intracerebral arterioles, and then developed arteriopathy characterized by a decrease in cerebral blood flow, intimal thickening, and neointima formation of the internal elastic lamina. Based on this finding, the inventors found that administering a drug that suppresses the accumulation of fibronectin or TIMP3 in the intima improves the symptoms of arteriopathy, and thus completed the present invention.

[0007] That is, the present invention includes the following aspects. (1) A pharmaceutical composition for preventing or treating cerebral small vessel diseases caused by hypertension and diabetes, and cerebral small vessel diseases accompanied by the accumulation of fibronectin in the cerebral small vessel intima, excluding Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, and containing a fibronectin accumulation inhibitor as an active ingredient, The pharmaceutical composition, wherein the fibronectin accumulation inhibitor is an angiotensin II type 1 receptor antagonist. (2) The pharmaceutical composition according to (1), wherein the angiotensin II type 1 receptor antagonist is candesartan or telmisartan. (3) The pharmaceutical composition according to (1) or (2), wherein the angiotensin II type 1 receptor antagonist is telmisartan. (4) The pharmaceutical composition according to any one of (1) to (3), wherein the cerebral small vessel disease is a hereditary cerebral small vessel disease. (5) The pharmaceutical composition according to any one of (1) to (4), wherein the cerebral small vessel disease is Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Advantages of the Invention

[0008] According to the pharmaceutical composition of the above aspect, cerebral small vessel diseases can be prevented or treated.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] Hereinafter, a pharmaceutical composition for preventing or treating cerebral small vessel disease according to an embodiment of the present invention will be described in detail.

[0011] <Cerebral small vessel disease> Generally, "small vessel disease (SVD)" refers to pathological changes in the small blood vessels of the brain or the resulting microlesions, and is a pathological condition that encompasses the relatively homogeneous pathology and clinical manifestations caused by these. Cerebral small vessel disease is classified into sporadic cerebral small vessel disease caused by hypertension or diabetes, and hereditary cerebral small vessel disease. Sporadic cerebral small vessel disease includes pathological conditions such as multiple lacunar infarcts, Binswanger's disease, diffuse white matter lesions (leukoaraiosis), amyloid angiopathy, etc. Examples of hereditary cerebral small vessel disease include, for example, Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (onset due to mutations in the HTRA1 gene), Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (onset due to mutations in the Notch3 gene), HERNS (Hereditary endotheliopathy with retinopathy, nephropathy, and stroke), HANAC (Hereditary angiopathy with nephropathy, aneurysms and muscle cramps), Familial Cerebral Amyloid Angiopathy, Familial British Dementia, mitochondrial leukoencephalopathy with lactic acidosis and stroke like episodes (MELAS), Fabry disease, etc.

[0012] These cerebral small vessel diseases can be the main cause of vascular dementia. In addition, the involvement of cerebral small vessel disease is also suspected in degenerative diseases such as Alzheimer's dementia and amyotrophic lateral sclerosis, and in Alzheimer's dementia, there are many cases with a high probability of coexisting with cerebral small vessel disease.

[0013] Among them, as shown in the examples described later, the pharmaceutical composition of the present embodiment is particularly effective for the prevention or treatment of sporadic cerebral small vessel disease, CARASIL, or CADASIL, which is accompanied by the accumulation of fibronectin (FN) or tissue metalloprotease inhibitor 3 (TIMP3). Further, the pharmaceutical composition of the present embodiment is also effective for the prevention or treatment of vascular dementia caused by cerebral small vessel disease accompanied by the accumulation of FN or TIMP3, and Alzheimer's dementia complicated with the cerebral small vessel disease.

[0014] <Pharmaceutical composition for preventing or treating cerebral small vessel disease> The pharmaceutical composition for preventing or treating cerebral small vessel disease of the present embodiment (hereinafter, may be simply abbreviated as "the pharmaceutical composition of the present embodiment") contains a fibronectin (FN) accumulation inhibitor or a tissue metalloprotease inhibitor 3 (TIMP3) accumulation inhibitor as an active ingredient.

[0015] In the present specification, "containing as an active ingredient" means containing a therapeutically effective amount of an FN accumulation inhibitor or a TIMP3 accumulation inhibitor. Here, the "therapeutically effective amount" means the amount of an FN accumulation inhibitor or a TIMP3 accumulation inhibitor, or the amount of a combination of an FN accumulation inhibitor or a TIMP3 accumulation inhibitor and one or more active agents that induces a biological, medical effect or response required by a physician, clinician, veterinarian, researcher, or other appropriate professional when administered according to a desired therapeutic measure. A preferred therapeutically effective amount is an amount that improves the symptoms of cerebral small vessel disease. Further, the "therapeutically effective amount" includes an amount effective for prevention, that is, an amount suitable for preventing a disease state.

[0016] So far, the treatment target of cerebral small vessel disease has not been known, and a drug effective for treatment has been demanded. In contrast, the inventors have for the first time found that in HTRA1-deficient mice, which are CARASIL model mice, FN and FN-binding proteins, as well as TIMP3, accumulate in the intima of leptomeningeal arteries and intracerebral arterioles, and then develop arteriopathy characterized by a decrease in cerebral blood flow, intimal thickening, and internal elastic lamina neogenesis. In addition, since the accumulation of FN and TIMP3 is also observed in CARASIL patients and CADASIL patients, as shown in the examples described later, it has been found that by suppressing the accumulation of FN or TIMP3, a decrease in cerebral blood flow, intimal thickening, and internal elastic lamina neogenesis can be suppressed, leading to the completion of the present invention. Note that the pharmaceutical composition of the present embodiment suppresses the accumulation of FN or TIMP3, but does not suppress the expression of TGF-β (Transforming growth factor β), as shown in the examples described later. That is, the molecular mechanism newly discovered by the inventors this time is completely different from the conventional pathological mechanism of CARASIL that focuses on the regulatory mechanism of TGF-β signal by HTRA1.

[0017] According to the pharmaceutical composition of the present embodiment, cerebral small vessel disease can be effectively prevented or treated.

[0018] [Fibronectin (FN)] Generally, fibronectin (FN) is a cell-adhesive glycoprotein that binds to collagen, fibrin, and heparin, which are components of the extracellular matrix protein (ECM). Fibronectin exists in a soluble form in plasma and is composed of two 250 kDa subunits linked by disulfide bonds. Insoluble fibronectin is a large complex in which the subunits are cross-linked. There are several isoforms of fibronectin, all of which are synthesized from a single gene. The structures of these isoforms are made up of three types of repetitive internal regions (modules I, II, III), and these regions are distinguished by differences in length and the presence or absence of disulfide bonds. Alternative splicing of pre-mRNA leads to combinations of the three types of regions and also creates variable regions.

[0019] The FN targeted by the FN accumulation inhibitor may be at least any one of the above three isoforms, but among them, it is preferable to inhibit the accumulation of all three isoforms.

[0020] [Tissue metalloprotease inhibitor 3 (TIMP3)] Generally, tissue metalloprotease inhibitor 3 (TIMP3) is a protein contained in the matrix metalloprotease inhibitor (TIMP) family, binds to the ECM, and promotes the inhibition of matrix metalloprotease (MMP). TIMP3 has been reported to be an important protein in the pathophysiology of CADASIL (see, for example, Reference 1: “Monet-Lepretre M et al., “Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.”, Brain: a journal of neurology, Vol. 136, Issue 6, pp. 1830-1845, 2013.”).

[0021] [FN accumulation inhibitor or TIMP3 accumulation inhibitor] Specific examples of the FN accumulation inhibitor or TIMP3 accumulation inhibitor include, for example, compounds having an activity to decompose FN or TIMP3, expression inhibitors of FN or TIMP3, specific binding substances of FN or TIMP3, and the like. Further, examples of the FN accumulation inhibitor include pUR4 known as a fibronectin fibrosis inhibitory peptide (see, for example, Reference 2: “Tomasini-Johansson BR et al., “PEGylated pUR4 / FUD peptide inhibitor of fibronectin fibrillogenesis decreases fibrosis in murine Unilateral Ureteral Obstruction model of kidney disease.”, PLoS One, Vol. 13, Issue 10: e0205360, 2018.”), which can also be used. Among them, the FN accumulation inhibitor or TIMP3 accumulation inhibitor is preferably an inhibitor that inhibits the accumulation of both FN and TIMP3, and more preferably an expression inhibitor of FN and TIMP3.

[0022] (Expression inhibitor of FN or TIMP3) The expression inhibitor of FN or TIMP3 may be any one that inhibits the expression of FN or TIMP3, and examples thereof include siRNA, shRNA, miRNA, ribozyme, antisense nucleic acid, low molecular weight compound, and the like. By administering these expression inhibitors of FN or TIMP3, the expression level of FN or TIMP3 can be decreased, and the symptoms of cerebral small vessel disease can be improved. That is, by administering these expression inhibitors of FN or TIMP3, cerebral small vessel disease can be prevented or treated. Among them, the expression inhibitor of FN or TIMP3 preferably inhibits the expression of both FN and TIMP3.

[0023] In addition, as shown in the examples described below, since the fibrosis of FN in the ECM is controlled by the expression of Adamtsl2 present upstream thereof, as an FN accumulation inhibitor, it is preferably an inhibitor of the expression of FN or Adamtsl2, and more preferably an inhibitor of the expression of FN and Adamtsl2.

[0024] siRNA (small interfering RNA) is a short double-stranded RNA of 21 to 23 base pairs used for gene silencing by RNA interference. The siRNA introduced into cells binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves the mRNA having a sequence complementary to the siRNA. Thereby, the expression of the gene is suppressed sequence-specifically.

[0025] siRNA can be prepared by synthesizing sense-strand and antisense-strand oligonucleotides with a DNA / RNA automatic synthesizer, respectively, and annealing them, for example, by denaturing them at about 90°C to 95°C for about 1 minute in an appropriate annealing buffer and then annealing them at about 30°C to 70°C for about 1 hour to 8 hours.

[0026] siRNA, shRNA, miRNA, ribozyme, and antisense nucleic acid may contain various chemical modifications in order to improve stability and activity. For example, in order to prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Also, at least a part thereof may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).

[0027] As the low-molecular compound that suppresses the expression of FN, it is generally preferable to use a known drug for which an FN expression-suppressing effect has been reported in the peripheral vascular system such as the cardiovascular system. By using such a drug, cerebral small vessel disease can be safely and surely prevented or treated.

[0028] Examples of such drugs include calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II type 1 receptor antagonists (ARBs), and the like.

[0029] Examples of calcium channel blockers include dihydropyridine compounds, benzothiazepine compounds, and the like. Examples of dihydropyridine compounds include nifedipine, amlodipine, efonidipine, cilnidipine, nicardipine, nisoldipine, nitrendipine, nilvadipine, barnidipine, felodipine, benidipine, manidipine, azelnidipine, aranidipine, and the like. Examples of benzothiazepine compounds include diltiazem and the like. Among them, dihydropyridine compounds are preferred, and amlodipine is more preferred.

[0030] Examples of angiotensin-converting enzyme (ACE) inhibitors include captopril, enalapril, alacepril, delapril, cilazapril, lisinopril, benazepril, imidapril, temocapril, quinapril,trandolapril, perindopril erbumine, and the like.

[0031] Examples of angiotensin II type 1 receptor antagonists (ARBs) include losartan, candesartan, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, and the like. Among them, candesartan or telmisartan is preferred.

[0032] Among them, as shown in the examples described below, since they have the effect of suppressing the expression of FN, Adamtsl2, and TIMP3, calcium channel blockers or angiotensin II type 1 receptor antagonists are preferred, angiotensin II type 1 receptor antagonists are more preferred, and candesartan is even more preferred.

[0033] In addition, these drugs include pharmaceutically acceptable esters, salts, or solvates of the above-exemplified compounds.

[0034] As used herein, the term "pharmaceutically acceptable ester" means an ester that is hydrolyzed in vivo, and includes those that are easily decomposed in the human body to release the above-exemplified compounds or their salts. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic acids, alkenoic acids, cycloalkanoic acids, and alkanedioic acids (each alkyl or alkenyl group preferably having 6 or fewer carbon atoms). Specific examples of esters include formate, acetate, propionate, butyrate, acrylate, ethyl succinate, and cyclohexyl-1-hydroxyethyl carbonate (sirexetil).

[0035] Also, the term "pharmaceutically acceptable salt" means a non-toxic salt of the above-exemplified compounds, and generally can be prepared by reacting the free acid thereof with a suitable organic or inorganic base. Examples of the salt forms of the above-exemplified compounds include, for example, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, calcium edetate, camsilate, carbonate, chloride, clubranate, citrate, dihydrogen chloride, edetate, edisytrate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, oleate, oxalate, pamoate, palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, sabsylate, succinate, tannate, tartrate, theoclate, tosylate, triethiodide, valerate, and mixtures thereof, but are not limited thereto.

[0036] In addition, "pharmaceutically acceptable solvate" means the above-exemplified compound combined with a solvent. The solvent is volatile, non-toxic, and acceptable for administration to animals including humans. Preferred solvates include, for example, hydrates.

[0037] (Specific binding substance of FN or TIMP3) Examples of the specific binding substance of FN or TIMP3 include those that specifically bind to FN or TIMP3 and inhibit the function of FN or TIMP3, such as antibodies, antibody fragments, aptamers, etc. Antibodies can be produced, for example, by immunizing animals such as mice with FN or TIMP3 or a fragment thereof as an antigen. Alternatively, they can be produced, for example, by screening a phage library. Examples of antibody fragments include Fv, Fab, scFv, etc. The above antibodies are preferably monoclonal antibodies. Also, commercially available antibodies may be used.

[0038] An aptamer is a substance having specific binding ability to a target substance. Examples of aptamers include nucleic acid aptamers, peptide aptamers, etc. Nucleic acid aptamers having specific binding ability to a target peptide can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method or the like. Also, peptide aptamers having specific binding ability to a target peptide can be selected, for example, by the Two-hybrid method using yeast or the like.

[0039] [Pharmaceutically acceptable carrier] The pharmaceutical composition of this embodiment can further contain a pharmaceutically acceptable carrier.

[0040] As the pharmaceutically acceptable carrier, those usually used in the formulation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid; solvents for injections such as water, ethanol, glycerin, etc.; adhesives such as rubber-based adhesives, silicone-based adhesives, etc. can be mentioned.

[0041] The pharmaceutical composition of this embodiment may further contain additives. Examples of the additives include lubricants such as calcium stearate, magnesium stearate, etc.; sweeteners such as sucrose, lactose, saccharin, maltitol, etc.; flavoring agents such as peppermint, perilla oil, etc.; stabilizers such as benzyl alcohol, phenol, etc.; buffers such as phosphates, sodium acetate, etc.; solubilizing agents such as benzyl benzoate, benzyl alcohol, etc.; antioxidants; preservatives, etc.

[0042] The pharmaceutical composition of this embodiment can be formulated by appropriately combining the above FN accumulation inhibitor or TIMP3 accumulation inhibitor with the above pharmaceutically acceptable carrier and additives and mixing them in a unit dosage form required for generally recognized pharmaceutical practice.

[0043] The pharmaceutical composition of this embodiment may be in a dosage form for oral use or in a dosage form for parenteral use, but a dosage form for oral use is preferred. Examples of the dosage form for oral use include tablets, capsules, elixirs, microcapsules, etc. Examples of the dosage form for parenteral use include injections, ointments, patches, etc.

[0044] The pharmaceutical composition of this embodiment may be used in combination with therapeutic agents for other diseases. For example, by using the above FN accumulation inhibitor or TIMP3 accumulation inhibitor in combination with an antihypertensive drug or the like, hypertension can be prevented or treated while improving the symptoms of cerebral small vessel disease. The above FN accumulation inhibitor or TIMP3 accumulation inhibitor and other drugs may be in the same formulation or in separate formulations. Also, each formulation may be administered by the same administration route or by separate administration routes. Furthermore, each formulation may be administered simultaneously, sequentially, or separately with a certain time or period in between. In one embodiment, the above FN accumulation inhibitor or TIMP3 accumulation inhibitor and other drugs may be in the form of a kit containing these.

[0045] <Administration method> The subjects to be administered are not limited, and examples include humans, monkeys, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, and their cells, etc. Among them, mammals or mammalian cells are preferred, and humans or human cells are particularly preferred.

[0046] Administration to a patient or diseased animal can be carried out, for example, by methods known to those skilled in the art such as intrathecal injection, intra-arterial injection, intravenous injection, subcutaneous injection, intranasal, transbronchial, intramuscular, percutaneous, or oral. The dosage varies depending on the patient's weight, age, patient's symptoms, administration method, etc., but those skilled in the art can appropriately select an appropriate dosage.

[0047] The dosage of the pharmaceutical composition of this embodiment, as the amount of the above FN accumulation inhibitor or TIMP3 accumulation inhibitor, in the case of oral administration, generally in adults (assuming a body weight of 60 kg), is considered appropriate to administer about 0.1 mg or more and 10 g or less, preferably about 0.05 mg or more and 5 g or less, more preferably about 1 mg or more and 3 g or less per day, once a day or divided into several times.

[0048] When administering parenterally, for example, in the form of an injection, generally in adults (assuming a body weight of 60 kg), it is considered appropriate to administer about 0.01 mg or more and 3000 mg or less, preferably about 0.05 mg or more and 300 mg or less, more preferably about 0.1 mg or more and 100 mg or less per day, once a day or divided into several times.

[0049] <Other Embodiments> In one embodiment, the present invention provides a method for preventing or treating cerebral small vessel disease, which includes administering an effective amount of the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor to a patient in need of treatment. Here, examples of the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor include those similar to those described above. Also, examples of cerebral small vessel disease include those similar to those described above.

[0050] In one embodiment, the present invention provides the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor for preventing or treating cerebral small vessel disease. Here, examples of the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor include those similar to those described above. Also, examples of cerebral small vessel disease include those similar to those described above.

[0051] In one embodiment, the present invention provides the use of the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor for manufacturing a pharmaceutical composition for preventing or treating cerebral small vessel disease. Here, examples of the FN accumulation inhibitor or TIMP3 accumulation inhibitor include those similar to those described above. Also, examples of cerebral small vessel disease include those similar to those described above.

Examples

[0052] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0053] [Materials and Methods] (Experimental Animals) The high-temperature requirement serine protease A1 gene-deficient mice (HTRA1- / - mice) were obtained from Lexicon Pharmaceuticals. The HTRA1- / - mice were backcrossed more than 10 times with C57BL / 6JJcl mice. The genotype of the mice was determined by PCR using DNA extracted from the tail. HTRA1+ / + littermates were used as controls. Male HTRA1+ / + mice and HTRA1- / - mice were used to analyze cerebral circulation. In other experiments, both female and male mice were used to equalize the sex ratio of HTRA1+ / + mice and HTRA1- / - mice. All animal experiments were approved by the Animal Use and Management Committee of Niigata University and were conducted in accordance with the guidelines of the National Institutes of Health (USA).

[0054] (Collection of blood vessels and brain tissues) Blood was removed from the whole body of the mice by cardiac perfusion with Hank's balanced salt solution (HBSS). Subsequently, a portion of the middle cerebral artery with medium-sized branches (referred to as the pial artery) was isolated from the mouse brain excised in ice-cold Eagle's minimum essential medium (MEM) under a dissecting microscope, immediately frozen in dry ice, and stored at -80°C. The cerebral cortex was incised and rolled on dry filter paper to remove the pia mater.

[0055] (Preparation of extracellular matrix) The extracellular matrix was prepared using conventional methods. Specifically, first, the leptomeningeal artery was homogenized with sodium deoxycholate lysis buffer (150 mM NaCl, 1 w / v% Triton X-100, 0.5 w / v% sodium deoxycholate, 50 mM Tris-HCl [pH 7.5]) and clarified by centrifugation. After collecting the soluble tissue material, the insoluble fraction (ECM fraction) was homogenized with sodium dodecyl sulfate (SDS) lysis buffer (62.5 mM Tris-HCl [pH 6.8], 2 w / v% SDS, 0.5 w / v% NP-40). After homogenization, 2-mercaptoethanol was added to the lysate at a concentration of 5 w / v% and incubated at room temperature for 30 minutes. After centrifugation, the ECM fraction solubilized with SDS was subjected to immunoblotting described below. In the case of LTBP, the sodium deoxycholate-insoluble material was digested with 0.3 U / mL plasmin (Sigma) in phosphate-buffered saline (PBS) containing 1 mM MgCl2, 1 mM CaCl2, and 0.1 w / v% n-octyl-β-D-glucopyranoside (Sigma) at 37°C for 2 hours. The reaction was stopped by adding a protease inhibitor, and the reaction solution was clarified by centrifugation at 8,000×g for 30 minutes.

[0056] (Immunoblotting) Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The amount of ECM fraction sample used was determined according to the protein content of the sodium deoxycholate-soluble fraction. The membrane was probed with the following antibodies: mouse anti-LTBP-4 antibody (AF2885, 1:2000; R&D System), rabbit anti-LTBP-1 antibody (22065-1-AP, 1:500; Proteintech), anti-LTBP-2 antibody (sc-166199, 1:500; Santa Cruz Biotech), rabbit anti-FN antibody (ab2413, 1:1000; Abcam), rabbit anti-TINAGL-1 antibody (12077-1-AP, 1:800; Proteintech), rabbit anti-Nidogen-1 antibody (LS-B9259, 1:500; LifeSpan BioScience), goat anti-LAPβ1 (AF-246-NA, 1:100, R&D System), rabbit anti-TIMP3 antibody (ab58804, 1:200; Abcam), rabbit anti-biglycan antibody (16409-1-AP, 1:500; proteintech), mouse anti-β-actin antibody (M177-3, 1:5000; Medical and Biological Laboratories Co.), rabbit anti-phosphorylated SMAD2 antibody (#3108, 1:500; Cell Signaling Tech), or rabbit anti-phosphorylated SMAD3 antibody (ab52903, 1:1000; Abcam). The immunoreactivity of the membrane was detected by peroxidase-conjugated anti-immunoglobulin antibody followed by chemiluminescence reaction. Images were taken using Image Quant LAS-4000 (Fujifilm).

[0057] (Preparation of Fixed Mouse Brain Tissue) At 37 °C and 75 mmHg for 10 minutes, Ca 2+Blood was removed from the whole body of mice by transcardial perfusion using HBSS without []. The perfusion pressure was monitored with a manometer. Brain samples were fixed with 4 w / v% paraformaldehyde. For the preparation of frozen sections, the brain was immersed in 30 w / v% sucrose in 0.1 M PBS, embedded in optimal cutting temperature compound (OCT) (Sakura Finetek), and sectioned at a thickness of 14 μm with a cryostat (CM1950; Leica). For paraffin embedding, the brain was processed after dehydration and sectioned at a thickness of 4 μm in the coronal plane. For brain capillary analysis, the fixed brain was embedded in agarose and cut using a vibratome (Leica) at a thickness of 50 μm. Isoflurane was used as an anesthetic in the vascular morphometric analysis. Halothane was also used in other experiments.

[0058] (Immunohistochemistry) For mouse brain tissue analysis, antigens were retrieved by boiling with sodium citrate buffer (pH 6.0) in a microwave oven for αSMA, CD31 (PECAM1), and FN staining, or by proteinase K treatment for LTBP-4, LAPβ1, and elastin staining. Next, the sections were incubated overnight at 4 °C with the following antibodies: biotinylated anti-αSMA antibody (LS-C87562, 1:100; LifeSpan BioScience), rat anti-CD31 (PECAM1) antibody (DIA-310, 1:20; Optistain), rabbit anti-FN antibody (mouse tissue: ab2413, 1:250; Abcam), rabbit or goat anti-LTBP-4 antibody (mouse tissue: sc-33144, 1:20; Santa Cruz Biotech, or AF2885, 1:50; R&D System), rabbit anti-elastin antibody (PR-387, 1:500; Elastin Products), or goat anti-LAPβ1 antibody (AF-246-NA, 1:50; R&D System). Next, the sections were incubated with Alexa Fluor-labeled secondary antibodies. To detect FN and LTBP-4 simultaneously, the primary antibodies were fluorescently labeled using Zenon Antibody Labeling Kits (Thermo Fisher Scientific). DyLight-594-labeled tomato lectin was also used for visualization of endothelial cells. Fluorescence microscope images were acquired using an all-in-one microscope (Keyence; BioRevo BZ-9000) or a confocal laser microscope (LSM710; Carl Zeiss). Vibratome sections were blocked and then incubated with the following antibodies: rat anti-CD13 antibody (R3-63, 1:50; AbD Serotec), rat CD31 (PECAM1) antibody (DIA-310, 1:20; Optistain), or rabbit anti-fibrinogen antibody (A0080, 1:250; Dako). Three-dimensional images were obtained using a confocal laser microscope. Vessel length was measured using the filament tracer function of Imaris software (version 6.2.0, Bitplane).

[0059] (Drug Therapy) Treatment with candesartan cilexetil (Takeda Pharmaceutical Company, Osaka, Japan) and amlodipine (Wako) was initiated at 4 months of age via drinking water at doses equivalent to those that exert a blood pressure-lowering effect. Candesartan and amlodipine were dissolved and diluted to final concentrations of 37.5 mg / L or 125 mg / L, respectively. Based on prior studies, the daily water intake of adult mice (6 months of age) was determined to be approximately 2.5 mL. Therefore, the daily doses of candesartan and amlodipine were approximately 3 mg / kg / day or 10 mg / kg / day, respectively. These doses are similar to those used in the clinical treatment of human blood pressure. At either dose, blood pressure was reduced by approximately 15 - 20%.

[0060] (Vascular Morphometric Analysis) In the morphometric analysis of pial arteries, microscopic images of cross-sections of pial arteries (anterior cerebral artery: luminal diameter, 80 - 100 μm in HTRA1+ / + mice) in coronal brain slices (bregma: +0.86 to -2.30) were selected and analyzed at equal intervals, and 5 - 7 images per animal were used in each analysis to avoid arterial branch points. Cerebral vascular endothelial cells and vascular smooth muscle cells (SMCs) were visualized by immunostaining using antibodies against CD31 (PECAM1) and αSMA (see the above immunohistochemistry). The inner perimeter of the lumen was measured using a macro in ImageJ (https: / / www.ipmc.cnrs.fr / ~duprat / scripts / ) using the CD31 image. Similarly, the inner and outer perimeters were measured using the inner and outer boundaries of the αSMA-positive region. The vascular inner diameter was calculated from the luminal inner perimeter. The inner thickness was measured using the inner inner diameter and outer diameter calculated from the inner inner perimeter and outer perimeter, respectively. The inner cross-sectional area (CSA) was measured using the αSMA-positive area. The intimal thickness was measured using the inner boundary of the αSMA-positive region and the CD31 layer. The intimal thickness of each image was determined by measuring 20 - 30 points per image and averaging. The CSA and intimal thickness were analyzed using Imaris software (version 6.2.0, Bitplane).

[0061] (MRI Measurement) MRI experiments were performed using a 7T horizontal magnet with an 18 cm bore (Magnex Scientific, Abingdon, UK) and a Varian Unity-INOVA-300 system (Varian Inc., Palo Alto, USA) with an active shield gradient. In this experiment, a birdcage-type transmit coil with an inner diameter of 6 cm and an orthogonal receive coil were used. Cerebral blood flow was evaluated by continuous arterial spin labeling (CASL) with a centered ordered variable tip angle gradient echo (VTE-GRE). Prior to the VTE-GRE sequence, 3 seconds of radio frequency (RF) at positions ±10 mm from the imaging slice was alternately irradiated with an axial gradient of 1 g / cm in 0.8 seconds. A total of 64 sets of images were combined to improve the signal-to-noise ratio. Magnetization transfer ratio was evaluated under the same conditions as cerebral perfusion measurements, but without an adiabatic inversion axial gradient. T1 measurements were performed using a hyperbolic secant inversion pulse and an inversion delay of 32 points (20 - 2000 milliseconds) with a centered ordered snapshot flash. According to the theory reported by Ewing et al. (Reference 3: Ewing JR et al., “Direct comparison of local cerebral blood flow rates measured by MRI arterial spin-tagging and quantitative autoradiography in a rat model of experimental cerebral ischemia.”, Journal of Cerebral Blood Flow & Metabolism, Vol. 23, Issue 2, pp. 198 - 209, 2003.), quantitative cerebral blood flow maps were calculated from cerebral blood flow images, T1 maps, and magnetization transfer (MTR) maps. The maps were calculated using MRI image calculation software (MR Vision, MR Vision Co., Menlo Park, CA, USA). The animals were anesthetized with 3 - 5 w / v% isoflurane for induction and then with 1.2 w / v% for maintenance. Cerebral blood flow in the normocapnic state was measured at 1 L / min of 30 v / v% O2:70 v / v% N2O under spontaneous breathing through a face mask.The state of hypercapnia was induced with 30 v / v% O2:60 v / v% N2O:10 v / v% CO2 at 1 L / min. Cerebral blood flow in the hypercapnic state was measured 10 minutes after the induction of the state of hypercapnia under spontaneous breathing. Rectal temperature was maintained at 37 ± 0.5 °C throughout the measurement using a homemade air conditioning system.

[0062] (Transcriptome (RNA seq.) analysis) Polyadenylated mRNA was enriched with oligo dT probes. RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit according to the instructions of the library kit. Quality control and quantification of the libraries were performed using an Agilent Bioanalyzer and the KAPA Library Quantification Kit (KAPA Biosystems). All nine libraries were sequenced on a single flow cell lane using an Illumina HiSeq 2500 (high output mode, 50-base single reads). The read counts (raw reads) of the raw data (immediately after measurement) were trimmed with adapters, quality-filtered using the FASTX-Toolkit (http: / / hannonlab.cshl.edu / fastx_toolkit / index.html), and reads derived from N or ribosomal RNA were excluded. The read counts (clean reads) after data cleaning were mapped to the mouse reference genome sequence (GRCm38 / mm10) using TopHat. The mapped read counts were measured with HTseq. Differentially expressed genes with a p-value of less than 0.05 were identified using DESeq2. Gene ontology and KEGG pathway enrichment analysis were performed with the clusterProfiler R package.

[0063] (qRT-PCR) qRT-PCR was performed using SYBR Green Premix ExTaq II (Takara Bio Inc). The expression levels of target genes relative to two housekeeping genes were determined using the ΔΔCT method.

[0064] (ddPCR) cDNA was synthesized using the superScript VILO TM cDNA Synthesis Kit (Thermo Fisher Scientific). The Bio-Rad QX200 ddPCR System (Bio-Rad, Hercules, CA) was used. Each reaction consisted of a 20 μL solution containing 10 μL of ddP TM Supermix (Bio-Rad), 1 μM primer, 250 nM probe, and template cDNA. Droplets encapsulating the PCR mix were formed using a Bio-Rad QX-100 emulsifier. After the PCR cycle, the droplets were immediately analyzed by QuantaSoft v.1.6 (Bio-Rad). Appropriate housekeeping genes were selected using the Mouse Housekeeping Gene Primer Set (Takara Bio Inc.) and geNorm software. Pre-designed primers and probes (5’ / FAM / ZEN / IBFQ / 3’) were purchased from Integrated DNA Technologies (Coralville, IA).

[0065] (Statistical analysis) Statistical calculations were performed using IBM SPSS 22. The data were first subjected to the Shapiro-Wilk test (to fit a Gaussian distribution) and Levene's test (for equal variance). For data with a Gaussian distribution and equal variance, one-way analysis of variance (ANOVA) or two-sided unpaired t-tests were employed. Subsequently, the Bonferroni test was applied to the data as a post hoc test. Alternatively, the Steel-Dwass test or Mann-Whitney U test was applied to data with unequal variances. P < 0.05 was considered statistically significant. Hereinafter, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. All data are presented as mean ± standard deviation (s.d.).

[0066] [Example 1] (Inhibitory Effect of Candesartan and Amlodipine Treatment on Fibronectin Accumulation) From the analysis of HTRA1 gene-deficient mice (HTRA1- / - mice), which are model mice for hereditary cerebral small vessel disease CARASIL, the inventors discovered that significant accumulation of fibronectin occurs in cerebral small vessels under HTRA1 deficiency. They also discovered that the accumulation of fibronectin is accompanied by the accumulation of fibronectin-binding extracellular matrix proteins and induces the formation of abnormal elastic laminae and the like. Accumulation of fibronectin in cerebral small vessels is also observed in patients with sporadic cerebral small vessel disease and in hypertensive or diabetic model animals, and furthermore, this finding has been reported even in CADASIL, which is caused by TIMP3 gene mutation and is different from CARASIL. The accumulation of fibronectin is seen to be consistent with the pathological finding of arteriolosclerosis, a typical representative of intimal thickening of blood vessels. Based on these findings, the inventors hypothesized that the suppression of fibronectin accumulation could be a very effective therapeutic target for cerebral small vessel disease. Based on this hypothesis, candesartan cilexetil, an angiotensin II type 1 receptor antagonist known to be a suppressor of FN expression, and amlodipine, a dihydropyridine calcium channel antagonist, were administered to HTRA1- / - mice to attempt to inhibit the accumulation of fibronectin.

[0067] Figure 1 is a graph showing each mRNA level quantified by ddPCR using total RNA extracted from pial arteries of 4-month-old HTRA1- / - mice and HTRA1+ / + mice that had or had not undergone 1-week candesartan or amlodipine treatment. Each mRNA level is shown as a relative amount to HTRA1+ / + mice.

[0068] As shown in Fig. 1, in HTRA1- / - mice treated with candesartan or amlodipine for one week, the mRNA levels of Fn1 and EDA (extra domain A)+Fn1 were significantly decreased compared with those in untreated HTRA1- / - mice. The mRNA levels of Ltbp-4 and Bgn were also decreased similarly. The decrease in each mRNA level was particularly remarkable in HTRA1- / - mice treated with candesartan. However, the mRNA levels of elastin and Tgfβ1 did not change. Also, the mRNA level of Timp3 was decreased to the same extent in HTRA1- / - mice treated with candesartan and amlodipine.

[0069] In previous animal experiments, since accumulation of fibronectin and LTBP-4 (latent TGF-β binding protein) was observed in 4-month-old HTRA1- / - mice and deteriorated with aging, administration of candesartan or amlodipine was started in 4-month-old HTRA1- / - mice, and the effects of these drug administrations were examined at 24 months of age. Figs. 2A and 3A are images showing detection of fibronectin (Fig. 2A) or LTBP-4 (Fig. 3A) in the leptomeningeal arteries of each 24-month-old mouse by immunohistochemical staining with visualization of the inside of blood vessels by α-SMA immunostaining. The scale bar is 50 μm in Figs. 2A and 3A. Figs. 2B and 3B are graphs showing the results of quantifying the immunoreactive areas of cross-sectional fibronectin or LTBP-4 from the images of Figs. 2A and 3A, respectively (n = 5 - 6 per mouse group). In Figs. 2B and 3B, the quantitative values were normalized for each outer circumference to exclude the influence of vasodilation in 24-month-old HTRA1- / - mice.

[0070] From Figs. 2A - 3B, it became clear that the accumulation of fibronectin and LTBP-4 was suppressed in HTRA1- / - mice treated with candesartan or amlodipine. The decrease in the accumulation amounts of fibronectin and LTBP-4 was particularly remarkable in the treatment with candesartan rather than in the treatment with amlodipine.

[0071] Figures 4A, 5A, 6A, and 7A are images showing the results of immunoblotting analysis using antibodies against fibronectin, LTBP-4, EDA, TIMP3, elastin, LAPβ1, or phosphorylated SMAD3 of leptomeningeal arteries of 24-month-old HTRA1- / - and HTRA1+ / + mice that were or were not treated with candesartan from 4 months to 24 months of age (n = 4 mice per mouse group). Figures 4B, 5B, 6B, and 7B are graphs quantifying the bands shown in the images of Figures 4A, 5A, 6A, and 7A, respectively, that is, the expression levels of each protein.

[0072] As shown in Figures 4A to 7B, a decrease in fibronectin and LTBP-4 was confirmed in HTRA1- / - mice treated with candesartan. Furthermore, a decrease in EDA+fibronectin, elastin, LAPβ1, TIMP3, and phosphorylated SMAD3 was also confirmed.

[0073] [Example 2] (Suppressive Effect of Candesartan and Amlodipine Treatments on Vascular Structural Changes) Next, the suppressive effect of candesartan and amlodipine treatments on vascular structural changes was examined. Specifically, administration of candesartan or amlodipine was started in HTRA1- / - mice at 4 months of age, and the leptomeningeal arteries at 24 months of age were examined. Figure 8 is a representative image of PECAM1 and αSMA stained cross-sections of the leptomeningeal arteries of each 24-month-old mouse. In Figure 8, the scale bar is 50 μm. Also, the lower image is a high-magnification image of the upper micrograph. In Figure 9, the leftmost graph is the intimal thickness of the leptomeningeal arteries in each 24-month-old mouse, the second graph from the left is the inner diameter of the leptomeningeal arteries in each 24-month-old mouse, the third graph from the left is the thickness of the inner vascular wall of the leptomeningeal arteries in each 24-month-old mouse, and the fourth (rightmost) graph from the left is the cross-sectional area (CSA) of the inner side of the leptomeningeal arteries in each 24-month-old mouse measured from the image (n = 5 - 7 mice per mouse group).

[0074] As shown in FIGS. 8 and 9, in HTRA1 - / - mice, it was found that the diameter of the leptomeningeal artery was significantly increased and the intimal thickness was significantly decreased compared to HTRA1 + / + littermate mice. Vascular smooth muscle cells showed a multi - layer cell structure in HTRA1 + / + littermate mice. However, in HTRA1 - / - mice, the vascular smooth muscle cell layer was partially monolayered and absent in some areas. The increase in the diameter of the vascular lumen and the decrease in the thickness of the inner vascular wall were observed at 24 months of age but not at 16 months of age (not shown). The increase in diameter and the decrease in the thickness of the inner vascular wall were suppressed by candesartan but not by amlodipine.

[0075] [Example 3] (Suppressive effect of candesartan treatment on reduction of cerebral blood flow) Next, using candesartan, for which the therapeutic effect was particularly remarkable, the suppressive effect of drug treatment on reduction of cerebral blood flow was examined. Specifically, administration of candesartan was started in HTRA1 - / - mice from 4 months of age, and cerebral blood flow at rest (CO2 0%) and under hypercapnic conditions (CO2 10%, for 10 minutes) was measured by continuous arterial spin - labeling method (CASL) using MRI at 16 - 20 months of age.

[0076] FIG. 10 is an MRI image of cerebral blood flow of each mouse at 16 - 20 months of age. FIG. 11 is a graph showing the resting cerebral blood flow (left side) and the increase in cerebral blood flow in the cerebral cortex in response to hypercapnic conditions (right side) of each mouse calculated from the image of FIG. 10.

[0077] As shown in FIGS. 10 and 11, a significant decrease in cerebral blood flow was observed in HTRA1 - / - mice. In HTRA1 - / - mice treated with candesartan, the decrease in cerebral blood flow was suppressed and improved to a level equivalent to that of HTRA1 + / + littermate mice.

[0078] [Example 4] (Search for genes whose expression changed by candesartan treatment) Next, candesartan treatment decreased fibronectin expression, and genes whose expression changed other than fibronectin were searched for by candesartan treatment. To exclude the influence of age, analysis was performed using mice from 4 months to 12 months old. Specifically, changes in gene expression in HTRA1 - / - mice treated with candesartan for a short period (1 week) or a long period (from 4 months to 12 months old) were comprehensively analyzed.

[0079] Figures 12A and 12B are volcano plots of gene expression analysis generated by DESeq2 using candesartan - treated and untreated HTRA1 - / - mice.

[0080] In HTRA1 - / - mice treated with candesartan for a short period (1 week) or a long period (from 4 months to 12 months old), 10 genes and 84 genes whose expression changed were respectively identified. Among them, attention was paid to the Adamtsl2 gene, which is one of the genes encoding extracellular matrix (ECM) proteins, as a gene that leads to a decrease in the expression level of fibronectin in vivo.

[0081] Next, the expression levels of the Adamtsl2 gene were confirmed in 4 - month - old HTRA1 - / - and HTRA1 + / + mice with or without 1 - week candesartan or amlodipine treatment, and in 24 - month - old HTRA1 - / - and HTRA1 + / + mice with or without candesartan or amlodipine treatment from 4 months to 24 months old. Figure 13 is a graph showing the mRNA expression levels of Adamtsl2 in each mouse at 4 months (left) and 24 months (right) old (n = 5 - 6 mice per mouse group).

[0082] As shown in Figure 13, the expression of the Adamtsl2 gene decreased also by amlodipine treatment, but the decrease by candesartan treatment was more significant.

[0083] [Discussion] Candesartan or amlodipine treatment improved the accumulation of fibronectin and fibronectin-binding proteins in the intima of pial arteries and parenchymal arterioles, but did not suppress the decrease in pericyte coverage, etc. These results suggest that the accumulation of HTRA1 substrates, such as fibronectin and fibronectin-binding proteins such as LTBP-4 in the intima, is strongly involved in the development of arteriopathy in HTRA1- / - mice. In addition, it was revealed for the first time that short-term (1 week) or long-term (20 months) treatment with candesartan or amlodipine reduced the mRNA expression of Adamtsl2 gene. ADAMTSL2 is known to be involved in both microfibril network and fibronectin network formation, and it is also known that fibronectin mRNA expression level is reduced in Adamtsl2 gene knockout (KO) mice. From these findings, it was inferred that candesartan or amlodipine treatment reduced fibronectin accumulation via a decrease in the mRNA expression level of the Adamtsl2 gene.

[0084] In addition, TIMP3 has been reported to be an important protein in the pathophysiology of CADASIL. In this study, the inventors confirmed a significant accumulation of TIMP3 in the leptomeningeal arteries of HTRA1- / - mice. In addition, for the first time, a significant accumulation of TIMP3 was discovered in the cerebral small blood vessels of CARASIL patients. Furthermore, candesartan or amlodipine treatment suppressed the accumulation of TIMP3 in the leptomeningeal arteries of HTRA1- / - mice. TIMP3 is the only TIMP that strongly binds to the extracellular matrix (ECM). In the above experiments, various ECM proteins whose accumulation was confirmed in the leptomeningeal arteries of HTRA1- / - mice were substrates of HTRA1, but it has not been proven that only TIMP3 binds directly or indirectly to fibronectin. TIMP3 may bind and accumulate to fibronectin via intervening proteins such as sulfated glucosaminoglycan proteins, or may accumulate independently of fibronectin. In the analysis results of gene expression of various ECM proteins, the mRNA expression level of TIMP3 was suppressed by both candesartan treatment and amlodipine treatment, but the mRNA expression levels of other ECM proteins were significantly suppressed by candesartan treatment. Therefore, further studies will be conducted on the mechanism of TIMP3 accumulation and the mechanism of suppression of TIMP3 accumulation by candesartan treatment and amlodipine treatment.

[0085] [Example 5] (Effect of telmisartan treatment on suppressing vascular structural changes) Next, in order to confirm that an equivalent therapeutic effect can be obtained for cerebral small vessel disease with angiotensin II type 1 receptor antagonists other than candesartan, a test was conducted using telmisartan, which is known as an angiotensin II type 1 receptor antagonist. Specifically, telmisartan administration was started in HTRA1- / - mice at 4 months of age, and the leptomeningeal arteries at 24 months of age were examined. The daily dose of telmisartan was 15 mg / kg / day. This dose is similar to the dose used in the clinical treatment of human blood pressure. Figure 14 shows the cross-sectional images of PECAM1 and αSMA staining of the leptomeningeal arteries of each mouse at 24 months of age, and the internal diameters of the leptomeningeal arteries in each mouse at 24 months of age were measured (n = 5-7 mice per mouse group). In Figure 14, "HTRA1+ / +" is the HTRA1+ / + mouse group without telmisartan administration, "HTRA1- / -" is the HTRA1- / - mouse group without telmisartan administration, and "HTRA1- / - + Telm." is the HTRA1- / - mouse group administered with telmisartan.

[0086] As shown in Figure 14, in HTRA1- / - mice, the diameter of the leptomeningeal artery was significantly increased. On the other hand, in HTRA1- / - mice administered with telmisartan, the increase in the diameter of the leptomeningeal artery was suppressed in the same manner as in the candesartan-administered group.

[0087] From the above, it was shown that the symptoms of cerebral small vessel disease can be improved by using angiotensin II type 1 receptor antagonists such as candesartan and telmisartan.

Industrial Applicability

[0088] According to the pharmaceutical composition of the present embodiment, cerebral small vessel disease can be prevented or treated.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of cerebral small vessel diseases caused by hypertension and diabetes, and cerebral small vessel diseases accompanied by the accumulation of fibronectin in the intima of cerebral small vessels associated with aging, excluding Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, comprising a fibronectin accumulation inhibitor as an active ingredient, The pharmaceutical composition, wherein the fibronectin accumulation inhibitor is candesartan or telmisartan. Claim 2. The pharmaceutical composition according to claim 1, wherein the fibronectin accumulation inhibitor is a compound having an Adamtsl2 expression inhibitory effect. Claim 3. The pharmaceutical composition according to claim 1 or 2, wherein the fibronectin accumulation inhibitor is telmisartan. Claim 4 The pharmaceutical composition according to any one of claims 1 to 3, wherein the cerebral small vessel disease is a hereditary cerebral small vessel disease. Claim 5 The pharmaceutical composition according to any one of claims 1 to 4, wherein the cerebral small vessel disease is Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

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