JP7708438B2 - Pharmaceutical composition for preventing or treating cerebral small vessel disease - Google Patents

Description

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 on May 13, 2020, the contents of which are incorporated herein by reference.

Cerebral small vessel disease, which develops as a result of cerebrovascular degeneration, with high blood pressure and diabetes as risk factors, can lead to dementia and gait disorders. The mechanism by which cerebral small vessel disease develops is unknown, and an effective treatment for this disease has not yet been developed. One of the main reasons for this is the lack of an appropriate animal model that functionally and pathologically reproduces the pathology. Research on vasculopathy with similar risk factors is limited to peripheral large vessels, and the pathology of these vessels is predominantly atherosclerotic, which differs 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 that takes into account the pathology of this disease.

There are not only sporadic cerebral small vessel diseases, 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 a mutation leading to the expression or inactivation of high-temperature-requiring serine protease A1 (HTRA1), a type of serine protease, is known to exhibit a disease form very similar to sporadic cerebral small vessel diseases, such as arteriosclerosis of cerebral small vessels and widespread 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, 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, it remains unclear which sites in the cerebral arteries these substrates initially accumulate in, or which substrates contribute to arterial damage.

Ito S et al., “Histopathologic Analysis of Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CARASIL): A Report of a New Genetically Confirmed Case and Comparison to 2 Previous Cases.”, J Neuropathol Exp Neurol., Vol. 75, Issue 11, pp. 1020-1030, 2016. Hara K et al., “Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease.”, N Engl J Med., Vol. 360, Issue 17, pp. 1729-1739, 2009. Zellner A et al., “CADASIL brain vessels show a HTRA1 loss-of-function profile.”, Acta Neuropathol., Vol. 136, Issue 1, pp. 111-125, 2018.

The present invention has been made in consideration of the above circumstances, and provides a novel pharmaceutical composition that is effective in preventing or treating cerebral small vessel disease.

As a result of extensive research conducted by the inventors to achieve the above-mentioned objective, they found that fibronectin, fibronectin-binding protein, and tissue inhibitor of metalloproteinase 3 (TIMP3) accumulate in the intima of cerebral pial arteries and cerebral parenchymal arterioles in HTRA1-deficient mice, and subsequently developed arteriopathy characterized by reduced cerebral blood flow, intima thickening, and internal elastic lamina neoformation. They also found that administering a drug that inhibits the accumulation of fibronectin or TIMP3 in the intima improves the symptoms of arteriopathy, thus completing the present invention.

That is, the present invention includes the following aspects.
(1) A pharmaceutical composition for preventing or treating cerebral small vessel disease, except for cerebral small vessel disease due to hypertension or diabetes, and cerebral autosomal recessive arteriopathy with subcortical infracts and leukoencephalopathy, which contains a fibronectin accumulation inhibitor as an active ingredient, and which is accompanied by accumulation of fibronectin in the intima of cerebral small vessels,
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 arteriopathies with subcortical infarcts and leukoencephalopathy.

The pharmaceutical composition of the above aspect can prevent or treat cerebral small vessel disease.

1 is a graph showing each mRNA level quantified by ddPCR using total RNA extracted from the pial artery of each mouse at 4 months of age in Example 1. 1 shows images of fibronectin in the pial artery of each 24-month-old mouse in Example 1, detected by immunohistochemical staining with visualization of the inside of the blood vessels by α-SMA immunostaining. FIG. 2B is a graph showing the results of quantification of fibronectin immunopositive areas in cross sections from the images in FIG. 2A. 1 shows images of LTBP-4 in the pial artery of each mouse at 24 months of age in Example 1, detected by immunohistochemical staining accompanied by visualization of the inside of the blood vessels by α-SMA immunostaining. FIG. 3B is a graph showing the results of quantification of LTBP-4 immunopositive areas in cross sections from the images in FIG. 3A. 1 is an image showing the results of immunoblotting analysis of the pial artery of each of the 24-month-old mice in Example 1 using antibodies against fibronectin, LTBP-4, or EDA. 4B is a graph showing the quantification of the bands shown in the image of FIG. 4A, i.e., the expression levels of each protein. 1 is an image showing the results of immunoblotting analysis of the pial artery of each mouse at 24 months of age in Example 1 using an antibody against TIMP3. This is a graph quantifying the expression level of the band shown in the image of Figure 5A, i.e., TIMP3. 1 is an image showing the results of immunoblotting analysis of the pial artery of each 24-month-old mouse in Example 1 using an antibody against elastin. This is a graph quantifying the expression level of the band shown in the image of Figure 6A, i.e., elastin. 1 is an image showing the results of immunoblotting analysis of the pial artery of each mouse at 24 months of age in Example 1 using an antibody against LAPβ1 or phosphorylated SMAD3. 7B is a graph showing the quantification of the bands shown in the image of FIG. 7A, i.e., the expression levels of each protein. 1 shows representative images of PECAM1 and αSMA stained cross sections of pial arteries from 24-month-old mice in Example 2. This is a graph showing the results of measuring the intima thickness of the pial artery of each 24-month-old mouse in Example 2 (far left), the inner diameter of the pial artery of each 24-month-old mouse (second from the left), the inner vascular wall thickness of the pial artery of each 24-month-old mouse (third from the left), and the inner cross-sectional area (CSA) of the pial artery of each 24-month-old mouse (fourth from the left (far right)) from the images shown in Figure 8. MRI images of cerebral blood flow in each mouse aged 16 to 20 months in Example 3. 11 is a graph showing the cerebral blood flow at rest (left side) and the increase in cerebral blood flow in the cerebral cortex in response to hypercapnia (right side) for each mouse, calculated from the image of FIG. 10 in Example 3. 13 is a volcano plot of gene expression analysis generated by DESeq2 using candesartan-treated and untreated HTRA1−/− mice in Example 4. 13 is a volcano plot of gene expression analysis generated by DESeq2 using candesartan-treated and untreated HTRA1−/− mice in Example 4. 13 is a graph showing the mRNA expression levels of Adamtsl2 in 4-month-old (left) and 24-month-old (right) mice in Example 4. 13 is a graph showing the results of measuring the inner diameter of the pial artery of each 24-month-old mouse from PECAM1 and αSMA stained cross-sectional images of the pial artery of each 24-month-old mouse in Example 5.

Below, we will provide a detailed description of a pharmaceutical composition for the prevention or treatment of cerebral small vessel disease in one embodiment of the present invention.

<Cerebral small vessel disease>
In general, "cerebral small vessel disease (SVD)" refers to pathological changes in small blood vessels in the brain or the resulting microlesions, and is a pathological condition that encompasses relatively homogeneous pathology and clinical features resulting from them. Cerebral small vessel disease is classified into isolated cerebral small vessel disease caused by hypertension or diabetes and hereditary cerebral small vessel disease. Isolated cerebral small vessel disease includes pathological conditions such as multiple lacunar infarctions, Binswanger's disease, diffuse white matter lesions (leukoaraiosis), and amyloid angiopathy. Examples of hereditary cerebral small vessel diseases include cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (due to a mutation in the HTRA1 gene), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (due to a mutation 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, and the like.

These cerebral small vessel diseases can be the main cause of vascular dementia. Cerebral small vessel disease is also suspected to be involved in degenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis, and there is a high probability that Alzheimer's dementia is accompanied by cerebral small vessel disease.

In particular, the pharmaceutical composition of this embodiment is particularly effective for preventing or treating sporadic cerebral small vessel disease, CARASIL, or CADASIL, which are accompanied by accumulation of fibronectin (FN) or tissue inhibitor of metalloproteinase 3 (TIMP3), as shown in the Examples described below. In addition, the pharmaceutical composition of this embodiment is also effective for preventing or treating vascular dementia caused by cerebral small vessel disease accompanied by accumulation of FN or TIMP3, and Alzheimer's dementia complicated by the cerebral small vessel disease.

<Pharmaceutical composition for preventing or treating cerebral small vessel disease>
The pharmaceutical composition for preventing or treating cerebral small vessel disease of this embodiment (hereinafter sometimes simply referred to as "the pharmaceutical composition of this embodiment") contains a fibronectin (FN) accumulation inhibitor or a tissue inhibitor of metalloproteinase 3 (TIMP3) accumulation inhibitor as an active ingredient.

In this specification, "containing as an active ingredient" means containing a therapeutically effective amount of an FN accumulation inhibitor or a TIMP3 accumulation inhibitor. In this specification, "therapeutically effective amount" means an amount of an FN accumulation inhibitor or a TIMP3 accumulation inhibitor, or an amount of a combination of an FN accumulation inhibitor or a TIMP3 accumulation inhibitor and one or more active agents, that induces a biological or medical effect or response desired by a physician, clinician, veterinarian, researcher, or other appropriate professional when administered in accordance with a desired treatment regimen. A preferred therapeutically effective amount is an amount that improves the symptoms of cerebral small vessel disease. In addition, "therapeutically effective amount" includes a prophylactically effective amount, i.e., an amount suitable for preventing a disease state.

Until now, no therapeutic target for cerebral small vessel disease was known, and effective therapeutic drugs were needed.
In response to this, the inventors have found for the first time that FN, FN-binding protein, and TIMP3 accumulate in the intima of cerebral pial arteries and cerebral intraparenchymal arterioles in HTRA1-deficient mice, which are CARASIL model mice, and then develop arteriopathy characterized by reduced cerebral blood flow, intima thickening, and internal elastic lamina neoformation. In addition, since accumulation of FN and TIMP3 is also seen in CARASIL patients and CADASIL patients, as shown in the Examples below, it has been found that reduced cerebral blood flow, intima thickening, and internal elastic lamina neoformation can be suppressed by inhibiting the accumulation of FN or TIMP3, and the present invention has been completed. Note that, as shown in the Examples below, the pharmaceutical composition of this embodiment inhibits the accumulation of FN or TIMP3, but does not inhibit the expression of TGF-β (Transforming growth factor β). In other words, the above molecular mechanism discovered by the inventors for the first time this time is completely different from the conventional pathological mechanism of CARASIL that focuses on the control mechanism of TGF-β signaling by HTRA1.

The pharmaceutical composition of this embodiment can effectively prevent or treat cerebral small vessel disease.

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

The FN targeted by an FN accumulation inhibitor may be at least one of the three isoforms listed above, but it is preferable to inhibit the accumulation of all three isoforms.

[Tissue inhibitor of metalloproteinase 3 (TIMP3)]
In general, tissue inhibitor of metalloproteinase 3 (TIMP3) is a protein that belongs to the matrix metalloproteinase (TIMP) family and binds to ECM to promote inhibition of matrix metalloproteinases (MMPs).
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).

[FN accumulation inhibitor or TIMP3 accumulation inhibitor]
Specific examples of FN accumulation inhibitors or TIMP3 accumulation inhibitors include compounds having activity of decomposing FN or TIMP3, FN or TIMP3 expression inhibitors, FN or TIMP3 specific binding substances, etc. In addition, as the FN accumulation inhibitor, for example, pUR4, which is known as a fibronectin fibrillogenesis inhibitor peptide (for example, see 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. ") can also be used.
Among these, the FN accumulation inhibitor or TIMP3 accumulation inhibitor is preferably an inhibitor of the accumulation of both FN and TIMP3, and is more preferably an inhibitor of the expression of FN and TIMP3.

(FN or TIMP3 expression inhibitor)
The expression inhibitor of FN or TIMP3 may be any that inhibits the expression of FN or TIMP3, and examples thereof include siRNA, shRNA, miRNA, ribozyme, antisense nucleic acid, low molecular weight compound, etc. By administering these expression inhibitors of FN or TIMP3, the expression level of FN or TIMP3 can be reduced, and 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.
In particular, it is preferable that the FN or TIMP3 expression inhibitor inhibits the expression of both FN and TIMP3.

Furthermore, as shown in the examples described below, fibrosis of FN in the ECM is controlled by the expression of Adamtsl2, which is located upstream of FN. Therefore, the FN accumulation inhibitor is preferably an inhibitor of FN or Adamtsl2 expression, and more preferably an inhibitor of FN and Adamtsl2 expression.

siRNA (small interfering RNA) is a small double-stranded RNA with 21 to 23 base pairs that is used for gene silencing by RNA interference. When siRNA is introduced into cells, it binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA with a sequence complementary to the siRNA, thereby suppressing gene expression in a sequence-specific manner.

siRNA can be prepared by synthesizing sense and antisense oligonucleotides using an automatic DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at approximately 90°C to 95°C for approximately 1 minute, and then annealing them at approximately 30°C to 70°C for approximately 1 hour to 8 hours.

siRNA, shRNA, miRNA, ribozymes, and antisense nucleic acids may contain various chemical modifications to improve their stability and activity. For example, to prevent degradation by hydrolases such as nucleases, phosphate residues may be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, at least a portion of them may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).

As a low molecular weight compound that suppresses the expression of FN, it is usually preferable to use a known drug that has been reported to have an effect of suppressing the expression of FN in the peripheral vascular system, such as the cardiovascular system. By using such a drug, cerebral small vessel disease can be prevented or treated safely and reliably.

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

Examples of calcium channel antagonists include dihydropyridine compounds, benzothiazepine compounds, etc. Examples of dihydropyridine compounds include nifedipine, amlodipine, efonidipine, cilnidipine, nicardipine, nisoldipine, nitrendipine, nilvadipine, barnidipine, felodipine, benidipine, manidipine, azelnidipine, aranidipine, etc. Examples of benzothiazepine compounds include diltiazem, etc.
Among these, dihydropyridine compounds are preferred, and amlodipine is more preferred.

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

Examples of angiotensin II type 1 receptor antagonists (ARBs) include losartan, candesartan, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, and the like.
Of these, candesartan or telmisartan is preferred.

Among these, as shown in the examples described below, calcium channel antagonists or angiotensin II type 1 receptor antagonists are preferred because they have the effect of suppressing the expression of FN, Adamtsl2, and TIMP3, with angiotensin II type 1 receptor antagonists being more preferred, and candesartan being even more preferred.

These agents also include pharma- ceutically acceptable esters, salts, or solvates of the compounds exemplified above.

As used herein, "pharmacologically acceptable ester" refers to an ester that is hydrolyzed in vivo, and includes those that are easily decomposed in the human body to release the above-listed compounds or salts thereof. Suitable ester groups include, for example, those derived from pharma- ceutically 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, ethylsuccinate, and cyclohexyl-1-hydroxyethyl carbonate (cilexetil).

In addition, "pharmacologically acceptable salts" refers to non-toxic salts of the compounds exemplified above, which can generally be prepared by reacting the free acid with a suitable organic or inorganic base. Examples of salt forms of the compounds exemplified above include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolyl arsenilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthalene, iodide, and 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, sabastate, succinate, tannate, tartrate, theoclate, tosylate, triethiodide, valerate, and mixtures thereof.

"Pharmaceutically acceptable solvates" refers to the compounds listed above combined with a solvent. The solvent is volatile, non-toxic, and acceptable for administration to animals, including humans. Suitable solvates include, for example, hydrates.

(Substances that specifically bind to FN or TIMP3)
The specific binding substance of FN or TIMP3 includes substances that specifically bind to FN or TIMP3 and inhibit the function of FN or TIMP3, such as antibodies, antibody fragments, aptamers, etc. The antibody can be prepared, for example, by immunizing an animal such as a mouse with FN or TIMP3 or a fragment thereof as an antigen. Alternatively, the antibody can be prepared, for example, by screening a phage library. The antibody fragment can include Fv, Fab, scFv, etc. The above antibody is preferably a monoclonal antibody. It may also be a commercially available antibody.

An aptamer is a substance that has a specific binding ability to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers that have a specific binding ability to a target peptide can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers that have a specific binding ability to a target peptide can be selected, for example, by the two-hybrid method using yeast.

Pharmaceutically acceptable carriers
The pharmaceutical composition of this embodiment may further contain a pharma- ceutically acceptable carrier.

As the pharma- ceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular restrictions. More specifically, examples of such carriers include binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, and glycerin; and adhesives such as rubber-based adhesives and silicone-based adhesives.

The pharmaceutical composition of the present embodiment may further contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.

The pharmaceutical composition of this embodiment can be formulated by appropriately combining the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor with the above-mentioned pharma- ceutically acceptable carriers and additives, and mixing them in a unit dose form required for generally accepted pharmaceutical practice.

The pharmaceutical composition of this embodiment may be in a dosage form for oral use or a dosage form for parenteral use, but is preferably in a dosage form for oral use. Dosage forms for oral use include, for example, tablets, capsules, elixirs, microcapsules, etc. Dosage forms for parenteral use include, for example, injections, ointments, patches, etc.

The pharmaceutical composition of the present embodiment may be used in combination with other disease treatment drugs. For example, the FN accumulation inhibitor or TIMP3 accumulation inhibitor is used in combination with antihypertensive drugs, etc., to prevent or treat hypertension while improving symptoms of cerebral small vessel disease.
The FN accumulation inhibitor or TIMP3 accumulation inhibitor and other drugs may be in the same preparation or in separate preparations. Also, each preparation may be administered by the same administration route or by separate administration routes. Furthermore, each preparation may be administered simultaneously, sequentially, or separately at a certain time or period. In one embodiment, the FN accumulation inhibitor or TIMP3 accumulation inhibitor and other drugs may be in the form of a kit that includes them.

Administration Method
Subjects to which the antibody is administered include, but are not limited to, humans, monkeys, dogs, cows, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, and cells thereof. Among these, mammals or mammalian cells are preferred, and humans or human cells are particularly preferred.

Administration to patients or animals can be by methods known to those skilled in the art, such as intrathecal injection, intraarterial injection, intravenous injection, subcutaneous injection, intranasal injection, transbronchial injection, intramuscular injection, transdermal injection, or oral injection. The dosage varies depending on the patient's weight and age, the patient's symptoms, the method of administration, etc., but a person skilled in the art can select an appropriate dosage.

The dosage of the pharmaceutical composition of this embodiment, in terms of the amount of the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor, is generally considered appropriate for oral administration to an adult (body weight 60 kg) at a dose of about 0.1 mg to 10 g, preferably about 0.05 mg to 5 g, and more preferably about 1 mg to 3 g, administered once a day or in divided doses.

When administered parenterally, for example in the form of an injection, it is generally considered appropriate to administer to an adult (body weight 60 kg) approximately 0.01 mg to 3,000 mg per day, preferably approximately 0.05 mg to 300 mg, and more preferably approximately 0.1 mg to 100 mg, once a day or in divided doses.

<Other embodiments>
In one embodiment, the present invention provides a method for preventing or treating cerebral small vessel disease, comprising administering an effective amount of the FN accumulation inhibitor or TIMP3 accumulation inhibitor to a patient who needs treatment.Here, the FN accumulation inhibitor or TIMP3 accumulation inhibitor can be the same as those described above.In addition, the cerebral small vessel disease can be the same as those described above.

In one embodiment, the present invention provides the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor for the prevention or treatment of cerebral small vessel disease. Here, examples of the above-mentioned FN accumulation inhibitor or TIMP3 accumulation inhibitor include those similar to those described above. Furthermore, examples of cerebral small vessel disease include those similar to those described above.

In one embodiment, the present invention provides use of the FN accumulation inhibitor or TIMP3 accumulation inhibitor for producing 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. Examples of cerebral small vessel disease include those similar to those described above.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

Materials and Methods
(Laboratory animals)
High-temperature-requiring serine protease A1 gene-deficient mice (HTRA1-/- mice) were obtained from Lexicon Pharmaceuticals. HTRA1-/- mice were backcrossed with C57BL/6JJcl mice more than 10 times. The genotypes of the mice were determined by PCR using DNA extracted from the tails. HTRA1+/+ littermates were used as controls. Male HTRA1+/+ and HTRA1-/- mice were used to analyze cerebral circulation. In other experiments, both female and male mice were used to ensure an equal sex ratio of HTRA1+/+ and HTRA1-/- mice.
All animal experiments were approved by the Niigata University Animal Use and Care Committee and followed the guidelines of the National Institutes of Health (USA).

(Collection of blood vessels and brain tissue)
Blood was removed from the whole mouse body by transcardial perfusion with Hank's balanced salt solution (HBSS). A portion of the middle cerebral artery with medium-sized branches (called the pial artery) was then isolated from the excised mouse brain in ice-cold Eagle's minimum essential medium (MEM) under a dissecting microscope, immediately frozen on dry ice, and stored at -80°C. The cerebral cortex was dissected and rolled with dry filter paper to remove the pial membrane.

(Preparation of extracellular matrix)
Extracellular matrix preparation was performed using conventional methods. Specifically, pial arteries were first homogenized in 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 in sodium dodecyl sulfonate (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 min. After centrifugation, the SDS-solubilized ECM fraction was subjected to immunoblotting as described below. For LTBP, sodium deoxycholate-insoluble material was digested with 0.3 U/mL plasmin (Sigma) in phosphate-buffered saline (PBS) containing ₁ mM MgCl , ₁ mM CaCl , and 0.1 w/v% n -octyl-β- d -glucopyranoside (Sigma) for 2 h at 37°C. The reaction was stopped by the addition of protease inhibitors, and the reaction was clarified by centrifugation at 8,000 × g for 30 min.

(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. Membranes were probed with the following antibodies: mouse anti-LTBP-4 (AF2885, 1:2000; R&D System), rabbit anti-LTBP-1 (22065-1-AP, 1:500; Proteintech), anti-LTBP-2 (sc-166199, 1:500; Santa Cruz Biotech), rabbit anti-FN (ab2413, 1:1000; Abcam), rabbit anti-TINAGL-1 (12077-1-AP, 1:800; Proteintech), rabbit anti-Nidogen-1 (LS-B9259, 1:500; LifeSpan BioScience), goat anti-LAPβ1 (AF-246-NA, 1:100; R&D System), rabbit anti-TIMP3 (ab58804, 1:200; Abcam), rabbit anti-biglycan (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). Immunoreactivity of the membrane was detected by peroxidase-conjugated anti-immunoglobulin antibody followed by chemiluminescence reaction. Images were captured using an Image Quant LAS-4000 (Fujifilm).

(Preparation of fixed mouse brain tissue)
Blood was removed from the whole body of mice by transcardial perfusion with Ca2 ⁺ -free HBSS at 37°C and 75 mmHg for 10 min. The perfusion pressure was monitored by a manometer. Brain samples were fixed in 4 w/v% paraformaldehyde. For preparation of frozen sections, brains were 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 on a cryostat (CM1950; Leica). For paraffin embedding, brains were processed after dehydration and sectioned at a thickness of 4 μm in the coronal plane. For brain capillary analysis, fixed brains were embedded in agarose and cut using a vibratome (Leica) at a thickness of 50 μm. Isoflurane was used as anesthesia in vascular morphometric analysis. In other experiments, halothane was also used.

(immunohistochemistry)
For mouse brain tissue analysis, antigens were obtained by boiling in 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. Sections were then incubated overnight at 4°C with biotinylated anti-αSMA (LS-C87562, 1:100; LifeSpan BioScience), rat anti-CD31 (PECAM1) (DIA-310, 1:20; Optistain), rabbit anti-FN (mouse tissue: ab2413, 1:250; Abcam), rabbit or goat anti-LTBP-4 (mouse tissue: sc-33144, 1:20; Santa Cruz Biotech, or AF2885, 1:50; R&D Systems), rabbit anti-elastin (PR-387, 1:500; Elastin Products), or goat anti-LAPβ1 (AF-246-NA, 1:50; R&D Systems). Sections were then incubated with Alexa Fluor-labeled secondary antibodies. To simultaneously detect FN and LTBP-4, primary antibodies were fluorescently labeled using Zenon Antibody Labeling Kits (Thermo Fisher Scientific). DyLight-594-conjugated tomato lectin was also used to visualize endothelial cells. Fluorescence microscopy images were acquired using an all-in-one microscope (Keyence; BioRevo BZ-9000) or a confocal laser scanning microscope (LSM710; Carl Zeiss).
Vibratome sections were blocked and incubated with the following antibodies: rat anti-CD13 (R3-63, 1:50; AbD Serotec), rat CD31 (PECAM1) (DIA-310, 1:20; Optistain), or rabbit anti-fibrinogen (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).

(Drug Treatment)
Treatment with candesartan cilexetil (Takeda Pharmaceutical Co., Osaka, Japan) and amlodipine (Wako) was started at 4 months of age via drinking water at doses equivalent to exert a blood pressure lowering effect. Candesartan and amlodipine were dissolved and diluted to a final concentration of 37.5 mg/L or 125 mg/L, respectively. A preliminary study determined that the daily water intake of adult mice (6 months old) was 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. Both doses reduced blood pressure by approximately 15 to 20%.

(Vascular morphometric analysis)
For 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) at equal intervals were selected and analyzed, avoiding arterial branching points, using 5–7 images per animal for each analysis. Brain endothelial cells and vascular smooth muscle cells (SMCs) were visualized by immunostaining with antibodies against CD31 (PECAM1) and αSMA (see immunohistochemistry above). Luminal circumference was measured using a macro in ImageJ (https://www.ipmc.cnrs.fr/~duprat/scripts/) using CD31 images. Similarly, inner and outer circumferences were measured using the inner and outer borders of the αSMA-positive area. Vessel inner diameter was calculated from the luminal circumference. Medial thickness was measured using inner and outer diameters calculated from inner and outer circumferences, respectively. Medial cross-sectional area (CSA) was measured using the αSMA-positive area. Intimal thickness was measured using the internal border of the αSMA-positive area and the inner side of the CD31 layer. Intimal thickness for each image was determined by measuring and averaging 20–30 points per image. CSA and intimal thickness were analyzed using Imaris software (version 6.2.0, bitplane).

(MRI measurement)
MRI experiments were performed using a Varian Unity-INOVA-300 (Varian Inc., Palo Alto, USA) system with an 18-cm bore 7 T horizontal magnet (Magnex Scientific, Abingdon, UK) and active shielding gradients. A birdcage transmit coil with an inner diameter of 6 cm and a quadrature receive coil were used in this experiment. Cerebral blood flow was assessed with centrally sequenced variable tip angle gradient echo (VTE-GRE) by continuous arterial spin labeling (CASL). Prior to the VTE-GRE sequence, a 3-second radio frequency (RF) pulse was alternated with a 1 g/cm axial gradient for 0.8 seconds at ±10 mm from the imaging slice. Sixty-four pairs of images were summed to improve the signal-to-noise ratio. Magnetization transfer rate was assessed under the same conditions as for cerebral perfusion measurements, but without the axial gradient of adiabatic inversion. T1 measurements were performed using a centrally sequenced snapshot flash with a hyperbolic secant inversion pulse and a 32-point (20,000 ms) inversion delay. Quantitative cerebral blood flow maps were calculated from cerebral blood flow images, T1 maps, and magnetization transfer (MTR) maps 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.). Maps were calculated using MRI image calculation software (MR Vision, MR Vision Co., Menlo Park, CA, USA). Animals were anesthetized with 3-5 w/v% isoflurane for induction, followed by 1.2 w/v% for maintenance. Normocapnic cerebral blood flow was measured with 30% _O2 :70% _N2O at 1 L/min under spontaneous breathing via face mask. Hypercapnic conditions were induced with 30% _O2 :60% _N2O :10% _CO2 at 1 L/min. Hypercapnic cerebral blood flow was measured 10 min after induction of hypercapnic conditions under spontaneous breathing. Rectal temperature was kept at 37 ± 0.5°C throughout the measurements using a home-made air conditioning system.

(Transcriptome (RNA seq.) analysis)
Polyadenylated mRNA was enriched by oligo-dT probes. RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit, following the library kit instructions. Library quality control and quantification 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 on an Illumina HiSeq 2500 (high output mode, single reads of 50 bases). Raw reads were adapter trimmed and quality filtered using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) to exclude reads derived from N or ribosomal RNA. Reads after data cleaning (clean reads) were mapped to the mouse reference genome sequence (GRCm38/mm10) using TopHat. Mapped reads were counted using HTseq. DEseq2 was used to identify differentially expressed genes with p-values < 0.05. Gene ontology and KEGG pathway enrichment analysis were performed with the clusterProfiler R package.

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

(ddPCR)
cDNA was synthesized with the superScript VILO ^™ cDNA synthesis kit (Thermo Fisher Scientific). A Bio-Rad QX200 ddPCR system (Bio-Rad, Hercules, CA) was used. Each reaction consisted of 20 μL of a solution containing 10 μL of dddP ^™ Supermix for Probes (Bio-Rad), 1 μM primers, 250 nM probe, and template cDNA. Droplets encapsulating the PCR mix were formed using a Bio-Rad QX-100 emulsifier. After PCR cycling, the droplets were immediately analyzed by QuantaSoft v.1.6 (Bio-Rad). Appropriate housekeeping genes were selected using a mouse housekeeping gene primer set (Takara Bio) and geNorm software. Predesigned primers and probes (5'/FAM/ZEN/IBFQ/3') were purchased from Integrated DNA Technologies (Coralville, IA).

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

[Example 1]
(Inhibitory effect of candesartan and amlodipine treatment on fibronectin accumulation)
The inventors have found through analysis of HTRA1 gene-deficient mice (HTRA1-/- mice), a model mouse of the hereditary cerebral small vessel disease CARASIL, that significant accumulation of fibronectin occurs in cerebral small vessels under HTRA1 deficiency. They also found that accumulation of fibronectin is accompanied by accumulation of fibronectin-binding extracellular matrix proteins, inducing the formation of abnormal elastic lamina.
Accumulation of fibronectin in cerebral small vessels is also seen in patients with isolated cerebral small vessel disease and in hypertensive or diabetic animal models. Furthermore, unlike CARASIL, this finding has also been reported in CADASIL, which is caused by TIMP3 gene mutations. The accumulation of fibronectin is consistent with the pathological finding of arteriolosclerosis, which is a typical symptom, namely vascular intima thickening.
Based on these findings, the inventors hypothesized that inhibition of fibronectin accumulation would be a very effective therapeutic target for cerebral small vessel disease. Based on this hypothesis, they attempted to inhibit fibronectin accumulation by administering candesartan cilexetil, an angiotensin II type 1 receptor antagonist known to be an inhibitor of FN expression, and amlodipine, a dihydropyridine calcium channel antagonist, to HTRA1-/- mice.

Figure 1 shows the levels of each mRNA quantified by ddPCR using total RNA extracted from pial arteries of 4-month-old HTRA1-/- and HTRA1+/+ mice with or without 1 week of candesartan or amlodipine treatment. Each mRNA level is shown relative to the amount in HTRA1+/+ mice.

As shown in Figure 1, HTRA1-/- mice treated with candesartan or amlodipine for 1 week had significantly reduced mRNA levels of Fn1 and EDA (extra domain A) + Fn1 compared to untreated HTRA1-/- mice. The mRNA levels of Ltbp-4 and Bgn were also reduced. The reduction in each mRNA level was particularly pronounced in HTRA1-/- mice treated with candesartan. However, the mRNA levels of elastin and Tgfβ1 were unchanged. Furthermore, the mRNA level of Timp3 was reduced to the same extent in HTRA1-/- mice treated with candesartan and amlodipine.

Previous animal experiments had shown accumulation of fibronectin and LTBP-4 (latent TGF-β binding protein) in 4-month-old HTRA1-/- mice, which worsened with age. Therefore, we began administering candesartan or amlodipine to 4-month-old HTRA1-/- mice and examined the effects of these drug administrations at 24 months of age.
Figures 2A and 3A show images of fibronectin (Fig. 2A) or LTBP-4 (Fig. 3A) in pial arteries of 24-month-old mice using immunohistochemical staining with α-SMA immunostaining to visualize the inside of the vessels. Scale bars in Fig. 2A and 3A are 50 μm. Figures 2B and 3B show the results of quantifying the immunopositive areas of fibronectin or LTBP-4 in cross sections from the images in Fig. 2A and 3A, respectively (n=5-6 mice per group). In Fig. 2B and 3B, the quantification values were normalized by circumference to eliminate the effect of vasodilation in 24-month-old HTRA1−/− mice.

Figures 2A to 3B show that the accumulation of fibronectin and LTBP-4 was suppressed in HTRA1-/- mice treated with candesartan or amlodipine. The reduction in the amount of accumulated fibronectin and LTBP-4 was more remarkable in the case of candesartan treatment than in the case of amlodipine treatment.

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 in pial arteries from 24-month-old HTRA1-/- and HTRA1+/+ mice treated with or without candesartan from 4 to 24 months of age (n=4 mice per group). Figures 4B, 5B, 6B, and 7B are graphs quantifying the expression levels of the bands shown in the images of Figures 4A, 5A, 6A, and 7A, respectively.

As shown in Figures 4A to 7B, fibronectin and LTBP-4 were decreased in HTRA1-/- mice treated with candesartan. In addition, EDA+fibronectin, elastin, LAPβ1, TIMP3, and phosphorylated SMAD3 were decreased.

[Example 2]
(Inhibitory effect of candesartan and amlodipine treatment on vascular structural changes)
We next examined the inhibitory effect of candesartan and amlodipine treatment on vascular structural changes. Specifically, we started administering candesartan or amlodipine to HTRA1-/- mice from 4 months of age and examined the pial arteries at 24 months of age.
Figure 8 shows representative images of PECAM1 and αSMA stained cross sections of pial arteries from 24-month-old mice. In Figure 8, the scale bar is 50 μm. The lower images are higher magnification images of the upper micrographs.
In Figure 9, the leftmost graph shows the intima thickness of the pial artery in each 24-month-old mouse, the second graph from the left shows the inner diameter of the pial artery in each 24-month-old mouse, the third graph from the left shows the inner vascular wall thickness of the pial artery in each 24-month-old mouse, and the fourth graph from the left (rightmost) shows the inner cross-sectional area (CSA) of the pial artery in each 24-month-old mouse, measured from images (n = 5-7 per mouse group).

As shown in Figures 8 and 9, the diameter of the pial artery was significantly increased and the intimal thickness was significantly decreased in HTRA1-/- mice compared with HTRA1+/+ littermate mice. Vascular smooth muscle cells showed a multilayered cellular structure in HTRA1+/+ littermate mice. However, in HTRA1-/- mice, the vascular smooth muscle cell layer was monolayered in places and partially absent. An increase in the diameter of the vascular lumen and a decrease in the thickness of the inner vessel 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 vessel wall were inhibited by candesartan but not by amlodipine.

[Example 3]
(Inhibitory effect of candesartan treatment on the reduction of cerebral blood flow)
Next, we used candesartan, which showed a particularly pronounced therapeutic effect, to examine the inhibitory effect of drug treatment on the decrease in cerebral blood flow. Specifically, candesartan was administered to HTRA1-/- mice from 4 months of age, and cerebral blood flow was measured at rest ( ₀ % CO2) and under hypercapnic conditions (10% _CO2 , 10 minutes) by continuous arterial spin labeling (CASL) using MRI at 16-20 months of age.

Figure 10 shows MRI images of cerebral blood flow in each mouse aged 16 to 20 months. Figure 11 shows graphs calculated from the images in Figure 10 showing cerebral blood flow at rest (left side) and the increase in cerebral blood flow in the cerebral cortex in response to hypercapnia (right side).

As shown in Figures 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 the same level as in HTRA1+/+ littermate mice.

[Example 4]
(Search for genes whose expression is altered by candesartan treatment)
We then investigated genes other than fibronectin that were altered by candesartan treatment, despite the fact that candesartan treatment reduced fibronectin expression. To exclude the effect of age, we performed analyses using mice aged from 4 to 12 months. Specifically, we comprehensively analyzed changes in gene expression in HTRA1-/- mice treated with candesartan for short (1 week) or long (4 to 12 months) periods.

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

In HTRA1-/- mice treated with candesartan for a short period (1 week) or a long period (from 4 to 12 months of age), 10 and 84 genes, respectively, were identified whose expression was altered. Among these, the researchers focused on the Adamtsl2 gene, a gene encoding an extracellular matrix (ECM) protein, as a gene that leads to a decrease in the expression of fibronectin in the body.

Next, the expression levels of the Adamtsl2 gene were confirmed in 4-month-old HTRA1-/- and HTRA1+/+ mice that had been treated with or without candesartan or amlodipine for one week, and in 24-month-old HTRA1-/- and HTRA1+/+ mice that had been treated with or without candesartan or amlodipine from 4 to 24 months of age.
FIG. 13 is a graph showing the mRNA expression level of Adamtsl2 in 4-month-old (left) and 24-month-old (right) mice (n=5-6 mice per group).

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

[Consideration]
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, we have now demonstrated for the first time that short-term (1 week) or long-term (20 months) treatment with candesartan or amlodipine reduces the mRNA expression of Adamtsl2, which is known to be involved in both microfibril and fibronectin network formation, and that fibronectin mRNA expression levels are reduced in Adamtsl2 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.

In addition, TIMP3 has been reported to be an important protein in the pathophysiology of CADASIL. In this study, the inventors confirmed the significant accumulation of TIMP3 in the pial arteries of HTRA1-/- mice. In addition, for the first time, the inventors discovered the significant accumulation of TIMP3 in the cerebral small vessels of CARASIL patients. Furthermore, candesartan or amlodipine treatment suppressed the accumulation of TIMP3 in the pial arteries of HTRA1-/- mice. TIMP3 is the only TIMP that strongly binds to the extracellular matrix (ECM). In the above experiment, various ECM proteins confirmed to accumulate in the pial arteries of HTRA1-/- mice were substrates for HTRA1, but it has not been proven that only TIMP3 binds directly or indirectly to fibronectin. TIMP3 may bind to fibronectin via an intervening protein such as sulfated glycosaminoglycan protein and accumulate, or it may accumulate independently of fibronectin. Analysis of gene expression of various ECM proteins showed that the mRNA expression level of TIMP3 was suppressed by both candesartan and amlodipine treatment, whereas the mRNA expression levels of other ECM proteins were significantly suppressed by candesartan treatment.
Therefore, we will further investigate the mechanism of TIMP3 accumulation and the mechanism of inhibition of TIMP3 accumulation by candesartan and amlodipine treatment.

[Example 5]
(Suppression of vascular structural changes by telmisartan treatment)
Next, to confirm that angiotensin II type 1 receptor antagonists other than candesartan could provide the same therapeutic effect on cerebral small vessel disease, a test was conducted using telmisartan, which is known as an angiotensin II type 1 receptor antagonist. Specifically, telmisartan was administered to HTRA1-/- mice from 4 months of age, and the pial arteries at 24 months of age were confirmed. 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 measurement of the inner diameter of the pial artery in each 24-month-old mouse from the cross-sectional images of PECAM1 and αSMA staining of the pial artery in each 24-month-old mouse (n = 5 to 7 mice per mouse group). In Figure 14, "HTRA1+/+" is the HTRA1+/+ mouse group not administered telmisartan, "HTRA1-/-" is the HTRA1-/- mouse group not administered telmisartan, and "HTRA1-/- +Telm." is the HTRA1-/- mouse group administered telmisartan.

As shown in Figure 14, the diameter of the pial artery was significantly increased in HTRA1-/- mice. On the other hand, in HTRA1-/- mice administered telmisartan, the increase in the diameter of the pial artery was suppressed, similar to the candesartan-administered group.

From the above, it has been 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.

The pharmaceutical composition of this embodiment can prevent or treat cerebral small vessel disease.

Claims (5)

A pharmaceutical composition for preventing or treating cerebral small vessel disease, except for cerebral small vessel disease caused by hypertension or diabetes, and cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, which is accompanied by accumulation of fibronectin in the intima of cerebral small vessels due to aging , comprising a fibronectin accumulation inhibitor as an active ingredient,
A pharmaceutical composition, wherein the fibronectin accumulation inhibitor is candesartan or telmisartan . The pharmaceutical composition according to claim 1 , wherein the fibronectin accumulation inhibitor is a compound having an effect of inhibiting Adamtsl2 expression. The pharmaceutical composition according to claim 1 or 2, wherein the fibronectin accumulation inhibitor is telmisartan. 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. 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.