ATP-sensitive potassium channel inhibitor and pharmaceutical composition containing same

Abstract

The present invention addresses the problem of providing a new ischemic disease treatment strategy targeting pericytes. As a solution, the present invention provides an inflammation suppressant or a tissue protective agent for ischemic injury, each including an inhibitor which blocks ATP-sensitive potassium (KATP) channels present in a pericyte. In particular, the KATP channel is composed of pore-forming KIR6.1 and regulatory SUR2 subunits, and it is preferable for the inhibitor to act on the KIR6.1. The inflammation suppressant or the tissue protective agent for ischemic injury is preferably used in the treatment of ischemic diseases such as cerebral infarction or ischemic cardiac disease.

Description

ATP-sensitive potassium channel inhibitor and pharmaceutical composition containing the same

[0001] The present invention relates to inhibitors that close ATP-sensitive potassium channels expressed in pericytes (pericyte ATP-sensitive potassium channel inhibitors) and pharmaceutical compositions containing the same.

[0002] ATP-sensitive potassium channels (KATP channels) are inwardly rectifying potassium ions (KATP channels) that link intracellular metabolic status with plasma membrane excitability. ＋ ATP-sensitive potassium channels are composed of KIR6.X (KIR6.1 or KIR6.2), which form the channel pore, and sulfonylurea receptor SUR (SUR1, SUR2A, or SUR2B) (see Figure 10). The combination of KIR6.X and SUR that constitutes an ATP-sensitive potassium channel varies depending on the organ (tissue) in which the KATP channel-expressing cells are present. For example, KATP channels expressed in vascular smooth muscle cells are composed of KIR6.1 and SUR2B; KATP channels expressed in neurons and pancreatic beta cells are composed of KIR6.2 and SUR1; and KATP channels expressed in cardiac myocytes are composed of KIR6.2 and SUR2A (see Non-Patent Documents 1 and 2).

[0003] Pericytes are cells that line the outer periphery of capillaries (microvessels) and are present not only in the blood vessels of the brain but also in the blood vessels of other organs such as the heart, retina, and spinal cord. Pericytes in brain capillaries (intracerebropericytes) are said to be essential cells for maintaining brain homeostasis, responsible for the blood-brain barrier (BBB) ​​function that controls the exchange of substances between the blood and the brain. For example, Non-Patent Document 3 reports that the BBB is disrupted in mouse mutants lacking intracerebral pericytes.

[0004] The structure and function of KATP channels expressed in cerebral pericytes have also been elucidated. For example, Non-Patent Document 4 reports the results of analyzing the cell types in the cerebrovascular system and the genetic phenotypes of each cell; it has been reported that cerebral pericytes specifically express the KCNJ8 gene encoding KIR6.1 and the ABCC9 gene encoding SUR2 (i.e., cerebral pericytes express a KATP channel composed of KIR6.1 and SUR2). Furthermore, Non-Patent Document 5 discusses the relationship between KATP channels expressed in cerebral pericytes and the etiology of brain disorders.

[0005] Known inhibitors that close ATP-sensitive potassium channels include sulfonylureas (SUs), mitiglinide, and U37883A (also known as PNU-37883A). A compound called U37883A (4-morpholinecarboxyimidine-N-adamantyl-N'-cyclohexylhydrochloride) is known to close ATP-sensitive potassium channels by acting on KIR6.1 (see Non-Patent Documents 6, 7, 8, and 9). Sulfonylureas (SUs) may inhibit ATP-sensitive potassium channels by acting on the sulfonylurea receptor (SUR) that constitutes the ATP-sensitive potassium channel. Glibenclamide, a sulfonylurea (SU) drug, is known to potentially close ATP-sensitive potassium channels. Glibenclamide has also been reported to have anti-stroke effects (Non-Patent Document 10). It has been reported that mitiglinide may selectively act on SUR1 (but not on USR2) and inhibit ATP-sensitive potassium channels (Non-Patent Document 11).

[0006] On the other hand, ischemic diseases occur when arteriosclerosis, blood clots, etc. cause an insufficient supply of blood and oxygen to various organs. For example, in a stroke caused by ischemia (ischemic stroke), the brain is deprived of blood and oxygen, which can lead to cerebral infarction (necrosis of part of the brain tissue). In heart disease caused by ischemia (ischemic heart disease), the heart muscle is deprived of oxygen and nutrients, which can lead to myocardial infarction (necrosis of the heart muscle).

[0007] Cerebral and myocardial infarctions caused by ischemia are major causes of death worldwide and carry a high risk of requiring nursing care, so there is a strong desire to control them. However, once brain (central) and myocardial tissues are damaged, it is extremely difficult to induce regeneration, so it is necessary to establish a medical infrastructure that can reduce tissue damage as much as possible.

[0008] Functional and structural understanding of ATP-sensitive K+ (KATP) channel regulation (Japanese Pharmacological Journal, 126, 311-316, 2005) Structure and function of ATP-sensitive K+ channels in various organs: Aiming for improvement of QOL through drug control (Japanese Pharmacological Journal, 122, 243-250, 2003) Pericytes regulate the blood-brain barrier (Nature, 468, 557-561, 2010) A molecular atlas of cell types and zonation in the brain vasculature (Nature. 2018 Feb. 22; 554 (7693): 475-480) KCNJ8 / ABCC9-containing K-ATP channels modulate brain vascular smooth muscle development and neurovascular coupling (Development Cell, 57, 1383-1399, 2022) Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A (British Journal of Pharmacology, 128, 667-672, 1999)Different molecular sites of action for the KATP channel inhibitors,PNU-99963 and PNU-37883A (British Journal of Pharmacology (2003) 139, 122-128)Pharmacological Profiling of KATP Channel Modulators: An Outlook for New Treatment Opportunities for Migraine (Pharmaceuticals, 2023, 16, 225.)Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1 / SUR2B KATP Channels (Molecular Pharmacology, March 2024, 105 (3) 202-212)Profile of intravenous glyburide for the prevention of cerebral edema following large hemispheric infraction: evidence to date (Drug Design, Development and Therapy 2018:12 2539-2552)Effects of mitiglinide (S 21403) on Kir6.2 / SUR1, Kir6.2 / SUR2A and Kir6.2 / SUR2B types of ATP-sensitive potassium channel (British Journal of Pharmacology (2001) 132, 1542-1548).

[0009] The present inventors have discovered a new finding that controlling ATP-sensitive potassium (KATP) channels expressed in pericytes can suppress inflammation in tissues and inhibit the progression of tissue damage. Therefore, an objective of the present invention is to provide a new therapeutic strategy for ischemic diseases that targets pericytes. More specifically, an objective of the present invention is to provide a new treatment option for cerebral infarction, ischemic heart disease, and other conditions by suppressing inflammation or damage in tissues through closure of KATP channels expressed by pericytes.

[0010] A first aspect of the present invention relates to the following agents: <1> An anti-inflammatory agent or a tissue protective agent against ischemic injury, comprising an inhibitor that closes an ATP-sensitive potassium (KATP) channel expressed in pericytes.

[0011] The first aspect of the present invention preferably relates to the following drugs: <2> An inflammation suppressor or tissue protective agent according to <1>, wherein the ATP-sensitive potassium (KATP) channel is composed of a pore component KIR6.1 and a regulatory subunit SUR2, and the inhibitor acts on KIR6.1; <3> An inflammation suppressor or tissue protective agent according to <1> or <2>, wherein the inhibitor selectively closes an ATP-sensitive potassium (KATP) channel composed of a pore component KIR6.1 and a regulatory subunit SUR2; <4> An inflammation suppressor or tissue protective agent according to any of <1> to <3>, wherein the inhibitor inactivates the NF-kB pathway in pericytes or vascular endothelial cells.

[0012] The second aspect of the present invention relates to the following pharmaceutical compositions: <5> A pharmaceutical composition comprising any one of the agents <1> to <4>; <6> The pharmaceutical composition according to <5>, which is a therapeutic agent for cerebral infarction; <7> The pharmaceutical composition according to <5>, which is a therapeutic agent for ischemic heart disease.

[0013] The present invention provides a new therapeutic strategy for ischemic diseases that targets pericytes, and is therefore expected to lead to the development of therapeutic methods for ischemic diseases that have not been adequately addressed until now.

[0014] This figure is a supplementary explanatory diagram illustrating the procedure for mouse ischemic surgery to create a mouse cerebral ischemia (transient middle cerebral artery occlusion) model. This figure is a graph showing the effect of U37883A administration on cerebral infarction volume in control mice and Kcnj8 KO mice in a cerebral ischemia model. This figure is a graph showing the effect of U37883A administration on neurological disability scores in control mice and Kcnj8 KO mice in a cerebral ischemia model. This figure is a graph showing the effect of U37883A administration on the mouse locomotion rate (a) and distance traveled (b) in control mice and Kcnj8 KO mice in a cerebral ischemia model. This figure is a graph showing that an increase in cerebral infarction volume is suppressed in a cerebral ischemia model using p65(Rela) gene knockout mice. This figure is a graph showing that NF-kB activation in pericytes and vascular endothelial cells is suppressed in a mouse cerebral ischemia model when U37883A is administered. 1 is a graph showing that the expression of various cytokines was suppressed when U37883A was administered to a mouse cerebral ischemia model. 2 is a graph showing the expression levels of various cytokines when U37883A was administered to control mice and Kcnj8 KO mouse cerebral ischemia models. 3 is a graph comparing the neurological scores of mice administered U37883A with other KATP channel inhibitors or activators. 4 is a schematic diagram showing the structure of an ATP-sensitive potassium channel (KATP channel).

[0015] [1. Inhibitor that closes ATP-sensitive potassium (KATP) channels expressed in pericytes] The gist of the present invention is to provide an anti-inflammatory agent, a tissue protective agent, etc., which contain an "inhibitor that closes ATP-sensitive potassium (KATP) channels expressed in pericytes" as an active ingredient, and further to use the same as a pharmaceutical composition, etc.

[0016] [1-1. About Pericytes] Most blood vessels have vascular endothelial cells that line the inside of the blood vessels and pericytes that line the outside. Pericytes are a general term for vascular smooth cells or pericytes, each of which exists in different blood vessels. That is, vascular smooth cells exist in arterioles, arteries, veins, etc., while pericytes exist in capillaries, venules, etc. Pericytes are also present in various organs, and pericytes in the brain are involved in the function of the blood-brain barrier (BBB) ​​and the control of cerebral blood circulation; pericytes in the heart have been reported to affect cardiac function; and pericytes in the kidneys are involved in blood filtration function, etc.

[0017] The present invention is directed to controlling various functions of KATP channels by closing KATP channels expressed by pericytes present in various organs, and applying this to disease treatment and the like.

[0018] [1-2. About ATP-sensitive potassium (KATP) channels] KATP channels are inwardly rectifying potassium ion channels that link intracellular metabolic status with cell membrane excitability. KATP channels close when the intracellular ATP concentration increases, and conversely, open when the ATP concentration decreases or the intracellular NDP concentration increases.

[0019] KATP channels consist of a double-spanning membrane protein Kir6.X that forms the pore and sulfonylurea receptors (SURs) that belong to the ABC protein family (see Figure 10). Kir6.X, which constitutes KATP channels, has two subtypes, KIR6.1 (KCNJ8) and KIR6.2 (KCNJ11), and SUR has two subtypes, SUR1 (Abcc8) and SUR2 (Abcc9). SUR2 has two splicing variants, SUR2A and SUR2B.

[0020] The structure of KATP channels (the combination of Kir6.X and SUR) varies depending on the type of cell that expresses it and the organ in which the cell resides. KATP channels expressed in pericytes are composed of KIR6.1 and SUR2; KATP channels expressed in neurons and pancreatic beta cells are composed of KIR6.2 and SUR1; and KATP channels expressed in cardiac myocytes are composed of KIR6.2 and SUR2. Thus, KATP channels composed of KIR6.1 and SUR2 are said to be expressed specifically in pericytes.

[0021] The present invention is preferably applied to the treatment of diseases in various organs (preferably the brain or heart) by closing the "KATP channel composed of KIR6.1 and SUR2" that is expressed specifically in pericytes.

[0022] [1-3. Inhibitors of the Present Invention] The "inhibitor" of the present invention closes the KATP channel expressed in pericytes. Specifically, the "inhibitor" of the present invention acts on Kir6.X or SUR to close the KATP channel, i.e., inhibit potassium ion current.

[0023] The KATP channel expressed in pericytes is preferably a KATP channel that is "specifically" expressed in pericytes and is composed of KIR6.1 and SUR2. Therefore, the inhibitor of the present invention preferably closes the KATP channel by acting on KIR6.1 or SUR2, more preferably by acting on KIR6.1, and even more preferably by selectively acting on KIR6.1 (but not on Kir6.2).

[0024] KATP channels include not only "KATP channels composed of KIR6.1 and SUR2," but also "KATP channels composed of KIR6.1 and SUR1," "KATP channels composed of KIR6.2 and SUR1," and "KATP channels composed of KIR6.2 and SUR2." However, it is preferable that the inhibitors of the present invention "selectively" close "KATP channels composed of KIR6.1 and SUR2."

[0025] Drugs known to act on Kir6.X or SUR and inhibit KATP channels include the sulfonylurea drugs Glibenclamide (acts on SUR), Glimepiride (acts on SUR), Mitiglinide (selectively acts on SUR1), and U37883A (acts on KIR6.1).

[0026] Glibenclamide and glimepiride can act non-selectively on both SUR1 and SUR2, but it is said that the closure of the KATP channel is due to their action on SUR1 (see the literature below). (1) Nat Med, 2006 April; 12(4): 433-440. “Newly expressed SUR1-regulated NCCa-ATP channel mediates cerebral edema after ischemic stroke” (2) Journal of Cerebral Blood Flow & Metabolism (2013) 33, 356-364. “Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia” (3) Stroke. 2009 February; 40(2): 604-609. “Protective Effect of Delayed Treatment With Low-Dose Glibenclamide in Three Models of Ischemic Stroke” (4) Diabetes, 62: 1289-1296, 2013 “The DPP-4 Inhibitor Linagliptin Counteracts Stroke in the Normal and Diabetic Mouse Brain A Comparison With Glimepiride”

[0027] Mitiglinide strongly inhibits KATP channels consisting of Kir6.2 and SUR1, but only to a lesser extent inhibits KATP channels consisting of Kir6.2 and SUR2 (2A or 2B). Therefore, it is speculated that mitiglinide selectively acts on SUR1 to inhibit (close) KATP channels (see Non-Patent Document 11 mentioned above).

[0028] U37883A, the drug used as an inhibitor in the Examples herein, is known to inhibit the KATP channel composed of KIR6.1 and SUR2 by acting on KIR6-1, making it a preferred drug for use as an inhibitor in the present invention. Rosiglitazone has also been reported to inhibit the KATP channel composed of KIR6.1 and SUR2 by acting on KIR6-1 (see Non-Patent Document 8, Pharmaceuticals 2023, 16, 225). Furthermore, a compound designated VU0542270 (see formula below) has been reported to selectively inhibit the vascular KATP channel composed of Kir6.1 and SUR2B (see Non-Patent Document 9, Molecular Pharmacology, March 2024, 105 (3), 202-212).

[0029] The inhibitor in the present invention preferably inhibits the KATP channel by acting on KIR6.1 or SUR2, more preferably KIR6.1, but its modality is not particularly limited and may be a small molecule drug, an antibody, a nucleic acid, a cell, etc.

[0030] 2. Use of Inhibitors in the Present Invention In the present invention, inhibitors that close ATP-sensitive potassium (KATP) channels expressed in pericytes can be used as anti-inflammatory agents or tissue protective agents.

[0031] The term "inflammation suppressor" refers to a drug that suppresses the expression of cytokines (inflammatory cytokines). Examples of inflammatory cytokines whose expression is suppressed include, but are not limited to, interleukin 1, interleukin 6, chemokines, and TNF (tumor necrosis factor).

[0032] A tissue protective agent refers to a drug that protects tissue, particularly suppresses tissue damage. Tissue damage includes necrosis of tissue cells. The inhibitor of the present invention can preferably serve as a tissue protective agent against ischemic injury. Ischemia refers to a state in which blood is not sufficiently supplied to tissues or cells that are supplied with blood by blood vessels, resulting in a lack of oxygen in tissues or cells. Ischemic injury refers to necrosis of tissues or cells due to ischemia.

[0033] As will be shown in Example 1 below, the inhibitor of the present invention strongly suppresses the progression of brain tissue damage by closing pericyte KATP channels in a cerebral infarction model (an animal model in which cerebral arteries are occluded and blood supply to brain tissue is cut off).

[0034] The mechanism by which the inhibitors of the present invention suppress inflammation is not particularly limited, but may involve inactivating the NF-kB pathway in cells. NF-kB is a transcription factor that controls the expression of genes involved in inflammation and immune responses, such as cytokines, and is composed of a heterodimer of p65 (Rela) and p50 (NFkB1) in many cells, including pericytes. Therefore, activation of the NF-kB pathway induces the expression of various inflammatory cytokines. Furthermore, activation of the NF-kB pathway is triggered by the translocation of p50 and p65 into the nucleus of cells.

[0035] The inhibitors of the present invention have been confirmed to suppress the nuclear translocation of p65 in pericytes and vascular endothelial cells in the brain in Example 3 below, suggesting that they inactivate the NF-kB pathway. Therefore, the inhibitors of the present invention may suppress inflammation by inactivating the NF-kB pathway in pericytes and other cells. Furthermore, because tissue injury can be caused by inflammation, the inhibitors of the present invention may protect tissue by inactivating the NF-kB pathway.

[0036] [3. Pharmaceutical Composition] The inflammation suppressor or tissue protective agent of the present invention can be used as a pharmaceutical composition. That is, since the inflammation suppressor or tissue protective agent of the present invention contains an inhibitor that closes the KATP channel expressed in pericytes, it can be used to treat or prevent various diseases in tissues where pericytes are present. In particular, since it is known that the KATP channel composed of KIR6.1 and SUR2 is specifically expressed in pericytes in the brain, heart, retina, and spinal cord, it is preferable to use it for treating or preventing diseases in the brain, heart, retina, and spinal cord.

[0037] In particular, the inflammation suppressant or tissue protective agent of the present invention can be used as a therapeutic or preventive agent for cerebral infarction. Cerebral infarction is a disease in which cerebral arteries are blocked for some reason, resulting in a lack of blood supply to brain tissue, resulting in brain necrosis (tissue damage). In Example 1 described below, it has been shown that the inhibitor of the present invention potently suppresses the progression of brain tissue damage in a cerebral infarction model, indicating that it can be used as a therapeutic agent for cerebral infarction.

[0038] Cerebral infarction that can be treated with the pharmaceutical composition of the present invention may be any of lacunar infarction, atherothrombotic cerebral infarction, cardiogenic cerebral embolism, etc. Lacunar infarction is cerebral infarction caused by clogging of cerebral blood vessels that have narrowed due to arteriosclerosis; atherothrombotic cerebral infarction is cerebral infarction caused by the formation of a blood clot as a result of cholesterol accumulation in cerebral blood vessels; and cardiogenic cerebral embolism is cerebral infarction caused by a blood clot formed in another body part, such as the heart, flowing into a cerebral blood vessel and clogging it.

[0039] The stage of cerebral infarction that can be treated with the pharmaceutical composition of the present invention may be any of the acute, recovery, and chronic phases. In particular, treatment of cerebral infarction during the acute phase is important, and it is necessary to prevent the progression of tissue damage during the acute phase. The pharmaceutical composition of the present invention is expected to contribute to the treatment of cerebral infarction by suppressing the progression of tissue damage during the acute phase.

[0040] The inflammation suppressing agent or tissue protecting agent of the present invention can also be used as a therapeutic or preventive agent for ischemic diseases in other organs. Examples of ischemic diseases include ischemic heart diseases such as myocardial infarction, spinal cord ischemia such as spinal cord infarction, and ocular ischemic syndromes such as retinal artery occlusion.

[0041] The route of administration of the pharmaceutical composition of the present invention is not particularly limited and may be either oral or parenteral. Examples of parenteral administration include injection (intravenous injection, subcutaneous injection, intramuscular injection, etc.), transdermal administration, eye drops, etc. The dosage form of the pharmaceutical composition of the present invention is also not particularly limited and may be selected depending on the physical properties of the inhibitor and the route of administration, etc., and can be manufactured as a pharmaceutical in an appropriate dosage form such as tablets, capsules, granules, powders, eye drops, mouthwashes, ointments, creams, gels, poultices, patches, liniments, tapes, poultices, injections, or suppositories.

[0042] The present invention will be described in more detail below with reference to examples, but the scope of the present invention should not be construed as being limited by these examples.

[0043] In the following examples, U37883A (sometimes referred to as PNU-37883A) was used as an inhibitor of ATP-sensitive potassium (KATP) channels expressed in pericytes. U37883A is a drug with the molecular structure shown below. It has been reported that U37883A blocks KATP channels composed of Kir6.1 and SUR1 but not KATP channels composed of Kir6.2 and SUR1, indicating that it selectively acts on Kir6.1 (see the aforementioned non-patent document 6: British Journal of Pharmacology, 128, 667-672, 1999). It has also been reported that U37883A strongly inhibits (closes) the KATP channel consisting of Kir6.1 and SUR2B (see the aforementioned non-patent document 7: British Journal of Pharmacology (2003) 139, 122-128); and it has been reported that U-37783A selectively inhibits the Kir6.1 / SUR2B subtype of human Kir6.1 / SUR2B, Kir6.2 / SUR1, and Kir6.2 / SUR2A KATP channels (see the aforementioned non-patent document 8: Pharmaceuticals, 2023, 16, 225.).

[0044] Example 1 1. Inhibitory Effect of U37883A (KATP Channel Blocker) on Tissue Injury after Cerebral Infarction 1-A. Preparation of Kcnj8 Gene-Deficient Mice (Kcnj8KO Mice) Kcnj8 gene-deficient mice were prepared according to the method described in Development Cell, 57, 1383-1399, 2022 (see Non-Patent Document 5, cited above). Kcnj8 gene-deficient mice lack KIR6.1 in the KATP channel expressed in pericytes. Briefly, Kcnj8flox mice, in which exon 3 is flanked by loxP sites, were crossed with PGK-Cre mice to generate a constitutive Kcnj8 null allele, and heterozygous and homozygous Kcnj8 knockout (KO) mice were then bred.

[0045] 1-B. Mouse ischemic surgery (creation of a cerebral ischemia (transient middle cerebral artery occlusion) model) Control mice and Kcnj8 KO mice underwent ischemic surgery. Specifically, the cerebral ischemia model was created using the method described in "Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining" (JoVE, November 2012, 69, e4038, pages 1-5). The procedure is briefly described below.

[0046] The mouse was maintained under isoflurane anesthesia, and a midline neck incision was made and the surrounding soft tissue was separated. The left common carotid artery (CCA) was then freed from the surrounding tissue and permanently sutured with silk suture (see Figure 1A, 1). The branches of the left internal common carotid artery (ICA) and external common carotid artery (ECA) were then freed from the surrounding tissue. The ECA was permanently sutured (see Figure 1A, 2), and the left ICA was clipped with suture (see Figure 1A, 4) to prevent bleeding. A small hole (see Figure 1A, 5) was made in the CCA between the permanent suture (see Figure 1A, 1) and the clip (see Figure 1A, 4).

[0047] Next, a silicone-coated filament (see Figure 1B, 6) (0.17–0.23 mm diameter, 9–10 mm length) was inserted through a small hole (see Figure 1B, 5) into the CCA and then into the ICA. The suture clip (see Figure 1B, 4) was loosened, and the filament was inserted into the origin of the MCA. The suture clip (see Figure 1B, 4) was re-tied tightly to prevent the filament from moving. The skin was closed with sutures, and the mouse was placed on a heat pad for 45 minutes.

[0048] The mouse was re-anesthetized with isoflurane, the temporary suture (see 4 in Figure 1B) in the ICA was loosened, and the filament (see 6 in Figure 1B) was withdrawn to allow blood reperfusion. The wound was then sutured and placed in a cage with a heat pad for 1 hour.

[0049] 1-C. Administration of U37883A. Six hours after ischemic surgery, control mice and Kcnj8 KO mice were administered phosphate-buffered saline (PBS) or 10 μM U37883A (4-morpholinecarboxyimidine-N-adamantyl-N'-cyclohexyl hydrochloride) in PBS into the lateral ventricle. The procedure is briefly described below (see STAR Protocol, 2 (3), 2021, 100725).

[0050] Mice undergoing ischemic surgery were anesthetized with isoflurane, their heads fixed, and the scalp incised to remove the pericranium. Bregma and lambda were adjusted to be horizontal (to match the height). A small hole was drilled in the skull with a microdrill to allow the syringe needle to reach the brain surface. The syringe was refilled with drug (PBS or PBS containing U37883A), and the syringe needle was extended to the brain surface and penetrated a further 3 mm. 5 μl of drug was injected at a rate of 2.5 μl / min. After the needle was removed, the hole in the skull was covered with cement and the scalp was sutured.

[0051] 1-D. Measurement of Infarct Volume in Mouse Brain Tissue. Cerebral infarct volume was measured 24 hours after ischemic surgery. The results are shown in Figure 2. As shown in Figure 2, cerebral infarct volume was significantly reduced in the U37883A-treated group compared with the PBS-treated group in control mice, with a reduction rate of 68.9%. On the other hand, in Kcnj8 KO mice, cerebral infarct volume tended to be reduced in the U37883A-treated group compared with the PBS-treated group, but this reduction was not significant; the reduction rate was smaller than that in control mice.

[0052] 1-E. Discussion As shown in Figure 2, U37883A significantly reduced cerebral infarction volume in control mice, but not in Kcnj8 KO mice. This suggests that U37883A inhibited the progression of cerebral infarction by acting on KIR6.1, which constitutes the KATP channel in pericytes, and by blocking (closing) the KATP channel. Thus, it was demonstrated that inhibiting the KATP channel in pericytes can inhibit the progression of cerebral infarction.

[0053] Furthermore, when U37883A (see section 1-C above) was administered to mice 2 hours before ischemic surgery rather than after, U37883A significantly reduced cerebral infarction volume in control mice, but not in Kcnj8 KO mice.

[0054] [Example 2] 2. Reduction of Cerebral Dysfunction by U37883A For the cerebral ischemia model in Example 1 to which a drug (PBS or PBS containing U37883A) was administered, the neurological impairment score (Fig. 3), and the mobility rate and distance traveled (Fig. 4) were evaluated 24 hours after ischemic surgery.

[0055] Neurological injury scores were evaluated on a 5-point scale from 0 to 4. These scores are general evaluation scores described in "Stroke 1989, 20, 84-91, Reversible Middle Cerebral Artery Occlusion Without Craniectomy in Rats" and were evaluated according to the following criteria. The results are shown in Figure 3. Score 0: No symptoms of neurological injury are observed. Score 1: Mild symptoms of focal neurological injury are observed (when the tail is lifted, the rat turns its body in the direction opposite to the infarcted side (left side) and does not move the front paws on the infarcted side (left side)). Score 2: Moderate symptoms of focal neurological injury are observed (the rat always turns toward the infarcted side (left side)). Score 3: Severe symptoms of focal neurological injury are observed (it has difficulty walking on all fours and falls toward the infarcted side (left side)). Score 4: No spontaneous walking, decreased level of consciousness, or death.

[0056] Mice were moved from their home cages into a 30 cm square cage. Their behavior was tracked for 10 minutes after the transfer using a video behavior analysis system, EthoVision XT (Sophia Scientific), and the movement rate (Fig. 4(a)) and distance traveled (Fig. 4(b)) were quantified.

[0057] As shown in Figure 3, in the cerebral ischemia model of control mice, administration of U37883A improved the neurological impairment score compared to administration of the vehicle, but in the cerebral ischemia model of Kcnj8 KO mice, there was no significant difference between administration of the vehicle and administration of U37883A.

[0058] As shown in Figure 4, in the cerebral ischemia model of control mice, administration of U37883A increased the rate and distance traveled (i.e., increased activity) compared with vehicle administration. In the cerebral ischemia model of Kcnj8KO mice, administration of U37883A also increased the rate and distance traveled compared with vehicle administration; however, the rate of increase was lower than in control mice.

[0059] These results indicate that U37883A acts on KIR6.1, which constitutes the KATP channel in pericytes, and inhibits (closes) the KATP channel, thereby reducing brain dysfunction, improving neurological disability scores, and increasing activity levels.

[0060] [Reference Example] 3. Relationship between NF-kB pathway activation and progression of tissue damage in cerebral infarction 3-A. Preparation of p65 (Rela) gene knockout mice 8-12 week old CAG-CreER model mice TMp65(Rela)Flox / Flox (The Jackson Laboratory, https: / / www.jax.org / strain / 004682) mice were orally administered tamoxifen solution (1.0 mg / 100 μl corn oil / 10 g body weight) or its vehicle daily for 7 days and then maintained for an additional week. The tamoxifen-treated mice were knocked out of p65(Rela), a subunit of the Rela gene and a member of the NF-kB family. The p65(Rela) knocked-out mice exhibited blockage of the NF-kB pathway, resulting in inactivation.

[0061] In addition, Rela mice were used as control mice. Flox / Flox (See J. Clin. Invest. 117, 1490-1501 (2007).) Tamoxifen solution was administered daily for 7 days and then maintained for an additional week. Rela Flox / Flox mice were provided by Professor Roland M. Schmid (Technical University of Munich).

[0062] 3-B. Mouse ischemic surgery (preparation of cerebral ischemia (transient middle cerebral artery occlusion) model) Each mouse obtained in 3-A was subjected to 45 minutes of transient middle cerebral artery occlusion (tMCAO) treatment using the same procedure as in Example 1 above (1-B. Mouse ischemic surgery).

[0063] 3-C. Measurement of cerebral infarct volume in mouse brain tissue 24 hours after the treatment in 3-B, the cerebral infarct volume of each mouse was measured. The results are shown in Figure 5. As shown in Figure 5, TM p65(Rela) Flox / FloxMice that had p65(Rela) knocked out by administering tamoxifen (labeled "TAM" in Figure 5) showed a significant reduction in cerebral infarct volume compared to mice that did not receive tamoxifen (treated with the vehicle "VEH"). Furthermore, Rela Flox / Flox mice also showed a significant reduction in cerebral infarct volume compared to mice that received tamoxifen. These results demonstrate that blocking and inactivating the NF-kB pathway can delay the progression of tissue damage during the acute phase of cerebral infarction.

[0064] [Example 3] 4. Suppression of NF-kB Activation by U37883A As shown in the Reference Example above, blocking and inactivating the NF-kB (NF-kappa B) pathway (knock-acting p65(Rela)) was shown to slow the progression of tissue damage in the acute phase of cerebral infarction. Activation of the NF-kB pathway is known to be caused by the translocation of its component, p65, into the cell nucleus and the induction of cytokines and other expression. Therefore, it is thought that inhibiting the translocation of p65 into the nucleus would inactivate the NF-kB pathway, thereby slowing the progression of tissue damage in cerebral infarction. Based on this, Example 3 was carried out.

[0065] 4-A. Administration of U37883A: Mice underwent ischemic surgery on either the left or right hemisphere of the brain using the same procedure as in Example 1 (see "1-B. Ischemic surgery in mice"), and then U37883A or vehicle was administered (see "1-C. Administration of U37883A"). Mice administered U37883A or vehicle were euthanized 24 hours after the ischemic surgery.

[0066] 4-B. Evaluation of p65 Translocation into the Nuclei of Pericytes and Vascular Endothelial Cells. Brain tissue (cerebral cortex or striatum) from the ischemic side of mice euthanized in 4-A was collected. The collected brain tissue was treated with DAPI (4,6-diamidino-2-phenylindole) to selectively label and stain the nuclear regions of cells. The same brain tissue was also treated with CD31 antibody to selectively label vascular endothelial cells, and with PDGFRβ antibody to selectively label and stain pericytes. The same tissue was then treated with p65 antibody to detect p65 translocated into the nuclei of each cell. The percentage of pericytes and vascular endothelial cells in which p65 was detected is shown in Figure 6.

[0067] As shown in Figure 6, administration of U37883A significantly reduced the percentage of cells in which nuclear p65 was detected in both pericytes and vascular endothelial cells. This suggests that U37883A inhibits nuclear translocation of p65, thereby inactivating the NF-kB pathway and slowing the progression of tissue damage in cerebral infarction.

[0068] 4-C. Evaluation of Cytokine Expression. Furthermore, 1 mm coronal brain sections located between Bregma +2 and +1 mm were collected from both the ischemic and non-ischemic hemispheres of mice euthanized in 4-A. RNA was extracted from the brain sections, and a cDNA library was constructed by reverse transcription using the extracted RNA as a template. This cDNA library was used to quantitatively analyze various inflammatory markers by quantitative PCR. Figure 7 shows the quantitative results for mRNA expression of (a) interleukin-1b, (b) interleukin-6, (c) the chemokine Ccl2, and (d) the cytokine TNF-α.

[0069] In Figure 7, "I" indicates the ischemic hemisphere, and "C" indicates the non-ischemic hemisphere. Figure 7 compares the expression levels of each cytokine between the ischemic hemisphere and the vehicle-treated hemisphere. The y-axis in Figure 7 indicates the ratio of mRNA expression in the vehicle-treated hemisphere "C" to 1. As shown in Figure 7, no cytokine mRNA expression was observed in the non-ischemic hemisphere "C." In contrast, all cytokine mRNA expression was observed in the ischemic hemisphere "I," but the expression levels were significantly reduced with U37883A administration compared to the vehicle-treated hemisphere. This demonstrates that U37883A administration can suppress cytokine expression in cerebral infarction.

[0070] Example 4 As in Example 1, Kcnj8 gene-deficient mice and control mice were subjected to ischemic surgery and administered U37883A or vehicle (see 1-A to 1-C). The mice were then euthanized as in Example 3, and the expression of various cytokines in brain tissue was evaluated (see 4-C). Figure 8 shows the quantitative results for the expression levels of (a) interleukin-1b, (b) interleukin-6, (c) the chemokine Ccl2, and (d) the cytokine TNF-α.

[0071] As shown in Figure 8, in control mice (Ctrl), U37883A treatment suppressed the mRNA expression of various cytokines compared to vehicle treatment. The y-axis in Figure 8 represents the ratio of mRNA expression levels in vehicle-treated control mice to 1. In Kcnj8-deficient mice, U37883A treatment also tended to suppress the mRNA expression of various cytokines compared to vehicle treatment; however, the expression levels of various cytokines were lower in control mice treated with U37883A. This suggests that U37883A acts on KIR6.1 to close KATP channels, thereby suppressing inflammation.

[0072] [Example 5] Advantages of selectively inhibiting KATP channels specifically expressed in pericytes Neuropathy scores were assessed using drugs known to close (inhibit) KATP channels (5-HD, U37883A, mitiglinide, glibenclamide), drugs known to open (activate) KATP channels (diazoxide, nicorandil, pinacidil), or solvents (PBS or DMSO) as controls.

[0073] Diazoxide, Nicorandil, and Pinacidil are known to be drugs that open (activate) KATP channels, as shown in the following literature: Cerebrovascular Safety of Sulfonylureas: The Role of K ATP Channels in Neuroprotection and the Risk of Stroke in Patients With Type 2 Diabetes (Diabetes. 2016 Sep; 65(9): 2795-809.) ・MitoK(ATP) opener, diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat (Am J PhysiolHeart Circ Physiol. 2002Sep; 283(3): H1005-11.) ・Protective effect of delayed remote limbischemic post conditioning: role of mitochondrial K ATP channels in a rat model of focal cerebral ischemic reperfusion injury (Journal of Cerebral Blood Flow & Metabolism (2012) 32, 851-85) ・Cytomembrane ATP-sensitive K +channels in neurovascular unit targets of ischemic stroke in the recovery period (EXPERIMENTAL AND THERAPEUTIC MEDICINE 12: 1055-1059, 2016) (Nicorandil) ・The KATP channel opener, nicorandil, ameliorates brain damage by modulating synaptogenesis after ischemic stroke (PLoSOne. 2021 Jan 26; 16(1): e0246019.) ・Effects of nicorandil on neurobehavioral function, BBB integrity, edema and stereological parameters of the brain in thesub-acute phase of stroke in a rat model (J Biosciences (2020) 45-49) ・Effect of nicorandil on the spatial arrangement of primary motor cortical neurons in the sub-acute phase of stroke in a rat model (J Chem Neuroanat. 2021Nov: 117: 102000.) (Pinacidil) ・Pinacidil reduces neuronal apoptosis following cerebral ischemia-reperfusion in rats through both mitochondrial anddeath-receptor signal pathways (Neuroscience Bulletin May 30, 2007, 23(3):145-150)

[0074] Control mice underwent ischemic surgery as in Example 1 (see 1-B). Two hours before the ischemic surgery, the control mice were administered each drug or vehicle (see 1-C). The drug concentrations were as follows: 5-HD (100 mM), U37883A (10 mM), diazoxide (10 mM), nicolandil (10 mM), pinacidil (10 mM), mitiglinide (10 mM), and glibenclamide (10 mM). Twenty-four hours after the ischemic surgery, the mice were evaluated for neurological impairment scores as in Example 2 (see 1-C). The results are shown in Figure 9.

[0075] As shown in Figure 9, all drugs that open (activate) KATP channels worsened the neuropathy score compared to the control, DMSO. Mitiglinide (which acts on SUR1 to close KATP channels) also worsened the neuropathy score compared to the control, DMSO. 5-HD (5-hydroxydecanoate), which is thought to close KATP channels by acting nonselectively on SURs (by acting on both SUR1 and SUR2), did not significantly improve the neuropathy score compared to the control, PBS. Glibenclamide, which is also thought to close KATP channels by acting nonselectively on SURs (by acting on both SUR1 and SUR2), did not significantly improve the neuropathy score compared to the control, DMSO. In contrast, U37883A significantly improved the neuropathy score compared to the control, PBS.

[0076] Thus, U37883A, which selectively acts on KIR6.1 to close the KATP channel, improved the neuropathy score, suggesting that selective closure of the KATP channel consisting of KIR6.1 and SUR2 (a KATP channel specifically expressed in pericytes) is important for inflammation suppression and tissue protection.

[0077] The therapeutic strategies provided by the present invention are expected to lead to the development of new treatment options for ischemic diseases. In particular, cerebral infarction and myocardial infarction caused by ischemia are major causes of death worldwide and have a very high risk of requiring nursing care, so it is expected that new treatment methods for these diseases will be developed. More specifically, if a new drug that selectively acts on KIR6.1 is discovered, it is thought that it will be possible to develop it as a therapeutic drug for ischemic diseases.

Claims

1. An anti-inflammatory agent or tissue protective agent against ischemic injury, comprising an inhibitor that closes the ATP-sensitive potassium (KATP) channel expressed in pericytes.

2. The inflammation suppressor or tissue protective agent described in claim 1, wherein the ATP-sensitive potassium (KATP) channel is composed of a pore component KIR6.1 and a regulatory subunit SUR2, and the inhibitor acts on KIR6.

1.

3. The inflammation suppressor or tissue protective agent of claim 1, wherein the inhibitor selectively closes an ATP-sensitive potassium (KATP) channel composed of the pore component KIR6.1 and the regulatory subunit SUR2.

4. An inflammation suppressor or tissue protective agent according to any one of claims 1 to 3, wherein the inhibitor inactivates the NF-kB pathway in pericytes or vascular endothelial cells.

5. A pharmaceutical composition comprising an inflammation suppressant or a tissue protective agent against ischemic injury according to any one of claims 1 to 3.

6. The pharmaceutical composition according to claim 5, which is a therapeutic agent for cerebral infarction.

7. The pharmaceutical composition according to claim 5, which is a therapeutic agent for ischemic heart disease.

Images