Renal fibrosis inhibitor, renal fibrosis recovery agent, and pharmaceutical

Abstract

This renal fibrosis inhibitor contains a substance that interferes with pyruvate kinase M2.

Description

Renal fibrosis inhibitor, renal fibrosis reversal agent, and medicine

[0001] The present invention relates to a renal fibrosis-suppressing agent, a renal fibrosis-reversing agent, and a medicine.

[0002] Organ fibrosis is a significant group of diseases that can lead to chronic functional decline or dysfunction, including liver cirrhosis, pulmonary fibrosis, chronic heart failure, and skin sclerosis. Renal fibrosis is known as chronic kidney disease, a national disease affecting one in eight Japanese people. There is no effective treatment for chronic kidney disease, making it a typical unmet medical need. While efforts have been made to develop anti-fibrotic drugs that act by calming chronic inflammation and inhibiting extracellular matrix production, no truly effective therapeutic agents have yet been found.

[0003] On the other hand, the present inventors have found that cellular communication network factor 2 (hereinafter sometimes referred to as "CCN2") promotes the progression of acute kidney injury to chronic kidney disease (see, for example, Non-Patent Document 1).

[0004] A method for treating a patient suffering from a disease caused by organ / tissue fibrosis has been proposed, which includes a step of administering to a patient in need thereof a pharmaceutical composition containing an effective amount of a pyruvate kinase M2 (PKM2) activator (see, for example, Patent Document 1). However, although Patent Document 1 specifically describes that a PKM2 activator has reversed liver and lung fibrosis, it does not describe any data specifically confirming that renal fibrosis has been suppressed or reversed.

[0005] Inoue, T. et al. , Cellular communication network factor 2 (CCN2) promotes the progression of acute kidney injury to chronic kidney disease, Biochemical and Biophysical Research Communications, Volume 517, Issue 1, 10 September 2019, Pages 96-102

[0006] Special Publication No. 2023-517963

[0007] The present invention aims to solve the above-mentioned conventional problems and achieve the following objectives: Namely, the present invention aims to provide a renal fibrosis-suppressing agent capable of suppressing renal fibrosis, a renal fibrosis-reversing agent capable of reversing renal fibrosis, and a medicament containing at least one of the renal fibrosis-suppressing agent and the renal fibrosis-reversing agent.

[0008] As a result of extensive research conducted by the present inventors to achieve the above object, they have found that a substance that inhibits pyruvate kinase M2 has an excellent effect of suppressing renal fibrosis and an excellent effect of reversing renal fibrosis.

[0009] The present invention is based on the findings of the present inventors, and provides the following means for solving the above problems: <1> A renal fibrosis-inhibiting agent containing a substance that inhibits pyruvate kinase M2. <2> A renal fibrosis-reversing agent containing a substance that inhibits pyruvate kinase M2. <3> A pharmaceutical used for the prevention and / or treatment of chronic kidney disease, which contains at least one of the renal fibrosis-inhibiting agent described in <1> and the renal fibrosis-reversing agent described in <2>.

[0010] According to the present invention, the above-mentioned conventional problems can be solved, the above-mentioned object can be achieved, and a renal fibrosis inhibitor capable of suppressing renal fibrosis, a renal fibrosis reversal agent capable of reversing renal fibrosis, and a pharmaceutical containing at least one of the renal fibrosis inhibitor and the renal fibrosis reversal agent can be provided.

[0011] FIG. 1 is a diagram showing an example of a kidney collected from a mouse sacrificed on day 14 of the (II) solvent control group in Test Example 1. FIG. 2 is a diagram showing an example of a kidney cut in half collected from a mouse sacrificed on day 14 of the (II) solvent control group in Test Example 1. FIG. 3 is a diagram showing an example of a kidney collected from a mouse sacrificed on day 14 of the compound-represented by structural formula (1) (III) administration group-1 in Test Example 1. FIG. 4 is a diagram showing an example of a kidney cut in half collected from a mouse sacrificed on day 14 of the compound-represented by structural formula (1) (III) administration group-1 in Test Example 1. FIG. 5 is a diagram showing an example of a kidney collected from a mouse sacrificed on day 14 of the compound-represented by structural formula (1) (IV) administration group-2 in Test Example 1. FIG. 6 is a diagram showing an example of a kidney cut in half collected from a mouse sacrificed on day 14 of the compound-represented by structural formula (1) administration group-2 in Test Example 1. FIG. 7 shows an example of a kidney collected from a mouse sacrificed on day 14 in Group-3 administered with a compound represented by structural formula (1) (V) in Test Example 1. FIG. 8 shows an example of a kidney cut in half collected from a mouse sacrificed on day 14 in Group-3 administered with a compound represented by structural formula (1) (V) in Test Example 1. FIG. 9 is a graph showing the results of measuring the expression level of CCN2 mRNA in Test Example 1. FIG. 10 is a graph showing the results of measuring the expression level of type 1 collagen mRNA in Test Example 1. FIG. 11 is a graph showing the results of measuring the expression level of fibronectin mRNA in Test Example 1. FIG. 12 shows an example of the results of examining the fibrosis state of a diseased kidney collected from a mouse sacrificed on day 14 in Group-3 administered with a compound represented by structural formula (1) (V) in Test Example 1. FIG. 13 shows an example of the results of examining the fibrosis state of a diseased kidney collected from a mouse sacrificed on day 14 in Group-3 administered with a compound represented by structural formula (1) (V) in Test Example 1. Fig. 14 is a graph showing the results of measuring the expression level of CCN2 mRNA in Test Example 2. Fig. 15 is a graph showing the results of measuring the expression level of type 1 collagen mRNA in Test Example 2. Fig. 16 is a graph showing the results of measuring the expression level of fibronectin mRNA in Test Example 2.17 is a diagram showing an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 14 in group a in Test Example 2. FIG. 18 is a diagram showing an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 7 in group b in Test Example 2. FIG. 19 is a diagram showing an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 14 in group c in Test Example 2. FIG. 20 is a diagram showing an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 14 in group d in Test Example 2. FIG. 21 is a diagram showing an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 14 in group e in Test Example 2. FIG. 22 is a graph showing the results of measuring the expression level of CCN2 mRNA when a compound represented by structural formula (2) or a compound represented by structural formula (3) was added in Test Example 3. FIG. 23 is a graph showing the results of measuring the expression level of type 1 collagen mRNA when a compound represented by structural formula (2) or a compound represented by structural formula (3) was added in Test Example 3. FIG. 24 is a graph showing the results of measuring the expression level of CCN2 mRNA when a compound represented by structural formula (4) or a compound represented by structural formula (1) was added in Test Example 3. FIG. 25 is a graph showing the results of measuring the expression level of type 1 collagen mRNA when a compound represented by structural formula (4) or a compound represented by structural formula (1) was added in Test Example 3. FIG. 26 is a diagram showing an example of the results of Western blotting in Test Example 4-1. FIG. 27 is a diagram showing an example of the results of cross-linked Western blotting in Test Example 4-2. FIG. 28 is a diagram showing an example of the results of Western blotting in Test Example 4-3.

[0012] (Renal fibrosis inhibitor, renal fibrosis reversing agent) The renal fibrosis inhibitor of the present invention contains at least a substance that inhibits pyruvate kinase M2, and optionally further contains other components. The renal fibrosis reversing agent of the present invention contains at least a substance that inhibits pyruvate kinase M2, and optionally further contains other components.

[0013] <Substances that inhibit pyruvate kinase M2> The substance that inhibits pyruvate kinase M2 is not particularly limited and can be appropriately selected depending on the purpose, but is preferably a substance that inhibits the polymerization of pyruvate kinase M2 monomers, and more preferably a substance that inhibits the formation of pyruvate kinase M2 dimers.

[0014] Specific examples of the substance that inhibits pyruvate kinase M2 include a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salt of the compound represented by the general formula (I) is not particularly limited and can be appropriately selected depending on the purpose.

[0015] In the general formula (I), n represents an integer of 0 to 3. Preferably, n is 1.

[0016] In the general formula (I), R is hydrogen or a substituent located at any position on the benzene ring, and １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ The R is any one selected from the group consisting of a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group. Preferably, R is hydrogen.

[0017] In the general formula (I), R' and R" satisfy either of the following conditions (1) and (2): (1) R' and R" are each independently any one selected from the group consisting of hydrogen, an alkyl group, a haloalkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, and an aralkyl group. (2) R' and R" together with the N atom to which they are bonded form a 5- or 6-membered heterocycle, and the heterocycle is a substituted or unsubstituted group selected from the group consisting of a morpholino group, a thiomorpholinyl group, a thiazole alkyl group, an oxazolyl group, an isoxazolyl group, an imidazolidinyl group, a piperidinyl group, a piperazinyl group, and a pyrrolidyl group. The heterocycle may have one or more substituents, and the substituents are each independently any one selected from the group consisting of an alkyl group, an acyl group, an alkenyl group, an alkynyl group, a phenyl group, and a benzyl group.

[0018] The R′ and R″ are preferably the above (2), more preferably a 6-membered heterocycle, even more preferably a 6-membered heterocycle having no substituent, and particularly preferably a piperidinyl group.

[0019] In the general formula (I), R''' is hydrogen, C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ The R''' is any one selected from the group consisting of a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group. １－４ The alkyl group is preferably a methyl group.

[0020] Other preferred examples of the substance that inhibits pyruvate kinase M2 include compounds represented by any of the following structural formulas (1) to (4) or pharmaceutically acceptable salts thereof. The pharmaceutically acceptable salts of the compounds represented by any of the following structural formulas (1) to (4) are not particularly limited and can be appropriately selected depending on the purpose.

[0021] Among the compounds represented by any one of the following structural formulas (1) to (4) or pharmaceutically acceptable salts thereof, the compounds represented by the following structural formula (1) or (2) or pharmaceutically acceptable salts thereof are preferred.

[0022] <Compound Represented by Structural Formula (1)> The compound represented by the following structural formula (1) is a compound (CAS registration number: 94164-88-2) called "compound 3k" (also known as PKM2-IN-1), and is known as a PKM2 inhibitor. The compound represented by the following structural formula (1) is a preferred example of the compound represented by the general formula (I).

[0023] <Compound Represented by Structural Formula (2)> The compound represented by the following structural formula (2) is a compound called "compound 10i" (also known as PKM2-IN-3) (CAS registration number: 2408841-19-8), and is known as a PKM2 inhibitor.

[0024] The compound represented by the structural formula (1) and the compound represented by the structural formula (2) have in common the fact that they both target the allosteric site (FBP binding site) of pyruvate kinase M2. Therefore, preferred examples of substances that inhibit pyruvate kinase M2 include substances that target the allosteric site of pyruvate kinase M2. By targeting the allosteric site of pyruvate kinase M2, polymerization of pyruvate kinase M2 monomers can be inhibited.

[0025] <Compound Represented by Structural Formula (3)> The compound represented by the following structural formula (3) is a compound called "compound 7d" (also known as PKM2-IN-6) (CAS Registry Number: 771467-00-6), and is known as a PKM2 inhibitor.

[0026] <Compound Represented by Structural Formula (4)> The compound represented by the following structural formula (4) is a compound called "NPD10084" (CAS Registry Number: 1040706-91-9), and is known as a PKM2 inhibitor.

[0027] The substance that inhibits pyruvate kinase M2 may be used alone or in combination of two or more.

[0028] The substance that inhibits pyruvate kinase M2 may be a commercially available product or may be prepared as appropriate.

[0029] The content of the substance that inhibits pyruvate kinase M2 in the renal fibrosis-suppressing agent or the renal fibrosis-reversing agent is not particularly limited and can be appropriately selected depending on the purpose. Furthermore, the renal fibrosis-suppressing agent or the renal fibrosis-reversing agent may consist solely of the substance that inhibits pyruvate kinase M2.

[0030] <Other Components> The other components in the renal fibrosis suppressant or the renal fibrosis reversing agent are not particularly limited and can be selected appropriately depending on the purpose as long as they do not impair the effects of the present invention, and examples thereof include pharmaceutically acceptable carriers. The carrier is also not particularly limited and can be selected appropriately depending on the dosage form, etc. The other components may be used alone or in combination of two or more. The content of the other components in the renal fibrosis suppressant or the renal fibrosis reversing agent is not particularly limited and can be selected appropriately depending on the purpose.

[0031] <Dosage Form> The dosage form of the renal fibrosis suppressant or the renal fibrosis reversing agent is not particularly limited and can be appropriately selected depending on the desired administration method, etc. Examples include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations to be dissolved when used, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation powders, etc.

[0032] The oral solid preparation can be prepared by a conventional method by adding, for example, an excipient, and optionally additives such as a binder, disintegrant, lubricant, colorant, and flavoring agent, to the substance inhibiting pyruvate kinase M2 (hereinafter sometimes referred to as the "active ingredient"). Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of the disintegrant include dry starch, sodium alginate, powdered agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of the lubricant include purified talc, stearates, borax, and polyethylene glycol. Examples of the coloring agent include titanium oxide, iron oxide, etc. Examples of the flavoring agent include sucrose, orange peel, citric acid, tartaric acid, etc.

[0033] The oral liquid preparation can be produced by a conventional method by adding additives such as flavorings, buffers, and stabilizers to the active ingredient. Examples of flavorings include sucrose, orange peel, citric acid, and tartaric acid. Examples of buffers include sodium citrate. Examples of stabilizers include tragacanth, gum arabic, and gelatin.

[0034] The injection preparation can be prepared by adding, for example, a pH adjuster, a buffer, a stabilizer, an isotonicity agent, a local anesthetic, etc. to the active ingredient, and then using a conventional method to prepare an injection preparation for subcutaneous, intramuscular, intravenous, etc. Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate, etc. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc. Examples of the isotonicity agent include sodium chloride, glucose, etc. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride, etc.

[0035] The ointment can be prepared by, for example, blending the active ingredient with known bases, stabilizers, humectants, preservatives, etc., and mixing them in a conventional manner. Examples of the bases include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.

[0036] The patch can be produced, for example, by applying the ointment as a cream, gel, paste, etc. to a known support by a conventional method. Examples of the support include woven fabrics made of cotton, staple fiber, or chemical fiber, nonwoven fabrics, films made of soft vinyl chloride, polyethylene, polyurethane, etc., and foam sheets.

[0037] <Use> The administration method, dosage, administration timing, number of administrations, administration interval, administration period, and administration target of the renal fibrosis-suppressing agent or the renal fibrosis-reversing agent are not particularly limited and can be selected appropriately depending on the purpose.

[0038] The method of administration of the renal fibrosis inhibitor or the renal fibrosis reversing agent is not particularly limited, and either systemic administration or local administration can be selected depending on, for example, the dosage form of the renal fibrosis inhibitor or the renal fibrosis reversing agent, the age, weight, and presence or absence of disease of the individual to be administered. For example, in systemic administration (e.g., oral administration, intraperitoneal administration, administration into the blood, etc.), it is preferable to appropriately apply a conventionally known drug delivery technique so that the active ingredient is stably and efficiently delivered to the desired site. In addition, in local administration, the active ingredient can be administered by direct injection into the desired site. For the injection, conventionally known techniques such as injection can be appropriately used. The renal fibrosis inhibitor or the renal fibrosis reversing agent can be suitably used for oral administration.

[0039] The dosage, number of doses, administration interval, and administration period of the renal fibrosis suppressant or the renal fibrosis reversal agent are not particularly limited and can be appropriately selected depending on the age, body weight, presence or absence of disease, etc. of the individual to be administered. For example, the dosage may be in the range of 0.01 to 500 mg per kg of body weight of the individual, in terms of the amount of the substance inhibiting pyruvate kinase M2. The number of doses and administration interval may be, for example, once every 1 to 5 days. The administration period can be appropriately selected depending on the period required for suppressing renal fibrosis or reversing renal fibrosis, and can include, for example, one day or more, one week or more, one month or more, one year or more, or ten years or more.

[0040] The timing of administration of the renal fibrosis-suppressing agent or the renal fibrosis-reversing agent is not particularly limited and can be appropriately selected depending on the purpose. For example, the agent may be administered prophylactically or therapeutically against renal fibrosis.

[0041] The individual (species) targeted by the renal fibrosis suppressant or the renal fibrosis reversal agent is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs, cats, sheep, goats, and birds.

[0042] The renal fibrosis-suppressing agent or the renal fibrosis-reversing agent may be used in combination with other drugs.

[0043] The renal fibrosis-suppressing agent can effectively suppress renal fibrosis. Therefore, the present invention also relates to a method for suppressing renal fibrosis, which comprises administering the renal fibrosis-suppressing agent to an individual.

[0044] The renal fibrosis reversal agent can effectively reverse fibrotic renal tissue. Therefore, the present invention also relates to a method for reversing renal fibrosis, which comprises administering the renal fibrosis reversal agent to an individual. In this specification, reversal of renal fibrosis refers to restoring fibrotic renal tissue to a state before fibrosis. The degree of recovery is not important as long as the renal tissue is brought closer to the state before fibrosis.

[0045] (Medicine) The medicine of the present invention is a medicine used for the prevention and / or treatment of chronic kidney disease, and contains at least one of the renal fibrosis-suppressing agent of the present invention and the renal fibrosis-reversing agent of the present invention, and may further contain other ingredients as necessary.

[0046] Chronic kidney disease (CKD) is defined as (1) kidney damage evident by urinary abnormalities, imaging, blood, or pathology, or (2) an estimated glomerular filtration rate (GFR) of 60 mL / min / 1.73 m ２ or less than 1 month, and either one or both of these symptoms persists for three months or more.

[0047] As used herein, "prevention of chronic kidney disease" refers to preventing the occurrence of renal fibrosis and preventing or delaying the onset of chronic kidney disease. Furthermore, as used herein, "treatment of chronic kidney disease" refers to delaying the progression of chronic kidney disease or alleviating symptoms in patients with chronic kidney disease that has already developed, by suppressing the progression of renal fibrosis, reducing or removing renal fibrotic tissue, or maintaining tissue with improved renal fibrosis.

[0048] <At least one of renal fibrosis inhibitor and renal fibrosis reversing agent> The renal fibrosis inhibitor is the same as the renal fibrosis inhibitor of the present invention described above. The renal fibrosis reversing agent is the same as the renal fibrosis reversing agent of the present invention described above. Either the renal fibrosis inhibitor or the renal fibrosis reversing agent may be used alone, or both may be used in combination.

[0049] The total content of the renal fibrosis-suppressing agent and the renal fibrosis-reversing agent in the medicament is not particularly limited and can be appropriately selected depending on the purpose. In addition, the medicament may consist of at least one of the renal fibrosis-suppressing agent and the renal fibrosis-reversing agent.

[0050] <Other Components> The other components in the pharmaceutical are not particularly limited and can be selected appropriately depending on the purpose, as long as they do not impair the effects of the present invention, and examples thereof include the same as the other components in the above-mentioned renal fibrosis suppressant or renal fibrosis reversal agent. The content of the other components in the pharmaceutical is not particularly limited and can be selected appropriately depending on the purpose.

[0051] <Dosage Form> The dosage form of the pharmaceutical is not particularly limited and can be appropriately selected depending on the desired administration method, etc. Examples include dosage forms similar to those of the above-mentioned renal fibrosis-suppressing agent or renal fibrosis-reversing agent.

[0052] <Use> The administration method, dosage, administration timing, number of administrations, administration interval, administration period, and administration target of the pharmaceutical are not particularly limited and can be selected appropriately depending on the purpose, and can be the same as those described in the "Use" section of the above-mentioned renal fibrosis inhibitor or renal fibrosis reversing agent. Note that the "period during which inhibition of renal fibrosis or reversal of renal fibrosis is required" in the "Use" section of the renal fibrosis inhibitor or renal fibrosis reversing agent should be read as "period during which prevention and / or treatment of chronic kidney disease is required."

[0053] The species (individual) targeted by the pharmaceutical is not particularly limited and can be appropriately selected depending on the purpose, and examples include humans, mice, rats, cows, pigs, monkeys, dogs, cats, sheep, goats, and birds.

[0054] The above medicines may be used in combination with other medicines.

[0055] The medicament can effectively suppress renal fibrosis and effectively restore fibrotic renal tissue. Therefore, the present invention also relates to a method for preventing and / or treating chronic kidney disease, which comprises administering the medicament to an individual.

[0056] Test examples of the present invention will be explained below, but the present invention is not limited to the following test examples.

[0057] Test Example 1 The effect of the compound represented by structural formula (1) was tested using a mouse model of chronic kidney disease as follows.

[0058] [Compound Represented by Structural Formula (1)] The compound represented by structural formula (1) used was manufactured by Selleck.

[0059] C57BL / 6 mice (CLEA Japan, Inc.) were housed at Saitama Medical University in a specific pathogen-free (SPF) environment with a 12-hour light / 12-hour dark cycle and optimal temperature. Male mice weighing 18-24 g and aged 6-8 weeks were used in the experiment.

[0060] <Unilateral Renal Ischemia-Reperfusion Model> A unilateral renal ischemia-reperfusion injury (IRI) model was used as a chronic kidney disease model. The IRI model was prepared as follows. The mouse's body temperature was monitored by rectal temperature and controlled at 37°C using an adjustable heating pad. Necessary treatments were performed under isoflurane inhalation (induction: 4-5%, maintenance: 2-3%), and the mouse was carefully observed until recovery after surgery. Necessary treatments were performed as follows. A 2-cm incision was made on the back of the mouse to expose the left kidney. The capsule was then peeled off to expose the renal artery, and the kidney was clipped to maintain renal ischemia for 18 minutes. After confirming the resumption of renal blood flow, the clip was removed and the incision was sutured. The right kidney was not treated in any way.

[0061] [Drug Administration Protocol] Male mice were randomly assigned to the following five groups (I) to (V). All mice in groups (II) to (V) underwent the unilateral renal ischemia-reperfusion surgery described above (18 minutes). Mice in group (I) underwent a sham operation (the vessels were exposed, and the wound was left unclipped for 18 minutes before closure). Mice were sacrificed on day 14, and kidney samples were collected and analyzed. (I) Sham operation group: 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) was orally administered every other day for 14 days. (II) Vehicle control group: 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) was orally administered every other day for 14 days. (III) Compound of structural formula (1) Administration Group-1: The compound of structural formula (1) was dissolved in 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) at a concentration of 1 mg / kg and orally administered once every two days for 14 days. (IV) Compound of structural formula (1) Administration Group-2: The compound of structural formula (1) was dissolved in 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) at a concentration of 3 mg / kg and orally administered once every two days for 14 days. (V) Compound of structural formula (1) Administration Group-3: The compound of structural formula (1) was dissolved in 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) at a concentration of 5 mg / kg and orally administered once every two days for 14 days.

[0062] [Analysis] Figures 1 to 8 show kidneys collected from mice sacrificed on day 14. Figures 1 and 2 show examples of kidneys collected from mice in the (II) solvent control group (vehicle group), Figures 3 and 4 show examples of kidneys collected from mice in the (III) compound-represented by structural formula (1) administration group-1 (1 mg / kg), Figures 5 and 6 show examples of kidneys collected from mice in the (IV) compound-represented by structural formula (1) administration group-2 (3 mg / kg), and Figures 7 and 8 show examples of kidneys collected from mice in the (V) compound-represented by structural formula (1) administration group-3 (5 mg / kg). In Figures 1 to 8, A shows the right kidney (no renal ischemia treatment), and B shows the left kidney (referred to as the "diseased kidney" below). Figures 2, 4, 6, and 8 show kidneys cut in half.

[0063] <RT-qPCR> Total RNA was extracted from the collected diseased kidney samples using TRIzol reagent, and cDNA was prepared using reverse transcriptase and the ReverTra Ace qPCR RT Kit (Toyobo). The obtained cDNA was subjected to PCR using DNA polymerase and Thunderbird Next qPCR Mix (Toyobo) on a Step One Plus real-time PCR system (Applied Biosystems).

[0064] The results of RT-qPCR are shown in Figures 9 to 11. Figure 9 is a graph showing the results of measuring the mRNA expression level of CCN2, a profibrotic humoral factor; Figure 10 is a graph showing the mRNA expression level of type 1 collagen (Col1a), an extracellular matrix protein; and Figure 11 is a graph showing the mRNA expression level of fibronectin (FN), a glycoprotein present in the extracellular matrix. In Figures 9 to 11, the horizontal axis represents, from left to right, (I) the sham-operated group, (II) the solvent control group, (III) the compound represented by structural formula (1) administration group-1, (IV) the compound represented by structural formula (1) administration group-2, and (V) the compound represented by structural formula (1) administration group-3. The vertical axis represents the relative mRNA expression level using GAPDH as an internal standard. In Figures 9 to 11, "*," "**," "***," and "****" all indicate statistically significant (p<0.05) differences in expression levels.

[0065] As shown in Figure 9, CCN2 mRNA expression was significantly increased in the kidney of the unilateral renal ischemia-reperfusion model (Figure 9 (II)). On the other hand, administration of the compound represented by structural formula (1) dose-dependently suppressed CCN2 mRNA expression.

[0066] In normal kidneys, type 1 collagen expression is controlled at a low level, and only the amount necessary to maintain normal tissue structure and function is synthesized. On the other hand, in diseased kidneys, excessive production of type 1 collagen occurs, which leads to the formation of fibrotic tissue and impaired kidney function. As shown in Figure 10, administration of the compound represented by structural formula (1) suppressed the expression of type 1 collagen mRNA.

[0067] Furthermore, as shown in FIG. 11, administration of the compound represented by structural formula (1) suppressed the expression of fibronectin mRNA.

[0068] <Histology> The collected diseased kidney samples were immersion fixed in 4% paraformaldehyde at 4°C for 24 hours, dehydrated, and then paraffin blocks were prepared. The paraffin blocks were sectioned at 5 μm and stained with Masson's trichrome. Observations and records were made using a bright-field microscope to examine the state of fibrosis in the diseased kidneys histologically.

[0069] (II) Figure 12 shows an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 14 in the solvent control group, and (V) Figure 13 shows an example of the results of examining the fibrosis state of diseased kidneys collected from mice sacrificed on day 14 in group-3 administered with the compound represented by structural formula (1).

[0070] 12 and 13, it was confirmed that the interstitial fibrotic area was reduced in group (V) administered with the compound represented by structural formula (1) compared to the solvent control group (II). Renal fibrosis corresponds to tissue changes in chronic kidney disease.

[0071] As described above, oral administration of the compound represented by structural formula (1) to chronic kidney disease model mice resulted in a significant decrease in mRNA production of CCN2, type 1 collagen, and fibronectin, and histologically demonstrated that the progression of kidney damage (fibrosis) was suppressed. This indicates that the compound represented by structural formula (1) can be used as a preventive and / or therapeutic agent for chronic kidney disease.

[0072] Test Example 2 A test was carried out in the same manner as in Test Example 1, except that the drug administration protocol in Test Example 1 was changed as follows.

[0073] [Drug Administration Protocol] Male mice were randomly assigned to five groups: a to e. All mice in groups b to e underwent the unilateral renal ischemia-reperfusion surgery described above (18 minutes). Mice in group a underwent a sham surgery (the vessels were exposed, then left unclipped for 18 minutes before closure). Mice were sacrificed on days 7 and 14, and kidney samples were collected and analyzed. Group a (sham-operated group): After the sham-operated unilateral renal ischemia-reperfusion surgery, 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) was orally administered every other day for 14 days. Group b (vehicle control group (vehicle group)-1): After the unilateral renal ischemia-reperfusion surgery, 100 μL of solvent (DMSO:corn oil = 1:9 (volume ratio)) was orally administered every other day for 7 days. Group c (vehicle control group-2): Starting from the unilateral renal ischemia-reperfusion surgery, 100 μL of solvent (DMSO:corn oil=1:9 (volume ratio)) was orally administered once every two days for 14 days. Group d (group-1 administered with the compound represented by structural formula (1)): Starting from the unilateral renal ischemia-reperfusion surgery, the compound represented by structural formula (1) was dissolved in 100 μL of solvent (DMSO:corn oil=1:9 (volume ratio)) at a concentration that gave a dose of 5 mg / kg, and this was orally administered once every two days for 14 days. Group e (group 2 administered with the compound represented by structural formula (1)): Starting 7 days after the unilateral renal ischemia-reperfusion surgery, the compound represented by structural formula (1) was dissolved in 100 μL of a solvent (DMSO:corn oil=1:9 (volume ratio)) at a concentration to give a dose of 5 mg / kg, and the solution was orally administered once every two days for 7 days.

[0074] [Analysis] <RT-qPCR> In the same manner as in Test Example 1, RT-qPCR was performed on diseased kidney samples collected from mice sacrificed on the 7th or 14th day.

[0075] The results of RT-qPCR are shown in Figures 14 to 16. Figure 14 is a graph showing the mRNA expression level of CCN2, a profibrotic humoral factor; Figure 15 is a graph showing the mRNA expression level of type 1 collagen (Col1a), an extracellular matrix protein; and Figure 16 is a graph showing the mRNA expression level of fibronectin (FN), a glycoprotein present in the extracellular matrix. In Figures 14 to 16, the horizontal axis represents, from left to right, Group a (sham-operated group), Group b (solvent control group (vehicle group)-1), Group c (solvent control group (vehicle group)-2), Group d (compound represented by structural formula (1)-administered group-1), and Group e (compound represented by structural formula (1)-administered group-2). The vertical axis represents the relative mRNA expression level using GAPDH as an internal standard. In Figures 14 to 16, "*" indicates a statistically significant (p<0.05) difference in expression level.

[0076] As shown in Figures 14 to 16, the expression of CCN2 mRNA, type 1 collagen mRNA, and fibronectin mRNA was suppressed in both cases, when the compound represented by structural formula (1) was administered immediately after the creation of the unilateral renal ischemia-reperfusion model (group d), and when the compound represented by structural formula (1) was administered after the unilateral renal ischemia-reperfusion model had progressed (group e).

[0077] <Histology> In the same manner as in Test Example 1, the collected diseased kidney samples were subjected to Masson's trichrome staining.

[0078] Examples of the results of examining the state of fibrosis in diseased kidneys collected from mice sacrificed on the 14th day in groups a, c, d, and e, and on the 7th day in group b, are shown in Figures 17 to 21.

[0079] As shown in Figures 17 to 21, it was confirmed that the interstitial fibrotic area was reduced compared to the solvent control group in both cases where the compound represented by structural formula (1) was administered immediately after the creation of the unilateral renal ischemia-reperfusion model (group d) and where the compound represented by structural formula (1) was administered after the unilateral renal ischemia-reperfusion model had progressed (group e).

[0080] As described above, it was confirmed that oral administration of the compound represented by structural formula (1) to chronic kidney disease model mice not only exerts a renal fibrosis inhibitory effect to suppress the onset and progression of chronic kidney disease, but also exerts a renal fibrosis recovery effect to cure or return to normal renal fibrosis in already advanced chronic kidney disease.

[0081] Test Example 3 The effects of the compounds represented by structural formulas (1) to (4) were tested using a cell damage model as follows.

[0082] [Compounds Represented by Structural Formulas (1) to (4)] Commercially available products were used as the compounds represented by structural formulas (1) to (4).

[0083] [Cell Culture] Human renal proximal tubule epithelial cell line Human Kidney-2 (HK-2, ATCC catalog number: CRL-2190) was cultured in Ham's F-12 / DMEM (growth medium) containing 5% fetal calf serum (FCS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO until confluent in a 100 mm dish. ２ The cells were cultured in an incubator. To synchronize the cell cycle, the medium was changed to serum-free medium and cultured for 24 hours, and then the cells were seeded again onto a 12-well plate (50,000 cells / well). After culturing in growth medium for 72 hours, the cells were used for each experiment.

[0084] [OGDR] The cytotoxicity model OGDR (oxygen glucose deprivation (OGD) / re-oxygenation) was used as a model mimicking the in vivo condition. Specifically, cells were cultured in a hypoxic environment (1% O ). ２ After 24 hours of exposure to glucose-free medium under normal oxygen concentration (20% O), ２ When the medium was replaced with a growth medium under the conditions described above, the compounds represented by structural formulas (1) to (4) were added. To the control group, the same concentration of DMSO used to dissolve the compounds represented by structural formulas (1) to (4) was added.

[0085] The test groups were as follows (N=4-6): Group 1: Cells treated with hypoxia for 24 hours. Group 2: Control group. Group 3: Group administered with 1 μM of the compound represented by structural formula (2). Group 4: Group administered with 3 μM of the compound represented by structural formula (2). Group 5: Group administered with 10 μM of the compound represented by structural formula (2). Group 6: Group administered with 1 μM of the compound represented by structural formula (3). Group 7: Group administered with 3 μM of the compound represented by structural formula (3). Group 8: Group administered with 10 μM of the compound represented by structural formula (3). Group 9: Group administered with 0.1 μM of the compound represented by structural formula (4). Group 10: Group administered with 1 μM of the compound represented by structural formula (4). Group 11: Group administered with 3 μM of the compound represented by structural formula (4). Group 12: Group administered with 1 μM of the compound represented by structural formula (1). Group 13 Group 13: A group administered with the compound represented by structural formula (1) at a concentration of 3 μM. Group 14: A group administered with the compound represented by structural formula (1) at a concentration of 10 μM.

[0086] [RT-qPCR] Total RNA was extracted from cells 48 hours after reoxygenation using TRIzol reagent, and cDNA was prepared using reverse transcriptase and the ReverTra Ace qPCR RT Kit (Toyobo). The resulting cDNA was subjected to PCR using DNA polymerase and Thunderbird Next qPCR Mix (Toyobo) on a Step One Plus real-time PCR system (Applied Biosystems).

[0087] The results of RT-qPCR are shown in Figures 22 to 25. Figures 22 and 23 show the results when a compound represented by structural formula (2) or a compound represented by structural formula (3) was added (Figure 22: CCN2 mRNA expression level, Figure 23: type 1 collagen (Col1a) mRNA expression level), while Figures 24 and 25 show the results when a compound represented by structural formula (4) or a compound represented by structural formula (1) was added (Figure 24: CCN2 mRNA expression level, Figure 25: type 1 collagen (Col1a) mRNA expression level). In Figures 22 to 25, the numbers on the horizontal axis represent the numbers for each group described above, and the vertical axis represents the relative mRNA expression level using GAPDH as an internal standard. In Figures 22 to 25, an "*" indicates a statistically significant (p<0.05) difference in expression level.

[0088] 22 to 25, the expression of CCN2 mRNA was suppressed when the compound represented by structural formula (2) or the compound represented by structural formula (1) was administered. On the other hand, the expression of type 1 collagen (Col1a) mRNA was suppressed when any of the compounds represented by structural formulas (1) to (4) was administered.

[0089] Test Example 4 The mechanism of action of the compound represented by structural formula (1) was investigated.

[0090] <Test Example 4-1> Using HK-2 cells, the expression of PKM1, PKM2, CCN2, and GAPDH was examined when siRNA targeting PKM2 was added. The results of Western blotting are shown in Figure 26.

[0091] As shown in Figure 26, when PKM2-targeting siRNA was added ("siPKM2" in Figure 26), PKM2 expression in HK-2 cells was specifically and efficiently suppressed immediately after addition, compared to mock ("mock" in Figure 26), and this effect continued for more than 72 hours. Furthermore, under the same conditions, CCN2 expression was also significantly suppressed.

[0092] Test Example 4-2 Using HK-2 cells, the expression of PKM2 was examined when the compound represented by structural formula (1) was added. The results of cross-linked Western blotting are shown in FIG.

[0093] As shown in Figure 27, when the compound represented by structural formula (1) was added ("3K" in Figure 27), the PKM2 monomer increased and the nuclear translocation of PKM2 was significantly suppressed. Note that "vehicle" in Figure 27 represents the solvent control group.

[0094] Test Example 4-3 Using HK-2 cells, the expression of PKM2 was examined when the compound represented by structural formula (1) was added. The results of Western blotting using whole cells as a sample are shown in Figure 28.

[0095] As shown in Figure 28, when the compound represented by structural formula (1) was added ("3K" in Figure 28), it was confirmed that the total amount of PKM2 protein remained unchanged, but the expression level of CCN2 was significantly reduced. Note that "vehicle" in Figure 28 represents the solvent control group.

[0096] These results suggest that the compound represented by structural formula (1) inhibits the polymerization of PKM2 monomers, suppressing the nuclear translocation of PKM2 and resulting in a decrease in CCN2 expression. It was also revealed that PKM1, which is normally expressed in renal tubular epithelial cells, is not involved in CCN2 expression.

[0097] Examples of aspects of the present invention include the following. <1> A renal fibrosis inhibitor containing a substance that inhibits pyruvate kinase M2. <2> The renal fibrosis inhibitor according to <1>, wherein the substance that inhibits pyruvate kinase M2 is a substance that inhibits polymerization of pyruvate kinase M2 monomers. <3> The renal fibrosis inhibitor according to <1> or <2>, wherein the substance that inhibits pyruvate kinase M2 is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: (In the general formula (I), n represents an integer of 0 to 3; R represents hydrogen or a substituent located at any position on the benzene ring, and C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４R' and R'' are any one selected from the group consisting of a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group; R' and R'' satisfy either of the following (1) and (2): (1) R' and R'' are each independently any one selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, and an aralkyl group; (2) R' and R'' together with the N atom to which they are bonded form a 5- or 6-membered heterocycle, and the heterocycle is a substituted or unsubstituted group selected from the group consisting of a morpholino group, a thiomorpholinyl group, a thiazole alkyl group, an oxazolyl group, an isoxazolyl group, an imidazolidinyl group, a piperidinyl group, a piperazinyl group, and a pyrrolidyl group; The heterocycle may have one or more substituents, each of which is independently selected from the group consisting of an alkyl group, an acyl group, an alkenyl group, an alkynyl group, a phenyl group, and a benzyl group; R''' is hydrogen, C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ (4) The renal fibrosis inhibitor according to (1) or (2), wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (1) or (2) or a pharmaceutically acceptable salt thereof: <5> The renal fibrosis-suppressing agent according to <1> or <2>, wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (3) or (4) or a pharmaceutically acceptable salt thereof: <6> A renal fibrosis reversing agent comprising a substance that inhibits pyruvate kinase M2. <7> The renal fibrosis reversing agent according to <6>, wherein the substance that inhibits pyruvate kinase M2 is a substance that inhibits polymerization of pyruvate kinase M2 monomers. <8> The renal fibrosis reversing agent according to <6> or <7>, wherein the substance that inhibits pyruvate kinase M2 is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: (In the general formula (I), n represents an integer of 0 to 3; R represents hydrogen or a substituent located at any position on the benzene ring, and C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ R' and R'' are any one selected from the group consisting of a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group; R' and R'' satisfy either of the following (1) and (2): (1) R' and R'' are each independently any one selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, and an aralkyl group; (2) R' and R'' together with the N atom to which they are bonded form a 5- or 6-membered heterocycle, and the heterocycle is a substituted or unsubstituted group selected from the group consisting of a morpholino group, a thiomorpholinyl group, a thiazole alkyl group, an oxazolyl group, an isoxazolyl group, an imidazolidinyl group, a piperidinyl group, a piperazinyl group, and a pyrrolidyl group; The heterocycle may have one or more substituents, each of which is independently selected from the group consisting of an alkyl group, an acyl group, an alkenyl group, an alkynyl group, a phenyl group, and a benzyl group; R''' is hydrogen, C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４(9) The agent for renal fibrosis recovery according to the above item (6) or (7), wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (1) or (2) or a pharmaceutically acceptable salt thereof: <10> The renal fibrosis reversal agent according to <6> or <7>, wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (3) or (4) or a pharmaceutically acceptable salt thereof: <11> A pharmaceutical used for the prevention and / or treatment of chronic kidney disease, comprising at least one of the renal fibrosis suppressant described in any of <1> to <5> and the renal fibrosis reversing agent described in any of <6> to <10>. <12> A method for suppressing renal fibrosis, comprising administering to an individual the renal fibrosis suppressant described in any of <1> to <5>. <13> A method for reversing renal fibrosis, comprising administering to an individual the renal fibrosis reversing agent described in any of <6> to <10>. <14> A method for preventing and / or treating chronic kidney disease, comprising administering to an individual the pharmaceutical described in <11>.

[0098] This application claims priority based on Japanese Patent Application No. 2024-152717, filed on September 4, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. A renal fibrosis inhibitor characterized by containing a substance that inhibits pyruvate kinase M2.

2. The renal fibrosis inhibitor according to claim 1, wherein the substance that inhibits pyruvate kinase M2 is a substance that inhibits polymerization of pyruvate kinase M2 monomers.

3. The renal fibrosis inhibitor according to claim 1, wherein the substance that inhibits pyruvate kinase M2 is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: (In the general formula (I), n represents an integer of 0 to 3; R represents hydrogen or a substituent located at any position on the benzene ring, and C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ R' and R'' are any one selected from the group consisting of a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group; R' and R'' satisfy either of the following (1) and (2): (1) R' and R'' are each independently any one selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, and an aralkyl group; (2) R' and R'' together with the N atom to which they are bonded form a 5- or 6-membered heterocycle, and the heterocycle is a substituted or unsubstituted group selected from the group consisting of a morpholino group, a thiomorpholinyl group, a thiazole alkyl group, an oxazolyl group, an isoxazolyl group, an imidazolidinyl group, a piperidinyl group, a piperazinyl group, and a pyrrolidyl group; The heterocycle may have one or more substituents, each of which is independently selected from the group consisting of an alkyl group, an acyl group, an alkenyl group, an alkynyl group, a phenyl group, and a benzyl group; R''' is hydrogen, C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group.

4. The renal fibrosis inhibitor according to claim 2, wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (1) or (2) or a pharmaceutically acceptable salt thereof.

5. The renal fibrosis inhibitor according to claim 1, wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (3) or (4) or a pharmaceutically acceptable salt thereof.

6. A renal fibrosis recovery agent characterized by containing a substance that inhibits pyruvate kinase M2.

7. The renal fibrosis reversal agent according to claim 6, wherein the substance that inhibits pyruvate kinase M2 is a substance that inhibits polymerization of pyruvate kinase M2 monomers.

8. The renal fibrosis reversal agent according to claim 6, wherein the substance that inhibits pyruvate kinase M2 is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: (In the general formula (I), n represents an integer of 0 to 3; R represents hydrogen or a substituent located at any position on the benzene ring, and C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ R' and R'' are any one selected from the group consisting of a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group; R' and R'' satisfy either of the following (1) and (2): (1) R' and R'' are each independently any one selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, and an aralkyl group; (2) R' and R'' together with the N atom to which they are bonded form a 5- or 6-membered heterocycle, and the heterocycle is a substituted or unsubstituted group selected from the group consisting of a morpholino group, a thiomorpholinyl group, a thiazole alkyl group, an oxazolyl group, an isoxazolyl group, an imidazolidinyl group, a piperidinyl group, a piperazinyl group, and a pyrrolidyl group; The heterocycle may have one or more substituents, each of which is independently selected from the group consisting of an alkyl group, an acyl group, an alkenyl group, an alkynyl group, a phenyl group, and a benzyl group; R''' is hydrogen, C １－４ alkyl group of C １－４ Alkoxy groups, halogens, C １－４ a haloalkyl group, a hydroxy group, a cyano group, a mercapto group, a nitro group, and an amino group.

9. The renal fibrosis reversal agent according to claim 7, wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (1) or (2) or a pharmaceutically acceptable salt thereof.

10. The renal fibrosis recovery agent according to claim 6, wherein the substance that inhibits pyruvate kinase M2 is at least one of a compound represented by the following structural formula (3) or (4) or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical used for the prevention and / or treatment of chronic kidney disease, characterized in that it contains at least one of a renal fibrosis inhibitor described in any one of claims 1 to 5 and a renal fibrosis reversal agent described in any one of claims 6 to 10.

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