The present invention relates to an adenosine triphosphate secretion inhibitor, a hepatic stellate cell activation inhibitor, a hepatic stellate cell deactivator, a hepatic fibrosis therapeutic agent, a non-alcoholic steatohepatitis therapeutic agent, a psoriasis therapeutic agent, and a use thereof for preparing the adenosine triphosphate secretion inhibitor, the hepatic stellate cell activation inhibitor, the hepatic stellate cell deactivator, the hepatic fibrosis therapeutic agent, the non-alcoholic stellate hepatitis therapeutic agent, and the psoriasis therapeutic agent. Use of compounds of non-alcoholic steatohepatitis therapeutic agent and psoriasis therapeutic agent

Abstract

The invention discloses an adenosine triphosphate secretion inhibitor which comprises imeglumine, metformin, phosphoenolpyruvic acid, an analogue of phosphoenolpyruvic acid, or pharmaceutically acceptable salts of the imeglumine, the metformin, the phosphoenolpyruvic acid and the analogue of the phosphoenolpyruvic acid.

Description

Technical Field

[0001] This invention relates to the use of compounds for preparing adenosine triphosphate (ATP) secretion inhibitors, hepatic stellate cell activation inhibitors, hepatic stellate cell deactivators, liver fibrosis treatment agents, non-alcoholic steatohepatitis (NAH) treatment agents, psoriasis treatment agents, and the use of these compounds. Background Technology

[0002] Purinergic chemical transport is a cell-to-cell communication mechanism in which adenosine triphosphate (ATP), filled in secretory vesicles, is secreted in response to various stimuli and transmits signals by binding to purine receptors. While ATP's role as the intracellular energy currency is well-known, it functions as a signal transduction substance in purinergic chemical transport.

[0003] ATP secretion is accomplished through the following steps: First, ATP is filled into secretory vesicles, the vesicles fuse with the cell membrane, and ATP is released extracellularly (exocytosis). The filling of ATP vesicles involves vesicular nucleotide transporters (VNUTs) and vacuolar ATPases (V-ATPases). V-ATPase transports H+... + Delivered into the vesicles, as a result, H + The driving force accumulates within the vesicles. Then, VNUT utilizes H... + The driving force transports ATP into the vesicles. According to Non-Patent Literature 1, ATP-filled secretory vesicles fuse with the cell membrane through the action of a group of SNARE (soluble NSF attachment protein receptor) proteins, thereby releasing ATP via exocytosis.

[0004] In recent years, purinergic chemical transport has been known to be involved in various diseases and symptoms, including inflammation, neuropathic pain, ulcerative colitis, non-alcoholic hepatitis, hepatocellular fibrosis, type 2 diabetes, chronic inflammation, blood coagulation disorders, lower urinary tract dysfunction, and pruritus caused by chemical substances or bacterial infections. These symptoms have been reported to be significantly alleviated by inhibiting ATP secretion in purinergic chemical transport. Furthermore, research involving purine receptors also suggests potential applications in Parkinson's disease, Alzheimer's disease, epilepsy, atopic dermatitis, liver fibrosis, psoriasis, atherosclerosis, and cancer.

[0005] In detail, purine chemical transport also plays an important role in fibrosis in various organs, including the liver. Extracellular ATP and its metabolites act as signaling molecules via purine receptors, thereby influencing tissue repair, healing, and scarring processes. In particular, adenosine generated from extracellular ATP by exonucleases, CD39, and CD73 promotes fibrosis in the skin, heart, liver, and lungs.

[0006] Fibrosis is characterized by the excessive accumulation of collagen and other components in the extracellular matrix (ECM) during wound healing and tissue processes. In liver fibrosis, the myofibroblasts that produce ECM mainly originate from hepatic stellate cells (HSCs) located in the perisinusoidal space of the liver. That is, when HSCs are stimulated by various molecules released from hepatocytes and immune cells due to some liver injury, HSCs transform into proliferative ECM-producing myofibroblasts, which actively synthesize and secrete ECM, fibrosis factors, chemokines, and cytokines. These cause further liver damage. As shown in Non-Patent Literature 2, HSC activation is the main response causing liver fibrosis, and it is believed that inhibiting and deactivating HSC activation is directly related to fibrosis prevention.

[0007] Non-Patent Literature 3 shows that VNUT-deficient mice significantly improve inflammatory cell infiltration and fibrosis in a high-fat diet-induced non-alcoholic steatohepatitis (NASH) model. Furthermore, Non-Patent Literature 4 discloses that administration of clodronate, a VNUT inhibitor, improves NASH activity scores, inflammatory cell infiltration, and liver fibrosis in a methionine and choline deficiency (MCD) diet-induced NASH model.

[0008] Furthermore, a portion of the ATP secreted by cells is metabolized extracellularly into ADP and adenosine. Extracellular ATP and its metabolites, acting as intercellular signaling mediators, transmit pain stimuli to target cells via purine receptors, enhancing the secretion of inflammatory cytokines and thus inducing an inflammatory response. Since increased ATP secretion is accompanied by increased cytokine secretion, ATP secretion triggers an inflammatory response.

[0009] Therefore, ATP secretion inhibitors with fewer side effects are useful for the treatment or relief of various diseases and symptoms. Patent Document 1 describes a pharmaceutical composition comprising clophosphonic acid, etc., which are compounds with VNUT inhibitory activity.

[0010] (Existing technical documents) (Patent Documents) (Patent Document 1) Japanese Patent Application Publication No. 2019-014684 (Non-patent literature) Non-patent literature 1: Nao Hasuzawa et al., 2020, Physiopathological roles of vesicular nucleotide transporter (VNUT), an essential component for vesicular ATP release, Biochim Biophys Acta Biomembranes, 1862(12), 183408 Non-patent literature 2: Juliane S Troeger et al., 2012, Deactivation of hepaticstellate cells during liver fibrosis resolution in mice, Gastroenterology, 143(4), 1073-1083 Non-patent literature 3: Keita Tatsushima et al., 2021, Vesicular ATP release from hepatocytes plays a role in the progression of nonalcoholic steatohepatitis, Biochem Biophys Acta MolBasis of Dis. 1867 (3), 166013. Non-patent literature 4: Nao Hasuzawa et al., 2021, Clodronate, an inhibitor of thevesicular nucleotide transporter, ameliorates steatohepatitis and acute liver injury, Sci Rep, 11(1), 5192 Summary of the Invention Technical problems to be solved However, clodronic acid is known to have extremely low absorption, bone accumulation, and cause a serious side effect known as osteonecrosis of the jaw. Therefore, the pharmaceutical industry is seeking compounds with ATP secretion-inhibiting effects but without side effects. Furthermore, while clodronic acid has long been used as a medication for osteoporosis in the UK and Canada, there is no proven track record of its use in Japan, the US, and China, thus necessitating renewed approval as a pharmaceutical product.

[0011] The present invention was made in view of the above-mentioned actual situation, and its purpose is to provide an ATP secretion inhibitor, a hepatic stellate cell activation inhibitor, a hepatic stellate cell deactivator, a liver fibrosis treatment agent, a non-alcoholic steatohepatitis treatment agent, and a psoriasis treatment agent that can inhibit ATP secretion in purinergic chemical transport and have very few side effects.

[0012] Solution to the problem The inventors identified a compound that inhibits ATP secretion in purinergic chemical transport, thus completing this invention. Furthermore, the inventors discovered that this compound inhibits or deactivates HSCs, thereby being effective in treating liver fibrosis and NASH. In addition, the inventors also found that this compound is effective in treating psoriasis and inhibiting recurrence.

[0013] The first aspect of the present invention is an inhibitor of adenosine triphosphate secretion, comprising imagliptin, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof.

[0014] At this point, the adenosine triphosphate secretion inhibitors mentioned in the first aspect above can be used to prevent, alleviate or treat diseases or symptoms related to purinergic chemical transmission.

[0015] In addition, the analogues may be L-2-phosphoglyceric acid, D-2-phosphoglyceric acid, or 3-phosphoglyceric acid.

[0016] A second aspect of the present invention is an inhibitor of hepatic stellate cell activation, comprising imagliptin, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof.

[0017] A third aspect of the present invention is a hepatic stellate cell deactivator comprising imagliptin, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof.

[0018] A fourth aspect of the present invention is a liver fibrosis treatment agent comprising imagliptin, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof.

[0019] The fifth aspect of the present invention is a treatment agent for non-alcoholic steatohepatitis, comprising imagliptin, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof.

[0020] The sixth aspect of the present invention is a psoriasis treatment agent comprising imagliptin, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof.

[0021] The seventh aspect of the present invention is the use of imatinib, metformin, phosphoenolpyruvate, phosphoenolpyruvate analogues, or pharmaceutically acceptable salts thereof, for the manufacture of adenosine triphosphate secretion inhibitors, hepatic stellate cell activation inhibitors, hepatic stellate cell deactivators, liver fibrosis treatment agents, non-alcoholic steatohepatitis treatment agents, or psoriasis treatment agents.

[0022] The effects of the invention According to the present invention, there are provided ATP secretion inhibitors, hepatic stellate cell activation inhibitors, hepatic stellate cell deactivators, liver fibrosis treatment agents, non-alcoholic steatohepatitis treatment agents, and psoriasis treatment agents that can inhibit ATP secretion in purinergic chemical transport and have very few side effects. Attached Figure Description

[0023] Figure 1 This is a diagram showing ATP secretion in Example 1.

[0024] Figure 2 This is a diagram showing ATP secretion in Example 2.

[0025] Figure 3 This is a diagram showing ATP secretion in Example 3.

[0026] Figure 4 This is a diagram showing ATP secretion in Example 4.

[0027] Figure 5 This is a graph showing the ATP uptake in Example 5.

[0028] Figure 6 This is a graph showing the ATP uptake in Example 6.

[0029] Figure 7 This is a diagram showing ATP secretion in Example 7.

[0030] Figure 8 This is a diagram showing ATP secretion in Example 8.

[0031] Figure 9 This is a graph showing the quantitative amount of ATP in Example 9.

[0032] Figure 10This is a graph showing the ATP uptake in Example 10.

[0033] Figure 11 This is a graph showing the dopamine intake in Example 10.

[0034] Figure 12 This is a graph showing the relative proton transport activity in Example 10.

[0035] Figure 13 This is a diagram showing a microscope image of human epidermal keratinocytes in Example 11.

[0036] Figure 14 This is a diagram showing a microscope image of human epidermal keratinocytes in Example 12.

[0037] Figure 15 This is a graph showing the comparison of VNUT gene expression levels in the liver based on microarray data in Example 13.

[0038] Figure 16 This is a diagram showing the immunostaining results in Example 13.

[0039] Figure 17 This is a diagram showing an immunofluorescence microscope image from Example 13.

[0040] Figure 18 This is a graph showing the Pearson correlation coefficients between the location of VNUT and the reactivity of each protein in Example 13.

[0041] Figure 19 This is a diagram showing an image obtained by correlated optical electron microscopy (CLEM) in Example 13.

[0042] Figure 20 This is a graph showing the change in ATP secretion in LX-2 cells over time induced by the addition of ionomycin in Example 14.

[0043] Figure 21 This illustrates the temperature dependence of ATP secretion and Ca in Example 14. 2+ Dependency diagram.

[0044] Figure 22 This is a graph showing the VNUT gene expression level induced by RNAi in Example 14.

[0045] Figure 23 This is a diagram illustrating the inhibition of ATP secretion caused by the suppression of VNUT gene expression in Example 14.

[0046] Figure 24 This is a diagram showing the inhibition of ATP secretion caused by clophosphamide in Example 14.

[0047] Figure 25 This is a diagram showing the ATP secretion induction induced by serotonin and the ATP secretion inhibition induced by clophosphamide in Example 14.

[0048] Figure 26 This is a diagram showing the inhibition of ATP secretion caused by imagramine in Example 14.

[0049] Figure 27 This is a diagram showing the results of hematoxylin-eosin (HE) staining, Sirius red (PSR) staining, and immunostaining in Example 15.

[0050] Figure 28 This is a graph showing the fibrosis score in Example 15.

[0051] Figure 29 This is a diagram showing the immunostaining results of liver sections for α-SMA in Example 16.

[0052] Figure 30 This is a diagram showing the immunostaining and fluorescence microscopy images of liver sections with α-SMA in Example 16.

[0053] Figure 31 This is a graph showing the results of Western blotting in Example 16.

[0054] Figure 32 This is a diagram showing ATP secretion in Example 17.

[0055] Figure 33 This is a diagram showing the morphology of the mouse ear in Example 18.

[0056] Figure 34 This is a graph showing the change in mouse ear thickness over time in Example 18.

[0057] Figure 35 This is a diagram showing tissue stained with HE in Example 18.

[0058] Figure 36 This is a diagram showing the localization of transglutaminase 1 (TMG1) positive cells in a mouse auricle section in Example 18.

[0059] Figure 37 This is a graph showing the change in mouse ear thickness over time in Example 19.

[0060] Figure 38 This is a graph showing the change of erythema score over time in Example 19.

[0061] Figure 39 This is a graph showing the change of desquamation score over time in Example 19.

[0062] Figure 40This is a graph showing the change of the infiltration score over time in Example 19.

[0063] Figure 41 This is a graph showing the change of the Psoriasis Area and Severity Index (PASI) score over time in Example 19.

[0064] Figure 42 This is a diagram showing the localization of transglutaminase 1 (TMG1) positive cells in a mouse ear section on day 8 in Example 19.

[0065] Figure 43 This is a graph showing the change in mouse ear thickness over time in Example 20.

[0066] Figure 44 This is a graph showing the change in mouse ear thickness over time in Example 21. Detailed Implementation

[0067] The embodiments of the present invention will now be described in detail. However, the present invention is not limited to the embodiments and accompanying drawings described below. Furthermore, in the following embodiments, expressions such as "having," "comprising," or "containing" also include the meanings of "composed of" or "constituted by."

[0068] The ATP secretion inhibitors according to this embodiment include imatinib, metformin, phosphoenolpyruvic acid (PEP), PEP analogs, or pharmaceutically acceptable salts thereof.

[0069] The structure of Imegliemin is shown below.

[0070]

[0071] The structure of metformin is shown below.

[0072]

[0073] The ATP secretion inhibitors, including imatinib or metformin, can be any salt such as hydrochloride, acetate, or sulfate, as long as they have an inhibitory effect on ATP secretion in purinergic chemical transport. For example, as shown in the following examples, imatinib hydrochloride and metformin hydrochloride have an inhibitory effect on ATP secretion in purinergic chemical transport.

[0074] Imegliptin hydrochloride was marketed in Japan in 2022 as a treatment for type 2 diabetes. However, due to the extremely low translocation rate of imegliptin, a daily dose of up to 1 g of imegliptin hydrochloride is required. It is known that when 1 g of imegliptin hydrochloride is administered daily for one week, the serum concentration of imegliptin reaches 9 μM. On the other hand, as shown in the examples below, ATP secretion in purinergic chemical transport is inhibited by 0.1 μM to 10 μM of imegliptin hydrochloride or metformin hydrochloride. Therefore, ATP secretion inhibitors can be administered at a serum concentration of 0.05 to 10 μM, preferably 0.1 to 5 μM.

[0075] The structure of a PEP is shown below.

[0076]

[0077] PEP is an intermediate metabolite of glycolysis, possessing the highest energy phosphate bond in living organisms at -62 kJ / mol. As shown in the examples below, it exhibits an inhibitory effect on ATP secretion in purine energy transfer. Furthermore, as shown in the examples below, L-2-phosphoglyceric acid, D-2-phosphoglyceric acid, and 3-phosphoglyceric acid, as analogs of PEP, also exhibit the same inhibitory effect on ATP transport in purine energy transfer. Therefore, ATP secretion inhibitors can include PEP or its analogs. "Analog" refers to a compound in which at least one position of the PEP structure is modified. For example, PEP analogs are compounds in which functional groups or atoms are added, removed, or substituted by different functional groups or atoms. Examples of PEP analogs include L-2-phosphoglyceric acid, D-2-phosphoglyceric acid, and 3-phosphoglyceric acid. PEP or its analogs, as long as they exhibit an inhibitory effect on ATP secretion in purine energy transfer, can be potassium salts, sodium salts, or other salts.

[0078] PEP exists in cells at extremely low concentrations (μM level), but because it can freely penetrate the cell membrane, cells can take up any concentration of PEP.

[0079] When the ATP secretion inhibitor contains PEP, it can be administered at a blood PEP concentration of 1–20 mM, preferably 3–10 mM. When the ATP secretion inhibitor contains a PEP analogue, it can be administered at a blood PEP analogue concentration of 1–20 mM, preferably 3–10 mM.

[0080] The ATP secretion inhibitor according to this embodiment can be a pharmaceutical product, an oral composition, or a cosmetic product, provided that it does not inhibit the inhibition of ATP secretion in purinergic chemical transfer, and may further contain other pharmaceutically acceptable ingredients in addition to imatinib, metformin, PEP, PEP analogs, or their pharmaceutically acceptable salts.

[0081] The form of the ATP secretion inhibitor in this embodiment is not particularly limited. Examples of forms of ATP secretion inhibitors include creams, lotions, gels, ointments, plasters, liquids, dispersants, tablets, granules, fine granules, powders, lozenges, and capsules. The ATP secretion inhibitor can be an injection, oral dosage form, topical dosage form, rectal suppository, vaginal suppository, nasal absorbent, dermal absorbent, pulmonary absorbent, or oral absorbent. Preferably, the ATP secretion inhibitor contains a pharmacologically acceptable carrier other than imatinib, metformin, PEP, PEP analogs, or their pharmaceutically acceptable salts. Pharmacologically acceptable carriers are various organic or inorganic carrier substances. Examples of pharmacologically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid dosage forms, and solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid dosage forms. Furthermore, preservatives, antioxidants, colorants, and sweeteners may be used as needed.

[0082] Examples of excipients include lactose, white sugar, D-mannitol, starch, crystalline cellulose, and light anhydrous silica. Examples of lubricants include magnesium stearate, calcium stearate, talc, and colloidal silica. Examples of binders include tragacanth gum, gum arabic, corn starch, gelatin, crystalline cellulose, white sugar, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Examples of disintegrants include starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, and sodium carboxymethyl starch.

[0083] Examples of solvents include water for injection, ethanol, propylene glycol, and polyethylene glycol. Examples of co-solvents include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, tris(hydroxymethyl)aminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate. Suspension agents are surfactants, hydrophilic polymers, etc., such as stearyltriethanolamine, sodium lauryl sulfate, dodecylaminopropionic acid, lecithin, benzalkonium chloride, benzyl chloride, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

[0084] Examples of isotonic agents include sodium chloride, glycerol, and D-mannitol. Examples of buffers include phosphate, acetate, carbonate, and citrate buffers. Examples of analgesics include benzyl alcohol. Examples of preservatives include parabens, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites and ascorbic acid.

[0085] The ATP secretion inhibitor according to this embodiment can be used to prevent, alleviate, or treat diseases or symptoms related to purinergic chemical transport by inhibiting ATP secretion in purinergic chemical transport. Diseases or symptoms related to purinergic chemical transport include, for example, inflammatory pain, neuropathic pain, ulcerative colitis, non-alcoholic hepatitis, hepatocellular fibrosis, type 2 diabetes, chronic inflammation, blood clotting disorders, lower urinary tract dysfunction, pruritus caused by chemical substances or bacterial infections, Parkinson's disease, Alzheimer's disease, epilepsy, atopic dermatitis, liver fibrosis, NASH, psoriasis, atherosclerosis, cancer, refractory dermatitis, refractory pain, and melanosis.

[0086] The ATP secretion inhibitor according to this embodiment can be used for the prevention, relief, or treatment of the aforementioned diseases or symptoms related to purinergic chemical transmission, and can also be applied to individuals with these diseases or symptoms. The ATP secretion inhibitor according to this embodiment is applied to humans and animals other than humans. Among animals other than humans, mammals are preferred; more specifically, examples include cats, dogs, cattle, pigs, horses, sheep, and deer.

[0087] The dosage of the ATP secretion inhibitor according to this embodiment is appropriately determined based on the subject's gender, age, weight, symptoms, etc. The ATP secretion inhibitor is administered in a manner that achieves a therapeutically effective amount of imatinib, metformin, PEP, a PEP analogue, or a pharmaceutically acceptable salt thereof. An effective amount refers to the amount of imatinib, metformin, PEP, a PEP analogue, or a pharmaceutically acceptable salt thereof required to achieve the desired outcome; it is the amount required to delay, inhibit, prevent, alleviate, reverse, or cure the progression of the treated or managed condition. The ATP secretion inhibitor can be administered once daily or divided into multiple doses. The ATP secretion inhibitor can be administered at various frequencies, such as daily, every other day, weekly, every other week, or monthly.

[0088] As illustrated in the following embodiments, the ATP secretion inhibitors according to this embodiment inhibit the activation of hepatic stellate cells (HSCs) or deactivate HSCs. Therefore, imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof are useful as hepatic stellate cell activation inhibitors and deactivators. Therefore, in another embodiment, hepatic stellate cell activation inhibitors and deactivators comprising imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof are provided.

[0089] Furthermore, as shown in the following embodiments, the ATP secretion inhibitor according to this embodiment is effective in treating liver fibrosis and NASH. Therefore, imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof are useful as therapeutic agents for liver fibrosis and NASH. Therefore, in another embodiment, a therapeutic agent for liver fibrosis and NASH comprising imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof is provided. Furthermore, this therapeutic agent for liver fibrosis can be used for the prevention of liver fibrosis, and this therapeutic agent for NASH can be used for the prevention of NASH.

[0090] Furthermore, as shown in the following embodiments, the ATP secretion inhibitor according to this embodiment is effective in treating psoriasis and inhibiting recurrence. Therefore, imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof are useful as psoriasis treatment agents. Therefore, in another embodiment, a psoriasis treatment agent comprising imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof is provided. Furthermore, this psoriasis treatment agent can be used for the prevention of psoriasis or the prevention of psoriasis recurrence.

[0091] In another embodiment, the use of imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof for the manufacture of ATP secretion inhibitors, hepatic stellate cell activation inhibitors, hepatic stellate cell deactivators, liver fibrosis treatment agents, NASH treatment agents, or psoriasis treatment agents is provided. In another embodiment, a method for inhibiting ATP secretion is provided. This method includes the step of administering imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof to a subject, particularly a subject having a disease or symptoms related to purinergic chemical transmission. Furthermore, in other embodiments, imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof are provided for the treatment of diseases or symptoms related to purinergic chemical transmission.

[0092] Furthermore, in another embodiment, methods for inhibiting hepatic stellate cell activation, deactivating hepatic stellate cells, treating liver fibrosis, treating NASH, and treating psoriasis are provided. These methods include administering imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof to a subject. Additionally, in other embodiments, imatinib, metformin, PEP, PEP analogs, or pharmaceutically acceptable salts thereof are provided for the treatment of liver fibrosis, NASH, and psoriasis.

[0093] In addition, in another embodiment, the use of imatinib, metformin, PEP, PEP analogues, or pharmaceutically acceptable salts thereof is provided for the treatment of ATP secretion inhibition, hepatic stellate cell activation inhibition, hepatic stellate cell deactivation, liver fibrosis, NASH, or psoriasis.

[0094] Example In the following embodiments, unless otherwise stated, sodium clophosphonate is used as "clophosphonic acid", imaglimide hydrochloride is used as "imaglimide", and metformin hydrochloride is used as "metformin".

[0095] (Example 1: The inhibitory effect of clophosphamide on ATP secretion) Epidermal keratinocytes are the primary source of eATP in the skin. Similar to nerve and hormone-secreting cells, ATP accumulates in secretory vesicles through the action of VNUT and V-ATPase, and is secreted via exocytosis induced by ultraviolet (UV) radiation, mechanical stimulation, or chemical stimulation. Clophosphonic acid is a known inhibitor of VNUT.

[0096] [Culturing human epidermal keratinocytes] Following the instructions, human epidermal keratinocytes (cryopreserved HEK batch number 1758, Cell Applications) were cultured using the EpiVita Keratinocyte Growth Medium Kit. The steps are as follows: 1. Add supplemental medium to the hydrocortisone-containing basal medium in the culture medium preparation kit to prepare complete medium. 2. Aspirate the medium from the culture dish, wash with 5 ml of BSS (Gbico), and aspirate again. 3. Add 5–6 ml of trypsin / EDTA solution (Gbico) and incubate at 37°C for 2 minutes. 4. Add 5 ml of trypsin neutralization buffer, transfer the cell suspension to two 15 ml Falcon tubes, centrifuge at 200 × g for 5 minutes, aspirate the supernatant, and resuspend the pelleted cell pellet in 2 ml of medium, adjusting the volume to 4.5 × 10⁻⁶. 5 Cells / ml, a portion was frozen for preservation, and the remainder was cultured for assays.

[0097] [ATP secretion assay] ATP secretion in human epidermal keratinocytes (hereinafter referred to as "keratinocytes") was measured. As a control solution, Krebs phosphate supplemented with 0.2% BSA and 25 mM glucose was used. Ringer-HEPES buffer (hereinafter, also referred to as "Krebs / Iono(-)") was used as a negative control, and a solution further supplemented with 5 μM iomycin (hereinafter, also referred to as "Krebs / Iono(+)") was used as a positive control for ATP secretion. Iomycin is known to be a calcium-based antibiotic. 2+ Ion carriers promote extracellular calcium absorption when added to cells. 2+ The intracellular inflow of [something] causes intracellular Ca2+ to [become more abundant]. 2+ The increased concentration resulted in increased ATP secretion. The test solutions were Krebs / Iono(-) and Krebs / Iono(+) solutions with clophosphonic acid added to bring the final concentration to 0.1 μM (hereinafter referred to as "Clo / Iono(-)" and "Clo / Iono(+)" respectively) (N=3).

[0098] Keratinocytes were sized at 2.5 × 10⁻⁶. 4Cells were cultured at the specified density in 24-well plates at 37°C and 5% CO2 for 4 days. The culture medium was then aspirated and replaced with Krebs / Iono(-) or Clo / Iono(-), and incubated at 37°C for 6 hours. Next, these solutions were aspirated, and the wells incubated with Krebs / Iono(-) were replaced with 300 μl of control solution (Krebs / Iono(-) or Krebs / Iono(+)), while the wells incubated with Clo / Iono(-) were replaced with 300 μl of test solution (Clo / Iono(-) or Clo / Iono(+)). The wells were then incubated at 37°C for 20 minutes, and 200 μl of supernatant was collected. The collected supernatant was immediately heated on a heating block at 95°C for 10 minutes to inactivate the exonuclease. ATP in the supernatant was quantified using the Dojindo ATP assaykit-Luminescence assay kit (manufactured by Dojindo Corporation).

[0099] (result) like Figure 1 As shown, ATP secretion increased in Krebs / Iono(+). On the other hand, the amount of ATP secretion in Clo / Iono(+) was almost unchanged compared to that in Krebs / Iono(-) and Clo / Iono(-). This indicates that iodine promotes ATP secretion in keratinocytes, while clophosphamide, a known VNUT inhibitor, inhibits ATP secretion promoted by iodine. Therefore, the use of keratinocytes to verify the effect of ATP secretion inhibition is justified.

[0100] (Example 2: The ATP secretion inhibition effect of imagramine) Keratinocytes were cultured in the same manner as in Example 1, and ATP secretion in the keratinocytes was measured. Solutions containing 10 μM emegligamine, 0.1 μM eicosapentaenoic acid (EPA), 1 μM EPA, and 10 μM clophosphonic acid were used in Krebs / Iono(-) or Krebs / Iono(-) solutions (N=4). EPA is a known substance that, like clophosphonic acid, inhibits ATP secretion.

[0101] After culturing keratinocytes, the culture medium was aspirated and replaced with Krebs / Iono(-) or Krebs / Iono(-) supplemented with 10 μM emegleemine, 0.1 μM EPA, 1 μM EPA, and 10 μM clophosphonic acid, respectively, and incubated for 6 hours. Then, the medium was replaced with control solution (Krebs / Iono(-) or Krebs / Iono(+)) or test solution (Krebs / Iono(+) supplemented with 10 μM emegleemine, 0.1 μM EPA, 1 μM EPA, and 10 μM clophosphonic acid, respectively), and incubated at 37°C for 20 minutes. 200 μl of the supernatant was collected. The collected supernatant was immediately heated on a heating block at 95°C for 10 minutes to inactivate exonucleases. ATP in the supernatant was quantified using an ATP chemiluminescence assay kit (manufactured by Dojindo Corporation, Japan).

[0102] (result) like Figure 2 As shown, both test solutions inhibited ATP secretion promoted by iodine. This indicates that 10 μM imatinib has an ATP secretion-inhibiting effect equivalent to 1 μM EPA or 10 μM clophosphamide. Furthermore, the survival rate of keratinocytes in this example was 79%, and no difference in survival rate was observed between cells treated with the control and test solutions.

[0103] (Example 3: ATP secretion inhibition effect of various concentrations of imatinib and metformin) Keratinocytes were cultured in the same manner as in Example 1, but with Krebs / Iono(-) or Krebs / Iono(-) supplemented with 0.1 μM, 1 μM, and 10 μM emegglimepiride, and 0.1 μM, 1 μM, and 10 μM metformin, respectively, and incubated for 6 hours. Then, the solutions were replaced with control solutions (Krebs / Iono(-) or Krebs / Iono(+)) or test solutions (Krebs / Iono(+) containing 0.1 μM, 1 μM, and 10 μM emegglimepiride, and 0.1 μM, 1 μM, and 10 μM metformin, respectively), and incubated at 37°C for 20 minutes. 200 μl of the supernatant was then collected. The collected supernatant was immediately heated on a heating block at 95°C for 10 minutes to inactivate the exonuclease. The ATP in the supernatant was quantified using the ATP chemiluminescence assay kit from Dojindo Corporation, Japan (N=3).

[0104] (result) like Figure 3 As shown, imatinib tends to have a slightly higher ATP secretion inhibitory effect than metformin. However, both show ATP secretion inhibitory effects even at low concentrations such as 0.1 μM. The inhibitory effect weakens with increasing metformin concentration.

[0105] (Example 4: Inquiry into the effect of incubation time on the ATP secretion inhibition effect of imagramine) Keratinocytes were cultured in the same manner as in Example 1, but with Krebs / Iono(-) or Krebs / Iono(-) supplemented with 1 μM emegligamine, and incubated for 2 or 6 hours. Then, the solutions were replaced with control solutions (Krebs / Iono(-) or Krebs / Iono(+)) or test solutions (Krebs / Iono(+) supplemented with 1 μM emegligamine), and incubated at 37°C for 20 minutes. 200 μl of the supernatant was collected. The collected supernatant was immediately treated on a heating block at 95°C for 10 minutes to inactivate the exonuclease. ATP in the supernatant was quantified (N=3) using an ATP chemiluminescence assay kit from Dojindo Chemical Co., Ltd.

[0106] (result) Figure 4 The ratio of RLU (relative luminescence) corresponding to ATP secretion using the test solution to the RLU corresponding to ATP secretion using the control solution is shown. After replacing the culture medium with the test solution and incubating for 2 hours, ATP secretion was approximately 85% of that of the control solution, with no significant ATP secretion inhibition observed. On the other hand, after incubation for 6 hours, ATP secretion was approximately 40% of that of the control solution, showing a significant ATP secretion inhibition effect. This indicates that the ATP secretion inhibition effect of imatinib does not reach its maximum immediately after addition to cells, but requires approximately 6 hours to exhibit a strong inhibitory effect.

[0107] (Example 5: Inhibition of ATP transport in chromaffin granule membranes) Chromaffin granules in the adrenal medulla are representative ATP-filled granules containing approximately 0.1 M ATP. Stimulation of chromaffin cells releases catecholamines and ATP from within the chromaffin granules. It is well known that 1–10 mM PEP inhibits ATP uptake in isolated chromaffin granules. However, whether ATP transport activity in the chromaffin granule membrane is mediated by VNUT remains unclear. Therefore, the effect of PEP on ATP transport activity in the isolated chromaffin granule membrane was first investigated.

[0108] [Preparation of Chromophilic Particle Membrane Vesicles] Chromaffin granule membranes were prepared using adrenal glands collected from slaughtered cattle according to reference A (Nathan Nelson et al., 1988, Chromaffin Granule Proton Pump, Methods in Enzymology, 157, 619-633). The chromaffin granule membranes prepared by sucrose density gradient centrifugation were suspended in 2 ml SME buffer (20 mM MOPS-tris (pH 7.0), 0.3 M sucrose, 5 mM EDTA, 10 μg / ml leucopeptide, 10 μg / ml aprotinin) + 25% glycerol, aliquoted, and stored at -80°C. Under these conditions, ATP transport activity, V-ATPase activity, and catecholamine transport activity remained completely intact for at least one year.

[0109] The ATP transport inhibition experiment in chromaffin granule membranes was performed according to reference B (Yoshinori Moriyama et al., 2022, Is the vesicular nucleotide transporter a molecular target of eicosapentaenoic acid?), Frontiers in Pharmacology, 103389. The assay was conducted in 0.5 ml of assay solution (20 mM MOPS-tris (pH 7.0), 0.3 M sucrose, 5 mM potassium chloride, 5 mM magnesium acetate, 5 mM creatine phosphate, 10 units of creatine kinase, 1 mM...). 3 Approximately 20 μg of chromophilic particulate membrane was added to [H]-ATP (15 KBq / assay) and incubated at 30 °C for 1 hour. 0.2 mL of the reaction solution was filtered through a Millipore MCE membrane filter with a pore size of 0.45 μm. After filtration, the filter was washed with 10 mL of 20 mM MOPS-tris (pH 7.0), 0.3 M sucrose, 5 mM potassium chloride, and 5 mM magnesium acetate. The solution was dissolved in 10 mL of Clear-sol II (Nacalai Tesque) and the radioactivity was determined using a liquid scintillation counter (N=3).

[0110] The reaction was achieved by adding 50 mM 3 The process begins with [H]-ATP to achieve a final concentration of 1 mM. 50 mM 3 [H]-ATP is obtained by mixing [2,8-] in an ATP-tris solution (made by Sigma-Aldrich). 3[H] Adenosine triphosphate tetrasodium salt (26.0 Ci / mmol) (manufactured by New England Nuclear Corporation) was used as the test reagent, and pyruvate was used as the negative control. PEP was dissolved in 20 mM MOPS-tris (pH 7.0), 0.3 M sucrose, 5 mM potassium chloride, and 5 mM magnesium acetate to obtain final concentrations of 0.02, 0.05, 0.1, 0.2, 0.4, and 1 mM, and pyruvate was dissolved in 0.5 and 1 mM, respectively. The solutions were added 5 minutes before ATP was added to the assay solution.

[0111] The catecholamine transport inhibition assay was performed following the ATP transport assay. However, the assay solution used contained 20 mM MOPS-tris (pH 7.0), 0.1 M sucrose, 0.1 M potassium chloride, 5 mM magnesium acetate, and approximately 5 μg of chromaffin particulate membrane. 2 mM ATP was added to 0.5 ml of this assay solution, and after incubation at 30°C for approximately 5 minutes, a final concentration of 10 μM was added. 3 [H]-dopamine was incubated at 30°C for 15 minutes. 0.2 ml of the reaction solution was then filtered through a Millipore MCE membrane filter with a pore size of 0.45 μm, and the radioactivity on the filter was measured (N=3). [H]-dopamine was used as the substrate. 3 [H]-Dopamine will convert 3,4-[RING-2,5,6-] 3 [H] Dihydroxyphenylethylamine (dopamine) (30 Ci / mmol) (manufactured by New England Nuclear) is mixed with dopamine hydrochloride (manufactured by LKT Laboratories) and adjusted to a final concentration of 0.1 mM for use.

[0112] ATP-dependent proton transport via V-ATPase was performed according to reference B above. Proton transport activity was measured using a fluorescence spectrophotometer with an excitation wavelength of 492 nm and a fluorescence wavelength of 540 nm, using fluorescence quenching of acridine orange as an indicator (N=2). Membrane potential difference formation was measured using a fluorescence spectrophotometer with an excitation wavelength of 580 nm and a fluorescence wavelength of 630 nm, using fluorescence quenching of oxonol-V as an indicator (N=2). 2 ml of acridine assay solution contained 20 mM MOPS-tris (pH 7.0), 0.2 M sucrose, 0.1 M potassium chloride, 5 mM magnesium acetate, 0.2 μg valinemycin, 2 μM acridine orange, and approximately 20 μg chromaffin particles. 2 ml of oxonol-V assay solution contained 20 mM MOPS-tris (pH 7.0), 0.3 M sucrose, 5 mM magnesium acetate, 5 μM oxonol-V, and approximately 20 μg chromaffin particles.

[0113] (result) ATP transport activity is expressed as ATP uptake per 1 mg of protein. Figure 5 .like Figure 5 As shown, PEP uses IC 50 (50% inhibition concentration) = 25 μM inhibited ATP transport, but pyruvate did not inhibit ATP transport at concentrations up to 1 mM.

[0114] On the other hand, PEP did not inhibit the transport of V-ATPase and catecholamines up to 5 mM (not illustrated). Specifically, in the control study, the ATP-dependent dopamine transport activity was (11.07 ± 0.129) nmol / mg protein, while the ATP-dependent dopamine transport activity in the presence of 5 mM PEP was (12.41 ± 0.250) nmol / mg protein (N=3). Furthermore, it was confirmed that ATP-dependent dopamine transport was almost completely inhibited by 1 μM reserpine, exhibiting typical characteristics of vesicular catecholamine transport.

[0115] The fluorescence quenching of acridine orange caused by the addition of ATP (relative to the fluorescence intensity before ATP addition being 100) was 62%. This quenching degree remained unchanged even with the addition of 5 mM PEP (N=2). The fluorescence quenching of oxonol-V caused by the addition of ATP (relative to the fluorescence intensity before ATP addition being 100) was 40%. This quenching degree remained unchanged even with the addition of 5 mM PEP (N=2).

[0116] Among PEP analogues, in addition to PEP, L-2-phosphoglycerate, D-2-phosphoglycerate, and 3-phosphoglycerate have also been shown to inhibit ATP transport, IC50, and ATP delivery. 50 The concentrations were 0.18, 0.18, and 0.45 mM, respectively. On the other hand, glucose-6-phosphate, fructose-6-phosphate, and pyruvate (refer to...) Figure 5 L-lactic acid, malic acid, oxaloacetic acid, citric acid and other intermediate metabolites of the TCA cycle, acetyl-CoA and NAD. + No inhibitory activity was confirmed.

[0117] (Example 6: Inhibitory effect of human VNUT) Valine can be used to fill ATP in liposomes containing human VNUT. The effect of PEP on inhibiting this ATP filling was investigated as follows.

[0118] [Preparation of human VNUT] Human VNUT was prepared according to reference C (Keisuke Sawada et al., 2008, Identification of a vesicular nucleotide transporter, Proc Natl Acad Sci USA., vol.105, no.15, 5683-5686). Baculoviruses integrating the human VNUT expression line were infected with insect cells (HIGH-FIVE cells) at MOI=1. HIGHFIVE cells expressing VNUT (1×10⁻⁶ cells) were collected using a cell scraper. 8 Cells were prepared and suspended in 20 mM Tris-HCl (pH 8.0), 0.1 M potassium acetate, 10% glycerol, 10 μg / ml aprotinin A, and 10 μg / ml leucopeptide to a concentration of approximately ~8 mg / ml. Cells were lysed using an ultrasonic generator (Sonics Material). The cell lysate was centrifuged at 700 × g for 10 minutes to remove unlysed cells, and then ultracentrifuged at 160,000 × g for 1 hour, collecting the cell membrane as a pellet. The pellet was resuspended in the same buffer solution, and 20% octyl glucoside was added to a final concentration of 2%. The mixture was vortexed and incubated on ice for 30 minutes. The solution was then ultracentrifuged again at 160,000 × g for 1 hour, and the crude VNUT fraction containing soluble VNUT was recovered from the supernatant.

[0119] [Preparation of VNUT-containing reconstructed liposomes] Soybean phospholipid liposome solution was prepared as follows: 0.5 g of Type II L-α-phospholipids from soybean (manufactured by Sigma) was added to 25 ml of 20 mM MOPS-tris (pH 7.0) and 1 mM dithiothreitol, and the solution was sonicated using an ultrasonic generator (manufactured by Sonics Material) until homogeneous. The solution was then aliquoted and stored at -80°C.

[0120] The VNUT (20 μg protein) prepared according to the above method was mixed with liposomes (200 μg lipids) and incubated at -80°C for 30 minutes. Next, the mixture was rapidly thawed at 37°C, then diluted 20 times with 20 mM MOPS-Tris (pH 7.0), 0.15 M sodium acetate, and 5 mM magnesium acetate. After standing on ice for 10 minutes, it was centrifuged at 160,000 × g at 4°C for 1 hour to obtain a clear precipitate (reconstructed liposomes). The reconstructed liposomes were resuspended in 20 mM MOPS-Tris (pH 7.0), 0.15 M sodium acetate, and 5 mM magnesium acetate and incubated on ice. The resulting reconstructed liposomes were subjected to SDS-polyacrylamide gel electrophoresis on an ePAGEL E-T520 2-10% gradient gel (Atto Corporation) and stained with Coomassie Brilliant Blue. The major band was identified at approximately 68 kDa. Proteins electrophoresed on ePAGEL E-T520 2-10% gradient gels (Atto) were transferred to nitrocellulose membranes and analyzed using Western blot with an anti-human SLC17A9 (VNUT) rabbit polyclonal antibody (Proteintech). The results confirmed that the bands were positive for VNUT antibody, and almost all of the added VNUT was recovered into the reconstructed liposomes.

[0121] [ATP transport activity assay] The ATP transport activity of the VNUT-containing reconstructed liposomes prepared according to the above method was determined. The assay solution contained 0.5 ml of 20 mM MOPS-Tris (pH 7.0), 0.1 M potassium acetate, 10 mM potassium chloride, 5 mM magnesium acetate, 0.1 μg valamicin (dissolved in ethanol and prepared to a concentration of 0.1 mg / ml for addition), and 0.1 mM radiolabeled solution. 3 H-ATP (15 KBq / assay). 0.1 mM PEP was used as the test reagent; 2 mM pyruvate was used as a negative control for ATP filling inhibition; and 1 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control for ATP filling inhibition. CCCP is known to inhibit ATP filling induced by valine in human VNUT. PEP or pyruvate was dissolved in 20 mM MOPS-Tris (pH 7.0), 0.1 M potassium acetate, 10 mM potassium chloride, and 5 mM magnesium acetate, and then added to the assay solution. 3The H-ATP reaction was initiated. The assay solution was incubated at 30°C for 2 minutes, and the entire volume was filtered through a 0.22 μm Millipore filter. The filter was washed with 10 mL of 20 mM MOPS-Tris (pH 7.0), 0.1 M potassium acetate, 10 mM potassium chloride, and 5 mM magnesium acetate. The residual radioactivity on the filter was measured using a liquid scintillation counter (N=3). Ethanol (1 μL) was added instead of valine as background in the absence of membrane potential.

[0122] (result) like Figure 6 As shown, 0.1 mM PEP strongly inhibited ATP filling into liposomes. On the other hand, 50 times the concentration (5 mM) of pyruvate did not inhibit ATP filling into liposomes. Therefore, it is concluded that PEP has a VNUT inhibitory effect.

[0123] (Example 7: Inhibition of ATP secretion by PEP in keratinocytes) Example 6 demonstrated that PEP has a VNUT inhibitory effect, but its effect on ATP secretion was further investigated. Keratinocytes were cultured in the same manner as in Example 1, and ATP secretion from keratinocytes was measured. However, in the ATP secretion assay, the incubation time was set to 20 minutes when replacing the control or test solutions. As test solutions, solutions containing 5 mM and 10 mM PEP, respectively, were used (N=4) in Krebs / Iono(+).

[0124] (result) like Figure 7 As shown, 5 mM and 10 mM PEP inhibited ATP secretion promoted by ionomycin. Furthermore, although not illustrated, it was confirmed that PEP at concentrations above 3 mM had a stronger inhibitory effect on ATP secretion.

[0125] In the above embodiments and figures, the values ​​are expressed as the mean ± experimental error (SE) for the number of trials shown by N. A t-test (Student's T-test) was performed, with a significance level set at 0.01.

[0126] (Example 8: The inhibitory effect of PEP on ATP secretion in keratinocytes) Skin tissue is stimulated by external factors such as bacterial infection and UV radiation, triggering various immune responses. Keratinocytes express receptors such as CD4 and Toll-like receptor 4, which induce various immune responses by binding to lipopolysaccharide (LPS), a component of the bacterial outer membrane. Inflammatory responses induced by LPS stimulation are a standard method for evaluating anti-inflammatory drugs. In this example, we investigated ATP secretion induced by LPS, a physiological stimulant for keratinocytes.

[0127] Keratinocytes were cultured in the same manner as in Example 1. The keratinocytes were cultured at a density of 2.5 × 10⁶ cells / year. 4 Cells were cultured at the specified density in 24-well plates at 37°C and 5% CO2 for 4 days. The culture medium was then replaced with the test solution (Hanks' balanced salt solution (HBSS) containing imatinib, metformin, or clophosphamide, manufactured by Gibco). After incubation at 37°C and 5% CO2 for 18 hours, LPS (10 μg / ml) was added, and the plates were incubated for another 10 minutes. 200 μl of the supernatant was then collected. The supernatant was immediately heated on a heating block at 95°C for 10 minutes, and the ATP in the supernatant was quantified using an ATP chemiluminescence assay kit (manufactured by Dojindo) (N=8).

[0128] (result) like Figure 8 As shown, ATP was secreted from keratinocytes upon LPS stimulation. This secretion was almost completely inhibited by 1 μM, 10 μM emegligamine, 10 μM metformin, or 10 μM clodronate. Furthermore, RLU 1.0 × 10⁻⁶ 4 This is equivalent to ATP at 29 nm. The survival rate of keratinocytes after measurement was over 99.9%. Incubation with HBSS significantly improved the survival rate of keratinocytes.

[0129] (Example 9: Inhibition of ATP secretion by emegligamine in macrophage-like cells) Macrophages take up foreign substances such as bacteria, secrete cytokines, and activate immune function. Macrophages are cells that secrete ATP and transmit inflammatory information, playing a major role in innate immunity. The known mechanism of ATP secretion in macrophages is through vesicle ATP filling and exocytosis via VNUT (Reference D; Hayato Sakaki, 2013, Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor, PLoSOne, 8(4):e59778). The inhibitory effect of emegligamine on ATP secretion in macrophages was investigated.

[0130] Human acute monocyticle leukemia cells (THP-1 cells (ACTC)) were cultured according to literature D. THP-1 cells were cultured for 1 day at 37°C and 5% CO2 in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. Further, THP-1 cells were cultured for 1 day at 37°C and 5% CO2 in RPMI 1640 medium containing 1 μg / ml E. coli scrotype 055:B5 (Sigma-Aldrich) and 1000 units / ml mouse IFN-γ (R&D Systems) to differentiate them into macrophages.

[0131] Differentiated macrophages were divided into groups of 2.5 × 10⁻⁶. 4 Cell / well density was determined in 24-well plates using RPMI 1640 medium containing 1 mg / ml E. coli Scrotype 055:B5 (Sigma-Aldrich) and 1000 units / ml mouse IFN-γ (R&D Systems), and cultured at 37°C and 5% CO2 for 4 days. The medium was then replaced with HBSS (Gibco) containing emegligamine or clophosphamide, and LPS (10 μg / ml) was added. After incubation for 10 minutes, 200 μl of the supernatant was collected. The supernatant was immediately heated on a heating block at 95°C for 10 minutes, and the ATP content in the supernatant was quantified using an ATP chemiluminescence assay kit (Dojindo) (N=10).

[0132] (result) like Figure 9 As shown, LPS stimulation resulted in the secretion of ATP from macrophages at a level comparable to that in reference D above. The amount of ATP secreted is expressed in the same manner as in reference D above. This ATP secretion was strongly inhibited by 10 μM emegligamine or 10 μM clodronate.

[0133] (Example 10: Inhibition of ATP filling of ATP-filled particles by fluorescent ATP derivatives) When the fluorescent ATP derivative 2' / 3'-O-(N-methyl-o-aminobenzoyl)-adenosine-5'-triethylammonium triphosphate (MANT-ATP, manufactured by Invitrogen) is incubated with keratinocytes, it is taken up into the cells and accumulates in ATP-filled granules. This accumulation does not occur when VNUT expression is inhibited via RNAi, therefore it is believed that this accumulation is a result of VNUT recognizing MANT-ATP as a substrate and transporting it into the granules (Reference E: Kaori Inoue et al., 2014, Mechanism Underlying ATP Release in Human Epidermal Keratinocytes, J. Invest. Dermatol., 134, 1465-1468). MANT-ATP also accumulates in the ATP-filled granules of macrophages (see Reference D). To confirm, using alternative methods, that MANT-ATP is a substrate for VNUT transport, the effect of MANT-ATP on ATP uptake in bovine adrenal medullary chromaffin granule membrane vesicles, which serve as ATP-filling particles, was investigated. In this experiment, MANT-ATP was expected to act as a competitive inhibitor of VNUT, thereby inhibiting ATP-dependent radioactive ATP uptake.

[0134] Chromophilic granular membrane vesicles were prepared in the same manner as in Example 5, and the uptake of radioactive ATP was measured. The transport activity of catecholamines (dopamine) and the ATP-dependent proton transport via V-ATPase were also measured in the same manner as in Example 5.

[0135] (result) like Figure 10 As shown, MANT-ATP strongly inhibited ATP transport (N=3). Its ID 50 Approximately 30mM. For example... Figure 11 As shown, MANT-ATP also had almost no effect on the catecholamine transport system (N=3). Figure 12As shown, MANT-ATP has no effect on ATP-dependent proton transport via V-ATPase, which drives ATP transport, at least up to 100 μM (N=1). Furthermore, the proton transport activity (relative 10%) is equivalent to 61.8% quenching of acridine orange fluorescence. V-ATPase can also recognize MANT-ATP as a substrate, hydrolyzing it and transporting protons. Upon addition of 0.1 mM MANT-ATP, 34% of acridine orange fluorescence was quenched; subsequent addition of ATP resulted in a total fluorescence quenching of 61.8%. That is, MANT-ATP does not inhibit V-ATPase. This indicates that MANT-ATP competitively inhibits ATP transport via VNUT and the vesicle filling process by which MANT-ATP is taken up by VNUT.

[0136] (Example 11: Detection of ATP-filling granules in keratinocytes by MANT-ATP and inhibition of MANT-ATP filling by emegligamine) To demonstrate that the target of imatinib's ATP secretion inhibition effect is ATP-filling vesicles, the effect of imatinib on intragranular accumulation of MANT-ATP was investigated. Keratinocytes were cultured in the same manner as in Example 8 above. Keratinocytes were cultured at 4 × 10⁶ cells / year. 4 Cells were cultured at a density of 1 / ml in 35mm glass-bottomed culture dishes (Matsunami Glass Co., Ltd.) at 37°C and 5% CO2 for 2 days. The culture medium was then replaced with MANT-ATP (Invitrogen) at a final concentration of 0.3mM, and incubated for another 4.5 hours. Next, imatinib was added to a final concentration of 10μM, and the cells were incubated at 37°C and 5% CO2 for 19 hours. Finally, the cells were washed twice with HBSS (Gibco) and observed using an inverted confocal laser microscope (FV-1000 OSR, Olympus Corporation).

[0137] (result) like Figure 13As shown, in the control group, vesicles accumulating MANT-ATP were mainly concentrated around the nucleus, while MANT-ATP accumulated within the granules of keratinocytes. This accumulation disappeared in cells treated with 10 μM imagragliflozin. This indicates that imagragliflozin targets ATP-filled vesicles. Based on the results of Example 4 and other experiments, neither imagragliflozin nor metformin showed a strong inhibitory effect on VNUT and V-ATPase (not illustrated). Therefore, it is believed that the inhibition of ATP filling within vesicles by imagragliflozin is not achieved through a short-term direct action on VNUT, but rather through changes in the ion permeability of the ATP-filled vesicle membrane, changes in energy state, post-translational modifications of VNUT and V-ATPase, and inhibition of VNUT expression.

[0138] (Example 12: Disappearance of intragranular MANT-ATP caused by ionomycin or LPS) As shown in Examples 1-4 and Example 8 above, ATP can be released from keratinocytes by iomycin or LPS. According to Examples 9 and 10 above, the released ATP originates from ATP filling the secretory granules. Therefore, MANT-ATP accumulated in the granules by iomycin or LPS should also be released extracellularly. This process was therefore observed using confocal microscopy.

[0139] Similar to Example 8 above, keratinocytes were prepared at a density of 2.5 × 10⁻⁶. 4 Cells were cultured at the specified density in 24-well plates at 37°C and 5% CO2 for 2 days. The culture medium was then replaced with medium supplemented with MANT-ATP (Invitrogen) to a final concentration of 0.3 mM, and the cells were incubated at 37°C and 5% CO2 for another 4.5 hours. Cells were washed twice with HBSS (Gibco), and after replacing the HBSS, LPS (10 μg / ml) was added. The disappearance of MANT-ATP was observed using a confocal microscope.

[0140] (result) like Figure 14 As shown, the granular MANT-ATP signal disappeared rapidly upon the addition of LPS (10 μg / ml). The same result was obtained upon the addition of 10 μM ionomycin. These results indicate that ATP secretion inhibitors such as emegligamine do not affect the exocytosis process of secretory vesicles.

[0141] (Example 13: Investigation of VNUT expression in hepatic stellate cells) According to reference F (Shanu Jain and Kenneth A. Jacobson, 2021, Purinergic signaling in Liver pathophysiology, Frontiers in Endocrinology, 12, 718429), HSC activation involves ATP signaling via purine receptors. Hepatocytes, macrophages, and platelets have been considered as sources of ATP, but the possibility of HSCs secreting ATP themselves has not been previously considered. If HSCs secrete ATP, ATP and its metabolites (adenosine) can effectively activate HSCs, and ATP signaling can be effectively blocked by ATP secretion inhibitors, resulting in effective inhibition and deactivation of HSC activation. Therefore, in this example and Example 14 below, it is shown that HSCs are ATP-secreting cells and targets of ATP secretion inhibitors.

[0142] First, the expression of the VNUT gene (SLC17A9) was compared between groups using microarray data from liver biopsies of obese patients (GEO database dataset; GDS4881). "Healthy control" and "Healthy Obese" were defined as healthy individuals, while "NAFLD steatohepatitis" and "NAFLD steatosis" were defined as patients with fatty liver disease. Figure 15 As shown, the expression of SLC17A9 in the liver of patients with fatty liver was statistically significantly increased compared with healthy individuals.

[0143] To demonstrate that VNUT is expressed in HSCs and that HSCs are ATP-secreting cells, the following experiments were performed. Mouse liver samples fixed with 4% paraformaldehyde were embedded in paraffin, sections were prepared, and immunostaining was performed using α-smooth muscle actin (α-SMA) monoclonal antibody (1:500, catalog number A5228, manufactured by Sigma-Aldrich) and VNUT polyclonal antibody (1:250, catalog number 26731-1-AP, manufactured by Proteintech).

[0144] Mice were used in combination with littermate wild-type mice (C57BL / 6J) and VNUT knockout mice (reference G: Shohei Sakamoto et al., 2014, Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity), Sci Rep., 4, 6689). Mice were housed in a pathogen-free environment (temperature: 22–24°C, humidity: 50–60%, 12-hour light-dark cycle) for 48 weeks on either a standard solid diet (5.4% fat, CRF-1, manufactured by Oriental Yeast Industrial Co., Ltd.) or a high-fat diet (24% fat, lard fat, 45 kcal fat, D12451, manufactured by Research Diet Co., Ltd.). After 48 weeks, mice were euthanized under free-feeding conditions, and liver samples were collected. Liver samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned.

[0145] Immortalized human hepatic stellate cell line LX-2 (Merck) was cultured in Dulbecco's modified Eagle's medium (DMEM, 4500 mg / L glucose, D5796, Sigma-Aldrich) in T25 or T75 culture flasks containing inactivated 2% fetal bovine serum (FBS), 10 IU / ml penicillin, and 10 IU / ml streptomycin at 37°C and 5% CO2.

[0146] For immunofluorescence microscopy examination of LX-2 cells, cells on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 minutes. Cells were washed with PBS and incubated for 20 minutes in the same buffer containing 0.1% Triton X-100. Primary antibody treatment was performed using the following antibodies.

[0147] α-SMA monoclonal antibody (1:500, catalog number A5228, manufactured by Sigma-Aldrich) VNUT polyclonal antibody (1:250, catalog number 26731-1-AP, manufactured by Proteintech) Vesicle-associated membrane protein (VAMP) 3 monoclonal antibody (1:200, catalog number sc-514843, manufactured by Santa Cruz Biotechnology). VAMP7 monoclonal antibody (1:200, catalog number sc-166394, manufactured by Santa Cruz Biotechnology) V-ATPase monoclonal antibody (1:200, catalog number sc-55544, manufactured by Santa Cruz Biotechnology) LAMP-1 monoclonal antibody (1:200, catalog number SMC-140, manufactured by StressMarq Biosciences) Furthermore, α-SMA is a contractile fibrous protein expressed during wound healing, representing the differentiation of fibroblasts into highly contractile myofibroblasts. It is present in hepatic hepatic chorionic villus subtilis (HSCs), and its expression increases with activation, thus serving as an indicator of activation and deactivation. VAMP exists in eight isoforms, from 1 to 8, and constitutes part of the membrane fusion apparatus SNARE. VAMP3 and VAMP7 are essential for secretory granule / endosome fusion / exocytosis. LAMP-1 is a lysosomal indicator protein.

[0148] Cell specimens were observed using a microscope (BZ-X700 series, Keyence) or a confocal laser microscope (FV1000, Olympus). In colocalization analysis, Pearson correlation coefficients were calculated using ImageJ's Coloc-2 analysis function.

[0149] In CLEM, wild-type mice were randomly fed and anesthetized with isoflurane. The liver was in situ perfused via the heart with 30 mM HEPES buffer containing 100 mM NaCl and 2 mM CaCl2, followed by fixation in 4% formaldehyde prepared in the same buffer at room temperature for 5 minutes. The tissue was then immediately cut into small pieces and further soaked for 4 hours. The specimens were dehydrated using an ethanol gradient and embedded in LR white resin (Polysciences). The embedded specimens were then cut into 1 μm thick sections using an ultramicrotome (Artos 3D, Leica Microsystems) and mounted on glass slides. The sections were incubated overnight at 4°C with VNUT polyclonal antibody (1:100, catalog number 26731-1-AP, Proteintech) and labeled with Alexa488-bound secondary antibody. The labeled sections were visualized using a confocal microscope (FV1000, Olympus). Next, the sections were stained with heavy metals (1% uranium acetate and Sato lead solution) and coated with a 2nm osmium layer (Neoc-Pro, MeiwaFosis). Finally, the sections were observed using a scanning electron microscope (JSM-IT800, NEC Corporation) to visualize the same areas previously observed with a confocal microscope.

[0150] (result) like Figure 16 As shown, the immunostaining results indicate that the positive sites for VNUT antibody and α-SMA monoclonal antibody are consistent in the livers of wild-type mice (WT). On the other hand, the VNUT polyclonal antibody response is absent in the livers of VNUT knockout mice (VNUT- / -). This suggests that VNUT is particularly strongly present in HSCs.

[0151] Figure 17 Images obtained through immunofluorescence microscopy of LX-2 cells are shown. In LX-2 cells, the specific reaction of the VNUT polyclonal antibody is particularly evident in its granular distribution in the cytoplasm surrounding the nucleus. This reaction is highly consistent with the reaction sites of VAMP3 monoclonal antibody and V-ATPase monoclonal antibody. Figure 18 Showing from Figure 17 The obtained VNUT localization and Pearson correlation coefficients with α-SMA, VAMP3, VAMP7, V-ATPase, or LAMP-1 reactivity were obtained. The correlation coefficients were approximately 0.8, which is a relatively high value. These results suggest that VNUT is present in acidic secretory vesicles containing V-ATPase.

[0152] Figure 19 Images obtained via CLEM are shown. VNUT expression in HSCs was confirmed at the electron microscopy level. Figure 19 The asterisk (*) indicates the nucleus of a stellate cell. VNUTs are present in vesicles surrounding intracellular lipid droplets.

[0153] (Example 14: Ca in LX-2 cells) 2+ (An exploration of ATP-dependent secretion and the effects of ATP secretion inhibitors) LX-2 cells were distributed at a rate of 2 × 10⁶ cells per well. 6 LX-2 cells were seeded at a density of [number] cells per well in 6-well culture dishes coated with collagen and containing DuPont modified Eagle medium (Sigma-Aldrich) supplemented with 10 IU / ml penicillin, 10 IU / ml and 2% fetal bovine serum, and incubated at 37°C with 5% CO2. After 3 days, LX-2 cells were washed twice and cultured with Krebs broth with or without 10 μM clophosphate. After pre-incubation with Ringer buffer (10 mM HEPES-Tris, pH 7.4, 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2.4 mM CaCl2, 10 mM glucose, 0.2% (w / v) bovine serum albumin), LX-2 cells were stimulated with Krebs-Ringer buffer containing iomycin (Cayman) with or without clophosphonic acid. Stimulation was performed with either 1 mM EGTA (Dojindo) or 10 μM serotonin (5-HT, Sigma-Aldrich). ATP concentration in the culture supernatant was measured as in Example 6.

[0154] The siRNA assay was performed using the TriFECTa RNAi kit (Integrated DNA Technologies). Randomized RNA (scRNA) or VNUT-specific siRNA, used as control siRNA, was incubated in OptiMEM reduced serum medium (#31985070, Gibco) with Lipofectamine 3000 reagent (Invitrogen) at room temperature for 15 minutes and then directly added to cultured cells. LX-2 cells were plated with the transfection mixture and cultured for 48 hours. The forward and reverse base sequences of the VNUT siRNA used in the above kit are shown in sequence numbers 1 and 2, respectively. An endogenous gene positive control (#51-01-08-02) and a negative control (#51-01-14-03) were used.

[0155] (result) like Figure 20As shown, by adding ionomycin, LX-2 cells secreted ATP over time (N=3). Figure 21 As shown, ATP secretion requires a constant temperature (37°C) and Ca2+. 2+ It flows into the cell. Ca is chelated with EGTA. 2+ At this temperature, ATP secretion was strongly inhibited. Furthermore, no ATP secretion was observed at 4°C, indicating a temperature dependence (N=6). Figure 22 As shown, RNAi suppressed SLC17A9 gene expression in LX-2 cells (N=6). Figure 23 As shown, ATP secretion was inhibited by suppressing SLC17A9 gene expression. On the other hand, ATP secretion was not inhibited in the control experiment using disordered RNA (N=5). These results indicate that ATP secretion induced by ionomycin in LX-2 cells occurs via exocytosis of ATP-filled vesicles.

[0156] like Figure 24 As shown, ATP exocytosis was inhibited by clophosphamide, a VNUT inhibitor (N=6). Figure 25 As shown, 5-HT, a physiological substance that activates hepatic stellate cells, also promotes ATP secretion in LX-2 cells. This secretion is strongly inhibited by clophosphamide (N=6). Figure 26 As shown, emegligamine inhibited ATP secretion in LX-2 cells (N=8).

[0157] (Example 15: Thioacetamide-induced liver fibrosis and its therapeutic effect with imatinib) Wild-type 2C57BL / 6J mice (manufactured by Charles River Pharmaceuticals, Japan) were allowed free access to a standard solid diet (5.4% fat, CRF-1, manufactured by Oriental Yeast Industrial Co., Ltd.) under a 12-hour light-dark cycle. Thioacetamide (manufactured by TAA, Fujifilm and Hikari Pure Chemical Industries, Ltd.) was dissolved in physiological saline at 0.9% w / v. Eight-week-old mice were randomly assigned to three groups and treated continuously for 9 weeks according to the following protocol. After 9 weeks, all mice were sacrificed 1 hour after administration of physiological saline or IMG.

[0158] Normal control group (N=6): Mice were administered physiological saline intraperitoneally 8 hours after administration 3 times a week, followed by oral administration of the drug carrier.

[0159] TAA group (N=6): Eight hours after administration of TAA (50 mg / kg, intraperitoneally), mice were orally administered saline. This treatment was performed three times a week for a total of nine weeks.

[0160] TAA+IMG group (N=6): Mice were initially administered saline orally for the first week. Subsequently, for 8 weeks, they were administered 200 mg / kg of imagliflozin (IMG) orally three times a week. IMG administration was performed 8 hours after intraperitoneal administration of TAA (50 mg / kg).

[0161] In immunohistochemical analysis, liver samples were fixed with 4% paraformaldehyde, embedded in paraffin to prepare sections, and stained with hematoxylin and eosin (HE) or pSR. Further, immunostaining was performed on paraffin-embedded sections using α-SMA monoclonal antibody (1:500, catalog number A5228, Sigma-Aldrich) and F4 / 80 monoclonal antibody (1:200, catalog number MCA497GA, AbD Serotec). Cell specimens were observed using a microscope (BZ-X700 series, Keyence) or a confocal laser microscope (FV1000, Olympus). The number of F4 / 80 positive cells was counted in four high-power fields on each section. The α-SMA-positive and PSR-positive areas were quantified using the Fiji-ImageJ (1.54f) image analysis software.

[0162] Histological scores were determined by blinded investigators using the NAFLD histological scoring system from the Clinical Research Network for Nonalcoholic Steatohepatitis. The total NAFLD activity score (NAS) was calculated as the sum of scores for steatosis (PSR staining), lobular inflammation (F4 / 80), and hepatocellular ballooning. Each parameter ranged from 0 to 2 (ballooning) or 3 (the other two scores), with 0 representing normal and 2 or 3 representing severe. Fibrosis staging was based on the NASH fibrosis staging system, with fibrosis stages ranging from 0 to 4 as determined by reference H (David E. Kleiner et al., 2005, Design and validation of a histological scoring system for nonalcoholic fatty liver disease, Hepatology, 41, 1313-1321).

[0163] All results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad software). Significance when comparing two groups was assessed using an unpaired two-tailed Student's t-test. Statistical significance among three or more groups was determined using a two-way analysis of variance (ANOVA) with Tukey's or Dunnett's post-hoc test. Coloc2 was used for image analysis, automatic thresholding, and pixel intensity-related statistical processing. A p-value < 0.05 was considered statistically significant.

[0164] (result) like Figure 27 As shown, in liver tissue stained with hematoxylin and eosin (HE), compared with the normal control group, the TAA group showed evidence of lobular structure destruction, ballooning degeneration of hepatocytes, and inflammatory cell infiltration. PSR-positive areas showing collagen fibers were significantly reduced in the TAA+IMG group compared to the TAA group. The α-SMA positive reaction showing hepatic stellate cell activation was particularly strong around the central and portal veins, and significantly increased by TAA, while this increase was strongly inhibited by IMG. The positive reaction of F4 / 80 positive macrophages was also significantly increased by TAA, while this increase was strongly inhibited by IMG. Figure 28 As shown, the fibrosis score was significantly higher in the TAA group than in the normal control group. This increase was inhibited by IMG (TAA+IMG group). IMG administration improved lobular structure destruction, ballooning degeneration of hepatocytes, and inflammatory cell infiltration, and reduced the fibrosis stage.

[0165] (Example 16: HSC activation induced by TAA and inhibition and deactivation of HSC activation by ATP secretion inhibitors) According to Example 15 above, liver sections from the normal control group, TAA group, TAA+IMG group, and TAA+Clo group (in the TAA+IMG group, clophosphamide 30 mg / kg was used instead of emegglitin and was mixed into drinking water and administered one week after the start of TAA administration) were subjected to α-SMA immunostaining and observation by fluorescence microscopy.

[0166] In addition, α-SMA was compared using Western blotting. Fresh mouse liver was homogenized in Western lysis buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.5% NP40) containing protease and phosphatase inhibitor tablets (Roche Applied Science), and centrifuged at 10000×g, 4°C for 5 min to obtain the supernatant. The protein content in the supernatant was quantified using a BCA protein assay kit (ThermoFisher Scientific). The sample was mixed with Laemmli sample buffer (1:1, Bio-Rad), heated at 95°C for 5 min, and 40 μg was loaded onto an e-PAGEL E-R520L (Atto) for SDS-PAGE in the presence of sodium dodecyl sulfate. The proteins in the gel were transferred to a PVDF membrane and reacted with primary antibody, followed by secondary antibody bound to horseradish peroxidase. The primary antibodies used were α-SMA monoclonal antibody (1:500, catalog number A5228, manufactured by Sigma-Aldrich) and β-actin monoclonal antibody (catalog number 4970-s, manufactured by Cell Signaling). Finally, the protein bands were visualized using an electrochemiluminescence (ECL) Western blot system (manufactured by GE Healthcare) and detected using an image analyzer LAS-4000 mini (manufactured by Fujifilm).

[0167] (result) α-SMA-positive cells (stellate cells) are mainly found around the central vein and portal vein in mouse liver tissue. For example... Figure 29 As shown, the α-SMA positive reaction was significantly increased in the entire liver of mice in the TAA group, but this increase was significantly reduced by clodronate. The α-SMA positive area ratio was 0.14±0.01 in the normal control group, 2.79±0.33 in the TAA group, and 0.71±0.10 in the TAA+Clo group (measured four times per group). Figure 30 As shown, emeclimide also exhibits the same effects as clophosphamide. Figure 31 The results of Western blot analysis of liver tissue are shown. Western blot analysis also confirmed that emegligamine significantly reduced the increase in α-SMA positivity induced by TAA. These results indicate that the ATP secretion inhibitor suppresses and deactivates HSCs.

[0168] (Example 17: Investigation of imiquimod-induced hyper-ATP secretion in keratinocytes and its inhibition by imagliflozin) Similar to Example 1 above, keratinocytes were prepared at a density of 2.5 × 10⁻⁶. 4Cells were cultured at the specified cell / well density in 24-well plates at 37°C and 5% CO2 for 4 days. The culture medium was then replaced with HBSS (Gibco) supplemented with 1 μM or 10 μM emegligamine. After incubation at 37°C and 5% CO2 for 18 hours, 1 μg / ml or 0.5 μg / ml imiquimod was added, and the plates were incubated for 15 minutes. 200 μl of the supernatant was collected. The supernatant was immediately heated on a heating block at 95°C for 10 minutes, and the ATP in the supernatant was quantified using an ATP chemiluminescence assay kit (Dojindo) (N=8).

[0169] (result) like Figure 32 As shown, imiquimod significantly promotes ATP secretion from keratinocytes. This ATP secretion is strongly inhibited by 1 μM and 10 μM emegligamine.

[0170] (Example 18: Investigation into the effect of imiquimod-induced psoriasis model) Experimental psoriasis symptoms were induced in the auricle of mice using imiquimod. Six-week-old male C57BL / 6 mice (N=5) were acclimatized for one week. From day 1 to day 7, 62.5 mg of 5% imiquimod cream (Beselna cream 5%) (3.125 mg of active ingredient, manufactured by Mochida Pharmaceutical Co., Ltd.) was applied daily to the right auricle of the mice (IMQ group). Following imiquimod application, the imatinib-treated group (IMQ+IMG group) received oral administration of 500 mg of Twymeeg tablets (manufactured by Sumitomo Pharmaceutical Co., Ltd.) dissolved in physiological saline once daily at a dose of 40 mg / kg. The control group received the same amount of saline. On day 8, the mice were euthanized by cervical dislocation, and the auricle thickness was measured using a dial-type thiometer (manufactured by Adachi Co., Ltd.).

[0171] Mouse auricles were fixed overnight at 4°C with 4% paraformaldehyde, replaced with ethanol, embedded in paraffin, and sectioned (5 mM thick) using a LEICA RM2165 rotary microtome for HE staining or immunohistoscopy. For immunohistoscopy, sections were dewaxed, washed in xylene, and hydrated with graded alcohols. Sections were incubated at 95°C for 30 minutes in a DAKO microtome. After blocking at room temperature for 60 minutes, the sections were reacted with TGMI anti-rabbit polyclonal antibody (catalog number 12912-3-AP, manufactured by Proteintech) at room temperature for 2 hours. The samples were washed and reacted with Alexa544 binding secondary antibody (goat anti-rabbit IgG) (manufactured by Molecular Probes) at room temperature for 60 minutes. After reacting with DAPI at room temperature for 10 minutes, the sections were mounted with fluorescent mounting medium and photographed using a fluorescence microscope (BZ-X710, manufactured by Keyence).

[0172] (result) like Figure 33 As shown, applying imiquimod directly to the auricle caused psoriasis-like symptoms, including partial peeling. Oral administration of imatinib eliminated the peeling and alleviated the psoriasis symptoms. Figure 34 As shown, by applying imiquimod directly to the auricle, the thickened auricle was suppressed by oral administration of imiquimod. Figure 35 and Figure 36 The images show the localization of HE-stained tissue and TMG1-positive cells. TMG1 is an enzyme expressed in a series of processes that contribute to the formation of epidermal sheet-like structures, including the division of keratinocytes (epidermal keratinocytes) in the basal layer, their passage through the spinous and granular layers to the stratum corneum, and their eventual shedding as dirt. TMG1 is an indicator of skin barrier function. The inhibitory effect of emeclimide on psoriasis symptoms is also supported by immunohistochemical images of TMG1 as an epidermal marker.

[0173] This embodiment demonstrates that imagleem inhibits the occurrence of psoriasis.

[0174] (Example 19: Comparison of the effects of imiquimod-induced psoriasis model with anti-TNFα antibody) Mice were shaved and had their hair removed from their backs (day 0). Following Example 18 above, 62.5 mg of 5% imiquimod cream (Beselna cream 5%) was applied once daily to the left ear and the shaved back of the mice for a total of 7 days (days 1-7) to induce imiquimod-induced psoriasis (IMQ group). In the IMQ+IMG group, after imiquimod application, imagliflozin (40 mg / kg) was administered orally once daily (days 1-7). In the IMQ+TNFα group, infliximab (manufactured by Nikko Pharmaceutical Co., Ltd.), an existing anti-TNFα antibody, was administered intravenously at 100 μg / mouse (equivalent to 5 mg / kg in psoriasis patients) on days 1, 3, and 5 (N=5). Auricular thickness was measured, and the degree of erythema, desquamation, and infiltration of the affected area were scored on days 1, 3, 5, 7, and 8 according to the PASI score of the DermNET Dermatology Resource. The scores were then calculated based on these scores.

[0175] Furthermore, the auricle removed on day 8 was subjected to immunohistochemical staining in the same manner as in Example 18 above, and auricle sections were photographed using a fluorescence microscope (BZ-X710, manufactured by Keyence).

[0176] (result) Figure 37 , Figure 38 , Figure 39 , Figure 40 and Figure 41 The changes in auricle thickness, erythema score, desquamation score, infiltration score, and PASI score over time are shown separately. Typical psoriasis symptoms induced by imiquimod treatment, except for erythema, were alleviated by infliximab and imagragliflozin. Figure 42 Immunohistochemical images (400x magnification) of auricular sections are shown. The inhibitory effects of infliximab and imeglitin on psoriasis symptoms are also supported by immunohistochemical images of TMG1 as an epidermal marker. The effects of infliximab and imeglitin are roughly equivalent, or imeglitin may be more effective.

[0177] (Example 20: Investigation on the relapse prevention effect of imiquimod in a psoriasis relapse model) Following Example 19 above, 62.5 mg of 5% imiquimod cream (Beselna cream 5%) was applied once daily to the left ear of mice for a total of 7 times (days 1-7) to induce imiquimod-induced psoriasis. Afterward, imiquimod treatment was interrupted for 2 weeks (days 8-22), and imagliflozin (40 mg / kg) was administered orally every other day (IMG). Mice not treated with imagliflozin were designated as IMQ. Imiquimod treatment was restarted on day 23, with 62.5 mg of 5% imiquimod cream (Beselna cream 5%) applied once daily for a total of 7 times (days 23-27), and imagliflozin (40 mg / kg) was administered orally to the IMQ.

[0178] (result) Figure 43 The change in auricle thickness over time is shown. By interrupting imiquimod treatment, psoriasis symptoms decreased dramatically, almost disappearing by day 11, reaching a state of remission. Subsequently, imiquimod treatment was restarted on day 23, inducing psoriasis symptoms faster and more strongly than the initial treatment. On the other hand, mice treated with imiquimod again while simultaneously receiving imatinib showed significantly reduced symptoms compared to IMQ. This example demonstrates that imatinib has a preventive effect against psoriasis relapse.

[0179] (Example 21: An investigation into the effects of topical clodronic acid and imatinib solution on imiquimod-induced psoriasis) On days 1, 2, 3, 5, and 6, 62.5 mg of 5% imiquimod cream (Beselna cream 5%) was applied to the auricles of wild-type mice (IMQ) or VNUT knockout mice (VNUT KO+IMQ) to induce psoriasis symptoms. After day 5, the auricles showed psoriasis symptoms and thickening (N=2). Applying imiquimod followed by a 20% glycerol solution (glycerol lotion) also induced psoriasis symptoms (IMQ+Gly). The negative control was treated with only a 20% glycerol solution. In IMQ+1% IMG and IMQ+0.1% IMG, a 20% glycerol solution containing 1% (w / v) emegglitinide and 0.1% (w / v) emegglitinide, respectively, was applied immediately after imiquimod application. In addition, in IMQ+1% Clo and IMQ+0.1% Clo, immediately after applying imiquimod, a 20% glycerin solution containing 1% (w / v) clophosphonic acid and 0.1% (w / v) clophosphonic acid were applied, respectively.

[0180] (result) Figure 44 The changes in auricular thickness over time are shown. No auricular thickening was observed in the negative control. Auricular thickening improved in IMQ+1% IMG, IMQ+1% Clo, and IMQ+0.1% Clo, and psoriasis symptoms were also alleviated. The effect of imiquimod was concentration-dependent, and no effect was observed in IMQ+0.1% IMG. Imiquimod's psoriasis induction was weak in VNUT knockout mice, with auricular thickness almost identical to that in IMQ+1% Clo, IMQ+0.1% Clo, and IMQ+1% IMG. These results indicate that the psoriasis-inducing effect of imiquimod requires ATP secretion, and that topical application of ATP secretion inhibitors is effective in the treatment of psoriasis.

[0181] This application is based on Japanese Patent Application No. 2023-074235, filed on April 28, 2023. The entire specification, claims and drawings of Japanese Patent Application No. 2023-074235 are incorporated herein by reference.

[0182] Industrial availability This invention has practical value in inhibiting ATP secretion in purine energy transfer.

Claims

1. An inhibitor of adenosine triphosphate secretion comprising imagliptin, metformin, phosphoenolpyruvate, an analogue of phosphoenolpyruvate, or a pharmaceutically acceptable salt thereof.

2. The adenosine triphosphate secretion inhibitor according to claim 1, wherein, The adenosine triphosphate secretion inhibitors are used to prevent, alleviate, or treat diseases or symptoms related to purinergic chemical transmission.

3. The adenosine triphosphate secretion inhibitor according to claim 1, wherein, The analogues are L-2-phosphoglyceric acid, D-2-phosphoglyceric acid, or 3-phosphoglyceric acid.

4. A hepatic stellate cell activation inhibitor comprising imagliptin, metformin, phosphoenolpyruvate, an analogue of phosphoenolpyruvate, or a pharmaceutically acceptable salt thereof.

5. A hepatic stellate cell deactivator comprising imagliptin, metformin, phosphoenolpyruvate, an analogue of phosphoenolpyruvate, or a pharmaceutically acceptable salt thereof.

6. A liver fibrosis treatment agent comprising imatinib, metformin, phosphoenolpyruvate, an analogue of phosphoenolpyruvate, or a pharmaceutically acceptable salt thereof.

7. A treatment agent for non-alcoholic steatohepatitis comprising imatinib, metformin, phosphoenolpyruvate, an analogue of phosphoenolpyruvate, or a pharmaceutically acceptable salt thereof.

8. A psoriasis treatment agent comprising imagliptin, metformin, phosphoenolpyruvate, an analogue of phosphoenolpyruvate, or a pharmaceutically acceptable salt thereof.

9. Use of imatinib, metformin, phosphoenolpyruvate, analogues of phosphoenolpyruvate, or pharmaceutically acceptable salts thereof in the manufacture of adenosine triphosphate secretion inhibitors, hepatic stellate cell activation inhibitors, hepatic stellate cell deactivators, liver fibrosis treatment agents, non-alcoholic steatohepatitis treatment agents, or psoriasis treatment agents.

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