Ferroptosis induction promoter

Abstract

[Problem] To provide a novel ferroptosis-inducing drug, and in particular, to provide a ferroptosis induction promoter that can effectively promote the induction of ferroptosis even in a cell having ferroptosis resistance even in a ferroptosis-inducing environment (for example, cancer cells and senescent cells). [Solution] Provided is a ferroptosis induction promoter comprising a drug that can reduce lysosomal pH.

Description

Ferroptosis inducer

[0001] The present disclosure relates to a novel ferroptosis-inducing agent, more specifically, to a ferroptosis-inducing agent that can effectively promote the induction of ferroptosis in cells that are resistant to ferroptosis (e.g., cancer cells and senescent cells) even under a ferroptosis-inducing environment.

[0002] When cells are subjected to various stresses, a state known as "cellular senescence" occurs, in which the cell cycle irreversibly stops. Senescent cells that accumulate in the body due to stress overexpress various inflammatory proteins and extracellular vesicles, secreting SASPs (Senescence-associated secretory phenotypes) into surrounding tissues, inducing chronic inflammation and causing many age-related diseases, including cancer and neurodegenerative diseases (Non-Patent Document 1). Therefore, controlling SASPs may lead to the suppression or treatment of disease onset (Non-Patent Document 2).

[0003] Senolytic drugs (senolytics), which selectively induce cell death by clarifying the mechanism by which senescent cells resist cell death, and senomorphic drugs (senomorphic drugs), which inhibit SASP in senescent cells, have the potential to extend healthy lifespan, and are currently being developed as preventive and therapeutic agents for the onset and progression of age-related pathologies (Non-Patent Document 3). To date, senolytics targeting the apoptosis resistance of senescent cells has been reported (Non-Patent Document 4).

[0004] Furthermore, some cancer cells, including those whose cellular senescence has been induced by chemotherapy or radiotherapy, are resistant to iron-dependent cell death called ferroptosis (Non-Patent Documents 5 and 6). Resistance to ferroptosis in pancreatic cancer, breast cancer, lung cancer, liver cancer, and other cancers has been reported to be involved in drug resistance and metastasis / recurrence of cancer (Non-Patent Documents 7 and 8). Therefore, inducing ferroptosis in cancer cells and senescent cells is expected to be a new cancer treatment or prevention method (Non-Patent Documents 9 and 10).

[0005] Gorgoulis V, et al, Cell, 179, 813-827, 2019Faget DV, et al, Nat Rev Cancer, 19, 439-453, 2019Chaib et al, Nat Med, 28, 1556-1568, 2024 Wakita et al. Nat Commun, 11, 1935, 2020 Dixon et al. Cell, 149, 1060-1072, 2012Jiang et al, Nat Rev Mol Cell Biol, 22, 266-282, 2021Badgley et al, Science, 368, 25-39, 2020Lei et al., Nat Rev Cancer, 22, 381-396, 2022Otsuki et al., Cancer Sci, 111, 127-136, 2020Chen et al. Nat Rev Clin Oncol 18, 280-296, 2021

[0006] The present disclosure aims to provide a novel ferroptosis-inducing agent, in particular a ferroptosis induction promoter that can effectively promote the induction of ferroptosis even in cells that are resistant to ferroptosis (e.g., cancer cells and senescent cells) even under a ferroptosis-inducing environment.

[0007] In the course of exploring the mechanism by which cancer cells and senescent cells acquire ferroptosis resistance, the present inventors have coincidentally found that in cells resistant to ferroptosis, the lysosomal pH is elevated compared to before the cells acquired ferroptosis resistance, and that this contributes to the conferral of ferroptosis resistance. The present disclosure is based at least on this finding and includes, but is not limited to, the following features.

[0008] [1] A ferroptosis induction promoter, comprising an agent capable of lowering lysosomal pH.

[0009] [2] The ferroptosis induction promoter according to [1], The agent capable of lowering lysosomal pH is a v-ATPase activator, an isolated v-ATPase subunit or a nucleic acid molecule encoding the same, a STING agonist, a cAMP-related compound, a TRPML1 agonist, a TPC2 antagonist, a TFEB activator, an mTOR inhibitor, a lysosome-targeted nanoparticle, an ATM inhibitor, aloisine A (CAS No. 496864-16-5), 4-aminopyridine (CAS No. 504-24-5), cAMP (CAS No. 60-92-4), SF-22 (CAS No. 824981-55-7), PI(3,5)P2 (CAS No. 2260795-31-9), tetrandrine (CAS No. 518-34-3), or rapamycin (CAS No. 53123-88-9). Ferroptosis induction promoter.

[0010] [3] The ferroptosis induction promoter according to [1], wherein the agent capable of lowering lysosomal pH is a v-ATPase activator.

[0011] [4] The ferroptosis induction promoter according to [3], wherein the v-ATPase activator is EN6, Schisandrol A, or C381.

[0012] [5] The ferroptosis induction promoter according to [4], wherein the v-ATPase activator is EN6.

[0013] [6] The ferroptosis induction promoter according to [2], wherein the v-ATPase subunit is ATP6V1 A.

[0014] [7] The ferroptosis induction promoter according to [2], wherein the STING agonist is C53. [8] The ferroptosis induction promoter according to [2], wherein the cAMP-related compound is cAMP or NKH-477; the TRPML1 agonist is SF-22; the TPC2 antagonist is tetrandrine; the TFEB activator is curcumin analog C1 or PF-11; the mTOR inhibitor is OSI-027 or PP242; the lysosome-targeted nanoparticles are poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), photo-activated nanoparticles (paNPs), acidic nucleolipid nanoemulsions (NL-NEs), or acidic nanoparticles (AcNPs); and / or the ATM inhibitor is KU-60019. Ferroptosis induction promoter.

[0015] [9] A pharmaceutical composition for treating or preventing an aging-related disease, comprising the ferroptosis induction promoter according to any one of [1] to [8] and a pharmaceutically acceptable carrier.

[0016]

[10] The pharmaceutical composition according to [9], further comprising a ferroptosis-inducing compound.

[0017]

[11] The pharmaceutical composition according to [9], which is used in combination with a ferroptosis-inducing compound.

[0018]

[12] The pharmaceutical composition according to

[10] or

[11] , wherein the ferroptosis-inducing compound is elastin, piperazine elastin, imidazole ketone elastin (IKE), sulfasalazine, sorafenib, glutamic acid, buthionine sulfoximine, artesunate, an artemisinin derivative, DPI2, cyst(e)inase, BAY87-2243, hemoglobin, or FeCl ２ , Hemin, (NH ４ ) ２ Fe(SO ４ )２ , non-thermal plasma, salinomycin, amino acid depletion and Cornell dot, lapatinib and siramesine, FIN56, FINO2, withaferin A, BAY11-7085, lanperisone, CIL41, CIL56, CIL69, CIL70, CIL75, CIL79, atorvastatin, simvastatin, cisplatin, neratinib, iFSP1, trigonelline, and brusatol.

[0019]

[13] The pharmaceutical composition according to [9], wherein the aging-related disease is a cardiac disease selected from cancer, age-related macular degeneration, cataract, glaucoma, presbyopia, chronic inflammation, chronic renal failure, interstitial pneumonia, atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary artery thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, hepatic fibrosis, cerebral aneurysm, autoimmune disease, neurodegenerative disease, and stroke.

[0020]

[14] The pharmaceutical composition according to

[13] , wherein the aging-related disease is cancer.

[0021]

[15] The pharmaceutical composition according to

[14] , further comprising an anticancer agent.

[0022]

[16] The pharmaceutical composition according to

[14] , which is used in combination with an anticancer agent.

[0023]

[17] The pharmaceutical composition according to

[15] or

[16] , wherein the anticancer drug is gemcitabine, doxorubicin, liposomal doxorubicin, docetaxel, carboplatin, paclitaxel, nab-paclitaxel, ifosfamide, etoposide, methotrexate, cyclophosphamide, pertuzumab, trastuzumab, amrubicin, vinblastine, dacarbazine, bleomycin, vincristine, prednisolone, eribulin, vincristine ... Relbine, actinomycin D, oxaliplatin, fluorouracil, irinotecan, anastrozole, exemestane, letrozole, palbociclib, abemaciclib, ribociclib, asparaginase, alectinib, cetuximab, pemetrexed, bevacizumab, irinotecan, gefitinib, epirubicin, oxaliplatin, nivolumab, capecitabine, cabazi Taxel, trastuzumab, emtansine, Keytruda, imatinib, crizotinib, ramucirumab, cytarabine, afatinib, regorafenib, sunitinib, capecitabine, lapatinib, dacarbazine, erlotinib, dasatinib, UFT, TS-1, temozolomide, docetaxel, vinorelbine, nimustine, sorafinib, nedaplatin, nogitecan, Herceptin, bilirubin A pharmaceutical composition selected from the group consisting of norelbine, veltuzumab, panitumumab, mitomycin, methotrexate, melphalan, rituxan, levofolinate, lenvatinib, trifluridine-tipiracil, abiraterone, ethinyl, enzalutamide, chlormazine, goserelin, degarelix, tamoxifen, toremifene, bicalutamide, flutamide, and leuprorelin.

[0024]

[18] The pharmaceutical composition according to

[14] , wherein the cancer is selected from the group consisting of pancreatic cancer, liver cancer, breast cancer, and lung cancer.

[0025]

[19] The pharmaceutical composition according to

[14] , wherein the cancer is pancreatic cancer.

[0026]

[20] The pharmaceutical composition according to

[15] or

[16] , wherein the cancer is pancreatic cancer, and the anticancer agent is gemcitabine.

[0027]

[21] The pharmaceutical composition according to

[14] , which is used in combination with radiation therapy.

[0028] According to the present disclosure, a novel ferroptosis-inducing agent, in particular a ferroptosis induction promoter, is provided that can effectively promote the induction of ferroptosis even in cells that are resistant to ferroptosis (e.g., cancer cells and senescent cells) even under a ferroptosis-inducing environment.

[0029] Figure 1 shows cell viability in cells in which cellular senescence was induced by X-ray (15 Gy) irradiation (IR) or subculture (Rep) under conditions of erastin addition and various ferroptosis inhibitors. Fer1, Trolox, DFO, and NAC represent ferrostatin 1, an antioxidant, an iron chelator (DFO), and N-acetylcysteine, respectively. Figure 2 shows cell viability in cells in which cellular senescence was induced by X-ray (15 Gy) irradiation (IR) or subculture (Rep) under conditions of cystine depletion and various ferroptosis inhibitors. Fer1, Trolox, DFO, and NAC represent ferrostatin 1, an antioxidant, an iron chelator (DFO), and N-acetylcysteine, respectively. Figure 3 shows the results of lysosomal pH measurements in cells in which cellular senescence was induced by X-ray irradiation (IR) or subculture (Rep). Figure 4 shows the effects of concanamycin C (ConC) treatment on lysosomal pH and on ferroptosis induced by erastin. Figure 5 shows the effects of tamoxifen (TMX) treatment on lysosomal pH and on ferroptosis induced by erastin. Figure 6 shows the effects of ATP6V1A expression suppression on lysosomal pH and on cell viability under ferroptosis-inducing conditions induced by erastin treatment. Figure 7 shows the results of lysosomal pH measurements after EN6 treatment in cells in which senescence was induced by X-ray irradiation (IR) or subculture (Rep). Figure 8 shows the effects of EN6 and various ferroptosis inhibitors on cell viability under ferroptosis-inducing conditions induced by erastin treatment in cells in which senescence was induced by X-ray irradiation (IR) or subculture (Rep). Figure 9 shows the effect of ATP6V1A overexpression on lysosomal pH and cell viability in a ferroptosis-inducing environment by erastin treatment in cells in which cellular senescence was induced by X-ray irradiation (IR). Figure 10 shows the effect of C53 treatment on lysosomal pH and cell viability in a ferroptosis-inducing environment by erastin treatment in cells in which cellular senescence was induced by subculture (Rep).Figure 11 shows the cell viability of human pancreatic stromal stellate cells (hPSCs) in which cellular senescence was induced by gemcitabine (GEM) under treatment with erastin and various ferroptosis inhibitors. Ferrostatin 1, Trolox, DFO, and NAC represent ferrostatin 1, an antioxidant, an iron chelator (DFO), and N-acetylcysteine, respectively. Figure 12 shows the effects of EN6 treatment on lysosomal pH and cell viability under ferroptosis-inducing conditions with erastin treatment in human retinal pigment epithelial cells (RPE-1) in which cellular senescence was induced by doxorubicin (DXR). Figure 13 shows the effects of EN6 treatment on lysosomal pH and cell viability under ferroptosis-inducing conditions with erastin treatment in human pancreatic cancer cells (PANC1) in which cellular senescence was induced by gemcitabine (GEM). Figure 14 shows the effect of EN6 treatment on lysosomal pH in ferroptosis-resistant pancreatic cancer cells (MIAPaCa2) and the effect on cell viability in a ferroptosis-inducing environment induced by erastin treatment. Figure 15 shows the tumor-suppressing effect of administration of the lysosomal pH-lowering agent EN6 in a pancreatic cancer induction model mouse. Figure 16 shows photographs of tumor cell-injected mice administered with vehicle, gemcitabine, or gemcitabine and EN6, as well as tumors excised from the mice, along with tumor weight and tumor volume. In the figure, GEM indicates gemcitabine. Figure 17 shows the change in tumor volume over time in tumor-injected mice administered with EN6. In the figure, GEM indicates gemcitabine. Figure 18 shows an outline of the experimental design for the animal experiment performed in Example 7. Figure 19 shows the change in relative tumor volume over time in the transplantation experiment performed in Example 7. In the figure, the arrows indicate the start date of EN6 or vehicle administration. Figure 20 shows photographs and weights of tumors 34 days after the start of administration of EN6 or vehicle in the transplantation experiment performed in Example 7. Figure 21 shows immunohistochemical staining of ferroptosis marker molecules (4-HNE, Ptgs2) using tumors 34 days after the start of administration of EN6 or vehicle in the transplantation experiment performed in Example 7. It can be seen that administration of EN6 significantly increases the positive area rate of ferroptosis marker molecules in the tumors.

[0030] Hereinafter, embodiments of the present disclosure will be described in detail.

[0031] In one aspect, the present disclosure relates to a ferroptosis induction promoter. The ferroptosis induction promoter according to the present disclosure does not induce ferroptosis by itself, but rather promotes its induction in an environment in which ferroptosis can occur. Therefore, for example, the present disclosure does not necessarily intend to induce ferroptosis in cells that do not meet the conditions necessary for ferroptosis, such as depletion of iron and cystine, and accumulation of lipid peroxides and reactive oxygen species, which are prerequisites for ferroptosis. The ferroptosis induction promoter according to the present disclosure is particularly preferably applied to cells that do not undergo ferroptosis despite meeting the conditions necessary for ferroptosis induction (also referred to herein as "ferroptosis-resistant cells"). Therefore, the ferroptosis induction promoter according to the present disclosure is effective against ferroptosis-resistant cells such as senescent cells or cancer cells.

[0032] The ferroptosis induction promoter according to the present disclosure is characterized in that it contains a drug capable of lowering lysosomal pH. In the present disclosure, the "drug capable of lowering lysosomal pH" may be any drug capable of lowering the lysosomal pH in target cells by 0.1 or more, preferably 0.2 or more, more preferably 0.3 or more, more preferably 0.4 or more, more preferably 0.5 or more, more preferably 0.6 or more, more preferably 0.7 or more, more preferably 0.8 or more, more preferably 0.9 or more, or more preferably 1.0 or more. In another embodiment of the present disclosure, the "drug capable of lowering lysosomal pH" may be a drug that lowers the lysosomal pH in target cells to 4.5 or less, preferably 4.4 or less, more preferably 4.3 or less, more preferably 4.2 or less, more preferably 4.1 or less, or more preferably 4.0 or less.

[0033] In one embodiment of the present disclosure, the agent capable of lowering lysosomal pH is a vacuolar ATPase (v-ATPase) activator. The v-ATPase is an ATP-driven proton pump that is involved in lysosomal pH regulation. The v-ATPase binds to the V-ATPase protruding from the membrane. １ Part and membrane embedded V Ｏ It is known that the v-ATPase is composed of a ATPase subunit and a ATPase subunit, and that the activity state is regulated by changes in the association state of these subunits. That is, in the activated state, these subunits are associated, and in the inactivated state, they are separated. The v-ATPase activators that can be used in the present disclosure are, but are not limited to, agents that can activate the function of v-ATPase by enhancing the activity of v-ATPase, promoting the association of v-ATPase subunits, or enhancing the expression of v-ATPase or its subunits.

[0034] Alternatively, in another embodiment, the v-ATPase activator that can be used in the present disclosure can be at least one of the isolated subunits of v-ATPase. １ The v-ATPase activator may be any of the subunits of the moiety, and may be at least one of ATP6V1 A to H. In one embodiment, the v-ATPase activator may be isolated ATP6V1 A.

[0035] In the present disclosure, agents known to those skilled in the art can be used as v-ATPase activators, and include, but are not limited to, for example, EN6 (CYS Chung et al., Nat Chem Biol, 2019), Schisandrol A (X Zhou et al., Acta Pharm Sin B, 2022), and C381 (R.T. Vest et al., Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration. Proc. Natl. Acad. Sci. 119, e2121609119(2022).)

[0036] In one embodiment, the v-ATPase activator used in the present disclosure is EN6 (CAS No. 1808714 73 9). EN6 is a small autophagy-activating molecule that covalently binds to cysteine ​​277 of the ATP6V1A subunit of the v-ATPase. Modification of ATP6V1A by EN6 is known to dissociate the v-ATPase from Rag, suppress mTORC1 signaling, and cause lysosomal acidification (Chung et al., Nat Chem Biol, 2019-2019; Guo et al., Neurochem Res, 2022; Zhang et al., Brain Res, 2022).

[0037] In another embodiment, the v-ATPase activator used in the present disclosure may include a nucleic acid molecule encoding at least one subunit constituting the v-ATPase. １The v-ATPase activator may be any of the subunits of the v-ATPase, and may be at least one of ATP6V1 A to H. In one embodiment, the v-ATPase activator may be a nucleic acid molecule encoding ATP6V1 A. The amino acid and nucleotide sequences of each subunit of the v-ATPase are known and can be obtained from NCBI. The sequence information for each subunit is as follows: ATP6V1A (NM_001690.4), ATP6V0D1 (NM_004691.5), ATP6V1B1 (NM_001692.4), ATP6V1B2 (NM_001693.4), ATP6V1C1 (NM_001695.5), ATP6V1C2 (NM_001039362.2), ATP6V1D (NM_015994.4), ATP6V1E1 (NM_001696.4), ATP6V1E2 (NM_001318063.2), ATP6V1F (NM_004231.4), ATP6V1G1 (NM_001693.4), and ATP6V1G2 (NM_001694.4). _004888.4), ATP6V1G2 (NM_130463.4), ATP6V1G3 (NM_001376861.1), ATP6V1 H (NM_015941.4), ATP6V0A1 (NM_001130021.3), ATP6V0A2 (NM_012463.4), AT P6V0A3 (NM_006019.4), ATP6V0A4 (NM_020632.3), ATP6V0D2 (NM_152565.1), ATP6V0C (NM_001694.4), ATP6V0B (NM_004047.5), ATP6V0E (NM_025272.2)). The desired nucleic acid molecule can be produced by any method known to those skilled in the art, for example, by using a nucleic acid synthesizer commonly used in the art. Many companies also offer custom synthesis of nucleic acids, making it easy to obtain nucleic acids with desired sequences from such companies.

[0038] The nucleic acid molecule of the present disclosure may be in any form known to those skilled in the art. For example, if the nucleic acid molecule of the present disclosure is a DNA molecule, it may be encoded in an available protein expression vector (including, but not limited to, gamma retrovirus, lentivirus, adenovirus, adeno-associated virus, etc.) or plasmid. Alternatively, if the nucleic acid molecule of the present disclosure is an RNA molecule (e.g., mRNA), it may be chemically modified to improve stability, as is known in the art.

[0039] The ferroptosis induction-promoting agent according to the present disclosure may optionally contain or be combined with a nucleic acid transfection agent for delivering a nucleic acid molecule to a subject. Examples of such nucleic acid transfection agents include, but are not limited to, lipid-based transfection agents (e.g., lipofection reagents), polymer-based transfection agents (e.g., cationic peptides and their derivatives (e.g., polylysine, polyornithine), linear or branched synthetic polymers (e.g., polybrene, polyethyleneimine), polysaccharide-based transfection molecules (e.g., cyclodextrin, chitosan), natural polymers (e.g., histone, collagen), and activated and inactivated dendrimers), magnetic particle-based transfection agents, exosomes for nucleic acid delivery, or viral proteins for nucleic acid delivery.

[0040] In another embodiment, the v-ATPase activator used in the present disclosure can be easily obtained using a method known in the art. For example, the activator can be obtained by measuring ATPase activity in the presence and absence of a candidate compound by the method described in a previous report (Iwate Ishakusha, Vol. 70, No. 4 (October 2018), pp. 139-146), and screening the candidate compounds for compounds that have the effect of enhancing v-ATPase activity.

[0041] In another embodiment of the present disclosure, the agent capable of lowering lysosomal pH may be an agent capable of retaining protons within the lysosome, including, but not limited to, a STING agonist such as C53 (CAS No. 2259624-71-8) (T. Huang et al., STING-induced noncanonical autophagy regulates endolysosomal homeostasis. Proc. Natl. Acad. Sci. 122, e2415422122 (2025)). In other embodiments of the present disclosure, other agents capable of lowering lysosomal pH may include cAMP-related compounds, TRPML1 agonists, TPC2 antagonists, TFEB activators, mTOR inhibitors, lysosome-targeted nanoparticles, ATM inhibitors, and the like. Specific examples of cAMP-related compounds include, but are not limited to, cAMP (H. Lee, J.-Y. Koh, Roles for H+ / K+-ATPase and zinc transporter 3 in cAMP-mediated lysosomal acidification in bafilomycin A1-treated astrocytes. Glia 69, 1110-1125 (2021)).), NKH-477 (C. J. Folts et al., Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity. PLoS Biol. 14, e1002583 (2016).Examples of TRPML1 agonists include, but are not limited to, SF-22 (C.-C. Chen et al., A small molecule restores function to TRPML1 mutant isoforms responsive for mucolipidosis type IV. Nat. Commun. 5, 4681 (2014)). Examples of TPC2 antagonists include, but are not limited to, tetrandrine (B. C.-K. Tong et al., Tetrandrine ameliorates cognitive deficits and mitigates tau aggregation in cell and animal models of tauopathies. J. Biomed. Sci. 29, 85 (2022)). Examples of TFEB activators include, but are not limited to, curcumin analog C1 (J. Song et al., A small molecule transcription factor EB activator ameliorate beta-amyloid precursor protein and Tau pathology in Alzheimer's disease models. Aging Cell 19, e13069 (2020)), PF-11 (X. C. Yao et al., Pseudoginsenoside-F11 alleviates oligomeric β-amyloid-induced endosome-lysosome defects in microglial. Traffic 20, 61-70 (2019).)Examples of mTOR inhibitors include, but are not limited to, OSI-027 and PP242 (both J. Zenge et al., Modulating lysosomal pH: a molecular and nanoscale materials design perspective. J. Life Sci. Westlake Village Calif 2, 25-37 (2020)). Examples of lysosome-targeting nanoparticles include, but are not limited to, poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), photo-activated nanoparticles (paNPs) (both J. Zeng et al., Modulating lysosomal pH: a molecular and nanoscale materials design perspective. J. Life Sci. Westlake Village Calif 2, 25-37 (2020)), and acidic nucleolipid nanoemulsion (NL-NE) (M. Brouillard et al. , Nucleolipid Acid-Based Nanocarriers Restore Neuronal Lysosomal Acidification Defects. Front. Chem. 9 (2021). ), acidic nanoparticles (AcNP) (J. Liu et al., Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4- / - Mice: Pharmacological Approaches and Functional Recovery. Ophthalmol. Vis. Sci. 49, 772-780 (2008).Examples of ATM inhibitors include, but are not limited to, KU-60019 (H. T. Kang et al., Chemical screening identifies ATM as a target for alleviating sence. Nat. Chem. Biol. 13, 616-623 (2017)).

[0042] In yet another embodiment, other agents capable of lowering lysosomal pH include, but are not limited to, aloisine A (CAS No. 496864-16-5), 4-aminopyridine (CAS No. 504-24-5), cAMP (CAS No. 60-92-4), SF-22 (CAS No. 824981-55-7), PI(3,5)P2 (CAS No. 2260795-31-9), tetrandrine (CAS No. 518-34-3), and rapamycin (CAS No. 53123-88-9).

[0043] In another aspect, the present disclosure relates to pharmaceutical compositions for treating or preventing aging-related diseases, including diseases that are at least partially caused by cellular senescence, diseases whose symptoms can be improved by removing senescent cells, or conditions such as chronic inflammation.

[0044] In the present disclosure, aging-related diseases include, but are not limited to, cancer, age-related macular degeneration, cataracts, glaucoma, presbyopia, chronic renal failure, interstitial pneumonia, atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary artery thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, hepatic fibrosis, cerebral aneurysm, autoimmune disease, neurodegenerative disease, or stroke.

[0045] The pharmaceutical composition according to the present disclosure includes the ferroptosis induction promoter according to the present disclosure. The pharmaceutical composition according to the present disclosure can be manufactured according to known methods, such as those described in the Japanese Pharmacopoeia (JP), the United States Pharmacopoeia (USP), or the European Pharmacopoeia (EP).

[0046] Pharmaceutical compositions according to the present disclosure may optionally include a pharmaceutically acceptable carrier (e.g., water, saline, phosphate buffered saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil) or other compounds.

[0047] There are no limitations on the subjects to which the pharmaceutical compositions according to the present disclosure can be administered; for example, the pharmaceutical compositions according to the present disclosure can be administered to humans or non-human mammals (monkeys, marmosets, mice, rats, rabbits, dogs, cows, horses, goats, etc.).

[0048] The method of administering the pharmaceutical composition according to the present disclosure to a subject (administration route, dosage, number of administrations per day, timing of administration, etc.) is not particularly limited, and can be appropriately determined by a person skilled in the art (e.g., a physician) depending on the subject's health condition, height / weight, sex, severity of disease, type of concomitant drug, etc.

[0049] In one embodiment, the pharmaceutical composition according to the present disclosure further comprises a ferroptosis-inducing compound. In the present disclosure, the ferroptosis-inducing compound is any compound that can itself induce ferroptosis in normal cells, and typically may include any ferroptosis inducer known to those skilled in the art, or a combination thereof. Thus, for example, ferroptosis-inducing compounds that can be used in the pharmaceutical composition according to the present disclosure include, but are not limited to, elastin, piperazine elastin, imidazole ketone elastin (IKE), sulfasalazine, sorafenib, glutamic acid, buthionine sulfoximine, artesunate, artemisinin derivatives, DPI2, cyst(e)inase, BAY87-2243, hemoglobin, FeCl ２ , Hemin, (NH ４ ) ２ Fe(SO ４ )２ , non-thermal plasma, salinomycin, amino acid depletion and Cornell dot, lapatinib and siramesine, FIN56, FINO2, withaferin A, BAY11-7085, lanperisone, CIL41, CIL56, CIL69, CIL70, CIL75, CIL79, atorvastatin, simvastatin, cisplatin, neratinib, iFSP1, trigonelline, or brusatol.

[0050] The pharmaceutical composition according to the present disclosure may be characterized by being used in combination with a ferroptosis-inducing compound. In this case, the pharmaceutical composition according to the present disclosure can be administered to a subject simultaneously or at different times with the ferroptosis-inducing compound. Furthermore, the pharmaceutical composition according to the present disclosure can be administered to a subject via the same or different administration route as the ferroptosis-inducing compound.

[0051] In one embodiment of the present disclosure, the aging-related disease is cancer. The cancer to be treated is not particularly limited, and examples thereof include pancreatic cancer, liver cancer, breast cancer, and lung cancer. In one embodiment, the pharmaceutical composition according to the present disclosure is used for treating pancreatic cancer.

[0052] In one embodiment, when the target of treatment is any cancer, the pharmaceutical composition according to the present disclosure can further contain an anticancer agent. As shown in the examples below, the pharmaceutical composition according to the present disclosure can effectively promote the induction of ferroptosis even in cells that have acquired ferroptosis resistance due to the progression of cellular senescence caused by the anticancer agent, and therefore can be particularly effectively used in combination with an anticancer agent. The anticancer agent used in the present disclosure can be selected appropriately by those skilled in the art depending on the type of cancer to be treated. For example, but not limited to, gemcitabine, doxorubicin, liposomal doxorubicin, docetaxel, carboplatin, paclitaxel, nab-paclitaxel, ifosfamide, etoposide, methotrexate, cyclophosphamide, pertuzumab, trastuzumab, amrubicin, vinblastine, dacarbazine, bleomycin, vincristine, prednisolone, eribulin, vinorelbine, actinomycin D, oxaliplatin, fluorouracil, irinotecan, anastrozole, exemestane, letrozole, palbociclib, abemaciclib, ribociclib, asparaginase, alectinib, cetuximab, pemetrexed, bevacizumab, irinotecan, gefitinib, epirubicin, oxaliplatin, nivolumab, capecitabine , cabazitaxel, trastuzumab, emtansine, Keytruda, imatinib, crizotinib, ramucirumab, cytarabine, afatinib, regorafenib, sunitinib, capecitabine, lapatinib, dacarbazine, erlotinib, dasatinib, UFT, TS-1, temozolomide, docetaxel, vinorelbine, nimustine, sorafinib, nedaplatin, nogitecan, herceptin, Anticancer drugs that can be used include vinorelbine, veltuzumab, panitumumab, mitomycin, methotrexate, melphalan, Rituxan, levofolinate, lenvatinib, trifluridine / tipiracil, abiraterone, ethinyl, enzalutamide, chlormazine, goserelin, degarelix, tamoxifen, toremifene, bicalutamide, flutamide, and leuprorelin.

[0053] In the present disclosure, the method of administering an anticancer agent to a subject (administration route, dosage, number of administrations per day, timing of administration, etc.) is not particularly limited, and can be appropriately determined by a person skilled in the art (e.g., a physician) depending on the symptoms, height / weight, sex, stage of cancer, etc. of the subject.

[0054] In another embodiment, when the subject of treatment is any cancer, the pharmaceutical composition according to the present disclosure is used in combination with an anticancer drug. In this case, the pharmaceutical composition according to the present disclosure can be administered to the subject simultaneously or at different times with the anticancer drug.

[0055] In one embodiment, when the subject of treatment is any cancer, the pharmaceutical composition according to the present disclosure is characterized by being used in combination with radiation therapy for cancer treatment. As shown in the examples below, the pharmaceutical composition according to the present disclosure can effectively promote the induction of ferroptosis even in cells that have acquired ferroptosis resistance due to the progression of cellular senescence caused by radiation exposure, and therefore can be particularly effectively used in combination with radiation therapy. In this case, the pharmaceutical composition according to the present disclosure can be administered to the subject before or after radiation therapy.

[0056] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise EN6 and are used to treat pancreatic cancer.

[0057] In another specific embodiment, the pharmaceutical composition of the present disclosure comprises EN6 and is used in combination with an anti-cancer agent.

[0058] In yet another specific embodiment, the pharmaceutical composition of the present disclosure comprises EN6, is used in combination with an anti-cancer agent, and is used to treat pancreatic cancer.

[0059] In yet another specific embodiment, the pharmaceutical composition of the present disclosure comprises EN6, is used in combination with an anti-cancer agent, and is used to treat pancreatic cancer, and the anti-cancer agent is gemcitabine.

[0060] It should be noted that the terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0061] Furthermore, the term "comprise" used in this specification intends that the described items (components, steps, elements, numbers, etc.) are present, unless the context clearly dictates otherwise, and does not exclude the presence of other items (components, steps, elements, numbers, etc.).

[0062] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0063] Although terms such as "first" and "second" may be used to describe various elements, it is understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another, and for example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the present disclosure.

[0064] The present disclosure will be described in more detail below with reference to examples. However, the present disclosure can be embodied in various forms and should not be construed as being limited to the examples set forth herein.

[0065] Unless otherwise noted, the following protocol was used throughout this example.

[0066] Cell Culture and Induction of Cell Senescence: Human normal fibroblasts (TIG-3) were obtained from the JCRB Cell Bank. Human retinal pigment epithelial cells (RPE-1) and human pancreatic cancer cell lines (PANC1 and MIAPaCa2) were obtained from the American Type Culture Collection (ATCC). Primary human pancreatic stromal stellate cells (hPSCs) were obtained from ScienCell and immortalized using pLV-hTERT-IRES-hygro (Addgene, 85140) to constitutively express hTERT. The cells used in this experiment were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Nacalai Tesque, 08458-16) containing 10% fetal bovine serum (FBS) (Gibco, 10437-028) and penicillin / streptomycin (Fujifilm Wako Chemicals, 168-23191) at 37°C and 5% CO. ２ TIG-3 cells with a PDL (Population Doubling Level) of less than 45 were used as proliferating cells (control cells), and TIG-3 cells with a PDL of more than 80 and cessation of proliferation were cultured for one month and used as senescent cells due to replicative senescence.

[0067] Induction of cellular senescence by X-ray irradiation was performed by irradiating TIG-3 cells with 15 Gy of X-rays using an X-ray irradiator CP-160 (Faxitron X-ray Corporation) and then irradiating the TIG-3 cells at a density of 2500 cells / cm. ２ Cells were seeded at a density of 100 μg / mL and cultured for 11 days. RPE-1 cells were cultured for 4 days in medium containing 150 ng / mL doxorubicin (Fujifilm Wako Chemicals, 040-21521) to induce cellular senescence. hPSCs were cultured for 10 days in medium containing 1 μM gemcitabine (Selleck, S1149) to induce cellular senescence. PANC1 and MIAPaCa2 cells were cultured for 10 days in medium containing 50 nM gemcitabine to induce cellular senescence.

[0068] To induce ferroptosis in cells, cells were cultured for 24 hours in a medium containing erastin (Selleck, S7242). Ferroptosis inhibitors, such as ferrostatin 1 (Fer1; Selleck, S7243), Trolox (Selleck, S3665), deferoxamine (Selleck, S5742), or NAC (Sigma, A9165), were co-administered with erastin and the cells were cultured for 24 hours. Additionally, concanamycin C (Adipogen Life Sciences, BVT-0254-C100), 5 μM tamoxifen (Abcam, AB141943), or 50 μM EN6 (Selleck, S6650), compounds that alter lysosomal pH, were co-administered with elastin and cells and cultured for 24 hours. 10 μM C53 (Cayman, 37354) was pretreated for 2 hours before co-administration with elastin and cells and cultured for 24 hours. To induce ferroptosis by cystine depletion, TIG-3 cells were cultured for 24 hours in a cystine-deficient medium (Gibco, 21013-024) containing 200 mM methionine (Sigma, M9625) and 4 mM glutamine (Life Technologies, 25030-081).

[0069] Measurement of Cell Viability Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7573) according to the manufacturer's protocol and measured using a plate reader Nivo (PerkinElmer).

[0070] Measurement of lysosomal pH LysoSensor is used to measure the lysosomal pH of cells. ＴＭ The experiment was carried out using a Yellow / Blue DND-160 (Thermo, L7545) according to the manufacturer's protocol, and measurements were made using a high-content image acquisition system, Operetta CLS (PerkinElmer).

[0071] Knockdown of ATP6V1A using siRNA. Suppression of ATP6V1A expression by siRNA was achieved by Lipofectamine. ＴＭTransfection was performed using RNAiMAX transfection reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. The siRNAs used were ON-TARGETplus siRNAs (Horizon Discovery, J-017590-09, J-017590-11) targeting the ATP6V1A mRNA sequence and a non-targeting control siRNA (Horizon Discovery, D-001810-01-20) at a final concentration of 1 nM for two days. Gene expression silencing efficiency was evaluated by RT-qPCR.

[0072] To construct a lentiviral vector for ATP6V1A, a Halo-tagged ATP6V1A gene was amplified from the plasmid pF1KB5105 (Kazusa DNA Res. Inst, FXC11629) by PCR using KOD plus DNA Polymerase (TOYOBO, KOD-201). The PCR product was then inserted into pENTR to create an entry vector. Then, using Gateway LR Clonase enzyme mix (Invitrogen, 11791-100), LR reaction was performed on the entry vector and pLenti CMV / TO Puro DEST (670-1) (Addgene, 17293) according to the manufacturer's protocol to produce a lentiviral vector (pLenti-Halo-ATP6V1A-Puro).

[0073] To produce lentivirus, HEK-293T cells were transfected with pLenti-Halo-ATP6V1A-Puro, pCMV-VSV-G (Addgene, 8454), and psPAX2 (Addgene, 12260). After 24 hours, the medium was replaced. After another 24 hours, the virus-containing medium was collected and concentrated 100-fold using a Lenti-X™ Concentrator (TAKARA, 631231) according to the manufacturer's protocol.

[0074] To overexpress ATP6V1A, cells induced to undergo cellular senescence by X-ray irradiation were cultured in DMEM (containing 10% FBS) containing 4 μg / mL polybrene and 0.001% concentrated virus solution, and the cells were infected with the virus. After 24 hours, the medium was replaced with new medium and cells were selected in medium containing puromycin (Fujifilm Wako Chemicals, 160-23151). The efficiency of overexpression was evaluated by RT-qPCR.

[0075] [Example 1] Induction of ferroptosis in senescent cells According to the above protocol, cellular senescence was induced in human normal fibroblasts (TIG-3) by X-ray (IR) or subculture (Rep), and then cell death was induced by adding or depleting elastin.

[0076] In normal cells (control), cell death induced by the addition of erastin was suppressed by the addition of lipid peroxidation inhibitors (ferrostatin 1; Fer1), antioxidants (Trolox), iron chelators (DFO), and N-acetylcysteine ​​(NAC), which are known to be ferroptosis inhibitors. This indicates that ferroptosis is induced in normal cells (control). On the other hand, TIG-3 cells induced by IR (irradiation with X-rays (15 Gy)) and IR (Rep) through subculture were shown to be resistant to erastin-induced ferroptosis (Figure 1).

[0077] Furthermore, in a system in which ferroptosis was induced by cystine depletion, as in the case of erastin addition, TIG-3 cells induced to undergo cellular senescence (IR) by X-ray irradiation (15 Gy) and cellular senescence (Rep) by subculture showed resistance to ferroptosis (Figure 2).

[0078] [Example 2] Evaluation of the relationship between cell death and lysosomal pH According to the above protocol, the lysosomal pH of the cells used in Example 1 was measured before and after induction of cell senescence by X-ray irradiation (IR) or subculture (Rep).

[0079] It was revealed that, compared with normal cells, lysosomal pH was elevated after induction of cell senescence by X-ray irradiation (IR) or subculture (Rep) (Figure 3).

[0080] Furthermore, to evaluate whether lysosomal pH is related to cell death, we used normal cells (TIG-3 cells) and evaluated whether cell death was induced by elastin when treated with the v-ATPase inhibitors concanamycin C or tamoxifen, both of which are known to be involved in increasing lysosomal pH, or by suppressing the expression of ATP6V1A, a component of v-ATPase.

[0081] All cells with elevated lysosomal pH, regardless of the means used, were rendered resistant to erastin-induced cell death (FIGS. 4-6).

[0082] To further confirm the relationship between cell death and lysosomal pH, we investigated whether adjusting lysosomal pH in cells that had been conferred ferroptosis resistance could confer ferroptosis sensitivity. Specifically, we used human normal fibroblasts (TIG-3) in which cellular senescence had been induced by X-ray irradiation (IR) or subculture (Rep) in the same manner as in Example 1, and investigated the effect of changes in lysosomal pH on cell death in an environment in which ferroptosis was induced by erastin.

[0083] In this experiment, EN6 was used as a means of changing the lysosomal pH. EN6 inhibits H ＋ EN6 is known to bind to the ATP6V1A subunit of the v-ATPase transporter, activating v-ATPase. Prior to this experiment, we confirmed that EN6 reduces lysosomal pH in human normal fibroblasts (TIG-3) induced by senescence (IR) or subculture (Rep) (Fig. 7).

[0084] Next, we investigated the effects of EN6 and various ferroptosis inhibitors on cell viability under erastin-induced ferroptosis-inducing conditions using the same cells (Figure 8). The results showed that treatment of senescent cells with EN6 increased their susceptibility to erastin-induced ferroptosis.

[0085] We also investigated the effect of overexpression of ATP6V1A induced according to the above protocol on cell viability in the same cells under the ferroptosis-inducing environment of erastin treatment (Figure 9).The results showed that overexpression of ATP6V1A in senescent TIG-3 cells decreased lysosomal pH and increased susceptibility to erastin-induced ferroptosis.

[0086] The above results suggest that an increase in lysosomal pH is involved in the resistance to ferroptosis in senescent cells.

[0087] We also conducted experiments using human normal fibroblasts (TIG-3) in which senescence was induced by subculture (Rep) and C53 was used as a means of altering lysosomal pH under an erastin-induced ferroptosis-inducing environment. C53 treatment was shown to decrease lysosomal pH in senescent cells. Furthermore, the decrease in lysosomal pH by C53 treatment was shown to increase ferroptosis sensitivity in ferroptosis-resistant senescent cells (Figure 10).

[0088] Example 3: Evaluation of the relationship between cellular senescence, lysosomal pH, and cell death in other cell types. To evaluate whether the increase in lysosomal pH and acquisition of ferroptosis resistance associated with cellular senescence are observed in other cell types, human pancreatic stromal stellate cells (hPSCs) or human retinal pigment epithelial cells (RPE-1) were used.

[0089] First, we induced senescence in human pancreatic stromal stellate cells (hPSCs) and assessed whether they exhibited resistance to ferroptosis induction by erastin. In this example, senescence was induced using a 10-day treatment with the anticancer drug gemcitabine (GEM). Cell viability assessment demonstrated that senescence-induced hPSCs conferred resistance to ferroptosis induction by erastin (Figure 11).

[0090] Furthermore, we induced senescence in human retinal pigment epithelial cells (RPE-1) and evaluated cell viability under lysosomal pH and ferroptosis-inducing conditions, with and without senescence induction. In this experiment, senescence was induced using 10 days of treatment with the anticancer drug doxorubicin (DXR), and EN6 was used to induce changes (decreases) in lysosomal pH. Similarly, in human retinal pigment epithelial cells (RPE-1), senescence induction increased lysosomal pH, which in turn suppressed ferroptosis under an elastin-inducing environment. However, suppression of the increase in lysosomal pH by administration of EN6 sensitized the cells to ferroptosis (Figure 12).

[0091] Example 4: Evaluation of the relationship between cellular senescence, lysosomal pH, and cell death in cancer cells. Human pancreatic cancer cells (PANC1 and MIAPaCa2) were used to evaluate whether cancer cells also exhibit increased lysosomal pH and acquired ferroptosis resistance associated with cellular senescence. Senescence was induced in human pancreatic cancer cells by treatment with gemcitabine (GEM) for 10 days. Furthermore, cell death induction by controlling lysosomal pH was evaluated in ferroptosis-resistant cancer cells.

[0092] Specifically, we evaluated human pancreatic cancer cells (PANC1) under senescence induction and under conditions of lysosomal pH and cell viability under ferroptosis induction. Similar to normal cells, senescence induction increased lysosomal pH in PANC1 (Figure 13) and PANC1 (Figure 14), and ferroptosis was suppressed under an elastin-induced environment. However, administration of EN6 suppressed the increase in lysosomal pH, making the cells more susceptible to ferroptosis.

[0093] Regarding ferroptosis-resistant pancreatic cancer cells (MIAPaCa2), it was shown that when the increase in lysosomal pH was suppressed by administration of EN6, both the cancer cells before and after induction of cell senescence became sensitive to ferroptosis (Figure 14).

[0094] Example 5: Cancer growth inhibitory effect of the lysosomal pH-lowering agent EN6 in a pancreatic cancer mouse model. BALB / c nude mice used in this study were purchased from Jackson Laboratory Japan, Inc. To construct pancreatic cancer model mice (LSL-Kras+ / G12D, Pdx1-Cre+ / -), Pdx1 ires-Cre mice were purchased from the RIKEN BioResource Research Center (RIKEN BRC) (H. Shibata et al., Nat Commun, 2018), and LSL-Kras G12D mice were obtained from Dr. Ryoji Yao of the Cancer Institute of the Japanese Foundation for Cancer Research. The two strains of mice were crossed. All mice were fed a normal diet (CE-2 feed from CLEA Japan, containing 12 kcal% fat, 29 kcal% protein, and 59 kcal% carbohydrates) under a 12-hour light / dark cycle in a specific pathogen-free (SPF) environment.

[0095] To induce pancreatic cancer, pancreatic cancer was induced in pancreatic cancer model mice (LSL-Kras+ / G12D, Pdx1-Cre+ / -) by administering cerulein (Fujifilm Wako Chemicals, 4030451.0001) at 50 mg / kg four times. EN6 (50 mg / kg) or vehicle was then administered intraperitoneally twice weekly. On day 25 after the start of administration, the mice were dissected and tumor weights were measured (Figure 15). The results showed that the lysosomal pH-lowering agent EN6 effectively suppressed tumor growth.

[0096] Example 6: Cancer growth suppression effect of combined use of anticancer drugs and lysosomal pH-lowering agent EN6 in a pancreatic cancer mouse model. BALB / c nude mice used in this study were purchased from Jackson Laboratory Japan. To construct pancreatic cancer model mice (LSL-Kras+ / G12D, Pdx1-Cre+ / -), Pdx1 ires-Cre mice were purchased from the RIKEN BioResource Research Center (RIKEN BRC) (H. Shibata et al., Nat Commun, 2018). LSL-Kras G12D mice were obtained from Dr. Ryoji Yao of the Cancer Institute of the Japanese Foundation for Cancer Research. The two strains of mice were crossed. All mice were fed a normal diet (CE-2 feed from CLEA Japan, containing 12 kcal% fat, 29 kcal% protein, and 59 kcal% carbohydrates) under a 12-hour light / dark cycle in a specific pathogen-free (SPF) environment.

[0097] To generate xenograft tumors in mice, 2 x 10 ６ A 25% Matrigel (Corning, 356237) solution containing 100 PANC1 cells was prepared and implanted subcutaneously into BALB / c nude mice. ３ After reaching the age of 18, the mice were intraperitoneally administered gemcitabine (100 mg / kg) every week, and then EN6 (50 mg / kg) or vehicle (10% DMSO / 35% PEG200 / 55% H ２ 0) was intraperitoneally administered every 3 days, and on the 25th day after the start of administration, the mice were dissected and the tumor weight was measured ( Figure 16 ). ３ ] = (tumor long diameter [mm] x tumor short diameter [mm]) ２ The tumor growth was measured weekly using the formula: 0.1 / 2 (FIG. 17). These results indicate that co-administration of the anti-cancer drug gemcitabine (GEM) and EN6 suppressed tumor growth.

[0098] [Example 7] Tumor-suppressing effect of EN6 and immunohistochemical staining To further investigate the tumor-suppressing effect of EN6 in vivo, further experiments were carried out as follows. ６PANC-1 cells were mixed with 25% Matrigel (Corning, 356237) / PBS (volume ratio) and subcutaneously injected into nude mice (15 weeks old). EN6 (50 mg / kg) or vehicle was administered intraperitoneally to the mice every 3 days for 34 days (Figure 18). On day 34, the mice were dissected and tumor weight was measured. Tumor size was measured with a caliper, and tumor volume (mm ３ ) was calculated using the formula (length × width × width) / 2. A significant decrease in tumor volume and tumor weight was observed in the EN6-administered group compared to the vehicle-treated control group (Figures 19 and 20).

[0099] To confirm whether tumor growth inhibition was due to ferroptosis, tumor tissue was analyzed by immunohistochemistry. Immunohistochemistry was performed using TaKaRa POD Conjugate Set (TaKaRa, anti-mouse MK200; anti-rabbit MK202) according to the manufacturer's instructions. The primary antibodies used for immunohistochemistry were anti-4-HNE (JAICA, MHN-100P) and anti-Ptgs2 (Abcam, ab15191). Immunofluorescence analysis was performed as previously reported (A. Takahashi et al., Nat. Commun. 9, 1249 (2018)). Image analysis was performed using ImageJ.

[0100] The suppression of tumor growth appeared to be due to the induction of ferroptosis, as judged by increased 4-hydroxynonenal (4-HNE) levels and prostaglandin endoperoxide synthase 2 (Ptgs2, also known as cyclooxygenase 2) expression in EN6-treated tumors (Figure 21).

[0101] According to the present disclosure, a ferroptosis induction promoter, in particular a ferroptosis inducer that can effectively promote the induction of ferroptosis even in cells that are resistant to ferroptosis (e.g., cancer cells or senescent cells) even under a ferroptosis-inducing environment, is provided.

Claims

1. A ferroptosis induction promoter, comprising an agent capable of lowering lysosomal pH.

2. The ferroptosis induction promoter according to claim 1, The agent capable of lowering lysosomal pH is a v-ATPase activator, an isolated v-ATPase subunit or a nucleic acid molecule encoding the same, a STING agonist, a cAMP-related compound, a TRPML1 agonist, a TPC2 antagonist, a TFEB activator, an mTOR inhibitor, a lysosome-targeted nanoparticle, an ATM inhibitor, aloisine A (CAS No. 496864-16-5), 4-aminopyridine (CAS No. 504-24-5), cAMP (CAS No. 60-92-4), SF-22 (CAS No. 824981-55-7), PI(3,5)P2 (CAS No. 2260795-31-9), tetrandrine (CAS No. 518-34-3), or rapamycin (CAS No. 53123-88-9). Ferroptosis induction promoter.

3. The ferroptosis induction promoter according to claim 1, wherein the agent capable of lowering lysosomal pH is a v-ATPase activator.

4. The ferroptosis induction promoter according to claim 3, wherein the v-ATPase activator is EN6, Schisandrol A, or C381.

5. The ferroptosis induction promoter according to claim 4, wherein the v-ATPase activator is EN6.

6. The ferroptosis induction promoter according to claim 2, wherein the v-ATPase subunit is ATP6V1A.

7. The ferroptosis induction promoter according to claim 2, wherein the STING agonist is C53.

8. The ferroptosis induction promoter according to claim 2, wherein the cAMP-related compound is cAMP or NKH-477; the TRPML1 agonist is SF-22; the TPC2 antagonist is tetrandrine; the TFEB activator is curcumin analog C1 or PF-11; the mTOR inhibitor is OSI-027 or PP242; the lysosome-targeted nanoparticles are poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), photo-activated nanoparticles (paNPs), acidic nucleolipid nanoemulsions (NL-NEs), or acidic nanoparticles (AcNPs); and / or the ATM inhibitor is KU-60019. Ferroptosis induction promoter.

9. A pharmaceutical composition for treating or preventing an aging-related disease, comprising the ferroptosis induction promoter according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, further comprising a ferroptosis-inducing compound.

11. The pharmaceutical composition according to claim 9, which is used in combination with a ferroptosis-inducing compound.

12. The pharmaceutical composition according to claim 10 or 11, wherein the ferroptosis-inducing compound is selected from the group consisting of elastin, piperazine elastin, imidazole ketone elastin (IKE), sulfasalazine, sorafenib, glutamic acid, buthionine sulfoximine, artesunate, artemisinin derivatives, DPI2, cyst(e)inase, BAY87-2243, hemoglobin, and FeCl ２ , Hemin, (NH ４ ) ２ Fe(SO ４ ) ２ , non-thermal plasma, salinomycin, amino acid depletion and Cornell dot, lapatinib and siramesine, FIN56, FINO2, withaferin A, BAY11-7085, lanperisone, CIL41, CIL56, CIL69, CIL70, CIL75, CIL79, atorvastatin, simvastatin, cisplatin, neratinib, iFSP1, trigonelline, and brusatol.

13. The pharmaceutical composition according to claim 9, wherein the aging-related disease is a cardiac disease selected from cancer, age-related macular degeneration, cataract, glaucoma, presbyopia, chronic inflammation, chronic renal failure, interstitial pneumonia, atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary artery thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, hepatic fibrosis, cerebral aneurysm, autoimmune disease, neurodegenerative disease, and stroke.

14. The pharmaceutical composition according to claim 13, wherein the aging-related disease is cancer.

15. The pharmaceutical composition according to claim 14, further comprising an anti-cancer agent.

16. The pharmaceutical composition according to claim 14, which is used in combination with an anticancer agent.

17. The pharmaceutical composition according to claim 15 or 16, wherein the anticancer drug is gemcitabine, doxorubicin, liposomal doxorubicin, docetaxel, carboplatin, paclitaxel, nab-paclitaxel, ifosfamide, etoposide, methotrexate, cyclophosphamide, pertuzumab, trastuzumab, amrubicin, vinblastine, dacarbazine, bleomycin, vincristine, prednisolone, eribulin, or vinofenone. Relbine, actinomycin D, oxaliplatin, fluorouracil, irinotecan, anastrozole, exemestane, letrozole, palbociclib, abemaciclib, ribociclib, asparaginase, alectinib, cetuximab, pemetrexed, bevacizumab, irinotecan, gefitinib, epirubicin, oxaliplatin, nivolumab, capecitabine, cabazi Taxel, trastuzumab, emtansine, Keytruda, imatinib, crizotinib, ramucirumab, cytarabine, afatinib, regorafenib, sunitinib, capecitabine, lapatinib, dacarbazine, erlotinib, dasatinib, UFT, TS-1, temozolomide, docetaxel, vinorelbine, nimustine, sorafinib, nedaplatin, nogitecan, Herceptin, bilirubin A pharmaceutical composition selected from the group consisting of norelbine, veltuzumab, panitumumab, mitomycin, methotrexate, melphalan, rituxan, levofolinate, lenvatinib, trifluridine-tipiracil, abiraterone, ethinyl, enzalutamide, chlormazine, goserelin, degarelix, tamoxifen, toremifene, bicalutamide, flutamide, and leuprorelin.

18. The pharmaceutical composition according to claim 14, wherein the cancer is selected from the group consisting of pancreatic cancer, liver cancer, breast cancer, and lung cancer.

19. The pharmaceutical composition according to claim 14, wherein the cancer is pancreatic cancer.

20. The pharmaceutical composition according to claim 15 or 16, wherein the cancer is pancreatic cancer and the anticancer agent is gemcitabine.

21. The pharmaceutical composition according to claim 14, characterized in that it is used in combination with radiation therapy.

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