Therapeutic compositions and methods

Abstract

The invention provides a pharmaceutical composition comprising a compound of formula (I): wherein A is a (C15-C23)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds and R1 is H or (C1-C6)alkyl, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient as well as methods for using the compounds of formula (I) and salts thereof to provide senolytic effects.

Description

[0001] THERAPEUTIC COMPOSITIONS AND METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 707,064 that was filed on October 14, 2024. The entire content of the applications referenced above is hereby incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under AG056278, AG063543, AG043376, and AG062413 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Aging is often accompanied by declines in health and physiological functions, resulting in an increase in the prevalence of numerous chronic diseases. One of the critical hallmarks or underlying mechanisms of aging is cellular senescence (Lopez-Otin, C., et al., Cell 153, 1194- 1217 (2013); Kennedy, B.K., et al. Cell 159, 709-713 (2014); and Lopez-Otin, et al., Cell 186, 243-278 (2023)). Senescence is a phenomenon in which cells become incapable of proliferating under various cellular stress conditions, leading to a state of stable cell cycle arrest (Hayflick, L. & Moorhead, P.S. Exp Cell Res 25, 585-621 (1961)). With age, senescent cells accumulate in many tissues and produce a range of deleterious signals that can negatively affect neighboring cells. These signals, known as senescence-associated secretory phenotype (SASP), consist of diverse pro-inflammatory cytokines, chemokines, and proteases (Coppe, J.P., et al., Annu Rev Pathol 5, 99-118 (2010); and Di Micco, R., et al., Genes Dev 34, 1565-1576 (2020)). The SASP released by senescent cells can result in tissue dysfunction and deterioration of tissue and even turn more healthy cells senescent, thereby further driving aging and promoting many age-related diseases and pathologies (Birch, J. & Gil, J. Genes Dev 34, 1565-1576 (2020); Gorgoulis, V., et al. Cell 179, 813-827 (2019); and Wiley, C.D. & Campisi, J. Nat Metab 3, 1290-1301 (2021)). The use of transgenic mouse models, where senescent cells can be genetically ablated, has established a key role of senescent cells in driving aging and age-related diseases (Baker, D.J., et al. Nature 479, 232-236 (2011); and Baker, D.J., et al. Nature 530, 184-189 (2016)).

[0008] Pharmacologically eliminating senescent cell burden using a class of drugs known as senolytics has been shown to be an effective strategy to extend healthspan and treat age-related diseases (Robbins, P.D., et al. Annu Rev Pharmacol Toxicol 61, 779-803 (2021); Prasnikar, E., Borisek, J. & Perdih, Ageing Res Rev 66, 101251 (2020); and Kirkland, J.L. & Tchkonia, T. J Intern Med 288, 518-536 (2020)).

[0009] Senolytics have emerged as an effective therapeutic approach to eliminate senescent cells and improve aging phenotypes and co-morbidities. Despite their promising potential, only a limited number of senolytics have been discovered. Fisetin has been demonstrated to reduce senescence, suppress age-related pathology, and extend healthspan in aged mice (Yousefzadeh, M.J., et al. EBioMedicine 36, 18-28 (2018)). However, its moderate potency and poor bioavailability have hindered its further clinical applications.

[0010] Currently there is a need for compounds that specifically induce ferroptosis in senescent cells. Such compounds would be useful for reducing the senescent cell burden that increases with aging and diseases, extending healthspan or lifespan, and / or treating age-related diseases.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect, senolytic compounds that induce ferroptosis in senescent cells are provided.

[0013] A pharmaceutical composition comprising a compound of formula (I): or a salt thereof, wherein:

[0014] A is a (Ci5-Ci9)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds and R1is H or (Ci-Ce)alkyl, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient is provided.

[0015] A pharmaceutical composition comprising a compound of formula (la): or a salt thereof, wherein:

[0016] R1is H or (Ci-Ce)alkyl or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient is provided.

[0017] In one embodiment, the pharmaceutical composition comprises a senolytic amount of the compound or the pharmaceutically acceptable salt. In one embodiment, the pharmaceutical composition is formulated as a unit dosage form.

[0018] A method for reducing the expression of one or more senescence markers in a cell comprising, contacting the cell with a compound of formula (I) or a salt thereof is also provided. The method can be carried out in vitro or in an animal.

[0019] A method for reducing senescence in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal is also provided.

[0020] A method for extending the healthspan or lifespan of an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal is also provided.

[0021] A method for treating age-related disease in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal is also provided.

[0022] A method for increasing senotherapeutic activity in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal is also provided.

[0023] A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medical therapy is also provided.

[0024] A compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing senescence, extending healthspan, treating an age-related disease, or increasing senotherapeutic activity is also provided.

[0025] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for reducing senescence, extending healthspan, treating an age-related disease, or increasing senotherapeutic activity is also provided.

[0026] A method for treating a disease or condition in an animal selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, tissue regeneration, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided. One embodiment provides a method of treatment as described herein, comprising the administration of a compound of formula (I) with one or more additional bioactive agents or pharmaceutical agents (e.g., combination therapy) such as a vaccine, an antiviral agent, an antibacterial agent, or an anticancer agent (e.g., a chemotherapeutic).

[0027] A method for treating a disease or condition in an animal selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, tissue regeneration, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0028] A method for improving response to chemotherapy in an animal, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0029] A method for improving response to radiation therapy in an animal, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0030] A method for improving recovery of a cancer survivor, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the cancer survivor is also provided.

[0031] A method for improving cognition in an animal, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0032] A method for reducing the negative effects of tobacco use (past or present) in an animal, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0033] A method for reducing one or more symptoms associated with long-haul COVID-19 in an animal, comprising administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0034] A method comprising contacting an organ (e.g., an organ from an older doner) with a compound of formula (I) a pharmaceutically acceptable salt thereof to improve the characteristics of the organ for transplantation is also provided.

[0035] Aa method for improving bone healing in an animal comprising, administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0036] A method for repairing damaged muscle in an animal comprising, administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0037] A method for improving tissue homeostasis comprising, contacting tissue with a compound of formula (I) a pharmaceutically acceptable salt thereof is also provided.

[0038] A method for improving immune response to a vaccine in an animal comprising, administering a compound of formula (I) a pharmaceutically acceptable salt thereof to the animal is also provided.

[0039] A compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a disease or condition selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, tissue regeneration, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection is also provided.

[0040] A compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a disease or condition selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, tissue regeneration, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection is also provided.

[0041] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving response to chemotherapy is also provided.

[0042] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving response to radiation therapy is also provided.

[0043] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving recovery of a cancer survivor is also provided.

[0044] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving cognition is also provided.

[0045] A compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing the negative effects of tobacco use (past or present) is also provided.

[0046] A compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing one or more symptoms associated with long-haul COVID-19 is also provided.

[0047] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving the characteristics of an organ for transplantation is also provided. A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving bone healing is also provided.

[0048] A compound of formula (I) or a pharmaceutically acceptable salt thereof for repairing damaged muscle is also provided.

[0049] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving tissue homeostasis is also provided.

[0050] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving immune response to a vaccine is also provided.

[0051] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating a disease or condition selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, tissue regeneration, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection in an animal is also provided.

[0052] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating a disease or condition selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, tissue regeneration, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection in an animal is also provided.

[0053] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving response to chemotherapy in an animal is also provided.

[0054] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving response to radiation therapy in an animal is also provided.

[0055] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving recovery of a cancer survivor is also provided.

[0056] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving cognition in an animal is also provided.

[0057] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for reducing the negative effects of tobacco use (past or present) in an animal is also provided.

[0058] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for reducing one or more symptoms associated with long-haul COVID-19 in an animal is also provided.

[0059] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving the characteristics of an organ for transplantation is also provided.

[0060] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving bone healing in an animal is also provided.

[0061] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for repairing damaged muscle in an animal is also provided.

[0062] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving tissue homeostasis in an animal is also provided.

[0063] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving immune response to a vaccine in an animal is also provided.

[0064] A method for improving the anti-tumor response in a cancer patient following treatment with radiation or chemotherapy by reducing the treatment-induced senescent tumor cells in the patient, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt to the patient is provided.

[0065] A method for treating ferroptosis-sensitive cancers in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the animal is provided.

[0066] A method for slowing the accelerated aging observed in a cancer survivor, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the cancer survivor is provided.

[0067] A method for slowing the accelerated aging in an HIV patient, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to the HIV patient is provided.

[0068] A compound of formula (I) or a pharmaceutically acceptable salt thereof for improving the anti-tumor response in a cancer patient following treatment with radiation or chemotherapy by reducing the treatment-induced senescent tumor cells in the patient is provided.

[0069] A compound of formula (I) or a pharmaceutically acceptable salt thereof for treating ferroptosis-sensitive cancers is provided.

[0070] A compound of formula (I) or a pharmaceutically acceptable salt thereof for slowing the accelerated aging observed in a cancer survivor is provided.

[0071] A compound of formula (I) or a pharmaceutically acceptable salt thereof for slowing the accelerated aging in an HIV patient is provided.

[0072] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for improving the anti-tumor response in a cancer patient following treatment with radiation or chemotherapy by reducing the treatment-induced senescent tumor cells in the patient is provided.

[0073] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating ferroptosis-sensitive cancers is provided.

[0074] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for slowing the accelerated aging observed in a cancer survivor is provided.

[0075] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for slowing the accelerated aging in an HIV patient is provided A kit comprising 1) a compound of formula (I) or a pharmaceutically acceptable salt thereof, 2) packaging material, and 3) instructions for administering the compound of formula (I) or the pharmaceutically acceptable salt thereof to an animal is also provided. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for reducing senescence, extending healthspan, treating an age-related disease, or increasing senotherapeutic. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for treating a disease or condition in an animal selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, tissue regeneration, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for improving response to chemotherapy. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for improving response to radiation therapy. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for improving recovery of a cancer survivor. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for improving cognition. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for reducing the negative effects of tobacco use (past or present). In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for reducing one or more symptoms associated with long-haul COVID-19. In one embodiment, the instructions provide for contacting an organ (e.g., an organ from an older doner) with the compound of formula (I) or the pharmaceutically acceptable salt thereof to improve the characteristics of the organ. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for improving bone healing. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for repairing damaged muscle. In one embodiment, the instructions provide for contacting tissue with the compound of formula (I) or the pharmaceutically acceptable salt thereof to improve tissue homeostasis. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for improving immune response to a vaccine. In one embodiment, the instructions provide for the administration of the compound of formula (I) or the pharmaceutically acceptable salt thereof to the animal for treating cancer. In one embodiment, the compound is present in a unit dosage form.

[0076] BRIEF DESCRIPTION OF DRAWINGS

[0077] Figs. 1A-1D. (Fig. 1 A) Schematic diagram of the senescent cell-based phenotypic drug screening platform for identifying novel senotherapeutics. Created in BioRender.com. (Fig. IB) Chemical structures of a-ESA and a-ESA-me. (Fig. 1C) Dose-response curves of senolytic activity of a-ESA and a-ESA-me in non-senescent proliferating WT MEF cells and senescent Erccl~ ~ MEFs. Error bars indicate SD for n = 3. (Fig. ID) Representative images of the CnFDG-based SA-P-gal senescence assay. Senescent cells were treated with a-ESA and a-ESA- me for 48 hours before imaging by Cytation 1 at 4X. Blue fluorescence indicates nucleus stained with Hoechst 33324, and green fluorescence indicates SA-P-gal positive senescent cells stained with C12FDG.

[0078] Figs. 2A-2K. Evaluation of the senolytic effects of a-ESA and a-ESA-me in models of accelerated aging and naturally aged mice. (Fig. 2A) WT C57BL / 6 mice (20-22 months old) were treated with 50 mg / kg of a-ESA or a-ESA-me by oral gavage for five consecutive days. Tissues were collected two days after the last dose for analysis. (Fig. 2B) Effects of a-ESA and a-ESA-me on reducing senescence across different tissues in aged WT C57BL / 6 mice. (Fig. 2C) WT C57BL / 6 mice (32 months old) were treated with 50 mg / kg of a-ESA-me by oral gavage for five consecutive days. Tissues were collected two days after the last dose for analysis. (Fig. 2D) Reduction of senescence markers by a-ESA-me in multiple tissues of 32-month-old WT mice, particularly in (Fig. 2E) the kidney. Error bars represent SEM for n = 6. (Fig. 2F) a-ESA-me reduced p21+ senescent 76 T cells in the spleen of the 32-month-old WT C57BL mice. Error bars represent SEM for n = 6. (Fig. 2G) ErccE ' progeria mice (17-20 weeks old) were treated with 25 mg / kg of a-ESA-me for three consecutive weeks by oral gavage. Mice were sacrificed two days after the last dose. (Fig. 2H) Acute a-ESA-me treatment reduced senescence in multiple tissues of Erccl~Iprogeria mice. (Fig. 21) ErccT ' progeria mice (10 weeks old) were treated with 50 mg / kg of a-ESA-me for six consecutive weeks by oral gavage. Weekly health assessments were conducted to score age-related symptoms, including tremor, kyphosis, dystonia, ataxia, gait disorder, hindlimb paralysis, and forelimb grip strength. (Fig. 2J) Chronic a-ESA-me treatment significantly improved composite health scores in ErccE1progeria mice. (Fig. 2K) Chronic a-ESA-me treatment reduced tissue senescence and SASP factors in ErccE1progeria mice. Error bars represent SEM for n = 6 (vehicle) and n = 4 (a-ESA-me).

[0079] Figs. 3A-3G. a-ESAs induced senolysis through ferroptosis. (Figs. 3A-3C) Senescent MEF cells were treated with a-ESA-me for 48 hours, with or without 1-hour pretreatment with the following compounds: sesamin (50 pM), SC26196 (200 nM), Q-VD-OPH (20 pM), Nec-ls (50 pM), Fer-1 (2 pM), deferoxamine (50 pM), or liproxstatin-1 (2 pM). Error bars represent SD for n = 3. (Fig. 3D) Cells were treated with a-ESA-me (5 pg / mL) for 6 hours, with or without 1- hour pretreatment with Fer-1 (2 pM). Ferrous iron and ROS levels were detected by FerroOrange and H2DCFDA, respectively. (Fig. 3E) Quantification of the ferrous iron and ROS levels. Error bars represent SD for n = 2. (Fig. 3F) Lipid peroxidation was detected by Cl 1 BODIPY after treatment with a-ESA-me (5 pg / mL) for 6 hours, with or without Fer-1 (2 pM). (Fig. 3G) Quantification of the lipid peroxidation. Error bars represent SD for n = 2.

[0080] Figs. 4A-4D. GSEA analysis of the transcriptome of a-ESA and a-ESA-me treated senescent cells. Senescent WT MEF cells induced by etoposide were treated with a-ESA (2 pg / mL) and a-ESA-me (2 pg / mL) for 24 hours, with or without 1-h pretreatment of Fer-1 (2 pM) prior to RNAseq analysis. Dot plots of enriched pathways determined from GSEA results illustrating hallmarks biological processes associated with (Fig. 4A) a-ESA treatment, (Fig. 4B) a-ESA treatment plus ferroptosis inhibitor Fer-1, (Fig. 4C) a-ESA-me treatment, and (Fig. D) a- ESA-me plus ferroptosis inhibitor Fer-1. The gene ratio is defined as the ratio of the count of core enrichment genes to the count of pathway genes, n = 3.

[0081] Figs. 5A-5B. Lipidomics analysis of cells treated with a-ESAs Non-senescent and etoposide-induced senescent MEF cells were treated with a-ESA (2 pg / mL) or a-ESA-me (2 pg / mL) for 8 hours, with or without 1-hour pretreatment of Fer-1 (2 pM) prior to lipidomic analysis. (Fig. 5 A) Changes in specific lipid species. Error bars represent SD for n = 4. (Fig. 5B) Changes in cholesterol esters containing nl8:3. Error bars represent SD for n = 4.

[0082] Figs. 6A-6D. Flexible docking results of ferroptosis associated target proteins with the a- ESAs and control compounds Native substrate or ligand and known inhibitor or activator drug for each protein target was used along with a randomly generated structural decoy / dud molecule. (Fig. 6 A) Binding free energy scores of best fitting conformers for each ligand within the selected receptor binding pockets, lower binding free energies correspond to stronger ligandreceptor interactions. Substrate binding pockets for all receptors were predefined except ALOX15 that used the interdomain cleft site for allosteric activation. (Fig. 6B) 3D molecular orientation of best fitting conformers of all simulated ligands in complex within the ALOX15 monomer allosteric activation pocket (color identifiers correspond to panel a) and 3D and 2D interaction plots of ligands in the activation pocket with neighboring pocket amino acid residues; ligand-residue interaction ligands are provided under the plots. (Fig. 6C) Protein backbone and hydrophobicity surface projections of ALOX15 monomer with the substrate binding (pink) pocket occupied by native crystalized arachidonic acid (ARA), and allosteric activation (red) pocket occupied by docked a-ESAs. (Fig. 6D) Binding free energy scores of best fitting conformers of ARA in the ALOX15 substrate binding pocket with or without (apo) lipid candidates or drugs docked into the allosteric activation.

[0083] Fig. 7. Proposed mechanisms of a-ESA and a-ESA-me inducing ferroptotic cell death in senescent cells. Senescent cells exhibit elevated levels of cellular ferrous iron and lipoxygenase expression. Both arms of the cascade lead to PUFA oxidation and generation of lipid-ROS through Fenton oxidation and iron-dependent enzymatic catalysis. a-ESA and a-ESA-me treatments exacerbate lipid ROS stress through direct oxidation and / or allosteric activation of ALOX15 activity, promoting the propagation of lipid peroxides. Fer-1 is an anti -ferroptotic agent that directly inhibits ALOX15 and suppresses a-ESA / a-ESA-me mediated lipid ROS generation. Interactions of reactive lipid species with ACSL and LPCAT enzymes lead to esterification, phospholipid (PL) membrane incorporation and destabilization. These processes sensitize senescent cells to membrane damage, ultimately resulting in ferroptotic cell death.

[0084] Created in BioRender.com.

[0085] Figs. 8A-8B. Conjugated PUFA senolytics (a-ESA / a-ESA-me) induce ferroptosis of senescent cells. (Fig. 8A) Structures and relative propagation rate constants of representative conjugated and unconjugated polyunsaturated fatty acids (PUFAs). Conjugated PUFAs such as a-ESA exhibit accelerated peroxidation kinetics compared to unconjugated PUFAs like a- linolenic acid (ALA) (Adapted from J Org Chem 2021, 86, 153). (Fig. 8B) Senolytic activity of different conjugated and unconjugated PUFAs in non-senescent and senescent IMR90 cells. Abbreviations: SnCs: C12FDG-positive senescent cells; TCs: Total Erccl- / - MEF cells; NSnCs: Non-senescent WT MEF cells; SI: Selectivity index. ECso: Concentration resulting in a 50% reduction in cell number compared to control. NA: ECso not available due to inactivity. Pharmacological inhibition of lipid peroxidation and ferroptosis regulators

[0086] Figs. 9A-9E. a-ESA / a-ESA-me induce ferroptosis of senescent cells by increasing ALOX15 activity. Pharmacological inhibition of lipid peroxidation and ferroptosis regulators (Fig. 9A) ACSL4, (Fig. 9B) LPCAT3, and (Fig. 9C) ALOX15 rescued a-ESA-induced cell death in etoposide- induced senescent IMR90 cells. (Fig. 9D) ALOX15 enzymatic assay demonstrated a-ESA directly activates ALOX15 in a dose-dependent manner. Data represent mean ± SD (n=3). (Fig. 9G) Unified model of a-ESA-mediated ferroptosis in senescent cells. In iron / ROS- rich senescent cells, conjugated PUFAs (a-ESA / a-ESA-me) amplify lipid peroxidation via two cooperating routes: (i) a non-enzymatic pathway driven by Fe2+-dependent Fenton chemistry and radical propagation, and (ii) an enzymatic pathway in which ACSL4 converts a-ESA to acyl-CoA, LPCAT3 incorporates it into membrane phospholipids, and ALOX15 peroxidizes these substrates. The resulting accumulation of PUFA-OOH phospholipids exceeds GPX4 detoxification capacity, compromises membrane integrity, and triggers ferroptotic senolysis.

[0087] Fig. 10. Structures of 9E,11E-CLA, Jacaric acid, and cis Parinaric acid.

[0088] DETAILED DESCRIPTION OF THE INVENTION

[0089] The term "alkyl" means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., (Ci-Cejalkyl means one to six carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n- butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, and n-hexyl. In one embodiment alkyl can comprise one or more double bonds (e.g., an alkenyl).

[0090] The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. For example, a (Cis-C23)alkenyl means 15-23 carbons and a (Ci7)alkenyl means 17 carbons. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl) and the higher homologs and isomers. Examples of such unsaturated alkyl groups also include (5E,7E,9Z)-heptadeca-5,7,9- triene, (7E,9E)-heptadeca-7,9-diene, (6Z,8E,10Z)-heptadeca-6,8,10-triene, and (3Z,5E,7E,9Z)- heptadeca-3 , 5 ,7, 9-tetraene .

[0091] The term “conjugated double bonds” refers to two or more double bonds separated by a single bond.

[0092] As used herein, the term "protecting group" refers to a substituent that is commonly employed to block or protect a particular functional group on a compound. For example, an "amino-protecting group" is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9- fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a "hydroxy-protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl and silyl. A "carboxy-protecting group" refers to a substituent of the carboxy group that blocks or protects the carboxy functionality. Common carboxy- protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2- (trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2- (diphenylphosphino)-ethyl, nitroethyl and the like. For a general description of protecting groups and their use, see P.G.M. Wuts and T.W. Greene, Greene's Protective Groups in Organic Synthesis 4thedition, Wiley-Interscience, New York, 2006.

[0093] As used herein a wavy line “ ” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.

[0094] The terms “treat”, “treatment”, or “treating” to the extent it relates to a disease or condition includes inhibiting the disease or condition, eliminating the disease or condition, and / or relieving one or more symptoms of the disease or condition. The terms “treat”, “treatment”, or “treating” also refer to both therapeutic treatment and / or prophylactic treatment or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as, for example, the development or spread of cancer. For example, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease or disorder, stabilized (i.e., not worsening) state of disease or disorder, delay or slowing of disease progression, amelioration or palliation of the disease state or disorder, and remission (whether partial or total), whether detectable or undetectable. “Treat”, “treatment”, or “treating,” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in which the disease or disorder is to be prevented. In one embodiment “treat”, “treatment”, or “treating” does not include preventing or prevention. In one embodiment the terms “improving, reducing, extending, increasing, slowing and repairing” refer to “improving, reducing, extending, increasing, slowing and repairing” with treatment as described herein compared to without treatment.

[0095] The phrase "therapeutically effective amount" or “effective amount” includes but is not limited to an amount of a compound of the that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0096] The term “animal” as used herein includes mammals.

[0097] The term “mammal” as used herein refers to humans, higher non-human primates, rodents, domestic, cows, horses, pigs, sheep, dogs and cats. In one embodiment, the mammal is a human. The term “patient” as used herein refers to any animal including mammals. In one embodiment, the patient is a mammalian patient. In one embodiment, the patient is a human patient.

[0098] The term “unit dosage form” includes pharmaceutical products that are in a form suitable for marketing and use. For example, a unit dosage form may comprise a specific mixture of an active ingredient and an excipient in a specific configuration that is suitable for packaging, sale, and administration. The unit dosage form may also comprise a specific dosage of the active ingredient. It is understood by one skilled in the art that this invention also includes any compound claimed that may be enriched at any or all atoms above naturally occurring isotopic ratios with one or more isotopes such as, but not limited to, deuterium (2H or D). As a non-limiting example, a -CH3 group may be substituted with -CD3.

[0099] The pharmaceutical compositions of the invention can comprise one or more excipients. When used in combination with the pharmaceutical compositions of the invention the term “excipients” refers generally to an additional ingredient that is combined with the compound of formula (I) or the pharmaceutically acceptable salt thereof to provide a corresponding composition. For example, when used in combination with the pharmaceutical compositions of the invention the term “excipients” includes, but is not limited to: carriers, binders, disintegrating agents, lubricants, sweetening agents, flavoring agents, coatings, preservatives, and dyes.

[0100] In cases where compounds are sufficiently basic or acidic, a salt of a compound of formula (I) can be useful as an intermediate for isolating or purifying a compound of formula (I). Additionally, administration of a compound of formula (I) as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a- ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

[0101] Salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0102] The compounds of formula (I) can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

[0103] Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0104] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0105] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0106] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0107] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0108] Useful dosages of the compounds of formula (I) can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0109] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0110] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0111] In one embodiment A is a (Cis-Ci8)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds.

[0112] In one embodiment A is a (Ci6-Ci7)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds.

[0113] In one embodiment A is a (Ci7)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds.

[0114] In one embodiment A the two or more double bonds are two or more conjugated double bonds.

[0115] In one embodiment A comprises three or more double bonds.

[0116] In one embodiment the three or more double bonds are three or more conjugated double bonds.

[0117] In one embodiment A is:

[0118] In one embodiment the compound of formula (I) is a compound of formula (la): or a pharmaceutically acceptable salt thereof, wherein R1is H or (Ci-Cejalkyl.

[0119] In one embodiment R1is H. In one embodiment R1is methyl, ethyl, propyl, or isopropyl.

[0120] In one embodiment A R1is methyl.

[0121] One embodiment provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

[0122] One embodiment provides a compound selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

[0123] The invention will now be illustrated by the following non-limiting Examples.

[0124] Examples

[0125] 9Z,l lE,13E-octadecatrienoic acid (CAS 506-23-0) is commercially available (e.g., from

[0126] Santa Cruz Biotechnology, Inc. (California, USA) or from Cayman Chemical (Michigan, USA)). The acid can be converted to esters of formula (I) using standard techniques and reagents that are well known. 9Z,1 lE,13E-octadecatrienoic acid methyl ester (CAS 4175-47-7) is also commercially available (e.g., from Santa Cruz Biotechnology, Inc. (California, USA) or from Cayman Chemical (Michigan, USA)).

[0127] Example 1. Biological Evaluations

[0128] Phenotypic screening of fatty acids

[0129] To identify novel senotherapeutics, a robust drug screening platform was developed based on the phenotype of senescence-associated beta-galactosidase (SA-P-gal) activity (Zhang, L., et al., J Clin lnvest 132, el58450 (2022); and Gorgoulis, V., et al., Cell 179, 813-827 (2019)). This platform allows for the induction of senescence in various cell types using different inducers. Both senescent and non-senescent control cells were seeded into separate 96- well plates and treated uniformly with the compounds under investigation. After treatment, cellular senescence was assessed using the fluorogenic dye C12FDG (Zhang, Y.Z., et al., Faseb j 5, 3108-3113 (1991)). This lipophilic dye produces a strong fluorescent signal upon cleavage by P-galactosidase and is widely used to detect SA-P-gal activity in senescent cells at pH 6 (Dimri, G.P., et al., Proc Natl Acad Set U S A 92, 9363-9367 (1995)). The platform is equipped with a high-content fluorescent imaging system, enabling rapid evaluation of two categories of senotherapeutics: senolytics, which selectively kill senescent cells and reduce the number of CnFDG-positive cells, and senomorphics, which reduce the senescence phenotype without inducing cell death (Fig. 1 A).

[0130] Using this screening platform, a focused library screen of various fatty acids was conducted given their health benefits and dietary functions (Kremmyda, L.S., et al., Biomed Pap MedFac Univ Palacky Olomouc Czech Repub 155, 195-218 (2011)). This library included medium-chain fatty acids with aliphatic tails of 6 to 12 carbons, long-chain fatty acids with tails of 13 to 21 carbons, and very long-chain fatty acids with tails of 22 or more carbons.

[0131] Primary F.rccl~ ~ mouse embryonic fibroblasts (MEFs) were used as the initial senescent cell model which was induced by oxidative stress (Niedernhofer, L.J., et al., Nature 444, 1038 (2006); and Fuhrmann-Stroissnigg, H., et al., Nat Commun 8, 422 (2017)). Wild-type (WT) primary MEF cells were used as the non-senescent, proliferative control. Both cell types were treated with compounds for 48 hours, followed by fixation and staining with C12FDG to detect SA-P-gal activity. Each compound was tested at various concentrations to determine the EC50 values, the effective concentration needed to achieve 50% reduction in cell number compared to the control (Table 1). Senolytic or senom orphic effects were assessed by comparing the ECso values of CnFDG-positive cells and the total number of senescent cells. The selectivity index (SI) was calculated based on the ratio of ECso values between non-senescent cells (NSnCs) and senescent cells (SnCs).

[0132] Table 1. Senescence screening of fatty acids in senescent Erccl1' MEFs.

[0133] NA: EC50 values are not available due to inactivity.

[0134] Structure-activity relationship (SAR) analysis

[0135] As summarized in Table 1, the senolytic potency and selectivity of the fatty acids are significantly influenced by their chemical structures and specific modifications.

[0136] Generally, medium-chain fatty acids and saturated fatty acids show no senolytic potential as exemplified by octanoic acid, decanoic acid, and pentadecanoic acid (cmpds 1-3). Fatty acids with longer chains such as those in the n20 and n22 series, including arachidonic acid (compound 32), EP A (compound 36), HP A (compound 38), DPA (compound 40), and DHA (compound 42), have important dietary functions (Kapoor, B., et al., Curr Nutr Rep 10, 232-242 (2021); and Zhang, Y., et al. The American Journal of Clinical Nutrition 103, 330-340 (2016)), however, they exhibit little to no senolytic activity. This suggests that increasing chain length alone does not necessarily enhance senolytic activity. Notably, many long-chain fatty acids with tails of 18 carbons show better senolytic activities than those with other chain lengths. For example, 9Z,1 IE-conjugated linoleic acid (CLA, compound 4) with two conjugated double bonds, punicic acid (compound 18), and a-eleostearic acid (ESA, compound 20) with three conjugated double bonds demonstrate exceptional senolytic activity.

[0137] Besides the carbon length, the double bond position and configuration of fatty acids also play critical roles in the senolytic activity. For instance, the CLAs can exist as either cis or trans configuration, and screening found that the 9E,11E-CLA isomer (compound 8) is highly potent and selective senolytic, whereas the 9Z,11E-CLA (compound 4), 9Z,11Z-CLA (compound 6), and 10E,12Z-CLA (compound 10) isomers are inactive. The configuration effect was also observed in ESA series where a-ESA (compound 20) with 9Z,1 lE,13E-conjugated isomer is more potent than its all-trans counterpart P-ESA (9E,11E,13E, compound 21).

[0138] Interestingly, esterification appears to be a key modification that can alter the senolytic activity of the fatty acids. For example, while unmodified DHA (compound 42) showed no significant activity, its methyl and ethyl esters (compounds 43 and 44) exhibit some senolytic properties. This trend was observed across several other fatty acids whose esterification improved selectivity, albeit sometimes at the cost of potency. For instance, 9E,1 lE-CLA-Me (compound 9) is less potent but 10-fold more selective than 9E,11E-CLA (compound 8). Another exceptional example is a-ESA methyl ester (ESAme, compound 22) which exhibited a striking selectivity index (SI) of 470, though less potent, compared to 1.94 for its non-esterified form a-ESA (compound 20).

[0139] Another major difference between these active and inactive unsaturated fatty acids is conjugation. Unconjugated fatty acids, though contain multiple double bonds, usually do not confer senolytic properties. For example, in the nl8 series of octadecanoids, all the inactive unsaturated fatty acids including alpha-linolenic acid (ALA, compound 12), gamma-linolenic acid (GLA, comp 13), pinolenic acid (compound 15), and stearidonic acid (compound 24) are methylene-interrupted polyenes. Same is true for the n20 series (e.g., mead acid or compound 26, DALA or compound 28, DGLA or compound 30, arachidonic acid or compound 32, EPA or compound 36), n21 series (e.g., HPA or compound 38), or the n22 VLCFAs (e.g., DPA or compound 40, DHA or compound 42), irrespective of whether they are characteristic of omega- 3 and omega-6 fatty acids.

[0140] Collectively, these SAR findings underscore the importance of molecular structures of fatty acids in determining the senolytic potency and selectivity. From the screening, the most selective senolytic, a-ESA methyl ester (compound 22, ECso = 3.228 pM, SI = 1.94), and the most potent senolytic, a-ESA (compound 20, ECso = 3.953 pM, SI = 470), were selected for further investigation (Fig. IB, C).

[0141] ESAs are novel lipid senolytics with broad spectrum of senolytic activity

[0142] Several senescent cell models were used to further confirm the senolytic activity of ESA and its derivative ESAme, These include WT MEF cells induced to senescence using oxidative stress (H2O2) and genotoxic stress (etoposide), human IMR90 fibroblast cells induced to senescence by genotoxic stress (etoposide), and human umbilical vein endothelial cells (HUVEC) induced by replicative passaging. Results showed that both ESA and ESAme demonstrate significant senolytic activity able to effectively reduce the CnFDG-positive senescent cells across a broad spectrum of senescent cell models (Fig. ID and Table 2). In all these senescent cell models, the senolytic EC50 values for ESA and ESAme are in the single micromolar range (Table 2).

[0143] Although ESA exhibits greater potency, ESAme shows higher selectivity indices, particularly in ErccT1' MEFs where the selectivity index (SI) reaches 470. Interestingly, in IMR90 cells, both compounds display comparable selectivity (Table 2), suggesting some cell type-specific variations in their senolytic effects.

[0144] Table 2. ESA and ESAme are broad spectrum of lipid senolytics.

[0145] To further dissect the senolytic actions of ESA and ESAme, their effects at various time points ranging from 12 to 48 hours were examined. The senolytic effect of ESA increases over time, with its ECso value decreasing from 12 to 36 hours (5.568 to 1.330 pM), indicating rapid action. However, its senolytic ECso value increases from 36 to 48 hours (1.330 to 2.195 pM). The ECso value of ESAme also decreased over time, being less potent than ESA but more sustained across all time points. These results suggest that ESA acts faster whereas ESAme provides a longer lasting senolytic effect.

[0146] Senolytic potential of the ESAs in vivo

[0147] Acute treatment of ESAs reduced tissue senescence in aged mice. To evaluate the senolytic potential of the lipid senolytics a-ESA and a-ESA-me in vivo, these compounds were in 20-22 months old WT C57BL / 6 mice. The mice were treated with 50 mg / kg of either a-ESA or a- ESA-me for five consecutive days. Two days after the last administration, the mice were sacrificed for tissue analysis (Fig. 2A). Molecular analysis revealed that a-ESA-me had a superior effect in reducing tissue senescence compared to a-ESA, particularly in the liver and heart, while a-ESA also demonstrated efficacy in reducing senescence in the brain (Fig. 2B). Further studies on 32-month-old C57BL / 6 mice treated under the same regimen (Fig. 2C) showed that a-ESA-me significantly reduced senescence and SASP factors in multiple tissues, including genes encoding plO^4*1, p21Cipl, TNFa, IL-6, CXCL1, MCP1, and IL-ip (Fig. 2D). The reduction was particularly pronounced in the kidney (Fig. 2E), liver, and lung. Additionally, a-ESA-me treatment led to a marked decrease in the proportion of p21Cipl-positive gamma delta (76) T cells in the spleen, suggesting a possible senolytic effect on specific senescent immune cells in aged mice (Fig. 2F).

[0148] Chronic treatment of ESAme extends healthspan in accelerated aging mice. Given the stronger senolytic effect of a-ESA-me compared to a-ESA in naturally aged mice, a-ESA-me was selected for further evaluation on healthspan in ErccT ' progeria mice, a well-established model of accelerated senescence and aging. Initially, to validate the senolytic effect of a-ESA- me in this model, a short-term, acute treatment of ErccT ' mice with a-ESA-me was performed for three days (Fig. 2G). RT-qPCR analysis of different tissues revealed a notable decrease in senescence markers and SASP factors, particularly in the kidney, liver, and gastrocnemius muscles (Fig. 2H).

[0149] To assess the long-term effects of a-ESA-me on healthspan, ErccT ' mice were treated with a-ESA-me orally three times per week for six weeks, starting at 10 weeks of age (Fig. 21). Weekly health assessments were conducted to monitor age-related symptoms, including tremor, kyphosis, dystonia, ataxia, gait disorder, hindlimb paralysis, and forelimb grip strength. The results indicated that a-ESA-me effectively reduced the composite score of aging symptoms without negatively affecting body weight, consistent with a lack of toxicity (Fig. 2J). In particular, it improved tremor and kyphosis in treated mice. Although no significant reduction in overall aging symptoms was observed by week 16 (Fig. 2J), RT-qPCR analysis continued to show a noticeable decrease in senescence and SASP markers in multiple tissues, including the kidney, liver, spleen, and muscle (Fig. 2K). These results further confirm the senolytic activity of a-ESA-me.

[0150] ESAs induce senescent cells death via ferropotosis

[0151] Whether the metabolites of ESAs contribute to the senolysis was evaluated to elucidate the mechanism by which ESA and ESAme induce cell death in senescent cells. ESA was reported to be quickly converted to conjugated linoleic acid (T Suzuki, T., et al., J Nutr 134, 2634-2639 (2004)) and further metabolized to gamma linolenic acid and arachidonic acid via A6 and A5 desaturase, respectively (Fig. 3B). To determine whether these metabolic pathways contributed to the senolytic effects of ESAs, senescent cells were pretreated with inhibitors of A6 desaturase (SC-26196) and A5 desaturase (sesamin) prior to ESA treatment. The results showed that neither SC-26196 nor sesamin prevented the senolytic effects of ESAs (Fig. 3C). Similar results were observed in senescent cells either induced by oxidative or genotoxic stress. These indicate that the observed senescent cell death is not mediated through the metabolism of ESAs.

[0152] Most, if not all, existing senolytics worked through inducing apoptosis (Zhang, L., et al., Meeh Ageing Dev 200, 111587 (2021)). To investigate whether the senolytic effects of ESA and ESAme involved apoptosis, senescent cells were pretreated with Q-VD-OPH, an irreversible pan-caspase inhibitor. Surprisingly, inhibition of apoptosis did not protect senescent MEF cells from the cell death induced by ESA or ESAme (Fig. 3D). Similarly, treatment with the necrosis inhibitor Nec-ls failed to prevent the senescent cell death. However, pretreatment with the ferroptosis inhibitor fer-1 completely blocked the senolytic effects of ESA and ESAme (Fig. 3D). Further investigation of this mechanism with other regulators of ferroptosis pathways, such as iron chelator deferoxamine and lipid peroxidation inhibitor liproxstain-1, showed that blocking the ferroptosis-related pathways could prevent the senescent cell death from ESA and ESAme. These findings revealed that ferroptosis, rather than apoptosis or necrosis, is likely the primary mode of cell death induced by the novel lipid senolytic ESAs.

[0153] Ferroptosis is an iron-dependent programed cell death triggered by the accumulation of reactive oxygen species (ROS) and lipid peroxidation (Dixon, Scott J., et al, Cell 149, 1060- 1072 (2012)). Further, it was found that senescent cells exhibit higher levels of ferrous iron and ROS compared to non-senescent cells, as detected by FerroOrange and H2DCFDA staining, respectively (Fig. 3E). Interestingly, ESAme treatment did not significantly alter the levels of iron or ROS. However, the addition of ferroptosis inhibitor Fer-1 inhibited a certain level of ROS (likely lipid ROS) but not ferrous iron (Fig. 3E). Lipid peroxidation is another critical precondition for ferroptosis. Increased lipid peroxidation was observed in senescent cells compared to non-senescent cells (Fig. 3F, G). Interestingly, ESAme further exacerbated this lipid peroxidation whereas ferroptosis inhibitor Fer-1 blocked this effect. A similar ferroptosis- dependent effect was also observed for ESA treatment.

[0154] This also brings a possibility of ferroptosis as a novel mechanism of senescent cell death. To further validate this hypothesis, senescent cells were treated with ferroptosis inducers, such as GPX4 inhibitor RSL3 and glutamate-cystine antiporter Xc inhibitor erasin. Indeed, these treatments resulted in significant senolysis in different senescent cell models, regardless of cell type or senescence inducers. Collectively, these findings suggest that the senescent cell death upon ESA or ESAme treatment is indeed through ferroptosis, unlike the conventional apoptotic senescent cell death. These results also support a new role of ferroptosis in senescent cell death. Transcriptomic analyses

[0155] To validate the mechanistic cues in experimental systems, RNA-Seq analysis was performed on non-senescent and senescent WT MEF cells treated with the two lipid senolytics, both in the presence and absence of the ferroptosis inhibitor Fer-1. Gene set enrichment analysis (GSEA) revealed a significant upregulation of genes associated with senescence and SASP in senescent MEF cells compared to non-senescent control. Additionally, there was an enrichment of NF-KB signaling and other inflammatory response pathways in the senescent cells, confirming their senescent phenotype. Upon treatment with a-ESA for 24 hours, the activation of pathways related to reactive oxygen species, heme metabolism, and cholesterol homeostasis (Fig. 4A) was observed. Pretreatment with the ferroptosis inhibitor Fer-1 significantly altered the transcriptomic response to a-ESA, resulting in the suppression of these pathways (Fig. 4B). Similarly, a-ESA-me treatment led to the activation of heme and fatty acid metabolism pathways (Fig. 4C), which were also suppressed upon Fer-1 pretreatment (Fig. 4D). These findings suggest that both a-ESA and a-ESA-me effectively initiate a ferroptosis-associated transcriptional program and ferroptosis plays a key role in the selective elimination of senescent cells by these compounds. mRNA expression levels of predicted target protein nodes that were shared between a- ESA and a-ESA-me were examined, as well as key ferroptotic proteins from the docking study. Among the candidate targets, the most substantial changes were seen in TOP2A, FABP4, and SLC7A11 (downregulated in senescent cells), and ALOX5, ALOX15, and FABP3 (upregulated in senescent cells). The expression levels of key ferroptotic nodes was verified using RT-qPCR, where ALOX15 and ALOX5 had the strongest upregulation in senescent cells. There were also minimal changes in ALOX15 expression in a-ESA / a-ESA-me treated cells compared to untreated senescent cells. Conversely, the greatest transcript-level changes were observed for SLC7A11 and PTGS2 when cells were treated with a-ESA and a-ESA-me treatment, but not in non-senescent vs senescent comparisons. These effects are reversed with Fer-1 pretreatment, suggesting these changes are likely dependent on activated ferroptotic processes.

[0156] Lipidomic analysis

[0157] To investigate the effect of ESAs on the lipidome, an undirected lipidomic analysis was performed in cell samples treated with ESAs in the presence or absence of ferroptosis inhibitor fer-1. Cells were treated with ESAs for eight hours and collected for lipidomics before overt cell death. In total, 310 lipid species of 20 classes were identified, with the most abundant lipid species identified being phosphatidylethanolamine (PE), phosphatidylcholine (PC), and free cholesterol. ESAme treatment did not significantly alter the overall composition of these lipids. However, an increase in oxidized polyunsaturated fatty acids (PUFAs), 4-hydroxy nonenal, and related products (such as 4-hydroxy hexenal (HHE) and 4-hydroxy nonenal (HNE)) was observed. These products are byproducts of lipid peroxidation and are hallmarks of ferroptosis (Fig. 5A). Additionally, there was an increase in phosphatidylserine (PS) levels in the senescent cells treated with ESAme. Pretreatment with the ferroptosis inhibitor Fer-1 mitigated the increase in 4-hydroxy nonenal and related products caused by ESAme but did not significantly affect the distribution of oxidized PUFAs. Deeper analysis of the lipidomics data found that the most dramatic change upon ESA or ESAme treatment is the increase in CE 18:3 (Cholesterol Ester). This increase is consistently observed in both non-senescent and senescent cells (Fig. 5B). Interestingly, the ferroptosis inhibitor fer-1 does not alter this increase. Collectively, these results suggest that the 18:3 fatty acids from ESA or ESAme are being integrated into cholesterol esters and this integration is upstream of ferroptosis.

[0158] Virtual screening and docking reveal potential targets for a-ESAs in ferroptosis pathways

[0159] To evaluate the interactions of the a-ESAs with key proteins in the ferroptosis signaling pathway, flexible molecular docking simulations were performed using a set of enzymes from the KEGG ferroptosis network based on enzymatic reactions contributing to ferroptosis progression and known drug targets. These targets include the System Xc- complex (SLC3 A2 encoding 4F2, and SLC7A11 encoding xCT) and GPX4 in the cystine-glutathione metabolism arm, as well as ACSL4, LPCAT3, and ALOX15 in the arachidonic acid metabolism arm of the ferroptosis pathway. System Xc- and GPX4 act as important inhibitors or mediators of ferroptosis initiation and progression, where the ACSL4 / LPCAT3 / ALOX15 axis acts as direct or indirect activator of ferroptotic processes.

[0160] Biophysical energetic estimations of the ligand-receptor interactions of the a-ESAs showed weak to moderate competition with the native substrates in the cystine / erastin-binding site in the 4F2 heavy chain of the Xc- complex, and the catalytic reducing site of GPX4; these were inconsequential when comparing to known inhibitors such as erastin and ML 162 (Fig. 6A). In case of ACSL4 and LPCAT3, a-ESA but not a-ESA-me show a relatively strong binding affinity towards the substrate catalytic sites outcompeting the native ligands arachidonic acid (ARA) and arachidonoyl-CoA (AR-CoA), although not as strongly as known inhibitor drugs. Looking at ligand-receptor interaction plots, this can be explained by the substrate-receptor and drug-receptor interactions primarily being driven by strong covalent, ionic, and hydrogen bonding events whereas a-ESA and a-ESA-me interactions are chemically preferential towards establishing weak hydrophobic pi-pi and pi-alkyl bonds. Finally, in case of the ALOX15 activation site, both lipid senolytics had substantially stronger binding affinities compared to other selected targets, with a-ESAs outperforming the known ALOX15 allosteric modulator PKUMDL MH IOOL The 3D ligand-receptor complex with the ALOX15 monomer showed that the binding free energies of the a-ESAs were primarily contributed by hydrogen bonds and a series of weak hydrophobic interactions with amino acid residues in the allosteric activation cavity including PHE88, TRP109, MET148, LEU172, ILE174, LYS175, LEU389, PHE399, ILE403, ARG407, TYR408 (Fig. 6B). In contrast to other targets, the abundance of these nonpolar amino acids in the allosteric activation site in the interdomain cavity neighboring the substrate binding site in ALOX15 constitute a moderately hydrophobic cleft contributing to a- ESA and a-ESA-me binding energetics (Fig. 6C). However, lipid molecules, such as arachidonic acid and linoleic acid, are actively bound and oxidized in the more hydrophobic catalytic active site or substrate binding site of ALOX15. If the a-ESAs do not have a preferential selection for binding sites, this can lead to promiscuity in terms of enzyme regulation and possible catalysis of the compounds by ALOX15.

[0161] To further investigate this, additional docking analysis was performed for a-ESA and a- ESA-me within a smaller simulation grid in the substrate binding pocket of ALOX15. The results showed that although a-ESA and a-ESA-me had weak to moderate affinity for the substrate binding site (Fig. 6D), they were not able to competitively inhibit ALOX15-ARA native substrate interactions effectively like the known inhibitor drug Zileuton. The binding energetics, when compared to the allosteric site, also revealed a preferential bias by both lipid senolytics towards the allosteric activation cavity. In all cases, a-ESA showed a stronger binding affinity to most targets compared to a-ESA-me, probably due to the steric hindrance and unfavorable donor-donor interactions with residues in the binding cavities by the additional methyl group on a-ESA-me.

[0162] Finally, whether there are any structural changes to the ALOX15-ARA catalytic binding due to occupancy of the allosteric pocket by a-ESA or a-ESA-me was investigated. Sequential docking of a-ESAs / PKUMDL_MH_1001 in the allosteric activation site were performed followed by iron cofactor and ARA binding in the substrate binding site of ALOX15. Consistent with previous literature reporting a PKUMDL MH 1001 mediated increase in arachidonic acid binding by ALOX15, the binding of the activator drug, as well as both lipid senolytics, were found to increase the binding affinity of ARA in the native substrate binding cavity. Particularly, a-ESA-mediated allosteric distortion of the interdomain cleft in the complex provided better access for the substrate fatty acids into the hydrophobic catalytic cavity of ALOX15 compared to the apo-enzyme (Fig. 6E).

[0163] Discussion

[0164] A new class of fatty acids, a-eleostearic acid (ESA) and its methyl ester derivative (ESAme), was identified as novel senolytics. ESA and ESAme were shown to effectively kill a broad spectrum of senescent cells. The senolytic potential of ESA and ESAme was further confirmed in naturally aged and accelerated aging mouse models where they significantly reduced tissue senescence and extended healthspan. Given that clearance of senescent cells mitigates a variety of age-related conditions (Childs, B.G., et al., Nat Med 21, 1424-1435 (2015); and He, S. & Sharpless, N.E., Cell 169, 1000-1011 (2017)), these novel lipid senolytics may have potential for the treatment of many age-related diseases driven by senescence.

[0165] Most existing senolytics induce senescent cell death through apoptosis (Zhang, L., et al., Meeh Ageing Dev 200, 111587 (2021)). For example, the first senolytic, the combination of dasatinib and quercetin, was discovered using bioinformatic screening based on anti-apoptotic networks (Zhu, Y., et aL, Aging Cell 14, 644-658 (2015)). Mechanistic studies have now revealed that the lipid senolytics ESA and ESAme induce senescent cell death via a novel mechanism of ferroptosis, rather than apoptosis or necrosis. Ferroptosis is an iron-dependent form of cell death characterized by the accumulation of ROS and lipid peroxides through the Fenton reaction (Dixon, Scott J., et al, Cell 149, 1060-1072 (2012)). Lipidomics analysis found that free 18:3 fatty acids from ESA or ESAme were mostly integrated into cholesterol esters, and this integration occurs upstream of ferroptosis. Senescent cells were also found to have elevated levels of ferrous iron and ROS compared to non-senescent cells. As illustrated in Figure 7, The iron overload in senescent cells facilitates the Fenton reaction to produce highly reactive hydroxyl radicals (RO»). These radicals initiate the peroxidation of polyunsaturated fatty acids to form lipid radicals. ESA and ESAme further amplify this process by contributing to the formation of lipid peroxides (PUFA-OO*) and hydroperoxides (PUFA-OOH). As a critical component of lipid membranes, cholesterol modulates the fluidity and stability of cell membranes. The integration of ESAs into cholesterol esters remodels lipid membranes in both non-senescent and senescent cells. However, the accumulated lipid peroxidation products in senescent cells promoted by ESAs lead to membrane damage and culminates in ferroptotic senescent cell death. The ferroptosis inhibitor fer-1, acting as a lipid peroxidation inhibitor, can reduce the oxidative stress and lipid peroxidation but does not alter the lipidome changes caused by ESA or ESAme treatment. The enzyme glutathione peroxidase 4 (GPX4) normally mitigates lipid peroxidation by reducing lipid hydroperoxides to their corresponding alcohols (PUFA-OH) using glutathione (GSH) as a cofactor. However, in senescent cells, the high levels of lipid peroxides upon treatment of ESAs overwhelm GPX4 activity. Using ferroptosis inhibitors, such as ferrostatin-1 and liproxstatin-1, as well as iron chelators like deferoxamine, effectively prevent ESA-induced senescent cell death. This confirms the pivotal role of ferroptosis in the senolytic action of ESAs. In contrast, inhibitors of apoptosis and necrosis pathways do not offer protection, further validating the unique mechanism of ferroptotic senescent cell death.

[0166] Iron dyshomeostasis and increased ROS levels have been observed in many senescent cells (Maus, M., et al., Nature Metabolism 5, 2111-2130 (2023); Admasu, T.D., et al., Cell Reports 42(2023); and Liao, C.M., et al., Am J Transplant 22, 2158-2168 (2022)). Due to this susceptibility, senescent cells are predisposed to ferroptosis. This suggests that inducing ferroptotic senescent cell death could be a novel therapeutic approach for senolytic interventions (Liao, C.M., et al., Am J Transplant 22, 2158-2168 (2022); and Go, S., et al., Tissue Engineering and Regenerative Medicine 18, 841-850 (2021)). Indeed, it was found that treatment with known ferroptosis inducers, such as the GPX4 inhibitor RSL3 or the glutamatecysteine antiporter inhibitor erastin, achieves senolysis albeit with low selectivity. It is foreseeable that repurposing other ferroptosis inducers could potentially provide more novel senolytics.

[0167] Based on a structure-activity relationship study, the ferroptosis-inducing activity of ESAs can be attributed to their structural features of conjugated polyunsaturated fatty acids, which are particularly prone to oxidation. In contrast, the double bonds of unconjugated PUFAs are disrupted by a methylene (CH2) moiety, thus losing the radical propagation ability. Although many unconjugated PUFAs exhibit various therapeutic functions, such as fish oils (DHA, EP A, and ALA) (Kapoor, B., et al., Curr Nutr Rep 10, 232-242 (2021); and Zhang, Y., et al. The American Journal of Clinical Nutrition 103, 330-340 (2016)), they showed no senolytic activity. SAR analysis also highlights that the configuration of double bonds (cis / trans) in the carbon chain of fatty acids impacts senolytic activity and selectivity. Additionally, the modification of these polyunsaturated fatty acids with ester groups influenced their senolytic effect. Esterification appears to decrease senolytic activity and but increase selectivity over nonsenescent cells.

[0168] Structure-based DSNN / DCNN machine leaming / ligand similarity models and molecular interaction simulations suggest that the a-ESAs represent novel ferroptosis inducers that are safe and bioavailable. They are predicted to exert their senolytic activity through interactions with multiple nodes in the arachidonic acid metabolic arm of the ferroptosis pathway. Recent studies parsing mechanisms underlying ferroptosis have highlighted Fenton chemistry and ALOX activity synergistically contribute to iron-dependent cell death induction and have inter-pathway crosstalk using oxidized metabolites (Feng, H., et al., P LOS Biology 16, e2006203 (2018); Feng, S., et al., Molecular Biomedicine 4, 33 (2023)). However, further mechanistic studies are needed to determine whether iron dependent oxidation of these lipid senolytics or enzymatic activation of lipid peroxidases is the primary or exclusive mechanism at work, or if the intermediate components of the interconnected pathways may orchestrate to push senescent cells towards ferroptosis. Multiple studies have shown that the expression of iron-dependent ALOX5 / ALOX15, along with their lipid peroxide products, increases during senescence programming (Hamsanathan, S., et al., Front Physiol 13, 796850 (2022); Liao, C.M., et al., Am J Transplant 22, 2158-2168 (2022); Wiley, C.D., et al., JCI Insight 4(24): el30056. (2019)). Reinforcing the prediction results, RSL3, a well-known ferroptosis inducer, was shown to selectively clear senescent cells, which was substantially diminished when LOX activity was inhibited by Zileuton (Wiley, C.D., et al., JCI Insight 4(24): el30056. (2019)). Cancer cells, which also accumulate high levels of cellular iron, have similarly shown susceptibility to ferroptotic cell death induced by a-ESA, which was attributed to membrane incorporation via ACSLs and increased lipid hydroperoxides (Beatty, A., et al., Nature Communications 12, 2244 (2021)). Interestingly, these cells had significant enrichment for lipid peroxides with arachidonate residues, suggesting that LOX-mediated PUFA peroxidation may drive ferroptosis in these cells. The mechanistic study also highlights a-ESAs to be moderately strong interactors in the ACSL4 and LPCAT3 substrate binding cavities, which may explain their observed integration into cholesterol esters, and potential to disrupt phospholipid membranes. Fer-1, an anti-ferroptosis agent, has been reported to exert its effects through direct inhibition of ALOX15 catalytic activity (Miotto, G., et al., Redox Biology 28, 101328 (2020)). In the experiments, a suppression of selective senolysis by a-ESAs in Fer-1 pre-treated cells was observed , suggesting the activity to be at least partially modulated by ALOX15. Transcriptomic analyses validated a significant upregulation of ALOX15 in senescent cells, and pro-ferroptotic programming upon treatment with the a-ESAs. These effects were reversed with Fer-1 pretreatment, corroborating ferroptosis as a novel mechanism of senescent cell death induced by the a-ESAs.

[0169] A proposed mechanism for how a-ESA and a-ESA-me induce ferroptotic cell death in senescent cells is shown in Fig. 7. Briefly, senescent cells exhibit elevated levels of cellular ferrous iron and lipoxygenase expression. This leads to PUFA oxidation and generation of lipid- ROS through Fenton oxidation and iron-dependent enzymatic catalysis. a-ESA and a-ESA-me treatments exacerbate lipid ROS stress through direct oxidation and / or allosteric activation of ALOX15 activity, promoting the propagation of lipid peroxides. Interactions of reactive lipid species with ACSL and LPCAT enzymes lead to esterification, phospholipid (PL) membrane incorporation and destabilization. These processes sensitize senescent cells to membrane damage, ultimately resulting in ferroptotic cell death.

[0170] In summary, a novel class of lipid senolytics has been identified through a senescent cellbased phenotypic screening of fatty acids. Conjugated polyunsaturated fatty acids, specifically a-eleostearic acid and its methyl ester derivative, effectively eliminated a broad range of senescent cells, reduced tissue senescence, and extended healthspan in aged mice. Importantly, these novel lipid senolytics induced senolysis through ferroptosis, rather than apoptosis or necrosis, by exploiting the elevated iron and ROS levels in senescent cells. The findings also demonstrated the vulnerability of iron-rich, ALOX 15 -overexpressing senescent cells to compounds that promote lipid peroxide generation and ferroptosis. This study not only expands understanding of senolytic mechanisms, but also introduces a new therapeutic strategy for targeting senescent cells.

[0171] Experiments

[0172] Compounds and reagents

[0173] Fatty acids were purchased from Cayman Chemical (Michigan, USA). Hoechst 33342 was purchased from Thermo Fisher (H1399). C12FDG was purchased from Setareh Biotech (7188). Formaldehyde 32% was purchased from Electron Microscopy Sciences (15714).

[0174] Cells and mice

[0175] Primary Erccl^ mouse embryonic fibroblasts (MEFs) and WT MEFs were isolated on embryonic day 12.5-13.5. In brief, mouse embryos were isolated from yolk sac followed by the complete removal of viscera, lung and heart if presented. Embryos were then minced into fine chunks, fed with MEFs medium, cultivated under 3% oxygen to reduce stresses. Cells were split at 1 :3 when reaching confluence. MEFs were grown at a 1 : 1 ratio of Dulbecco’s Modification of Eagles Medium (supplemented with 4.5 g / L glucose and L-glutamine) and Ham’s F10 medium, supplemented with 10% fetal bovine serum, penicillin, streptomycin and non-essential amino acid. To induce oxidative stress-mediated DNA damage, Erccl ' MEFs were switched to 20% oxygen for three passages. WT MEFs were induced senescence by treating with hydrogen peroxide H2O2 (200 pM) or etoposide (2 pM) for 24 h, followed by 5 days in normal culture media.

[0176] Human IMR90 lung fibroblasts were obtained from American Type Culture Collection (ATCC) and cultured in EMEM medium with 10% FBS and pen / strep antibiotics. To induce senescence, cells were treated with 20 pM etoposide for 24 hours, followed by five days in normal culture media.

[0177] Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC and cultured using Endothelial Cell Growth Media plus supplement (without vascular endothelial growth factor (VEGF)) and 1% pen / strep antibiotics. The cells were experimentally treated at late passage 13 to 15.

[0178] ErccE and ErccE ' mice from C57BL / 6J and FVB / n backgrounds were crossed to generate ErccTIAmice to prevent potential strain-specific pathology. Aged wild-type C57BL / 6J:FVB / NJ mice were generated by crossing C57BL / 6J and FVB / n inbred mice purchased from Jackson Laboratory. Mice were left to age for two years before being enrolled into the late life intervention study. Animal protocols used in this study were approved by University of Minnesota Institutional Animal Care and Use Committees.

[0179] Senotherapeutic screening

[0180] Senescence was evaluated based on SA-P-gal activity using C12FDG staining assay. Specifically, senescent ErccT^ MEFs were passaged for three times at 20% O2 to induce senescence then seeded at 3000 cells per well in black wall, clear bottom 96 well plates at least 16 hours prior to treatment. Following the addition of drugs, the MEFs were incubated for 48 hours at 20% O2. After removing the medium, cells were incubated in 100 nM Bafilomycin Al in culture medium for 60 min to induce lysosomal alkalinization, followed by incubation with 20 pM fluorogenic substrate C12FDG (7188, Setareh Biotech, USA) for 2 h and counterstaining with 2 pg / ml Hoechst 33342 (H1399, Thermo Fisher Scientific, MA, USA) for 15 min. Subsequently, cells were washed with PBS and fixed in 2% paraformaldehyde for 15 minutes. Finally, cells were imaged with 6 fields per well using a high content fluorescent image acquisition and analysis platform Cytation 1 (BioTek, VT, USA). Cells were seeded at 3000 cells per well in black-wall, clear-bottom 96-well plates at least 16 hours prior to treatment. Following the addition of drugs, cells were incubated for 48 hours. After removing the medium, cells were incubated in 100 nM Bafilomycin Al in culture medium for 1 hour to induce lysosomal alkalinization, followed by incubation with 20 pM fluorogenic substrate C12FDG (7188, Setareh Biotech, OR, USA) for 2 hours and counterstaining with 2 pg / mL Hoechst 33342 (H1399, Thermo Fisher Scientific, MA, USA) for 15 minutes. Subsequently, cells were washed with PBS and fixed in 2% paraformaldehyde for 15 minutes. Finally, cells were imaged with six fields per well using the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, VT, USA).

[0181] Cellular ferrous iron measurement

[0182] Cells were treated with ESA (10 pg / mL) or ESAme (20 pg / mL) for 6 hours with or without a 1-hour pretreatment with Fer-1 (2 pM). After treatment, cells were stained with 1 pM FerroOrange (SCT210, Sigma-Aldrich, USA) for 1 hour and 2 pg / mL Hoechst 33342 (H1399, Thermo Fisher Scientific, MA, USA) for 15 minutes. The staining solution was removed, and cells were washed with PBS. Cells were immediately imaged using the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, VT, USA).

[0183] Cellular reactive oxygen species detection

[0184] Cells were treated with ESA (10 pg / mL) or ESAme (20 pg / mL) for 6 hours with or without a 1-hour pretreatment with Fer-1 (2 pM). After treatment, cells were stained with 1 pM H2DCFDA (D399, Thermo Fisher Scientific, MA, USA) for 1 hour and 2 pg / mL Hoechst 33342 (H1399, Thermo Fisher Scientific, MA, USA) for 15 minutes. The staining solution was removed, and cells were washed once with PBS. Cells were immediately imaged using the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, VT, USA).

[0185] Lipid peroxidation assay

[0186] Cells were treated with ESA (10 pg / mL) or ESAme (20 pg / mL) for 6 hours with or without a 1-hour pretreatment with Fer-1 (2 pM). Following treatment, cells were stained with 10 pM Cl 1 BODIPY (D3861, Thermo Fisher Scientific, MA, USA) for 30 minutes and 2 pg / mL Hoechst 33342 (H1399, Thermo Fisher Scientific, MA, USA) for 15 minutes. The staining solution was removed, and cells were washed with PBS. Cells were immediately imaged using the Cytation 1 Cell Imaging Multi-Mode Reader (BioTek Instruments, VT, USA).

[0187] Health evaluation of Erccl'^ mice Health assessment of Erccl^ mice was conducted twice per week to evaluate age-related symptoms, including body weight, tremor, forelimb grip strength, kyphosis, hindlimb paralysis, gait disorder, dystonia and ataxia. Kyphosis, body condition and coat condition were used to reflect general health conditions. Ataxia, dystonia, gait disorder and tremor were used as indicators of aging-related neurodegeneration. Muscle wasting was studies by monitoring hindlimb paralysis and forelimb grip strength. All aging symptoms were scored based on a scale of 0, 0.5 and 1, with the exception of dystonia that has a scale from 0 to 5. The sum of aging scores of each group was used to determine the overall aging conditions, with zero means no symptom presented. RT-qPCR analysis

[0188] Total RNA was extracted from cells or snap frozen tissues using Trizol reagent (Thermo Fisher, USA). cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, USA). Quantitative PCR reactions were performed with PowerUp™ SYBR™ Green Master Mix (ThermoFisher, USA). The experiments were performed according to the manufacturer’s instructions. The sequences of the primers used were listed in Supplementary Table 3.

[0189] Table 3. List of primer sequences used for RT-qPCR analysis.

[0190] RNA-Seq and enrichment analysis

[0191] RNA samples were extracted using Trizol reagent (Thermo Fisher, USA). Samples were quantified using fluorimetry (RiboGreen assay) and RNA integrity was assessed using capillary electrophoresis, generating an RNA Integrity Number (RIN). All samples had at least 500ng mass and RIN of at least 8. Library preparation was carried out using the Illumina TruSeq Stranded Total RNA Library Prep kit, followed by sequencing on the NovaSeq 6000 using 150 PE flow cell, with a sequencing depth of 20 million reads per sample. Quality control on raw sequence data for each sample was performed with FastQC. Read mapping was performed via Hisat2 (v2.1.0) using the mouse genome (GRCm38.94) as reference. Gene quantification was done via Feature Counts for raw read counts. Differentially expressed genes (DEGs) were identified using the R package edgeR. Gene Set Enrichment Analysis (GSEA) and over representation analysis (ORA) was performed using the R Clusterprofiler package.

[0192] Pathway analysis

[0193] Gene Ontology (GO) biological processes, molecular function and cellular compartments and pathway analysis were determined by PANTHER classification system.32 Functional enrichment analysis was determined using WebGestalt (WEB-based Gene SeT AnaLysis Toolkit).33 Heatmap of iron metabolism related genes and ferroptosis related genes were generated by Morpheus (https: / / software.broadinstitute.org / morpheus).

[0194] Lipid extraction for mass spectrometry lipidomics

[0195] Cells were treated with ESAme (2 pg / mL) for 8 hours with or without 1-hour pretreatment with fer-1 (2 pM). Cell pellets with 3 * 106cells were collected by trypsinization with 0.25% trypsin-EDTA for lipidomic analysis. Lipids were extracted using a two-step chloroform / methanol procedure. Samples were spiked with internal lipid standard mixture containing: cardiolipin 16: 1 / 15:0 / 15:0 / 15:0 (CL), ceramide 18: l;2 / 17:0 (Cer), xxx, xxx. After extraction, the organic phase was transferred to an infusion plate and dried in a speed vacuum concentrator. 1st step dry extract was re-suspended in 7.5 mM ammonium acetate in chloroform / methanol / propanol (1 :2:4, V:V:V) and 2nd step dry extract in 33% ethanol solution of methylamine in chloroform / methanol (0.003:5: 1; V:V:V). All liquid handling steps were performed using Hamilton Robotics STARlet robotic platform with the Anti Droplet Control feature for organic solvents pipetting. Samples were analyzed by direct infusion on a QExactive mass spectrometer (Thermo Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were analyzed in both positive and negative ion modes with a resolution of Rm / z = 200 = 280,000 for MS and Rm / z = 200 = 17,500 for MSMS experiments, in a single acquisition. MSMS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments

[0019] , MS and MSMS data were combined to monitor CE, DAG and TAG ions as ammonium adducts; PC, PC O-, as acetate adducts; and CL, PA, PE, PE O-, PG, PI and PS as deprotonated anions. MS only was used to monitor LPA, LPE, LPE O-, LPI and LPS as deprotonated anions; Cer, HexCer, SM, LPC and LPC O- as acetate adducts.

[0196] Example 2. The following illustrate representative pharmaceutical dosage forms, containing a compound of formula (I) ('Compound X'), for therapeutic or prophylactic use in humans.

[0197] (i) Tablet 1 mg / tablet

[0198] Compound X= 100.0

[0199] Lactose 77.5

[0200] Povidone 15.0

[0201] Croscarmellose sodium 12.0

[0202] Microcrystalline cellulose 92.5

[0203] Magnesium stearate 3,0

[0204] 300.0 (ii) Tablet 2 mg / tablet

[0205] Compound X= 20.0

[0206] Microcrystalline cellulose 410.0

[0207] Starch 50.0

[0208] Sodium starch glycolate 15.0

[0209] Magnesium stearate 5,0

[0210] 500.0

[0211] (iii) Capsule mg / capsule

[0212] Compound X= 10.0

[0213] Colloidal silicon dioxide 1.5

[0214] Lactose 465.5

[0215] Pregelatinized starch 120.0

[0216] Magnesium stearate 3,0

[0217] 600.0

[0218] (iv) Injection 1 (1 mg / ml) mg / ml

[0219] Compound X= (free acid form) 1.0

[0220] Dibasic sodium phosphate 12.0

[0221] Monobasic sodium phosphate 0.7

[0222] Sodium chloride 4.5

[0223] 1.0 N Sodium hydroxide solution

[0224] (pH adjustment to 7.0-7.5) q.s.

[0225] Water for injection q.s. ad 1 mL

[0226] (v) Injection 2 (10 mg / ml) mg / ml

[0227] Compound X= (free acid form) 10.0

[0228] Monobasic sodium phosphate 0.3

[0229] Dibasic sodium phosphate 1.1

[0230] Polyethylene glycol 400 200.0

[0231] 1.0 N Sodium hydroxide solution

[0232] (pH adjustment to 7.0-7.5) q.s. Water for injection q.s. ad 1 mL

[0233] (vi) Aerosol mg / can

[0234] Compound X= 20.0 Oleic acid 10.0

[0235] Trichloromonofluoromethane 5,000.0

[0236] Dichlorodifluoromethane 10,000.0

[0237] Dichlorotetrafluoroethane 5,000.0 The above formulations may be obtained by conventional procedures well known in the pharmaceutical art.

[0238] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical composition comprising a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein A is a (Cis-Ci9)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds and R1is H or (Ci-Ce)alkyl, and a pharmaceutically acceptable excipient.2 The pharmaceutical composition of claim 1, wherein A is a (Cis-Ci8)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds.3 The pharmaceutical composition of claim 1, wherein A is a (Ci6-Ci7)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds.4 The pharmaceutical composition of claim 1, wherein A is a (Ci7)alkenyl comprising two or more (e.g., 2, 3, 4) double bonds.5 The pharmaceutical composition of any one of claims 1-4, wherein the two or more double bonds are two or more conjugated double bonds.6 The pharmaceutical composition of any one of claims 1-4, wherein A comprises three or more double bonds.7 The pharmaceutical composition of claim 6, wherein the three or more double bonds are three or more conjugated double bonds.8 The pharmaceutical composition of claim 1, wherein A is:9 The pharmaceutical composition of claim 1, wherein the compound of formula (I) is a compound of formula (la):or a pharmaceutically acceptable salt thereof, wherein R1is H or (Ci-C6)alkyl.10 The pharmaceutical composition of any one of claims 1-9, wherein R1is H.11 The pharmaceutical composition of any one of claims 1-9, wherein R1is methyl, ethyl, propyl, or isopropyl.12 The pharmaceutical composition of any one of claims 1-9, wherein R1is methyl.13 The pharmaceutical composition of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical composition of claim 1, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

15. The pharmaceutical composition of any one of claims 1-14, that comprises a senolytic amount of the compound or the pharmaceutically acceptable salt.

16. The pharmaceutical composition of any one of claims 1-15 that is formulated as a unit dosage form.

17. A method for reducing the expression of one or more senescence markers in a cell comprising, contacting the cell with a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16.

18. The method of claim 17, wherein the contacting occurs in vitro.

19. The method of claim 17, wherein the contacting occurs in an animal.

20. A method for reducing senescence in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

21. A method for extending the healthspan or lifespan of an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

22. A method for treating age-related disease in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

23. A method for increasing senotherapeutic activity in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

24. A method for treating a disease or condition in an animal selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 orwith a composition as described in any one of claims 1-16 to the animal.

25. A method for improving response to chemotherapy in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

26. A method for improving the anti-tumor response in a cancer patient following treatment with radiation or chemotherapy by reducing the treatment-induced senescent tumor cells in the patient, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the patient.

27. A method for treating ferroptosis-sensitive cancers in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

28. A method for improving response to radiation therapy in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

29. A method for slowing the accelerated aging observed in a cancer survivor, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the cancer survivor.

30. A method for improving recovery of a cancer survivor, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the cancer survivor.31 A method for slowing the accelerated aging in an HIV patient, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof asdescribed in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the HIV patient.

32. A method for improving cognition in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

33. A method for reducing the negative effects of tobacco use (past or present) in an animal, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

34. A method for reducing one or more symptoms associated with long-haul COVID-19 in an animal, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

35. A method comprising contacting an organ with a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to improve the characteristics of the organ for transplantation.

36. A method for improving bone healing in an animal comprising, administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

37. A method for repairing damaged muscle in an animal comprising, administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

38. A method for improving tissue homeostasis comprising, contacting tissue with a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 orwith a composition as described in any one of claims 1-16.

39. A method for improving immune response to a vaccine in an animal comprising, administering a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14 or with a composition as described in any one of claims 1-16 to the animal.

40. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for use in medical therapy.

41. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for reducing senescence, extending healthspan, treating an age-related disease, or increasing senotherapeutic activity.

42. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for reducing senescence, extending healthspan, treating an age-related disease, or increasing senotherapeutic activity in an animal.

43. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for treating a disease or condition selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection.

44. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceuticallyacceptable salt thereof for improving response to chemotherapy.

45. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving response to radiation therapy.

46. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving recovery of a cancer survivor.

47. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving cognition.

48. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for reducing the negative effects of tobacco use.

49. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for reducing one or more symptoms associated with long-haul CO VID-1950. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving the characteristics of an organ for transplantation.

51. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving bone healing.

52. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for repairing damaged muscle.

53. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving tissue homeostasis.

54. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving immune response to a vaccine.

55. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for treating a disease or condition selected from the group consisting of diabetes, obesity, age-related lipodystrophy, cardiac dysfunction including atrial fibrillation, vascular hyporeactivity, vascular calcification, AV fistulae, frailty, sarcopenia, sequellae of bone marrow transplantation, sequellae of organ transplantation, myeloma, MGUS, Alzheimer’s disease, Parkinson’s disease, cancer survivors, cancer, ALS, anxiety, renal dysfunction, osteoporosis, osteoarthritis, rheumatoid arthritis, COPD, idiopathic pulmonary fibrosis, hyperoxic lung damage, hepatic steatosis, liver cirrhosis, primary biliary cirrhosis, liver, kidney and lung fibrosis, progerias, critical illness myopathy, pre-eclampsia, uterine fibrosis, ovarian involution, cataracts, macular degeneration, glaucoma, prostatic hypertrophy, skin disorders, scleroderma, stem cell activation, progenitor cell dysfunction, improving immune function including an immune response to vaccines, COVID-19, a viral infection, and a bacterial infection in an animal.

56. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving response to chemotherapy in an animal.

57. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving response to radiation therapy in an animal.

58. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving recovery of a cancer survivor.

59. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving cognition in an animal.

60. The use of a compound of formula (I) as described in any one of claims 1-14 or apharmaceutically acceptable salt thereof to prepare a medicament for reducing the negative effects of tobacco use (past or present) in an animal.

61. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for reducing one or more symptoms associated with long-haul COVID-19 in an animal.

62. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving the characteristics of an organ for transplantation.

63. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving bone healing in an animal.

64. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for repairing damaged muscle in an animal.

65. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving tissue homeostasis in an animal.

66. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving immune response to a vaccine in an animal.

67. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for improving the anti-tumor response in a cancer patient following treatment with radiation or chemotherapy by reducing the treatment-induced senescent tumor cells in the patient.

68. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for treating ferroptosis-sensitive cancers.

69. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for slowing the accelerated aging observed in a cancer survivor.

70. A compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof for slowing the accelerated aging in an HIV patient.

71. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for improving the anti-tumor response in a cancer patient following treatment with radiation or chemotherapy by reducing the treatment-induced senescent tumor cells in the patient.

72. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for treating ferroptosis-sensitive cancers.

73. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for slowing the accelerated aging observed in a cancer survivor.

74. The use of a compound of formula (I) as described in any one of claims 1-14 or a pharmaceutically acceptable salt thereof to prepare a medicament for slowing the accelerated aging in an HIV patient.

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