Composition for selective elimination of senescent cells

Abstract

The invention relates to a composition comprising PRGL493 for use in selectively eliminating senescent cells and the treatment of selected diseases, disorders and conditions, wherein the senescent cells are characterized by increased intracellular levels of at least one of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid.

Description

[0001] COMPOSITION FOR SELECTIVE ELIMINATION OF SENESCENT CELLS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of senolytics and provides a composition comprising PRGL493 for the selective elimination of senescent cells and treatment of selected diseases, disorders and conditions.

[0004] BACKGROUND OF THE INVENTION

[0005] Senescent cells were found to accumulate in tissues and organs during the aging process at close proximity of age-related pathologies where they play a critical role in the development and progression of age-related diseases and disorders. Clearance of senescent cells in mouse models using either genetic or pharmacological approaches was shown to extend the health span, to prevent or delay the occurrence of senescence- associated diseases and disorders and the development of frailty. Since then, several pharmacological compounds have been identified which were able to selectively eliminate senescent cells and are generally referred to as “senolytics”.

[0006] Although cellular senescence is a tumor suppressive mechanism which plays an important role in embryonic development and wound healing, the chronic accumulation of senescent cells in organs and tissues during the aging process is believed to be a major driving force for the development and progression of age-related diseases and disorders. Senescent cells are terminally growth arrested either via the p53-p21CIP1or via the p16INK4a-Rb axis, accumulate senescence-associated P-galactosidase activity (SA-P-gal) and display a typical morphology. When chronically present, they negatively affect the surrounding tissue by secreting a pro-tumorigenic and pro-inflammatory mixture of cytokines, growth factors and proteases termed the senescence-associated secretory phenotype (SASP).

[0007] It has been previously described that senescent cells have decreased intracellular levels of arachidonic acid compared to young cells, due to an increase in the release of arachidonic acid into the extracellular medium (Lorenzini et al., 2001).

[0008] In contrast to these findings, WO2019070407A1 discloses a method of identifying elevated levels of senescent cells in a mammal by determining an indicator of senescent cells such as e.g. eicosanoids, or the eicosanoid precursor arachidonic acid.

[0009] Genetic mouse models using the p16INK4apromoter to visualize and selectively eliminate p16INK4apositive cells convincingly demonstrated that senescent cells accumulate during the aging process in vivo and that the clearance of p16INK4apositive cells increases the health span and impairs the development and progression of senescence-associated diseases and disorders (Baker et al., 2011 , 2016). There is compelling evidence for a causal relationship between senescent cells and several age- related diseases and disorders, such as atherosclerosis, idiopathic pulmonary fibrosis, osteoporosis, post-traumatic osteoarthritis, renal aging, skin aging, neurodegenerative diseases and impaired adipogenesis (Lammermann et al., 2018; Xu et al., 2015; Y. Zhu et al., 2015). Furthermore, it was shown that clearance of senescent cells attenuates the negative effects of irradiation- and chemotherapy-induced senescence and restores tissue functionality. In addition to age-related diseases and disorders, cellular senescence is also associated with tumor relapse following chemotherapy and the performance of transplant organs highlighting the potential of senolytic therapies.

[0010] The list of senolytic compounds and targets comprises inhibitors of the Bcl-2 family, Hsp90 inhibitors , dasatinib (Y. Zhu et al., 2015), FOXO4, OXR1 , glucose metabolism, mitochondria-targeted tamoxifen (MitoTam) or reduction of ATP synthase activity with oligomycin A and several plant derived compounds, such as quercetin (Y. Zhu et al., 2015), fisetin, piperlongumine and an alcoholic extract of solidago virgaurea.

[0011] Combinations of compounds targeting senescent cells have been described. WO20151 16735A1 describes methods for treatment of senescent cell associated diseases or disorders by administering a senolytic combination comprising for example dasatinib and quercetin.

[0012] However, many of the reported senolytics like navitoclax and dasatinib have serious side effects and most senolytics are not universally effective in all cell types.

[0013] Wiley and coworkers associated cellular senescence with an increased phospholipase A2 activity (Wiley et al., 2019).

[0014] W02020084105A2 discloses the use of inhibitors inhibiting, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase, enzymes with arachidonate- CoA ligase activity for eliminating senescent cells.

[0015] W02020170192A1 describes the synthesis of PRGL493 and its use for the treatment of tumors expressing acyl-CoA synthetase 4 (ACSL4).

[0016] Although several pharmacological compounds were able to eliminate senescent cells in vitro, many senolytic compounds and compositions can cause serious side effects and may have serious limitations in vivo. Therefore, there is still a strong need in the field for improved senolytic compounds combining effective and selective elimination of senescent cells in a subject without having negative side effects.

[0017] SUMMARY OF THE INVENTION

[0018] It is the objective of the present invention, to provide an improved composition capable of selectively eliminating senescent cells.

[0019] The problem is solved by the present invention.

[0020] It has been shown by the inventors that a composition comprising PRGL493 selectively eliminates senescent cells in vitro and in vivo. The inventors further demonstrated that, despite comparatively high in vitro half maximal effective concentrations (ECso), the administration of said composition was able to reduce or reverse signs of aging in mice without any side effects. It has further been shown by the inventors, that a composition comprising the synthetically synthesizable, hence chemically highly pure, PRGL493 can be administered in vivo using high dose without noticeable side effects.

[0021] Surprisingly, the composition comprising PRGL493 was even more effective than a composition comprising Triacsin C (a published ACSL4 inhibitor) or Navitoclax (a published senolytic drug) in eliminating senescent cells in vivo.

[0022] According to the invention there is provided a composition comprising PRGL493 or a pharmaceutically acceptable salt thereof for therapeutic use in selectively eliminating senescent cells, wherein the senescent cells are characterized by increased intracellular levels of at least one of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid.

[0023] According to a specific embodiment, PRGL493 or salt thereof, is in the form of a solvate.

[0024] According to a specific embodiment of the invention, the senescent cells have an at least 2-fold increased intracellular level of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid compared to a cell of the same type or age known to be non-senescent.

[0025] According to a specific embodiment of the invention, the elimination of senescent cells prevents or delays the onset of a senescence-related disease or condition, or prevents or delays the progression of a senescence-related disease or condition, or promotes the regression of a senescence-related disease or condition, or decreases age related frailty.

[0026] According to a specific embodiment of the invention, the senescence-related disease or condition is cardiovascular disease, atherosclerosis, osteoporosis, ischemic reperfusion injury, stroke, myocardial infarct, osteoarthritis, neurological disorders, neurodegenerative diseases, dementia, cataract, kidney disease, retinopathy, diabetes, lung fibrosis, liver fibrosis, non-alcoholic liver disease, liver steatosis, intervertebral disc degeneration, age-related muscular atrophy, age-related decline of the immune system and decreased efficacy of vaccination, delayed wound healing and chronic wounds, hair loss and loss of hair pigmentation or skin aging.

[0027] According to a further embodiment of the invention, the composition improves the performance of transplants.

[0028] According to a specific embodiment of the invention, the composition prevents or attenuates senescence-associated scar formation and fibrosis.

[0029] According to a specific embodiment of the invention, the composition ameliorates side effects of chemotherapy and prevents or delays tumor relapse.

[0030] According to a further embodiment of the invention, said composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable excipient.

[0031] According to a specific embodiment there is also provided a composition comprising PRGL493 for use in treating a subject suffering or going to suffer from a senescence-related disease by selectively eliminating senescent cells, wherein the senescent cells are characterized by increased intracellular levels of at least one of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid.

[0032] According to an embodiment, herein provided is also a method of treatment of a disease or condition mediated by senescent cells, wherein the senescent cells are characterized by increased intracellular levels of at least one of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid, and which comprises administering to a subject in need of such treatment an effective amount of PRGL493 or a pharmaceutically acceptable salt thereof.

[0033] According to a specific embodiment, herein provided is a method for treatment, wherein the disease or condition mediated by senescent cells is cardiovascular disease, atherosclerosis, osteoporosis, ischemic reperfusion injury, stroke, myocardial infarct, osteoarthritis, neurological disorders, neurodegenerative diseases, dementia, cataract, kidney disease, retinopathy, diabetes, lung fibrosis, liver fibrosis, non-alcoholic liver disease, liver steatosis, intervertebral disc degeneration, age-related muscular atrophy, age-related decline of the immune system and decreased efficacy of vaccination, delayed wound healing and chronic wounds, hair loss and loss of hair pigmentation, or skin aging.

[0034] FIGURES

[0035] Fig. 1 : Senolytic effect of PRGL493 in HLF, HDF, RPTEC and rKCC. Doxorubicin-induced premature senescent cells (SIPS) and quiescent control cells (Q) were treated with PRGL493. Data represents the average of three experiments.

[0036] Fig. 2: p16 mRNA expression in fat tissue of doxorubicin-aged mice after senolytic treatment. p16 mRNA expression was significantly reduced in doxorubicin- aged mice after senolytic treatment with PRGL493 when compared to other senolytics or combinations thereof, including Triacsin C (TrC).

[0037] Fig. 3: p21 mRNA expression in liver, kidney, and spleen tissue of naturally aged mice (24 months of age) after PRGL493 treatment. Treatment of naturally aged mice with PRGL493 resulted in a significant downregulation of p21 levels in liver, kidney, and spleen.

[0038] Fig. 4: SA-0-gal staining of fat tissue of naturally aged mice (24 months of age) after PRGL493 treatment with exemplary micrographs. The percentage of SA-p-gal positive cells was significantly reduced in fat tissue after treatment of naturally aged mice with PRGL493.

[0039] Fig. 5: Weight difference between baseline and time of dissection. The weight of naturally aged mice (27 months of age) was measured before and after the treatment with PRGL493. Mice treated with vehicle lost on average about 1 g during the 8-week treatment period, whereas the PRGL493 treated mice gained in average 1 g

[0040] Fig. 6: Total distance and maximum speed during open field test. Treatment of naturally aged mice (27 months of age) with PRGL493 was able to significantly increase both parameters in old mice indicating an improvement in exploratory behavior and physical fitness. Fig. 7: Anxiety-related behavior during open field test. (A) Quantification of path efficiency. (B) Representative track plots. PRGL493 treated naturally aged mice (27 months of age) showed an increased explorative behavior.

[0041] Fig. 8: Hanging time / impulse during hanging wire test. The PRGL493 treated naturally aged mice (27 months of age) nearly reached the level of young control mice indicating a rejuvenation of physical function in naturally aged mice after PRGL493 treatment.

[0042] Fig. 9: Distribution of pathologies in both treatment groups at dissection. Only 8 out of 27 (29,6 %) of the PRGL493 treated naturally aged mice (27 months of age) showed one of the assessed pathologies.

[0043] Fig. 10: Blood cell count analysis at time of dissection. White blood cell (WBC) and middle cell count were significantly increased in PRGL493 treated naturally aged mice (27 months of age).

[0044] Fig. 11 : SA-0-gal staining of the cortex. Representative micrographs of brain sections stained for SA-P-galactosidase and the quantification of the stained area as percentage of the total analyzed area. A global reduction of senescent cells could be observed after treatment of naturally aged mice (27 months of age) with PRGL493.

[0045] Fig. 12: SA-0-gal staining of the hippocampus. Representative micrographs of brain sections stained for SA-P-galactosidase and the quantification of the stained area as percentage of the total analyzed area. A global reduction of senescent cells could be observed after treatment of naturally aged mice (27 months of age) with PRGL493.

[0046] Fig. 13: SA-0-gal staining of the Purkinje cell layer. Representative micrographs of brain sections stained for SA-P-galactosidase and the quantification of the stained area as percentage of the total analyzed area. A global reduction of senescent cells could be observed after treatment of naturally aged mice (27 months of age) with PRGL493.

[0047] Fig. 14: Oil Red O staining of several brain regions. Representative micrographs of brain sections stained for lipid droplets with Oil Red O and the quantification of the stained area as percentage of the total analyzed area. A global reduction of senescent cells was observed after treatment of naturally aged mice (27 months of age) with PRGL493.

[0048] Fig. 15: p16 mRNA expression in kidney tissue of naturally aged mice after senolytic treatment. A significant reduction of the expression levels for the widely used senescence marker p16INK4awas observed after treatment of naturally aged mice (27 months of age) with PRGL493.

[0049] Fig. 16: p16 mRNA expression in spleen tissue of naturally aged mice after senolytic treatment. A significant reduction of the expression levels for the widely used senescence marker p16INK4awas observed after treatment of naturally aged mice (27 months of age) with PRGL493.

[0050] Fig. 17: p16 mRNA expression in skin tissue of naturally aged mice after senolytic treatment. A significant reduction of p16INK4amRNA expression levels was observed in skin cells after treatment of naturally aged mice (27 months of age) with PRGL493.

[0051] Fig. 18: Frailty index score at baseline and at week 8 of life span study using naturally aged mice (27 months of age). Vehicle treated mice showed a significant increase in the frailty index score, while PRGL493 treated mice showed a significant reduction in the frailty index score.

[0052] Fig. 19: Change of frailty index score after 24 weeks of life span study using naturally aged mice (27 months of age). (A) Time course of frailty index score, (B) representative pictures of a vehicle and a PRGL493 treated mouse at the age of 34 months and (C) change of sex separated frailty index score from baseline to week 24 time point. Even after 24 weeks of treatment, some of the PRGL493 treated naturally aged mice showed only a mild increase in the frailty index score as compared to the baseline.

[0053] Fig. 20: Survival curves of life span study using naturally aged mice (27 months of age). Treatment of naturally aged mice with PRGL493 is able to significantly increase the life span of mice.

[0054] Fig. 21 : Wound area of naturally aged mice after PRGL493 treatment and wounding. The wounds of PRGL493 treated naturally aged mice (20 months of age) were closing faster than from vehicle treated mice.

[0055] Fig. 22: Analysis of immune cells in the peripheral blood of naturally aged mice (20 months of age) after PRGL493 treatment. Analysis of immune cells in the peripheral blood revealed a significant increase of total immune cells (CD45+) as well as CD8+ T cells in PRGL493 treated naturally aged mice. In addition, the amount of SA-P-gal positive cells was significantly decreased after PRGL493 treatment in several subsets of T cells and B cells. Fig. 23: Analysis of immune cells in the spleen of naturally aged mice (20 months of age) after PRGL493 treatment. Analysis of immune cells in the spleen of naturally aged mice revealed an increase of total immune cells (CD45+) as well as B cells. In addition, the amount of SA-P-gal positive B cells was significantly decreased after PRGL493 treatment.

[0056] Fig. 24: Analysis of immune cells in the bone marrow (BM) of naturally aged mice (20 months of age) after PRGL493 treatment. Analysis of immune cells in the bone marrow of naturally aged mice revealed a significant increase of CD8+ central memory T cells and a simultaneously decrease in CD8+ effector memory T cells after treatment with PRGL493, indicating a favorable shift in the immune cell population.

[0057] Fig. 25: SA-0-gal staining of the choroid plexus 4thventricle and the periventricular region. Quantification of the stained area as percentage of the total analyzed area or total stained area. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=19 / 21 ; N=19 / 23). Amount of SA-0-gal positive cells was significantly decreased after PRGL493 treatment.

[0058] Fig. 26: Oil Red O staining of several brain regions. Representative micrographs of brain sections stained for lipid droplets with Oil Red O and the quantification of the stained area as percentage of the total analyzed area. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 (n=14 / 13; n=14 / 13; n=13 / 13; n=13 / 13). A global reduction of senescent cells was observed after treatment of naturally aged mice with PRGL493.

[0059] Fig. 27: Glial Fibrillary Acidic Protein (GFAP) staining of retrospinal cortex. Representative micrographs of brain sections stained for GFAP and the quantification of the number of positive cells per area and number of primary branches. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 (n=9 / 8; n=5 / 5). The number of GFAP positive cells was significantly reduced in the retrospinal cortex by the treatment of PRGL493.

[0060] Fig. 28: Immunohistological staining for p21 protein of liver tissue of naturally aged mice after senolytic treatment. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 . p21 protein of liver tissue of naturally aged mice was significantly reduced after treatment with PRGL493.

[0061] Fig. 29: Masson trichrome staining and immunohistological staining for a-SMA protein of liver tissue of naturally aged mice after senolytic treatment. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001. A massive reduction of interstitial fibrosis and a-SMA positive cells in liver could be observed after treatment with PRGL493.

[0062] Fig. 30: Masson trichrome staining of muscle tissue of naturally aged mice after senolytic treatment. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 . Interstitial fibrosis was significantly reduced in muscle tissue after treatment with PRGL493.

[0063] Fig. 31 : Overview of glomerulonephritis study and GFR (glomerular filtration rate) at day 32. Significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 (n=4 / 5 / 4). PRGL493 is highly effective in the treatment of chronic kidney disease by restoring the glomerular filtration rate to the level of young healthy mice.

[0064] DETAILED DESCRIPTION

[0065] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017), and Murphy & Weaver, "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.

[0066] Specific terms as used throughout the specification have the following meaning.

[0067] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0068] The term “about” as used herein refers to the same value or a value differing by +Z-5 % of the given value.

[0069] Singular and plural forms can be used interchangeably herein if not otherwise indicated.

[0070] As used herein, the term “subject” or “individual” or “patient” shall refer to a warmblooded mammalian, particularly a human being. Alternatively, it may also be an animal, for example mouse, rat, dog, cat, swine, bovine, or a non-human primate.

[0071] The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment for a senescence-associated condition or disease or are diagnosed of cellular senescence or a senescence-associated condition or disease.

[0072] The term “sample” generally refers to tissue or organ sample, blood, cell-free blood such as serum and plasma, platelet-poor plasma, lymph, urine, saliva, and biopsy probes.

[0073] “Senescence” refers to the gradual deterioration of functional characteristics. Cellular senescence occurs in culture and in vivo as a response to extracellular or intracellular stress and / or due to aging. The senescence response locks cells into a cellcycle arrest that prevents the propagation of damaged cells and precludes potential malignant transformation. Senescence refers to an array of changes that occurs over time. Compared to a reference cell (e.g., a cell or sample of the same type or age known to be non-senescent), a senescent cell is defined as a cell that shows one, two, three, four, five, six or more or all of any of the following features: a cessation of cell proliferation; an accumulation of lipofuscin (e.g., increase in lipofuscin accumulation); an increase in lysosomal beta-galactosidase activity; an increase in the secretion of members of the senescence-associated secretory phenotype (SASP); an excessive accumulation of lipid droplets; an increase of mitochondrial-derived reactive oxygen species; an increase in nuclear DNA damage foci; a critical shortening of telomeres; an increased expression of p16 or p21 or any combination thereof; or a cell or subject shows a process that causes those described above.

[0074] Senescent cells accumulate in tissues and organs in response to internal or external stress as well as during the aging process. They play a critical role in the development and progression of senescence-related diseases and disorders.

[0075] “Stress”, the natural and in vivo equivalent of which are unknown, causes a senescence arrest without significant telomere erosion. These stresses may include inappropriate substrate, e.g., tissue culture plastic, serum (most cells experience plasma, not serum, in vivo), serum withdrawal, DNA-damage inducing substances or oxidative stress, e.g., culture in atmospheric O2, which is hyperphysiological or exposure to substances that produce reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), paraquat or tert-Butyl hydroperoxide or ultraviolet or ionizing irradiation. Cells also enter senescence upon loss of the PTEN tumor suppressor, a phosphatase that counteracts pro-proliferative / pro-survival kinases. Additionally, ectopic expression of the cyclin-dependent kinase inhibitors (CDKis) that normally enforce the senescence growth arrest, notably p21WAF1and / or p16INK4a, may cause senescence.

[0076] “Aging” is a combination of processes of deterioration that follow the period of development of an organism. Aging is generally characterized by a declining adaptability to stress, increased homeostatic imbalance, increase in senescent cells, and increased risk of disease. Because of this, death is the ultimate consequence of aging. Environmental factors may affect aging, for example, overexposure to ultraviolet radiation accelerates skin aging. Different parts of the body may age at different rates. Two organisms of the same species can also age at different rates, making biological aging and chronological aging distinct concepts.

[0077] The term “senescent cells” as used herein specifically refers to cells that express a marker or combination of markers that are characteristic of senescence, i.e. having an at least 2-fold increased intracellular level of phospholipase A2 activity, lysophosphatidylcholine, specifically lyso PPC and / or lyso SPC, and / or arachidonic acid compared to a cell of the same type or age known to be non-senescent. In some embodiments, a senescent cell expresses other markers including but not limited to increased expression relative to a reference, such as a non-senescent cell, in the levels of DNA-damage response (DDR) markers, co-localization of DNA damage proteins like 53BP1 or gammaH2AX with telomeres, as well as the cell cycle inhibitors p16INK4A, p-|giNK4B p2ic|p1iand p53. DEC1 , DCR2, and PAI1 can also be used as senescence biomarkers. In one embodiment, senescent cells express SA-beta-Gal (senescence- associated beta galactosidase) to an extent that staining with X-Gal at pH=6 results in a blue color.

[0078] The senescent cell may further show a decrease in autophagy activity or a decrease in mitochondrial membrane potential or shows a process that causes those described above. Compared to a cell or subject such as a known senescent cell or subject, a non-senescent cell or subject may show an increase in cell proliferation ability, a decrease in lipofuscin accumulation, a decrease in P-galactosidase activity, or a combination thereof. Specifically, in the case of a human, a cell or sample that is taken from a person about 30 years or older, about 40 years or older, about 50 years or older, about 60 years or older, about 70 years or older, about 80 years or older, about 90 years or older, may be defined as a senescent cell or sample. In certain cases, a cell or sample that is taken from a person about 30 years or younger, 20 years or younger, 10 years or younger or even at the embryonic stage, may be defined as a senescent cell or sample. Specifically, a cell or sample that is taken from a human child suffering from or being at risk of developing a senescence-related disease or condition, such as for example diabetes, specifically type I diabetes, may be defined as a senescent cell or sample.

[0079] The terms “cellular senescence” and “senescent cell(s)” thus refer to the essentially irreversible growth arrest that occurs when cells that can divide encounter critically short telomeres, oncogenic stress, or DNA damage, or experience strong mitogenic signals, such as but not limited to oncogenes or highly expressed pro- proliferative genes and a senescent cell, which is a potentially persisting cell that is metabolically active and has undergone widespread changes in protein expression.

[0080] Acceleration of the rate of aging may be induced by stress conditions including, but not limited to chemical, physical, and biological stresses. For example, accelerated aging can be induced by stresses caused by UV and IR irradiation, drugs and other chemicals, chemotherapy, intoxicants, such as but not limited to DNA intercalating and / or damaging agents, oxidative stressors; mitogenic stimuli, oncogenic stimuli, toxic compounds, hypoxia, oxidants, exposure to environmental pollutants, for example, silica, exposure to an occupational pollutant, for example, dust, smoke, asbestos, or fumes. All these stressors alone or in combination can also cause cellular senescence. Specifically, senescence is induced by combinations of stresses, e.g., two or more chemical and physical stresses; two or more chemical and biological stresses; two or more physical and biological stresses; chemical, physical, and biological stresses in combination, etc.

[0081] Cellular senescence may also be caused by telomeric dysfunction (telomere uncapping) resulting from repeated cell division (termed replicative senescence), mitochondrial deterioration, oxidative stress, severe or irreparable DNA damage and chromatin disruption (genotoxic stress), and the expression of certain oncogenes (oncogene-induced senescence). Stresses that cause cellular senescence can be induced by external or internal chemical and physical insults encountered during the course of the life span, during therapeutic interventions (for example, X-irradiation or chemotherapy), or as a consequence of endogenous processes such as oxidative respiration and mitogenic signals. External mitogenic signals, for example growth- related oncogene alpha (GROa) secretion by tumor cells in close proximity to normal cells or circulating angiotensin II, have also been shown to induce cellular senescence. All somatic cells that have the ability to divide can undergo senescence. Regardless of the disparate mechanisms of senescence-inducing stresses, the senescence program is activated once a cell has sensed a critical level of damage or dysfunction. So far, the senescence growth arrest has been shown to depend on the activities of the major tumor-suppressor pathways controlled by p16INK4aand pRB (retinoblastoma protein), as well as by p53. Some of the molecules involved in pathways upstream and downstream of the senescence-associated phenotype have been used as markers to detect senescent cells in culture and in vivo.

[0082] As described herein, senescent cells have an altered lipid metabolism. Specifically, phospholipase A2 activity is upregulated. According to a specific example, the secretory phospholipase A2 receptor (PI-A2R1) is upregulated, which activates phospholipase A2 (PI-A2), specifically, cytosolic phospholipase A2 (cPI_A2) and thereby induces the production of lyso PCs, leading to a concurrent intracellular release of arachidonic acid (AA). Specifically, senescent cells thus have an increased AA formation rate. According to a further specific example, lysophosphatidylcholine is upregulated. In particular, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 lyso PC) and 1- palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso PC) are upregulated independent of whether the senescence inducer is telomere dependent (replicative senescence, aging) or independent (stress-induced premature senescence).

[0083] The term “phospholipase A2” or “PLA2” as used herein refers to a superfamily of lipases with PLA2 activity. PLA2 activity refers to the ability of PLA2 enzymes to hydrolyze the fatty acid from the sn-2 position of membrane phospholipids, such as for example phosphatidylcholine. The PLA2 superfamily comprises four main types of enzymes including the secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), calcium- independent PLA2 (iPLA2), and platelet activating factor (PAF) acetyl hydrolase / oxidized lipid lipoprotein associated PI_A2 ((Lp)PI_A2). Specifically, PI_A2s, in particular sPI_A2s and cPI_A2s, are known to hydrolyze phosphatidylcholines, which produce lysophosphatidylcholines and concurrently release arachidonic acid.

[0084] The term “lysophosphatidylcholine” or “lyso PC” as used herein refers to a chemical compound derived from phosphatidylcholine, which is normally located in the cell membrane. Lyso PC can be generated from phosphatidylcholines (PC) either non- enzymatically through oxidation processes or enzymatically through conversion by phospholipases with PLA2 activity. Specifically, Lyso PC can be generated by cytosolic phospholipase A2 (PLA2G4A), cytosolic phospholipase A2 gamma (PLA2G4C) and / or group XV phospholipase A2 (PLA2G15). Lyso PCs can have different combinations of fatty acids of varying lengths and saturation attached at the C-1 (sn-1) position. Fatty acids containing 16, 18 and 20 carbons are the most common. 18:0 lyso PC, also called lyso SPC, in particular, consists of one chain of stearic acid at the C-1 position. 16:0 lyso PC, also called lyso PPC, in particular, consists of one chain of palmitic acid at the C-1 position.

[0085] The term “arachidonic acid” or “AA” as used herein refers to a polyunsaturated omega-6 fatty acid. Specifically, AA is present in phospholipids, especially phosphatidylcholine, of membranes of the body's cells. In addition to being involved in cellular signaling as a lipid second messenger involved in the regulation of signaling enzymes, AA is a key inflammatory intermediate and can also act as a vasodilator. Specifically, AA is released into the cytosol from phosphatidylcholines upon hydrolysis of phosphatidylcholines into lysophosphatidylcholines by lipases with PLA2 activity.

[0086] Non-senescent cells may be proliferating cells or may be quiescent cells. In certain instances, exposure of non-senescent cells to the composition described herein may temporarily reduce the capability of a non-senescent cell to proliferate, however, the non-senescent cell is not destroyed.

[0087] Incorporation of free intracellular AA into phospholipids requires thioesterification of AA by enzymes belonging to the group of long-chain acyl-CoA synthetases.

[0088] “Long-chain-fatty acyl CoA synthetase”, also referred to as “long-chain-fatty-acid- CoA ligase”, in short “ACSL”, is an enzyme of the ligase family that activates oxidation and re-acylation of complex fatty acids. These enzymes produce acyl-CoAs from fatty acids that are at least 12 carbons in length. ACSL catalyzes the formation of fatty acyl- CoA by a two-step process proceeding through an adenylated intermediate, specifically, it catalyzes the activation of a long fatty acid chain to fatty acyl Coenzyme A (fatty acid CoA). There are several highly conserved areas and a 20-30 % amino acid sequence similarity between the members of this superfamily. Specifically, the enzymes in the family consist of a large N-terminal and a small C-terminal domain, with the catalytic site positioned between the two domains.

[0089] As referred to herein, an enzyme with arachidonate-CoA ligase activity is an enzyme capable of catalyzing the conversion of arachidonic acid and CoA into arachidonoyl-CoA. Specific examples of such enzymes are long-chain-fatty-acid-CoA 4 ligases (ACSL4).

[0090] Specifically, the term “ACSL” as used herein also refers to isozymes of the long- chain fatty-acid coenzyme A ligase family. There are five ACSL isoforms known in humans and rodents: ACSL1 , ACSL3, ACSL4, ACSL5, and ACSL6. Although differing in substrate specificity, subcellular localization, and tissue distribution, all isozymes of this family convert free long-chain fatty acids into fatty acyl-CoA esters, and thereby play a key role in lipid biosynthesis and fatty acid degradation.

[0091] Specifically, “ACSL4” is a member of the acyl-CoA synthetase family, which is required for the activation of free fatty acids with CoA, generating acyl-CoA, the precursor for the re-integration and degradation processes in the Lands cycle (Murphy et al., 2019). The Lands cycle encompasses the enzymatic hydrolysis of phospholipids, generating lysophospholipids and free fatty acids, such as AA, the activation of free fatty acids through acyl-CoA synthetases and the re-integration of acyl-CoA into lysophospholipids by acyl-CoA transferases. Alternatively, arachidonate-CoA can be degraded via P-oxidation or used by diacylglycerol O-acyltransferases to form diacyl- and triacylglycerols and by sterol O-acyltransferases to form cholesterol esters. Specifically, ACSL4 preferentially utilizes arachidonate as substrate. ACSL4 controls the intracellular level of arachidonic acid, because AA can induce apoptosis, specifically in senescent cells, thus ACSL4 has the ability to modulate apoptosis. Overexpression of ACSL4 has been shown to result in a higher rate of arachidonoyl- CoA synthesis, increased AA incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified AA.

[0092] ACSL4 is necessary for the induction of cell death via ferroptosis.

[0093] As used herein, the term “PRGL493” refers to an ACSL4 inhibitor, specifically to the compound (A / -(4-(3-(5-(methylfuran-2-yl) 1 -phenyl-1 / 7-pyrazol-4-yl)-3,4- dihydorbenzo[4,5]imidazo[1 ,2-a][1 ,3,5]triazin-2-yl)acetamide; CAS Registry Number: 2479378-45-3) of formula A, an enantiomer, or pharmaceutical acceptable salt and / or solvate thereof.

[0094] Pharmaceutically acceptable salts are described in e.g. Stahl, P. H., Wermuth, C. G, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich:Wiley-VCH / VHCA, 2002.

[0095] A salt of a compound of formula A, i.e. of PRGL493, includes an acid addition salt. Pharmaceutically acceptable acid addition salts include salts of the compound of formula A with an acid, e.g. fumaric acid, tartaric acid, sulphuric acid, p-toluene sulphonic acid, methane sulphonic acid, phosphoric acid, citric acid, L-malic acid, hippuric acid, D gluconic acid, L-lactic acid, benzoic acid, hydrogen maleic acid, hydrogen sulphuric acid, hydrogenphosphoric acid, hydrogen tartaric acid, hydrogen fumaric acid, hydrogen malic acid, hydrogen succinic acid, ethane-1 ,2-disulphonic acid, maleic acid, naphthalin 1 ,5 sulphonic acid, acetic acid, succinic acid, salicylic acid, azelaic acid, 2 [(2,6 dichlorophenyl)amino]benzene acetic acid, trifluoro acetic acid, hydrochloric acid, deuterochloric acid, preferably it is hydrochloric acid.

[0096] PRGL493 inhibits, prevents, or decreases the activity of any long-chain-fatty-acid- CoA 4 ligase (ACSL4). PRGL493 blocks cell proliferation and tumor growth in both breast and prostate cellular and animal models. PRGL493 is used for cancer research (Castillo et al., 2021 ; W02020170192A1).

[0097] Conversion of AA into arachidonoyl-CoA is essential because intracellular accumulation of AA has a toxic effect on cells. For example, AA released into the cytosol by phospholipase A2-activity can trigger apoptosis through the mitochondrial apoptotic pathway. As described herein, senescent cells have an increased AA formation rate, because PI_A2 activity and lyso PC formation are upregulated. Therefore, inhibition or reduction of the conversion of AA into arachidonoyl-CoA increases AA levels, specifically intracellular AA levels, of senescent cells to a greater extent than in non-senescent cells, which leads to selective elimination of senescent cells. Specifically, senescent cells are selectively eliminated by PRGL493 through cell death triggered by increased intracellular AA levels due to inhibition or reduction of the AA metabolization.

[0098] The term “selectively eliminating senescent cells” refers to the exposure of the cells or subjects to a senolytic composition comprising PRGL493, which induces lysis of senescent cells. Specifically, PRGL493 selectively eliminates senescent cells, preferably by killing senescent cells e.g. by inducing apoptosis in a biologically, clinically, and / or statistically significant manner compared with its capability to destroy or kill a nonsenescent cell. Specifically, PRGL493 alters the lipid biosynthesis and fatty acid degradation of senescent cells by targeting ACSL4 in a manner that induces (initiates, stimulates, triggers, activates, promotes) and results in (i.e. , causes, leads to) the death of the senescent cell.

[0099] According to a further embodiment of the invention, the composition described herein is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or excipient.

[0100] Specifically, the pharmaceutical composition comprises PRGL493 described herein in free form, in the form of a pharmaceutically acceptable salt, or in the form of a solvate and further comprising at least one pharmaceutical excipient or carrier.

[0101] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, amino acids such as glycine or histidine, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life.

[0102] Said pharmaceutical excipient can also be, but is not limited to a diluent, fillers, binders, disintegrators, flow conditioners, flow enhancers, glidants, lubricants, sugars and sweeteners, fragrances, taste maskers, preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for regulating osmotic pressure and / or buffers.

[0103] Such pharmaceutical compositions may be manufactured according, e.g. analogously, to a method as conventional, e.g. by mixing, granulating, coating, dissolving or lyophilizing processes.

[0104] PRGL493 used herein may be administered by any conventional route, for example enterally, parenterally and topically, e.g. in form of coated or uncoated tablets, capsules, injectable solutions or suspensions, e.g. in the form of ampoules, vials, in the form of creams, gels, pastes, inhaler powder, foams, tinctures, drops or sprays.

[0105] As used herein, “treatment”, “treat” or “treating” refers to a clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease / condition, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease / condition, decreasing the rate of disease progression or condition, amelioration or palliation of the disease state, and remission or improved prognosis.

[0106] An effective amount of a pharmaceutical preparation or drug is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit a disease, disease condition or disorder. Such an effective dose specifically refers to that amount of the compound sufficient to result in healing, prevention or amelioration of conditions related to diseases or disorders described herein. The amount of PRGL493 described herein that will correspond to such an effective amount will vary depending on various factors, such as the given drug or compound, the formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, the assessment of the medical situations and other relevant factors, but can nevertheless be routinely determined by one skilled in the art.

[0107] Preferably, the treatment is a short treatment period of 1 to 8 weeks, specifically up to 8 weeks, 7 weeks, 6, weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week leading to the selective elimination of senescent cells, further resulting in an improvement of the disease or condition that extends beyond the treatment period for at least 4 weeks, specifically at least 5, 6, 7, or 8 weeks, or even results in a permanent improvement of the disease or condition.

[0108] In general, for treatment of subjects, specifically larger mammals, for example humans, an indicated daily dosage is in the range from about 10 mg to 6 g of PRGL493, specifically in the range of 100 mg to 3 g. Specifically, PRGL493 can be administered in divided doses up to four times a day, e.g. once a day (QD), two times a day (BID), three times a day (TID) or four times a day (QID). Alternatively, PRGL493 can also be administrated continuously, e.g. via infusion.

[0109] The composition provided herein can be used for the treatment of a senescence- related disease or condition. Specifically, the composition provided herein is used for the prevention ( / .e. reduction of the likelihood of occurrence) or delay of the onset of a senescence-related disease or condition, for the prevention or delay of the progression of a senescence-related disease or condition or to promote the regression of a senescence-related disease or condition.

[0110] The term “senescence-related disease or condition” refers to conditions, diseases or disorders related to, associated with, or caused by cellular senescence, including age- related diseases and disorders. Cellular senescence is characterized herein by an at least 2-fold increased intracellular level of phospholipase A2 activity, lysophosphatidylcholine, and / or arachidonic acid of a senescent cell compared to a cell of the same type or age known to be non-senescent.

[0111] The senescence-related disease or condition can be a cardiovascular disease or condition, including but not limited to angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease (CAD), carotid artery disease, endocarditis, coronary thrombosis, carotid thrombosis, myocardial infarction (Ml), high blood pressure / hypertension, aortic aneurysm, brain aneurysm, cardiac fibrosis, cardiac diastolic dysfunction, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease, peripheral artery disease (PAD), cardiac stress resistance, and stroke.

[0112] Subjects suffering from cardiovascular disease can be identified using standard diagnostic methods known in the art for cardiovascular disease. Generally, diagnosis of atherosclerosis and other cardiovascular disease is based on symptoms (e.g., chest pain or pressure (angina), numbness or weakness in arms or legs, difficulty speaking or slurred speech, drooping muscles in face, leg pain, high blood pressure, kidney failure and / or erectile dysfunction), medical history, and / or physical examination of a patient. Subjects at risk of developing cardiovascular disease include those having a family history of cardiovascular disease and those having other risk factors such as high blood pressure, high cholesterol, diabetes, obesity and / or smoking. In a certain embodiment, the cardiovascular disease that is a senescence cell associated disease / disorder is atherosclerosis. Atherosclerosis is characterized by patchy intimal plaques (atheromas) that encroach on the lumen of medium-sized and large arteries; the plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue.

[0113] The effectiveness of a composition described herein for treating or preventing (i.e., reducing or decreasing the likelihood of developing or occurrence of) a cardiovascular disease (e.g., atherosclerosis) can readily be determined by a person skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiography, stress test, non-stress test), may be used for monitoring the health status of the subject. The effects of the treatment of a senolytic combination can be analyzed using techniques known in the art, such as comparing symptoms of patients suffering from or being at risk of cardiovascular disease that have received the treatment with those of patients without such a treatment or with placebo treatment.

[0114] The senescence-related disease or condition can be a neurological condition. Neurological conditions include, but are not limited to, Parkinson's disease, Alzheimer's disease, dementia, amyotrophic lateral sclerosis (ALS), bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, motor neuron dysfunction (MND), mild cognitive impairment (MCI), Huntington's disease, ocular diseases, macular degeneration (wet and dry), glaucoma, vision loss, presbyopia, cataracts, progressive muscular atrophy, lower motor neuron disease, spinal muscular atrophy (SMA), Werdnig-Hoffman Disease (SMA1), SMA2, Kugelberg-Welander Disease (SM3), Kennedy's disease, post-polio syndrome, hereditary spastic paraplegia, age-related memory decline, and depression and mood disorders. The effects of the senolytic composition described herein for use in the treatment or prophylaxis of a neurological condition can be analyzed by comparing symptoms of patients suffering from or being at risk of a neurological disease or disorder, such as e.g. Parkinson’s disease or Alzheimer’s disease, who have received the treatment with those of patients without such a treatment or with placebo treatment.

[0115] Parkinson's disease is the second most common neuro degenerative disease. It is a disabling condition of the brain characterized by slowness of movement (bradykinesia), shaking, stiffness, and in the later stages, loss of balance. Many of these symptoms are due to the loss of certain nerves in the brain, which results in the lack of dopamine. This disease is characterized by neuro degeneration, such as the loss of about 50 % to 70 % of the dopaminergic neurons in the substantia nigra pars compacta, a profound loss of dopamine in the striatum, and / or the presence of intracytoplasmic inclusions (Lewy bodies), which are composed mainly of alpha-synuclein and ubiquitin. Parkinson's disease also features locomotor deficits, such as tremor, rigidity, bradykinesia, and / or postural instability. These motor manifestations can also be accompanied by non-motor symptoms such as olfactory deficits, sleep impairment, and neuropsychiatric disorders. Generally, diagnosis of Parkinson's disease is based on symptoms, medical history, and neurological and / or physical examination of a patient. Subjects at risk of developing Parkinson's disease include those having a family history of Parkinson's disease and those exposed to pesticides (e.g., rotenone or paraquat), herbicides (e.g., agent orange), or heavy metals. Methods for detecting, monitoring or quantifying neuro degenerative deficiencies and / or locomotor deficits associated with Parkinson's diseases are known in the art, such as histological studies, biochemical studies, and behavioral assessment.

[0116] Alzheimer 's disease (AD) is a neurodegenerative disease that shows a slowly progressive mental deterioration with failure of memory, disorientation, and confusion, leading to profound dementia. Age is the single greatest predisposing risk factor for developing AD, which is the leading cause of dementia in the elderly. Early clinical symptoms show remarkable similarity to mild cognitive impairment, which is characterized by difficulty in remembering recent life experiences or people's names. As the disease progresses, impaired judgment, confusion, behavioral changes, disorientation, and difficulty in walking and swallowing occur. Alzheimer's disease is characterized by the presence of neurofibrillary tangles and amyloid (senile) plaques in histological specimens. The disease predominantly involves the limbic and cortical regions of the brain. Several behavioral and histopathological assays are known in the art for evaluating Alzheimer's disease phenotype, for characterizing therapeutic agents, and assessing treatment. Subjects suffering from Alzheimer's disease can be identified using standard diagnostic methods known in the art for Alzheimer's disease. Generally, diagnosis of Alzheimer's disease is based on symptoms (e.g., progressive decline in memory function, gradual retreat from and frustration with normal activities, apathy, agitation or irritability, aggression, anxiety, sleep disturbance, dysphoria, aberrant motor behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia), medical history, neuropsychological tests, neurological and / or physical examination of a patient. Cerebrospinal fluid may also be for tested for various proteins that have been associated with Alzheimer pathology, including tau, amyloid beta peptide, and AD7C- NTP. Genetic testing is also available for early-onset familial Alzheimer disease (eFAD), an autosomal-dominant genetic disease. Clinical genetic testing is available for individuals with AD symptoms or at-risk family members of patients with early-onset disease. In the U.S., mutations for PS2, and APP may be tested in a clinical or federally approved laboratory under the Clinical Laboratory Improvement Amendments. A commercial test for PS 1 mutations is also available (Elan Pharmaceuticals).

[0117] MCI is a brain-function syndrome involving the onset and evolution of cognitive impairments beyond those expected based on age and education of the individual, but which are not significant enough to interfere with this individual's daily activities. MCI is an aspect of cognitive aging that is considered to be a transitional state between normal aging and the dementia into which it may convert. MCI that primarily affects memory is known as "amnestic MCI”. A person with amnestic MCI may start to forget important information that he or she would previously have recalled easily, such as recent events. Amnestic MCI is frequently seen as prodromal stage of Alzheimer's disease.

[0118] MND is a group of progressive neurological disorders that destroy motor neurons, the cells that control essential voluntary muscle activity such as speaking, walking, breathing and swallowing. It is classified according to whether degeneration affects upper motor neurons, lower motor neurons, or both. Examples of MNDs include, e.g. Lateral Sclerosis (ALS), bulbar palsy, and spinal muscular atrophy (SMA). It can affect the arms, legs, or facial muscles. Patients with an MND show one or more motor deficits, including muscle weakness and wasting, uncontrollable twitching, spasticity, slow and effortful movements, and overactive tendon reflexes. Primary lateral sclerosis is a disease of the upper motor neurons, while progressive muscular atrophy affects only lower motor neurons in the spinal cord. In progressive bulbar palsy, the lowest motor neurons of the brain stern are most affected, causing slurred speech and difficulty chewing and swallowing. There are almost always mildly abnormal signs in the arms and legs. Methods for detecting, monitoring, quantifying, or assessing motor deficits and histopathological deficiencies associated with MND are known in the art, including histopathological, biochemical, and electrophysiological studies and motor activity analysis.

[0119] The senescence-related disease or condition can be an inflammatory condition. Inflammatory conditions include, but are not limited to, musculoskeletal diseases, osteoarthritis, osteoporosis, sarcopenia, lupus, interstitial cystitis, scleroderma, alopecia, oral mucositis, rheumatoid arthritis, inflammatory bowel disease, kyphosis, herniated intervertebral disc, ulcerative colitis, Crohn's disease, ulcerative asthma, renal fibrosis including post-transplant renal fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing including diabetes related wound healing, oral submucosa fibrosis, neurodegenerative diseases, intervertebral disc degeneration, age-related decline of the immune system and decreased efficacy of vaccination, atherosclerosis, dementia, cancer, myocardial infarct, age-related muscular atrophy, kidney disease, cardiovascular disease, stroke, diabetes, non-alcoholic liver disease, retinopathy, hair loss and loss of hair pigmentation or skin aging, liver steatosis, lung fibrosis, neurological disorders, delayed wound healing and chronic wounds, ischemic reperfusion injury, and cataract. The effects of the senolytic composition described herein for use in the treatment or prophylaxis of an inflammatory condition can be analyzed by comparing symptoms of patients suffering from or being at risk of an inflammatory disease or disorder, such as e.g. osteoarthritis, who have received the treatment with those of patients without such a treatment or with placebo treatment.

[0120] Osteoarthritis is a degenerative joint disease that is characterized by fibrillation of the cartilage at sites of high mechanical stress, bone sclerosis, and thickening of the synovium and the joint capsule. Symptoms of osteoarthritis include sore or stiff joints, particularly the hips, knees, and lower back, after inactivity or overuse; stiffness after resting that goes away after movement; and pain that is worse after activity or toward the end of the day. The effectiveness the composition described herein for treatment or prophylaxis of osteoporosis and monitoring of a subject who receives the composition can readily be determined by a person skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination (such as determining tenderness, swelling or redness of the affected joint), assessment and monitoring of clinical symptoms (such as pain, stiffness, mobility), and performance of analytical tests and methods described herein and practiced in the art (e.g., determining the level of inflammatory cytokines or chemokines; X-ray images to determine loss of cartilage as shown by a narrowing of space between the bones in a joint; magnetic resonance imaging (MRI), providing detailed images of bone and soft tissues, including cartilage), may be used for monitoring the health status of the subject.

[0121] Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and density that may lead to an increased risk of fracture. Bone mineral density (BMD) is reduced, bone microarchitecture deteriorates, and the amount and variety of proteins in bone are altered. Osteoporosis is typically diagnosed and monitored by a bone mineral density test. Post-menopausal women or women who have reduced estrogen are most at risk. While both men and women over 75 are at risk, women are twice as likely to develop osteoporosis than men.

[0122] Further inflammatory / autoimmune disorders that may be treated with PRGL493 includes irritable bowel syndrome (IBS) and inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. Inflammatory bowel disease (IBD) involves chronic inflammation of all or part of the digestive tract. In addition to life-threatening complications arising from IBD, the disease can be painful and debilitating. Ulcerative colitis is an inflammatory bowel disease that causes long-lasting inflammation in part of the digestive tract. Symptoms usually develop over time, rather than suddenly. Ulcerative colitis usually affects only the innermost lining of the large intestine (colon) and rectum. Crohn's disease is an inflammatory bowel disease that causes inflammation anywhere along the lining of the digestive tract, and often extends deep into affected tissues. This can lead to abdominal pain, severe diarrhea, and malnutrition. The inflammation caused by Crohn's disease can involve different areas of the digestive tract. Diagnosis and monitoring of the diseases is performed according to methods and diagnostic tests routinely practiced in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scan, MRI, endoscopy, and small intestine imaging.

[0123] The senescence-related disease or condition can be a dermatological condition. Dermatological conditions include, but are not limited to, psoriasis, eczema, rhytides, pruritis, dysesthesia, papulosquamous disorders, erythroderma, lichen planus, lichenoid dermatosis, atopic dermatitis, eczematous eruptions, eosinophilic dermatosis, rashes, photosensitivity and photoaging related diseases and disorders, reactive neutrophilic dermatosis, pemphigus, pemphigoid, immunobullous dermatosis, fibrohistocytic proliferations of skin, skin nevi, urticaria, hyperpigmentation, cutaneous lymphomas, psoriasis, and cutaneous lupus. In certain embodiments, the senescent cell associated disorder is an inflammatory disorder of the skin, such as by way of a non-limiting examples, psoriasis and eczema that may be treated or prevented using the composition described herein. Psoriasis is characterized by abnormally excessive and rapid growth of the epidermal layer of the skin. A diagnosis of psoriasis is usually based on the appearance of the skin. Skin characteristics typical for psoriasis are scaly red plaques, papules, or patches of skin that may be painful and itch.

[0124] Other immune disorders or conditions that may be treated with the composition comprising PRGL493 include conditions resulting from a host immune response to an organ transplant (e.g., kidney, bone marrow, liver, lung, or heart transplant), such as rejection of the transplanted organ. Specifically, the senolytic composition may be used for treating or reducing the likelihood of occurrence of graft-vs-host disease.

[0125] The senescence-related disease or condition can be a metabolic disorder. Metabolic disorders include, but are not limited to, type I and type II diabetes mellitus, diabetic ulcers, and obesity. The effects of the PRGL493 composition described herein for use in the treatment or prophylaxis of metabolic condition can be analyzed by comparing symptoms of patients suffering from or being at risk of a metabolic disease or disorder, such as e.g. diabetes, who have received the treatment with those of patients without such a treatment or with placebo treatment.

[0126] Diabetes is characterized by high levels of blood glucose caused by defects in insulin production, insulin action, or both. The great majority (90 to 95 %) of all diagnosed cases of diabetes in adults are type 2 diabetes, characterized by the gradual loss of insulin production by the pancreas. Subjects suffering from type 2 diabetes can be identified using standard diagnostic methods known in the art for type 2 diabetes. Generally, diagnosis of type 2 diabetes is based on symptoms (e.g., increased thirst and frequent urination, increased hunger, weight loss, fatigue, blurred vision, slow-healing sores or frequent infections, and / or areas of darkened skin), medical history, and / or physical examination of a patient. Subjects at risk of developing type 2 diabetes include those who have a family history of type 2 diabetes and those who have other risk factors such as excess weight, fat distribution, inactivity, race, age, prediabetes, and / or gestational diabetes.

[0127] Obesity and obesity-related are used to refer to conditions of subjects who have a body mass that is measurably greater than ideal for their height and frame. Body Mass Index (BMI) is a measurement tool used to determine excess body weight and is calculated from the height and weight of a subject. A human is considered overweight when the person has a BMI of 25-29; a person is considered obese when the person has a BMI of 30-39, and a person is considered severely obese when the person has a BMI of 40 or higher.

[0128] A condition or disorder associated with diabetes and senescence is a diabetic ulcer (i.e. , diabetic wound). An ulcer is a breakdown in the skin, which may extend to involve the subcutaneous tissue or even muscle or bone. These lesions occur, particularly, on the lower extremities. Patients with diabetic venous ulcer exhibit elevated presence of cellular senescence at sites of chronic wounds.

[0129] The senescence-related disease or condition can be a macular degeneration, dry (non-neovascular) or wet (neovascular) macular degeneration. Macular degeneration is a neurodegenerative disease that causes the loss of photoreceptor cells in the central part of retina, called the macula. Macular degeneration generally is classified into two types: dry type and wet type. The dry form is more common than the wet, with about 90 % of age-related macular degeneration (ARMD) patients diagnosed with the dry form. The wet form of the disease usually leads to more serious vision loss. Symptoms include perceived distortion of straight lines and, in some cases, the center of vision appears more distorted than the rest of a scene; a dark, blurry area or "white-out" appears in the center of vision; and / or color perception changes or diminishes. Diagnosing and monitoring of a subject with macular degeneration may be accomplished by a person skilled in the ophthalmic art according to art-accepted periodic eye examination procedures and report of symptoms by the subject. The senescent cell associated condition or disease can be a pulmonary condition. Pulmonary conditions include, but are not limited to, idiopathic pulmonary fibrosis (TPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, emphysema, age-related loss of pulmonary function, and age- associated sleep apnea. The effects of the senolytic composition described herein for use in the treatment or prophylaxis of pulmonary condition can be analyzed by comparing symptoms of patients suffering from or at risk of a pulmonary disease or disorder, such as e.g. COPD, who have received the treatment with those of patients without such a treatment or with placebo treatment.

[0130] COPD is a lung disease defined by persistently poor airflow resulting from the breakdown of lung tissue (emphysema) and the dysfunction of the small airways (obstructive bronchiolitis). Primary symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excess sputum production. COPD is most commonly caused by tobacco smoke (including cigarette smoke, cigar smoke, secondhand smoke, pipe smoke), occupational exposure (e.g., exposure to dust, smoke or fumes), and pollution, occurring over decades thereby implicating aging as a risk factor for developing COPD.

[0131] Pulmonary fibrosis is a chronic and progressive lung disease characterized by stiffening and scarring of the lung, which may lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with repair of epithelium. Subjects at risk of developing pulmonary fibrosis include those exposed to environmental or occupational pollutants, such as asbestosis and silicosis; who smoke cigarettes; having some typical connective tissue diseases such as rheumatoid arthritis, SLE and scleroderma; having other diseases that involve connective tissue, such as sarcoidosis and Wegener's granulomatosis; having infections; taking certain medications (e.g., amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin); those subject to radiation therapy to the chest; and those whose family member has pulmonary fibrosis. Symptoms of pulmonary fibrosis are known in the art and include shortness of breath, particularly during exercise; dry, hacking cough; fast, shallow breathing; gradual unintended weight loss; tiredness; aching joints and muscles; and clubbing (widening and rounding of the tips of the fingers or toes).

[0132] As used herein, “frailty” is defined as a clinically recognizable state of increased vulnerability resulting from aging-associated decline in reserve and function across multiple physiologic systems such that the ability to cope with everyday or acute stressors is compromised.

[0133] Specifically, frailty is one of the elderly person syndromes among the symptoms that appear along with aging, which is said to represent various physical and mental symptoms not necessarily considered illness. It refers to a “condition before developing health problems” (life dysfunction, etc.), which is generated by a variety of factors involved in elderly generation that reduce the spare capacity of various organs playing an important role in life activity, and reduce the ability to adapt to changes in not only the internal environment of the body but also external environment, as a result of which physical, mental, social functions are gradually lost.

[0134] In the absence of an established quantitative standard, frailty can been operationally defined as meeting three out of five phenotypic criteria indicating compromised energetics: (1) weakness (grip strength in the lowest 20% of population at baseline, adjusted for gender and body mass index), (2) poor endurance and energy (self-reported exhaustion associated with VO2 max), (3) slowness (lowest 20% of population at baseline, based on time to walk 5 meters, adjusting for sex and standing height), (4) low physical activity (weighted score of kilocalories expended per week at baseline, lowest quintile of physical activity identified for each sex; e.g., less than 383 kcal / week for males and less than 270 kcal / week for females), and / or (5) unintentional weight loss (4.5 kg in past year). A pre-frail stage, in which one or two of these criteria are present, identifies a high risk of progressing to frailty. (Fried et al., 2001)

[0135] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of a cardiovascular disease.

[0136] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of atherosclerosis.

[0137] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of osteoporosis.

[0138] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of ischemic reperfusion injury.

[0139] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of stroke.

[0140] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of myocardial infarct. According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of osteoarthritis.

[0141] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of neurological disorders.

[0142] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of neurodegenerative diseases.

[0143] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of dementia.

[0144] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of cataract.

[0145] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of kidney disease.

[0146] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of retinopathy.

[0147] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of diabetes.

[0148] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of lung fibrosis.

[0149] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of liver fibrosis.

[0150] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of non-alcoholic liver disease.

[0151] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of liver steatosis.

[0152] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of intervertebral disc degeneration.

[0153] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of age-related muscular atrophy.

[0154] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of age-related decline of the immune system and decreased efficacy of vaccination.

[0155] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of delayed wound healing. According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of chronic wounds.

[0156] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of hair loss and loss of hair pigmentation.

[0157] According to a specific embodiment, PRGL493 or its pharmaceutically acceptable salt or solvate is used for therapeutic treatment of skin aging.

[0158] According to a further specific embodiment, the PRGL493 composition provided herein is used to improve the performance of transplants. Specifically, presence of an increased number of senescent cells in the donor organ and / or in the recipient negatively influences the performance of a transplant. According to a specific embodiment, the composition described herein is used to selectively eliminate senescent cells in the donor organ and / or the recipient to improve the performance of the transplant.

[0159] According to a further specific embodiment, the PRGL493 composition provided herein is used to prevent or attenuate senescence-associated scar formation and fibrosis.

[0160] According to a further specific embodiment, the PRGL493 composition provided herein is used to ameliorate side effects of chemotherapy. According to yet a further specific embodiment, the composition provided herein is used to prevent or delay tumor relapse and the occurrence of senescence-related diseases and conditions. Therapy- induced senescence (TIS) is a common side effect of cancer therapy and causes premature aging in patients surviving the therapy. In addition, TIS induces senescence- associated sternness (SAS) in tumors, for example in Bcl2 lymphomas, thereby causing an increased tumor-initiation capacity and an increased risk for cancer relapse.

[0161] Removal or destruction of senescent cells may ameliorate acute toxicity, including acute toxicity comprising energy imbalance, of a chemotherapy or radiotherapy. Acute toxic side effects include but are not limited to gastrointestinal toxicity (e.g., nausea, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, malaise, low physical activity, hematological toxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, dermatological toxicity (e.g., rashes, dermatitis, hyperpigmentation, urticaria, photosensitivity, nail changes), mouth, gum or throat problems, or any toxic side effect caused by a chemotherapy or radiotherapy. For example, toxic side effects caused by radiotherapy or chemotherapy (see, e.g., National Cancer Institute website) may be ameliorated by the methods described herein. Accordingly, in certain embodiments, methods are provided herein for ameliorating (reducing, inhibiting, or preventing occurrence (i.e., reducing the likelihood of occurrence)) acute toxicity or reducing severity of a toxic side effect (i.e., deleterious side effect) of a chemotherapy or radiotherapy or both in a subject who receives the therapy, wherein the method comprises administering to the subject the composition as described herein that selectively kills, removes, or destroys or facilitates selective destruction of senescent cells. Administration of the senolytic PRGL493 composition for treating or reducing the likelihood of occurrence, or reducing the severity of a chemotherapy or radiotherapy side effect may be accomplished by the same treatment courses described above for treatment / prevention of metastasis. As described for treating or preventing (i.e., reducing the likelihood of occurrence of) metastasis, the senolytic combination is administered during the off-chemotherapy or off-radiotherapy time interval or after the chemotherapy or radiotherapy treatment regimen has been completed.

[0162] According to a specific embodiment, PRGL493 may be used for treating a subject suffering or going to suffer from a senescence-related disease by selectively eliminating senescent cells.

[0163] A “control”, “reference value”, or “reference level” are terms which can be used interchangeably herein, and are to be understood as a sample or standard used for comparison with the experimental sample. The control may include a sample obtained from a healthy subject or a subject, which is not at risk of or suffering from senescence or is not exposed to stress conditions inducing senescence or which is exposed to treatment with a senolytic. Reference level specifically refers to the level of lysophosphatidylcholine, arachidonic acid and / or PI_A2 activity quantified in a sample from a healthy subject, from a subject, which is not at risk of or suffering from senescence or is not exposed to stress conditions inducing senescence or, in case of senolytic treatment monitoring, said level could also be derived from a sample of a subject before starting senolytic treatment or during the course of senolytic treatment.

[0164] Specifically, levels of lysophosphatidylcholine, AA, and / or PI_A2 activity are measured using immunoassays such as ELISA, enzymatic assays, mass spectrometry or high-performance liquid chromatography (HPLC).

[0165] For example, the level, specifically intracellular level, of lysophosphatidylcholines can be measured by high performance liquid chromatography coupled to tandem mass spectrometry in the positive ion mode (Gruber et a , 2015) or by HPLC (Jeschek et al., 2016).

[0166] For example, the level of intracellular AA can be measured by an enzyme linked immunosorbent assay, such as for example the universal arachidonic acid ELISA Kit supplied by Novus Biologicals (NBP2-66372) or any other equivalent ELISA assay. According to further specific examples, the level of arachidonic assay can be measured by flow-injection electrospray ionization mass spectrometry (Gruber et al., 2015), with high performance liquid chromatography (Nishikiori et a , 2015) or with high performance liquid chromatography coupled to tandem mass spectrometry in the negative ion mode.

[0167] For example, PLA2 activity can be measured by an assay that comprises a PLA2 substrate that on cleavage by the phospholipase A2 activity yields a fluorescent product that can be quantified with a fluorimeter, e.g. bis-BODIPY®-FL-C11 -PC (Thermo Fisher), or with the EnzChek™ Phospholipase A2 Assay Kit (Thermo Fisher) or any other equivalent assay.

[0168] According to a specific embodiment, an increase by more than two or three standard deviations of the level, specifically the intracellular level, of at least one of the biomarkers lysophosphatidylcholine, arachidonic acid, or phospholipase A2 activity compared to the level of said biomarkers in a reference is indicative of the presence of senescent cells or cellular senescence.

[0169] According to a specific embodiment, a senescent cell is characterized by an at least 2-fold, specifically 2.5, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 fold increased intracellular level of at least one of the biomarkers lysophosphatidylcholine, arachidonic acid, or phospholipase A2 activity compared to the level of said biomarkers in a reference is indicative of the presence of senescent cells or cellular senescence.

[0170] A senescent cell is characterized by an at least 2-fold, specifically 2.5, 3.0, 3.1 ,

[0171] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 fold increased intracellular level of phospholipase A2 activity of a senescent cell compared to a cell of the same type or age known to be non-senescent.

[0172] A senescent cell is characterized by an at least 2-fold, specifically 2.5, 3.0, 3.1 ,

[0173] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 ,

[0174] 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 ,

[0175] 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0 fold increased intracellular level of lysophosphatidylcholine of a senescent cell compared to a cell of the same type or age known to be non-senescent.

[0176] A senescent cell is characterized by an at least 2-fold, specifically 2.5, 3.0, 3.1 ,

[0177] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 fold increased intracellular level of arachidonic acid of a senescent cell compared to a cell of the same type or age known to be non-senescent.

[0178] Specifically, a more than 2.0, 2.5, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 fold increase of the reference level, specifically the intracellular level, of any one or more of phospholipase A2 activity; lysophosphatidylcholine, or arachidonic acid obtained from a healthy subject or a group of healthy subjects compared to the level, specifically the intracellular level, of one or more of phospholipase A2 activity; lysophosphatidylcholine, or arachidonic acid obtained from a sample of a subject is indicative of the presence of senescent cells in said sample.

[0179] Specifically a more than 2.0, 2.5, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 fold decrease of the reference level, specifically the intracellular level, of any one or more of phospholipase A2 activity; lysophosphatidylcholine, or arachidonic acid obtained from a sample of a subject during or after senolytic treatment compared with a sample of the same subject from an earlier timepoint (intra-individual comparison or intra-individual difference) is indicative of positive response to senolytic treatment.

[0180] Additionally, a control may also be a standard reference value or range of values. The reference level can also be determined as the average level of any one or more of lysophosphatidylcholine, arachidonic acid and phospholipase A2 activity in a sample of a healthy subject and / or in a subject prior to a pharmacologic, dietary, or life-style intervention. As an alternative, also a pool of samples from one or more subjects may be used or a reference disclosed in literature.

[0181] An in vitro method of identifying senescent cells in a subject comprises the following steps: a) providing a sample of said subject, b) determining the intracellular level of at least one of lysophosphatidyl- choline, arachidonic acid and / or phospholipase A2 activity in said sample, c) comparing the level of b) to a reference level, wherein the reference level is the intracellular level of at least one of lysophosphatidylcholine, arachidonic acid and / or phospholipase A2 activity in non-senescent cells, and wherein an increase of at least 2-fold in the level of lysophosphatidylcholine, arachidonic acid and / or phospholipase A2 activity is indicative of the presence of senescent cells in said sample.

[0182] The method for the detection of senescent cells as described above specifically provides a diagnostic and predictive tool using a diagnostic signature or expression pattern as described herein, comprised of an increase in the level of any one or more or all of lysophosphatidylcholine, arachidonic acid and / or phospholipase A2, applicable over a broad range of senescence of various cell types including but not limited to fibroblasts, endothelial cells, kidney epithelial cells, liver cells, neuronal cells, skin cells, lung epithelial cells, or colon epithelial cells. In particular, detection of senescent cells in the tissue, blood or serum of young subjects or subjects not being exposed to senescence inducing stress conditions and old subjects or subjects being exposed to senescence inducing stress conditions provides a diagnostic and predictive tool that has a higher significance for early diagnosis, long-term prognosis, and screening of patients with cellular senescence. The method for the detection of senescent cells as described above also provides a diagnostic tool for monitoring treatment with PRGL493.

[0183] Specifically, the method of identifying senescent cells as described above can be performed as single measurement but may also be performed by repetitive determinations.

[0184] Specifically, use of the method for detecting senescent cells as described above also encompasses predicting transplant organ function or predicting organ transplant failure.

[0185] The above-described method can also be used for detecting a decline of senescent cells or reduction of cellular senescence, wherein the level of at least one of the biomarkers lysophosphatidylcholine, arachidonic acid and / or PLA2 activity is compared with the level of the corresponding lysophosphatidylcholine, arachidonic acid and / or PI_A2 activity prior to a treatment with senolytics, anti-aging agents or any antiaging intervention. Specifically, a decrease in the level of at least one of said biomarkers can indicate the removal of senescent cells e.g. during use of senolytics, anti-aging agents (e.g. rapamycin, spermidine, metformin), or any other anti-aging intervention like diet, exercise, etc. This signature can also be used to identify subjects that would benefit from any senolytic intervention.

[0186] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Different embodiments of the present invention have been described according to the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the invention.

[0187] EXAMPLES

[0188] The following materials and methods were used throughout the examples provided herein unless indicated otherwise.

[0189] Example 1 : In vitro experiment showing the senolytic effect of PRGL493 in HDF, HLF, RPTEC and rKCC.

[0190] Methods

[0191] Cell isolation

[0192] Human lung fibroblasts (HLFs) were isolated from donor lungs and obtained from the Ludwig Boltzmann Institute for Lung Vascular Research (Graz, Austria). Human dermal fibroblasts (HDFs) were isolated from skin biopsies of healthy female adult donors and obtained from Evercyte (Vienna, Austria). Human renal proximal epithelial cells (RPTECs) were obtained from Biopredic (Saint Gregoire, France). Rat knee chondrocytes (rKCCs) were isolated from a pool of knee cartilage tissue derived from 8 male adult Sprague Dawley rats as provided by the Ludwig Boltzmann Institute for Traumatology (Vienna, Austria).

[0193] Cell culture

[0194] HLFs and HDFs were cultured with DMEM / Ham’s F-12 (1 :1 mixture) (F4815, Biochrome) supplemented with 10 % FCS (F7524, Sigma-Aldrich) and 4 mM L- Glutamine (G7513, Sigma-Aldrich) under ambient oxygen, 7 % CO2 and 37 °C. rKCCs were cultured with DMEM- high glucose (41966, Gibco) supplemented with 10 % FCS (F7524, Sigma-Aldrich), 50 pg / mL L-Ascorbic acid 2-phosphate sesquimagnesium salt (A8960, Sigma-Aldrich), 10 mM HEPES (15630-056, Gibco), 5 pg / mL insulin (I6634, Sigma-Aldrich) and 2 mM L-Glutamine (25030, Gibco). Cells were detached by incubation with 0.1 % trypsin and 0.02 % EDTA at 37 °C for 3-5 minutes and split at ratios between 1 :2 and 1 :8 depending on cell type and growth rate. RPTECs were cultured with ProxUp (MHT-003, Evercyte) without G418 under ambient oxygen, 7 % CO2 and 37 °C. Cells were detached by incubation with 0.05 % Trypsin-EDTA solution (25300054, Gibco) at 37 °C for 3-5 minutes and split at ratios between 1 :2 and 1 :4 depending on growth rate.

[0195] Stress-induced premature senescent (SIPS)

[0196] Depending on cell type cells were seeded at a cell density of 3500-7000 cells / cm2one day prior to the treatment and stress-induced premature senescent (SIPS) was induced with 100-200 nM doxorubicin (D1515, Sigma-Aldrich) supplemented to the medium for 6 days.

[0197] Viability assay

[0198] Alamar blue (DAL1100, Thermo Fisher Scientific) assay was performed according to the manufacturer’s instructions. Cells were treated with PRGL493 for 9 days with a media change on day 0, 3, and 6. The control was treated with the respective concentration of the solvent (DMSO).

[0199] Results and Conclusion

[0200] Fig. 1 shows the senolytic effect of PRGL493 in HLF, HDF, RPTEC and rKCC. Doxorubicin-induced premature senescent cells (SIPS) and quiescent control cells (Q) were treated with PRGL493. Data represents the average of three experiments.

[0201] PRGL493 showed an EC50 of 23 pM and 94.2 pM in HLF SIPS cells and HLF Q cells, respectively. This is a 4.1 -fold change in EC50.

[0202] PRGL493 showed an EC50 of 2.06 pM and 17 pM in RPTEC9 SIPS cells and RPTEC9 Q cells, respectively. This is an 8.25-fold change in EC50.

[0203] PRGL493 showed an EC50 of 33 pM and 154 pM in rKCC SIPS cells and rKCC Q cells, respectively. This is a 4.67-fold change in EC50.

[0204] PRGL493 showed an EC50 of 30.3 pM and 72.9 pM in HDF SIPS cells and HDF Q cells, respectively. This is a 2.41 -fold change in EC50.

[0205] The excellent in vitro data and the clear senolytic effect of PRGL493 on various human senescent cells provide clear evidence that PRGL493 allows for a highly effective treatment of diseases. The studies using human lung fibroblasts (HLFs) can serve as model for the treatment of diseases such as lung fibrosis, idiopathic pulmonary disease and chronic obstructive pulmonary disease.

[0206] Renal proximal epithelial cells (RPTECs) can serve as model for the treatment of diseases such as chronic kidney disease, diabetic kidney disease, acute kidney injury and IgA nephropathy.

[0207] Rat knee chondrocytes (rKCCs) can serve as model for the treatment of osteoarthritis and intervertebral disc degeneration.

[0208] Human dermal fibroblasts (HDFs) can serve as model for the treatment of delayed wound healing and chronic wounds, as well as skin aging and other senescence- associated skin diseases and disorders such as chronic atopic dermatitis and psoriasis (H. Zhu et al., 2024).

[0209] Example 2: In vivo use of PRGL493 in doxorubicin aged mice

[0210] Senescent cells were induced with doxorubicin (2 * 10 mg / kg; i.p.) in young male mice at the age of 8 weeks (Baar et al., 2017) and subsequently treated with in vitro identified test compounds. The test compounds were administered on day 24 / 26 / 28 via oral gavage and doses were based on in vitro test results and existing data for test compounds. Mice treated with vehicle (10 % Dimethylacetamide / 50 % PEG-400 / 40 % 1 ,2-Propandiol) only served as a negative control and doxorubicin-aged mice treated with Navitoclax (50 mg / kg) served as a positive control.

[0211] The following doses for senolytic test compounds were selected:

[0212] • Triacsin C (20 mg / kg)

[0213] • Triacsin C (20 mg / kg) I Diclofenac (20 mg / kg) I MK-886 (10 mg / kg)

[0214] • PRGL493 (20 mg / kg)

[0215] • PRGL493 (20 mg / kg) I Diclofenac (20 mg / kg) I MK-886 (10 mg / kg)

[0216] Triacsin C is isolated from fungi and difficult to purify and is limited in availability and is subject to varying activity. Additionally, Triacsin C is highly lipophilic, unstable, shows undesired side effects such as vasodilation in vivo that are dose limiting.

[0217] Surprisingly, the ACSL4 inhibitor PRGL493 was the only treatment that significantly reduced p16 mRNA expression in doxorubicin aged mice (Fig. 2) despite the inferior in vitro ECso values, when compared to Triacsin C as described in W02020084105A2 (e.g. HLF ECso fold change of 9,19 and RPTEC ECso fold change of 354), and the present in vitro senolytic Example 1 for PRGL493 (Fig. 1 ; e.g. HLF ECso fold change of 4,1 and RPTEC ECso fold change of 8,25).

[0218] Fig. 2 shows p16 mRNA expression in fat tissue of doxorubicin-aged mice after senolytic treatment. Error bars in Fig. 2 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 and ***P < 0.001.

[0219] In addition, Triacsin C treated mice showed an apathetic behavior in the first 5-10 minutes after administration of the drug that can be attributed to the vasodilative properties of the drug (Yoshida etal., 1982) and thereby poses a dose limiting side effect which was not observed after PRGL493 treatment. Thus, PRGL493 has superior characteristics in vivo in terms of potency, side effects and overall stability of the molecule when compared to the ACSL4 inhibitor Triacsin C.

[0220] Surprisingly, the senolytic effect of PRGL493 was also enhanced (significantly reduced p16 mRNA expression) when compared to Navitoclax, a compound which was previously published as a senolytic drug.

[0221] Conclusion:

[0222] The significantly reduced p16mRNA expression in fat tissue using PRGL493 is an adequate model to confirm advantageousness of PRGL493 in the treatment of diabetes.

[0223] Example 3: In vivo use of PRGL493 in naturally aged mice — low dose

[0224] Naturally aged C57BL6 / J mice of more than 18 months of age typically display signs of aging and loss of physical functions which goes hand in hand with an increased load of senescent cells throughout the body (Baker et al., 2016; Xu et al., 2018). These naturally occurring senescent cells are closest to the situation faced in human clinical trials which is why naturally aged mice are considered the gold standard when it comes to preclinical models for testing senolytics. Either the ACSL4 inhibitor PRGL493 (Castillo et al., 2021) (20 mg / kg) or vehicle (10 % Dimethylacetamide / 40 % 1 ,2- Propanediol / 50 % PEG-400) only was administered on day 0 / 2 / 4 via oral gavage (o.g.) to ~24 months old female C57BL6 / J mice. 8-week-old female C57BL6 / J mice served as a benchmark for senescent cell load.

[0225] When mice were sacrificed on day 11 , samples from several tissues were analyzed for their mRNA expression of the cell cycle inhibitors and senescence markers p2lcipi / wafianc| pi6ink4a Naturally aged mice displayed a robust induction of senescent cells in all tested organs compared to young mice. Treatment with PRGL493 resulted in a significant downregulation of p21 levels in liver, kidney, and spleen (Fig. 3).

[0226] Fig. 3 shows the p21 mRNA expression in liver, kidney, and spleen tissue of naturally aged mice after PRGL493 treatment. Error bars in Fig. 3 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0227] In addition, the percentage of SA-p-gal positive cells was significantly reduced in fat tissue (Fig. 4).

[0228] Fig. 4 shows the SA-0-gal staining of fat tissue of naturally aged mice (24 months of age) after PRGL493 treatment with exemplary micrographs. Error bars in Fig. 4 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0229] The results presented in Fig. 3 and Fig. 4 are adequate models supporting the treatment with PRGL493 inter alia of kidney and liver diseases as well as diabetes.

[0230] Example 4: In vivo use of PRGL493 in naturally aged mice — high dose

[0231] The dosage of PRGL493 was 5 * 40 mg / kg o.g. per week and the duration of the treatment phase was 4 treatment weeks (biweekly in the course of 8 weeks). 31 and 30 female C57BL / 6 mice were included per condition (vehicle (10 % Dimethylacetamide / 40 % 1 ,2- Propanediol / 50 % PEG-400), PRGL493) at the age of around 116 weeks as well as 10 young female control mice at the age of 8 weeks as a benchmark for young healthy mice.

[0232] In addition to the quantification of senescent cells in different organs / tissues, the effect on age-associated physical dysfunctions (Xu etal., 2018) and several blood-based parameters indicative for organ functionality were analyzed. This included the following parameters: I) exploratory behavior with the open field test, II) muscle strength with the hanging wire test and III) blood cell counts. All non-invasive tests were performed at the beginning and end of the treatment phase. Weight

[0233] The weight of the mice (naturally aged; 27 months of age) was measured before and after the treatment. Mice treated with vehicle (10 % Dimethylacetamide / 40 % 1 ,2- Propanediol / 50 % PEG-400) lost on average about 1 g during the 8-week treatment period, whereas the PRGL493 treated mice gained in average 1 g (Fig. 5).

[0234] Fig. 5 shows the weight difference between baseline and time of dissection. Error bars in Fig. 5 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=26 / 27).

[0235] Exploratory behavior

[0236] Exploratory behavior was studied using an open field test with a standard protocol measuring several parameters shortly before dissection. Vehicle treated mice showed a pronounced reduction of the total distance travelled and the maximum speed compared to young control mice. Treatment with PRGL493 was able to significantly increase both parameters in old mice (naturally aged; 27 months of age) indicating an improvement in exploratory behavior and physical fitness (Fig. 6).

[0237] Fig. 6 shows the total distance and maximum speed during open field test. Error bars in Fig. 6 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Sidak's post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n= 10 / 27 / 25).

[0238] In addition, the vehicle treated mice showed an elevated level of anxiety-related behavior compared to young control mice as illustrated by an increased path efficiency and a preference for the peripheral area (thigmotaxis), which is also associated with many neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. PRGL493 treated naturally aged mice (27 months of age) in contrast remained explorative (Fig. 7).

[0239] Fig. 7 shows the anxiety-related behavior during open field test. Fig. 7A shows the quantification of path efficiency. Fig. 7B shows the representative track plots. Error bars in Fig. 7 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Sidak's post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n= 10 / 27 / 25). Muscle strength

[0240] Muscle strength was assessed with a hanging wire test shortly before dissection using a standard protocol. While the hanging time and the weight adjusted hanging impulse for the vehicle group was greatly reduced compared to young control mice, the PRGL493 treated mice showed a massive improvement in both parameters compared to the vehicle group. Intriguingly, the PRGL493 treated naturally age mice (27 months of age) nearly reached the level of young control mice indicating a rejuvenation of physical function in naturally aged mice after PRGL493 treatment (Fig. 8).

[0241] Fig. 8 shows the hanging time / impulse during hanging wire test. Error bars in Fig. 8 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Sidak's post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=10 / 26 / 27).

[0242] Pathological abnormalities at dissection

[0243] During dissection a clear difference in the prevalence of pathological abnormalities (cataracts, enlarged spleen, cysts and tumors in spleen, liver, ovaries) was observed between the two treatment groups. Only 8 out of 27 (29,6 %) of the PRGL493 treated naturally age mice (27 months of age) showed one of the assessed pathologies, whereas 18 out of 26 (69,2 %) mice of the vehicle treated group were positive for at least one (Fig. 9).

[0244] Fig. 9 shows the distribution of pathologies in both treatment groups at dissection. T-test and significance levels in Fig. 9 are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 (n=22 / 21). This result was statistically significant when analyzed with an unpaired Mann-Whitney U-test (p=0.025).

[0245] Blood cell count

[0246] A standardized blood cell count was carried out to assess the effects on white and red blood cells. White blood cell (WBC) count was significantly increased in PRGL493 treated naturally aged mice (27 months of age), whereas all other analyzed parameters were not significantly different between the treatment groups. The change in WBC could be attributed to the middle cells, as the number of lymphocytes and granulocytes were not significantly different (Fig. 10).

[0247] Fig. 10 shows the blood cell count analysis at the time of dissection. Error bars in Fig. 10 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P

[0248] < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=25).

[0249] This increase in immune cells stems from a rejuvenation of the hematopoietic stem cell system, as was observed by others after senolytic treatment (Chang et al., 2016).

[0250] Histological analysis of brain tissue

[0251] Histological analysis of brain tissues was performed on vehicle and PRGL493 treated aged mice.

[0252] Fig. 11 shows the SA-0-gal staining of the cortex. Representative micrographs of brain sections stained for SA-P-galactosidase and the quantification of the stained area as percentage of the total analyzed area. Error bars in Fig. 11 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T- test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p

[0253] < 0.0001 (n=20 / 25; n=19 / 24).

[0254] Fig. 12 shows the SA-0-gal staining of the hippocampus. Representative micrographs of brain sections stained for SA-P-galactosidase and the quantification of the stained area as percentage of the total analyzed area. Error bars in Fig. 12 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=20 / 23; n=20 / 24; n=20 / 23; n=18 / 22; n=18 / 24).

[0255] Fig. 13 shows the SA-0-gal staining of the Purkinje cell layer. Representative micrographs of brain sections stained for SA-P-galactosidase and the quantification of the stained area as percentage of the total analyzed area. Error bars in Fig. 13 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=21 / 24).

[0256] Figure 25 shows the SA-0-gal staining of the choroid plexus 4thventricle and the periventricular region. Representative micrographs of brain sections stained for SA-0- galactosidase and the quantification of the stained area as percentage of the total analyzed area or total stained area. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=19 / 21 ; N= 19 / 23). Fig. 14 shows the Oil Red O staining of several brain regions. Representative micrographs of brain sections stained for lipid droplets with Oil Red O and the quantification of the stained area as percentage of the total analyzed area. Error bars in Fig. 14 are presented as mean ± standard error of mean and significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=20 / 21 ; n=20 / 17; n=22 / 22; n=20 / 18).

[0257] First analysis of brain sections showed a global reduction of senescent cells in multiple regions after treatment with PRGL493, as quantified after SA-P-gal staining. Regions affected most comprise the cortex, specifically the orbitofrontal cortex (Fig. 11), several regions of the hippocampus that also showed a strong global trend (p = 0.09) towards reduced SA-0-gal staining (Fig. 12) and the Purkinje cell layer (Fig. 13). Senescent cells in these brain regions are associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (Bussian et al., 2018; Chinta et al., 2018; Herdy et al., 2022; Musi et al., 2018; Zhang et al., 2022).

[0258] In addition, cells with excessive lipid droplet accumulation (analyzed with Oil Red O staining), which is associated with cellular senescence (Lizardo etal., 2017) and many age-related diseases (Bresgen et al., 2023) including neurodegenerative diseases (Parkinson, Alzheimer), were also significantly reduced in multiple regions after PRGL493 treatment (Fig. 14 and 26).

[0259] Figure 14 and 26 show Oil Red O staining of several brain regions. Representative micrographs of brain sections stained for lipid droplets with Oil Red O and the quantification of the stained area as percentage of the total analyzed area. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=14 / 13; n=14 / 13; n=13 / 13; n=13 / 13).

[0260] Furthermore, the number of GFAP (Glial fibrillary acidic protein) positive cells was significantly reduced in the retrospinal cortex by treatment with PRGL493, indicating less pro-inflammatory astrocytes (Palmer et al., 2018). Analysis of the primary branches of the GFAP positive cells further revealed that the number of primary branches was higher in PRGL493 treated mice, indicating a less activated state of the astrocytes (Escartin et al., 2021). Together these results demonstrate a reduction of neuroinflammation in the retrospinal cortex after treatment with PRGL493 (Fig. 27).

[0261] Figure 27 shows GFAP staining of retrospinal cortex. Representative micrographs of brain sections stained for GFAP and the quantification of the number of positive cells per area and number of primary branches. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 (n=9 / 8; n=5 / 5).

[0262] Quantification of senescent cells in different organs / tissues

[0263] * Kidney

[0264] A significant reduction of the expression levels for the widely used senescence marker p16INK4acould be observed after treatment with PRGL493 (Fig. 15).

[0265] Fig. 15 shows the p16 mRNA expression in kidney tissue of naturally aged mice after senolytic treatment. Error bars in Fig. 15 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0266] * Spleen

[0267] A significant reduction of the expression levels for the widely used senescence marker p16INK4acould be observed after treatment with PRGL493 (Fig. 16).

[0268] Fig. 16 shows the p16 mRNA expression in spleen tissue of naturally aged mice after senolytic treatment. Error bars in Fig. 16 are presented as mean ± standard error of mean. Significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0269] * Skin

[0270] A significant reduction of p16INK4amRNA expression levels could be observed in skin cells after treatment with PRGL493 (Fig. 17).

[0271] Fig. 17 shows the p16 mRNA expression in skin tissue of naturally aged mice after senolytic treatment. Error bars in Fig. 17 are presented as mean ± standard error of mean and significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0272] * Liver

[0273] Using immunohistology in liver tissue sections, the results from the low dose study (Fig. 3) regarding the expression levels of p21Cip1 / Waf1were verified on protein expression level from the high dose study (Fig. 28).

[0274] Figure 28 shows immunohistological staining for p21 protein of liver tissue of naturally aged mice after senolytic treatment. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001.

[0275] The massive reduction of senescent cells after the PRGL493 treatment in kidney, spleen, skin and liver qualify PRGL493 as treatment option of related diseases. Furthermore, a massive reduction of interstitial fibrosis and a-SMA positive cells after PRGL493 treatment could be observed, that are indicative of pro-fibrotic myofibroblasts (Fig. 29).

[0276] Figure 29 shows Masson trichrome staining and immunohistological staining for a-SMA protein of liver tissue of naturally aged mice after senolytic treatment. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0277] * Muscle

[0278] In line with the pronounced effects seen on muscle strength with the hanging wire test (Fig. 8) interstitial fibrosis was significantly reduced in muscle tissue after treatment with PRGL493 (Fig. 30).

[0279] Figure 30 shows Masson trichrome staining of muscle tissue of naturally aged mice after senolytic treatment. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001.

[0280] The increased dosage and treatment period used in this study greatly enhanced the effects of the inventive senolytic treatment described herein.. The results show a drastic improvement of overall physical functionality which is manifested in increased weight, muscle strength, mobility, body mass, immune cells, and reduced occurrence of physical abnormalities, such as cataracts, cysts, and tumors. When compared to a similar study the effect sizes exceed the ones observed with the senolytic combination of dasatinib and quercetin (Xu etal., 2018). The data gained herewith are a strong model for use of PRGL493 for the treatment of age-related decline of immune system, age- related sarcopenia, cognitive decline and loss of organ function due to the chronic accumulation of senescent cells.

[0281] The global reduction of SA-P-gal positive cells and cells with excessive lipid droplet accumulation in the brain indicate that PRGL493 is able to reduce the senescent cell load in brain tissue, which makes it a possible treatment option for neurodegenerative diseases with a high unmet clinical need.

[0282] Furthermore, the massive reduction of fibrosis and a-SMA positive cells seen in muscle and liver tissue indicates a highly effective therapeutic treatment not only in liver disease and sarcopenia, but also in different fibrotic diseases due to the strong link between senescence, myofibroblast activation and fibrosis.

[0283] Example 5: In vivo use of PRGL493 in naturally aged mice — Life span analysis

[0284] 32 male and 33 female C57BL / 6 mice were used per condition (i.e. , vehicle (10 % Dimethylacetamide / 40 % 1 ,2- Propanediol / 50 % PEG-400) and PRGL493) at the age of around 113 weeks, due to large inter-gender differences in the life span of mice. The cohort was treated biweekly with 5x PRGL493 (40 mg / kg) or vehicle via oral gavage and the clinical frailty index will be determined every 1-2 months as long as the group size was sufficient for statistical analysis.

[0285] The clinical frailty index consists of 31 parameters, which can be measured non- invasively and without special equipment in 4-6 minutes per laboratory animal. This index correlates well with the life expectancy of the mice and a similar human frailty index (Kane et al., 2016; Whitehead et al., 2014).

[0286] Table 1 Parameters of the clinical frailty index (Whitehead et al., 2014). Frailty index

[0287] Analysis of the frailty index revealed a significant increase in the frailty index score of vehicle treated mice in the first 8 weeks of the life span experiment as expected. In contrast, the PRGL493 treated mice showed a significant reduction in the frailty index score (Fig. 18).

[0288] Fig. 18 shows the frailty index score at baseline and at week 8 of life span study using naturally aged mice (27 months of age). Error bars in Fig. 18 are presented as mean ± standard error of mean and significance was determined with a Wilcoxon matched-pairs signed rank test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=52; n=54).

[0289] The difference in the frailty index score in PRGL493 compared to vehicle treated mice continued to increase and was observable in both sexes, indicating a sex unspecific positive effect on the health span. The vehicle treated mice showed as expected a steady increase of the frailty index score over time, whereas the PRGL403 treated mice showed only a mild increase. Even after 24 weeks of treatment, some of the PRGL493 treated mice showed only a mild increase in the frailty index score as compared to the baseline (Fig. 19). These differences in the health conditions were also outwardly apparent when looking at the appearance of the mice as seen in Fig. 19B. The observable changes in the fur quality and pigmentation also clearly indicate the possible use of PRGL493 for the treatment of age-related hair loss and dyspigmentation.

[0290] Fig. 19 shows the change of frailty index score after 24 weeks of life span study using naturally aged mice (27 months of age). Fig. 19A shows the time course of overall frailty index score (both sexes combined), Fig. 19B shows representative pictures of a vehicle and a PRGL493 treated mouse at the age of 34 months and Fig. 19C shows the change of frailty index score from baseline to week 24 time point individually for female and male. Error bars are presented as mean ± standard error of mean. Significance was determined with an unpaired Mann-Whitney U-test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=64 / 61 , n= 52 / 51 , n=37 / 47, n=33 / 31 , n=24 / 25). Survival curves

[0291] In line with the reduction observed in the frailty index score, the survival curves of the life span experiments demonstrated that treatment with PRGL493 is indeed able to significantly increase the life span of mice (Fig. 20).

[0292] Fig. 20 shows the survival curves of life span study using naturally aged mice (27 months of age). Significance in Fig. 20 was determined with a Log-rank (Mantel-Cox) test (survival curves) or with an unpaired student’s T-test for the mean survival time and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001 (n=31 / 28).

[0293] Example 6: In vivo use of PRGL493 in naturally aged mice — Wound healing

[0294] To investigate the effect of PRGL493 on the regenerative potential and the immune system, a cutaneous wound healing study was performed. 18 male C57BL / 6 mice at the age of 18-20 months were used per condition (i.e., vehicle (10 % Dimethylacetamide / 40 % 1 ,2- Propanediol / 50 % PEG-400) and PRGL493). Animals were treated biweekly with 5x PRGL493 (40 mg / kg) or vehicle via oral gavage for a total of two treatment weeks. This was followed by a 12 days of washout phase. Subsequently mice were wounded with a 6 mm full thickness punch biopsy. Wound area was measured on day 0 / 7 / 15 post wounding. On day 15 post wounding, mice were sacrificed and immune cells from peripheral blood, spleen, bone marrow and brain were isolated and analyzed.

[0295] During the isolation of the immune cells from the bone marrow, the blinded operator observed a pronounced difference in the mechanical stability of the femur between the treatment groups. When snipping off the heads of the femur of vehicle treated aged mice using surgical scissors, the bone was less resistant and more brittle compared to young and aged PRGL493 treated mice. This clearly indicates that PRGL493 treatment was able to counteract age-related bone loss as was previously reported for other senolytic compounds (Farr et al., 2017) and might therefore be suitable for the treatment of age-related osteoporosis.

[0296] The wounds of PRGL493 treated naturally age mice (20 months of age) were closing faster than from vehicle treated mice (Fig. 21).

[0297] Fig. 21 shows the wound area of naturally aged mice after PRGL493 treatment and wounding. Significance in Fig. 21 was determined with a two-way ANOVA or with an unpaired student’s T-test for the T7 time point and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=9 / 8).

[0298] Analysis of immune cells in the peripheral blood revealed a significant increase of total immune cells (CD45+) as well as CD8+ T cells. In addition, the amount of SA-0- gal positive immune cells (B cells, T cells, CD4+ T cells, central memory T cells, CD8+ T cells, CD8+ naive T cells) was significantly decreased after PRGL493 treatment (Fig. 22).

[0299] Fig. 22 shows the analysis of immune cells in the peripheral blood of naturally aged mice (20 months of age) after PRGL493 treatment. Significance in Fig. 22 was determined with an unpaired student’s T-test and significance levels are denoted as: *P

[0300] < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=9 / 8).

[0301] Analysis of immune cells in the spleen revealed an increase of total immune cell s (CD45+) as well as B cells. In addition, the amount of SA-P-gal positive B cells was significantly decreased after PRGL493 treatment (Fig. 23).

[0302] Fig. 23 shows the analysis of immune cells in the spleen of naturally aged mice (20 months of age) after PRGL493 treatment. Significance in Fig. 23 was determined with an unpaired student’s T-test and significance levels are denoted as: *P < 0.05, **P

[0303] < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=9 / 8).

[0304] Analysis of immune cells in the bone marrow revealed a significant increase of CD8+ central memory T cells and a simultaneous decrease in CD8+ effector memory T cells, indicating a favorable shift in the immune cell population (Fig. 24).

[0305] Fig. 24 shows the analysis of immune cells in the bone marrow of naturally aged mice (20 months of age) after PRGL493 treatment. Significance in Fig. 24 was determined with an unpaired student’s T-test and significance levels are denoted as: *P

[0306] < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=9 / 8).

[0307] Example 7: Glomerulonephritis model (Chronic Kidney Disease (CKD) model)

[0308] Using a glomerulonephritis model (Mooslechner et al., 2022), it could further be demonstrated that PRGL493 is highly effective in the treatment of chronic kidney disease by restoring the glomerular filtration rate to the level of young healthy mice (~1000 pL / min / 100g BW). The effect observed was superior to the previously published senolytic compound Navitoclax (ABT-263) (Fig. 31). Figure 31 shows an overview of glomerulonephritis study and glomerular filtration rate at day 32. Significance was determined with a one-way ANOVA followed by a Dunnett’s post hoc test and significance levels are denoted as: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****P < 0.0001 (n=4 / 5 / 4).

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Claims

CLAIMS1 . A composition comprising PRGL493 or a pharmaceutically acceptable salt thereof of the formula A:for therapeutic use in selectively eliminating senescent cells, wherein the senescent cells are characterized by increased intracellular levels of at least one of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid.

2. The composition of claim 1 , wherein PRGL493 is in the form of a solvate.

3. The composition for use according to claim 1 or 2, wherein the senescent cells have an at least 2-fold increased intracellular level of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid compared to a cell of the same type or age known to be non-senescent.

4. The composition for use according to any one of claims 1 to 3, wherein the elimination of senescent cells prevents or delays the onset of a senescence-related disease or condition, or prevents or delays the progression of a senescence-related disease or condition, or promotes the regression of a senescence-related disease or condition, or decreases age related frailty.

5. The composition for use according to claim 4, wherein the senescence- related disease or condition is a cardiovascular disease, atherosclerosis, osteoporosis, ischemic reperfusion injury, stroke, myocardial infarct, osteoarthritis, neurological disorders, neurodegenerative diseases, dementia, cataract, kidney disease, retinopathy,diabetes, lung fibrosis, liver fibrosis, non-alcoholic liver disease, liver steatosis, intervertebral disc degeneration, age-related muscular atrophy, age-related decline of the immune system and decreased efficacy of vaccination, delayed wound healing and chronic wounds, hair loss and loss of hair pigmentation or skin aging.

6. The composition for use according to any one of claims 4, wherein the composition improves the performance of transplants.

7. The composition for use according to any one of claims 4, wherein the composition prevents or attenuates senescence-associated scar formation and fibrosis.

8. The composition for use according to any one of claims 4, wherein the composition ameliorates side effects of chemotherapy and prevents or delays tumor relapse.

9. The composition for use according to any one of claim 1 to 8, wherein said composition is a pharmaceutical composition, further comprising at least one pharmaceutically acceptable excipient and / or carrier.

10. A method of treatment of a disease or condition mediated by senescent cells, wherein the senescent cells are characterized by increased intracellular levels of at least one of phospholipase A2 activity, lysophosphatidylcholine, or arachidonic acid, and which comprises administering to a subject in need of such treatment an effective amount of PRGL493 or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein the disease or condition mediated by senescent cells is cardiovascular disease, atherosclerosis, osteoporosis, ischemic reperfusion injury, stroke, myocardial infarct, osteoarthritis, neurological disorders, neurodegenerative diseases, dementia, cataract, kidney disease, retinopathy, diabetes, lung fibrosis, liver fibrosis, non-alcoholic liver disease, liver steatosis, intervertebral disc degeneration, age-related muscular atrophy, age-related decline of the immune system and decreased efficacy of vaccination, delayed wound healing and chronic wounds, hair loss and loss of hair pigmentation, or skin aging.

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