(19) *EP003643305A1*

(11) EP 3 643 305 A1

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 29.04.2020 Bulletin 2020/18 A61K 31/196 (2006.01) A61K 31/403 (2006.01) A61K 31/405 (2006.01) A61K 31/616 (2006.01) (2006.01) (21) Application number: 18202657.5 A61P 35/00

(22) Date of filing: 25.10.2018

(84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • LAEMMERMANN, Ingo GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 1170 Wien (AT) PL PT RO RS SE SI SK SM TR • GRILLARI, Johannes Designated Extension States: 2102 Bisamberg (AT) BA ME • PILS, Vera Designated Validation States: 1160 Wien (AT) KH MA MD TN • GRUBER, Florian 1050 Wien (AT) (71) Applicants: • NARZT, Marie-Sophie • Universität für Bodenkultur Wien 1140 Wien (AT) 1180 Wien (AT) • Medizinische Universität Wien (74) Representative: Loidl, Manuela Bettina et al 1090 Wien (AT) REDL Life Science Patent Attorneys Donau-City-Straße 11 1220 Wien (AT)

(54) COMPOSITIONS FOR THE ELIMINATION OF SENESCENT CELLS

(57) The invention relates to a composition compris- ing senescent cells. The invention further relates to an ing one or more inhibitors capable of inhibiting at least in vitro method of identifying senescent cells in a subject two of cyclooxygenase-1 (COX-1), cyclooxygenase-2 and to a method of identifying candidate compounds for (COX-2) and lipoxygenase for use in selectively eliminat- the selective elimination of senescent cells. EP 3 643 305 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 3 643 305 A1 2

Description al. 2018; Lewis et al. 2011; Ressler et al. 2006; Yosef et al. 2016), neurodegenerative diseases (Bussian et al. FIELD OF THE INVENTION 2018) and impaired adipogenesis (Xu et al. 2015). Fur- thermore, it was shown that clearance of senescent cells [0001] The present invention relates to the field of se- 5 attenuates the negative effects of irradiation- and chem- nolytics and provides a composition useful for the selec- otherapy-induced senescence and restores tissue func- tive elimination of senescent cells and a method of tionality (Baar et al. 2017; Chang et al. 2016; Dörr et al. screening for senolytic compounds. 2013; Pan et al. 2017). In addition to age-related diseases and disorders, cellular senescence is also associated BACKGROUND OF THE INVENTION 10 with tumor relapse following chemotherapy (Milanovic et al. 2017) and the performance of transplant organs [0002] Senescent cells were found to accumulate in (Braun et al. 2012) highlighting the potential of senolytic tissues and organs during the aging process at close therapies. proximity of age-related pathologies where they play a [0005] The list of senolytic compounds and targets critical role in the development and progression of age- 15 comprises inhibitors of the Bcl-2 family (Chang et al. related diseases and disorders. Clearance of senescent 2016; Pan et al. 2017; Yosef et al. 2016; Zhu et al. 2017; cells in mouse models using either genetic or pharma- Zhu et al. 2016), Hsp90 inhibitors (Fuhrmann-Stroissnigg cological approaches was shown to extend the health et al. 2017), dasatinib (Roos et al. 2016; Schafer et al. span, to prevent or delay the occurrence of senescence- 2017; Zhu et al. 2015), FOXO4 (Baar et al. 2017), OXR1 associated diseases and disorders and the development 20 (Zhang et al. 2018), glucose metabolism (Dörr et al. of frailty. Since then several pharmacological com- 2013), mitochondria-targeted tamoxifen (MitoTam) or re- pounds have been identified which were able to selec- duction of ATP synthase activity with oligomycin A (Hu- tively eliminate senescent cells and are generally re- backova et al. 2018) and several plant derived com- ferred to as "senolytics". pounds, such as quercetin (Roos et al. 2016; Schafer et [0003] Although cellular senescence is a tumor sup- 25 al. 2017; Zhu et al. 2015), fisetin (Zhu et al. 2017), pip- pressive mechanism which plays an important role in em- erlongumine (Wang et al. 2016a) and an alcoholic extract bryonic development (Muñoz-Espín et al. 2013) and of solidago virgaurea (Lämmermann et al. 2018). wound healing (Demaria et al. 2014), the chronic accu- [0006] However, many of the reported senolytics like mulation of senescent cells in organs and tissue during navitoclax and dasatinib have serious side effects and the aging process is believed to be a major driving force 30 most senolytics are not universally effective in all cell for the development and progression of age-related dis- types. Therefore, there is a strong need in the field for eases and disorders. Senescent cells are terminally improved senolytics with less severe side effects and growth arrested either via the p53-p21CIP1 or via the broad range of applications. p16INK4a-Rb axis, accumulate senescence-associated β-galactosidase activity (SA-β-gal) and display a typical 35 SUMMARY OF THE INVENTION morphology (Campisi and d’Adda di Fagagna 2007). When chronically present, they negatively affect the sur- [0007] It is the objective of the present invention, to rounding tissue by secreting a pro-tumorigenic and pro- provide a composition capable of efficiently eliminating inflammatory mixture of cytokines, growth factors and senescent cells. It is a further objective of the present proteases (Acosta et al. 2013; Coppé et al. 2010; Krtolica 40 invention to provide a method to screen for compounds et al. 2001) termed the senescence-associated secretory useful for the selective elimination of senescent cells. phenotype (SASP). [0008] The problem is solved by the present invention. [0004] Genetic mouse models using the p16INK4a pro- [0009] The inventors have shown that senescent cells moter to visualize and selectively eliminate p16INK4a pos- comprise an altered lipid metabolism, which can be ex- itive cells convincingly demonstrated that senescent cells 45 ploited to selectively eliminate senescent cells. In partic- accumulate during the aging process in vivo and that the ular, lyso PC is upregulated in senescent cells, indicating clearance of p16INK4a positive cells increases the health an increased formation of arachidonic acid. Arachidonic span and impairs the development and progression of acid is metabolized to eicosanoids by lipoxygenases and senescence-associated diseases and disorders (Baker cyclooxygenases, thereby preventing cell death due to et al. 2016; Baker et al. 2011). There is compelling evi- 50 high intracellular levels of arachidonic acid. Accordingly, dence for a causal relationship between senescent cells inhibition of lipoxygenases or cyclooxygenases leads to and several age-related diseases and disorders, such as an increase in arachidonic acid in senescent cells and, atherosclerosis (Childs et al. 2016; Roos et al. 2016), thus, to selective elimination of senescent cells. idiopathic pulmonary fibrosis (Lehmann et al. 2017; [0010] According to the invention there is provided a Schafer et al. 2017), osteoporosis (Farr et al. 2017; Zhu 55 composition comprising one or more inhibitors capable et al. 2015), post-traumatic osteoarthritis (Jeon et al. of inhibiting at least two of cyclooxygenase-1 (COX-1), 2017), renal aging (Schmitt and Melk 2017; Valentijn et cyclooxygenase-2 (COX-2) and lipoxygenase, for use in al. 2018), skin aging (Baar et al. 2017; Lämmermann et selectively eliminating senescent cells.

2 3 EP 3 643 305 A1 4

[0011] Specifically, the one or more inhibitors are spe- fluriprofen methyl ester, metamizole, nitroaspirin, melox- cific inhibitors of COX-1 and/or COX-2. icam, flufenamic acid, oxaprozin, tiaprofenic acid, mag- [0012] Specifically, the composition provided herein nesium salicylate, diethylcarbamazine, lornoxicam, car- comprises one or more inhibitors and is capable of inhib- profen, phenylbutazone, nepafenac, antipyrine, antrafe- iting the enzymatic activity of at least two enzymes se- 5 nine, choline magnesium trisalicylate, triflusal, niflumic lected from the group consisting of COX-1, COX-2 and acid, dexibuprofen, aceclofenac, acemetacin, droxicam, lipoxygenase. loxoprofen, tolfenamic acid, dexketoprofen, talniflumate, [0013] According to a specific embodiment, the com- propacetamol, trolamine salicylate, phenyl salicylate, position provided herein comprises at least two inhibitors. bufexamac, glycol salicylate, menthyl salicylate, FK-506, Specifically, the composition provided herein comprises 10 lenalidomide, rofecoxib, valdecoxib, cimicoxib, chlorphe- at least one COX-1 or COX-2 inhibitor and at least one nesin, clodronic acid, seliciclib, drospirenone, triamci- lipoxygenase inhibitor. Specifically, the composition nolone, pomalidomide, parecoxib, firocoxib, aclofenac, comprises at least one COX-1 inhibitor and at least one adapalene, thalidomide, etoricoxib, robenacoxib, asaral- COX-2 inhibitor. Specifically, the composition provided dehyde, zaltoprofen, deracoxib, dexamethasone, pran- comprises a cyclooxygenase inhibitor, selected from the 15 oprofen, amfenac sodium monohydrate, ampiroxicam, group consisting of COX-1 inhibitor, COX-2 inhibitor and NS-398, bismuth subsalicylate, diclofenac diethylamine, COX-1/COX-2 inhibitor, and a lipoxygenase inhibitor. trometamol, rutaecarpine, salicin, fenbufen, xanthohu- Specifically, the composition provided herein comprises mol, flunixin meglumin and nimesulide. at least one COX-1 inhibitor and at least one lipoxygen- [0019] Specifically, one or more inhibitors comprised ase inhibitor, or at least one COX-2 inhibitor and at least 20 in the composition provided herein are lipoxygenase one lipoxygenase inhibitor, or at least one COX-1/COX- and/or FLAP (ALOX5AP) inhibitors selected from the 2 inhibitor and at least one lipoxygenase inhibitor. group consisting of MK886, zileuton, masoprocol, di- [0014] According to a further specific embodiment, the ethylcarbamazine, azelastine, benoxaprofen, nordihy- composition provided herein comprises at least one dual droguaiaretic acid, abietic acid, esculetin, montelukast, cyclooxygenase and/or lipoxygenase inhibitor. Specifi- 25 minocycline, MLN-977, rhein, diacerein, nabiximols, fos- cally, said composition comprises one dual cyclooxyge- tamatinib, AM103, DG031, fiboflapon, AA-861 and atre- nase and/or lipoxygenase inhibitor and optionally at least leuton. one cyclooxygenase or lipoxygenase inhibitor. Specifi- [0020] According to a preferred embodiment, the com- cally, the composition provided herein comprises at least position provided herein comprises at least one cycloox- one cyclooxygenase and at least one lipoxygenase in- 30 ygenase inhibitor selected from the group consisting of hibitor as combination treatment or it comprises at least acetylsalicylic acid, diclofenac, celecoxib, cyclosporin A one dual inhibitor of cyclooxygenase and lipoxygenase, and ibuprofen and at least one lipoxygenase inhibitor se- or any combination thereof. lected from the group consisting of MK886 and zileuton. [0015] According to a further specific embodiment, the Specifically, the composition provided herein comprises composition provided herein comprises one or more in- 35 acetylsalicylic acid and MK886. Specifically, the compo- hibitors capable of inhibiting COX-1 and COX-2. Specif- sition provided herein comprises diclofenac and MK886. ically, the composition provided herein comprises at least Specifically, the composition provided herein comprises one COX-1/COX-2 inhibitor or at least one COX-1 inhib- celecoxib and MK886. Specifically, the composition pro- itor and at least one COX-2 inhibitor. vided herein comprises acetylsalicylic acid, diclofenac or [0016] Specifically, the senescent cells are character- 40 celecoxib and cyclosporin A and MK886. ized by increased intracellular levels of at least one of [0021] Specifically, one or more inhibitors comprised lysophosphatidylcholine, arachidonic acid and phosphol- in the composition provided herein are dual cyclooxyge- ipase A2 activity. nase and lipoxygenase inhibitors, preferably selected [0017] Specifically, the lysophosphatidylcholine is se- from the group consisting of licofelone, darbufelone, CI- lected from the group consisting of 1-steraroyl-2-hy- 45 987, S-2474, KME-4, Chebulagic acid, balsalazide, me- droxy-sn-glycero-3-phosphocholine (LysoSPC) or 1- salazine, sulfasalazine, aminosalicylic acid, palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lys- meclofenamic acid, morniflumate diarylpyrazole deriva- oPPC). tives, thieno[2,3-b]pyridine derivatives, N-substituted 5- [0018] Specifically, one or more inhibitors comprised aminosalicylicylamides, flavocoxid, indolizine deriva- in the composition provided herein are COX-1 and/or 50 tives, LQFM-091, hyperforin, celastrol, BW755C, tepoxa- COX-2 inhibitors, selected from the group consisting of lin, b-boswellic acid, D-002, 2,3-diarylxanthones, pheni- acetylsalicylic acid, diclofenac, celecoxib, cyclosporin A, done and ER-34122. ibuprofen, acetaminophen, indomethacin, nabumetone, [0022] According to a further specific embodiment, the ketorolac, tenoxicam, tolmetin, piroxicam, fenoprofen, composition provided herein comprises an additional etodolac, naproxen, diflunisal, suprofen, bromfenac, ke- 55 compound capable of inhibiting intracellular conversion toprofen, dihomo-gamma-linolenic acid, icosapent, of arachidonic acid. Specifically, said additional com- fluriprofen, mefenamic acid, salsalate, sulindac, salicylic pound leads to an increase of arachidonic acid levels. acid, lumiracoxib, O-acetyl-L-serine, phenacetin, Specifically, said additional compound is any one of a

3 5 EP 3 643 305 A1 6 natural compound, an inhibitor of cytochrome P450, an mide, armodafinil, agomelatine, noscapine, clevidipine, inhibitor of long-chain-fatty-acid-CoA ligase 4 (ACSL4), sulconazole, gefitinib, ticagrelor, ceritinib, floxuridine, an inhibitor of a lysophosphatidylcholine acyltransferase lifitegrast, rhein, diacerein, zucapsaicin, stiripentol, or an inhibitor of a fatty acid elongase or any combination lobeglitazone, dosulepin, manidipine, cimicifuga race- thereof. 5 mose, curcumin, felbamate, piperine, safinamide, irpo- [0023] Specifically, the additional compound is a nat- niazid, oritavancin, masorpocol and pegvisomant. ural compound, preferably selected from the group con- [0025] Specifically, the additional compound is an in- sisting of turmeric, rosemary, ginger, oregano, resvera- hibitor of long-chain-fatty-acid-CoA ligase 4 (ACSL4), trol, curcumin, cannabinoids, ginseng, saponins, terpe- preferably selected from the group consisting of triascin noids, flavonoids, polyphenols, ginkgo biloba, capsaicin, 10 A, triascin B, triascin C, triascin D, troglitazone, ciglita- genistein, kaempferol and quercetin. zone, pioglitazone and rosiglitazone. [0024] Specifically, the additional compound is an in- [0026] Specifically, the additional compound is an in- hibitor of cytochrome P450 (CYP2J, CYP2C, CYP4A, hibitor of a lysophosphatidylcholine acyltransferase, spe- CYP4F), preferably selected from the group consisting cifically an inhibitor of LPCAT1, LPCAT2, LPCAT3, of sulfaphenazole, avasimibe, benzbromarone, rosiglita- 15 LPCAT4, MBOAT2 and/or MBOAT7, and wherein the zone, troglitazone, cervistatin, warfarin, pioglitazone, la- inhibitor of a lysophosphatidylcholine acyltransferase is patinib, trimethoprim, zafirlukast, amodiaquine, nica- preferably selected from the group consisting of N-phe- rdipine, simvastatin, fluvastatin, loratadine, ethinylestra- nylmaleimide derivatives, TSI-01 and thimerosal. diol, irbesartan, quinine, sorafenib, eltrombopag, losar- [0027] Specifically, the additional compound is an in- tan, licofelone, amitriptyline, atorvastatin, mefenamic ac- 20 hibitor of a fatty acid elongase, specifically an inhibitor of id, meloxicam, piroxicam, erlotinib, pazopanib, diethyl- ELOVL2, ELOVL4 and/or ELOVL5, and wherein the in- stilbestrol, enzalutamide, ponatinib, dabrafenib, enasi- hibitor of a fatty acid elongase is preferably selected from denib, lovastatin, montekulast, ketoconazole, felodipine, the group consisting of cycloate, adenosine 5’-hexade- candesartan cilexetil, clotrimazole, mometasone, salm- cylphosphate, endo-1k, (S)-1y and compound 37, 5,5- eterol, raloxifene, fenofibrate, levothyroxine, tamoxifen, 25 dimethyl-3-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1 H- quercetin, oxybutynin, medroxyprogesterone acetate, pyrazol-4-yl)-1-phenyl-3-(trifluoromethyl-3,5,6,7-tet- nifedipine, liotrix, amlodipine, bezafibrate, chloramphen- rahydro-1H-indole-2,4-dione) and (3-endo)-3-(phenyl- icol, cyclosporin, cimetidine, clopidogrel, cholecalciferol, sulfonyl)-N-[4-(trifluoromethyl)phenyl]-8-azabicyc- delavirdine, dextropropoxyphene, etoposide, isoniazid, lo[3.2.1]octane-8-carboxamide. ketoprofen, metronidazole, nilutamide, nilvadipine, par- 30 [0028] According to a further specific embodiment, the oxetine, phenelzine, pravastatin, propafenone, pyrimeth- composition comprises an additional compound capable amine, rofecoxib, rutin, saquinavir, sulfamethoxazole, of manipulating intracellular ATP levels. sulfinpyrazone, tegaserod, terfenadine, thioridazine, [0029] Specifically, the additional compound capable ticlopidine, tioconazole, triazolam, troleandomycin, val- of manipulating intracellular ATP levels is an inhibitor of proic acid, abiraterone, vismodegib, regorafenib, tra- 35 ATP synthases, preferably selected from the group con- metinib, idelalisib, lopinavir, celecoxib, efavirenz, rabe- sisting of oligomycin A, inositol nicotinate, bedaquiline, prazole, teriflunomide, crisaborole, belinostat, topiroxo- efrapeptins, leucinostatins, tentoxin, tnetoxin derivates, stat, candesartan, letermovir, rucaparib, opicapone, na- angiostatin, enterostatin, melittin, IF1, Syn-A2, Syn-C, bilone, fluvoxamine, fluticasone, fluticasone furoate, flu- resveratrol, piceatannol, diethylstilbestrol, 4-acetoami- ticasone propionate, bosutinib, cabozantinib, genistein, 40 do-4’-isothiocyanostilbene-2,2’-disulfonate, 4,4’-D-iso- lenvatinib, atazanavir, bexarotene, deferasirox, quini- thiocyanatostilbene-2,2-disulfonic acid, quercetin, dine, mifepristone, vemurafenib, sildenafil, diclofenac, kaempferol, morin, apigenin, genistein, biochanin A, fluoxetine, valdecoxib, voriconazole, etodolac, sertra- daidzein, epicatechin gallate, epigallocatechin gallate, line, glyburide, acenocoumarol, rosuvastatin, imatinib, proanthocyanidin, curcumin, phloretin, theaflavin, tannic clozapine, diazepam, progesterone, omeprazole, valsar- 45 acid, 4-hydroxy-estradiol, 2-hydroxy-estradiol, 17α-es- tan, bortezomib, nevirapine, azelastine, lornoxicam, phe- tradiol, 17β-estradiol, α-zearalenol, β-zearalenol, oligo- nylbutazone, etravirine, leflunomide, sitaxentan, ami- mycin, venturicidin, apoptolidin, ossamycin, cytovaricin, nophenazone, verapamil, etoricoxib, propofol, sulfamox- peliomycin, tributyltin chloride, tricyclohexyltin hydrox- ole, dicoumarol, diltiazem, histamine, moclobemide, se- ide, triethyltin sulfate, triphenyltin chloride, dimethyltin 3- legiline, parecoxib, doconexent, acetyl sulfisoxazole, flu- 50 hydroxyflavone chloride, diethyltin 3-hydroxyflavone conazole, pantoprazole, desloratadine, miconazole, chloride, dibuthyltin 3-hydroxyflavone bromide, dioctyltin amiodarone, gemfibrozil, probenecid, teniposide, sul- 3-hydroxyflavone chloride, diphenyltin 3-hydroxyflavone fadiazine, capecitabine, fluorouracil, tranylcypromine, chloride, diethyltin 3,5,7,2’,4’-pentahydroxyflavone chlo- anastrozole, atovaquone, cyclizine, dexfenfluramine, di- ride, dibutyltin 3,5,7,2’,4’-pentahydroxyflavone bromide, sulfiram, epinephrine, eprosartan, flecainide, indinavir, 55 diphenyltin 3,5,7,2’,4’-pentahydroxyflavone chloride, methazolamide, nelfinavir, olanzapine, pranlukast, pro- tributyltin 3-hydroxyflavone, triethyllead, aurovertin, cit- methazine, sulfadimethoxine, sulfamethizole, sulfanila- reoviridin, asteltoxin, rhodamine B, rhodamine 123, rhod- mide, sulfapyridine, methimazole, tolcapone, bicaluta- amine 6G, rosaniline, malachite green, brilliant green,

4 7 EP 3 643 305 A1 8 quinacrine, quinacrine mustard, acridine orange, cori- phosphono-butanamide, 2-phosphoglyceric acid, iodoa- phosphine, pyronin Y, dequalinium, safranin O, nile blue cetate, gossypol, bisphosphonate analogs of 1,3-bi- A, ethidium bromide, tetracaine, dibucaine, procaine, sphosphoglyceric acid, benzene hexacarboxylic acid, 3- lidocaine, chlorpromazine, trifluoperazine, procaina- phosphoglyceric acid, phosphonoacetohydroxamic acid, mide, propranolol, octyl guanidine, 1-dansyl amido-3- 5 2-phospho-D-glyceric acid, TLN-232 and CAP-232. dimethypropylamine compounds, cetyltrimethylammoni- [0032] According to a specific embodiment, the com- um, spermine, spermidine, bathophenan throline-metal position provided herein prevents or delays the onset of chelate, 4,4-diphenyl-2,2-bipyridine, 3-(2-pyridyl)-5,6- a senescence-related disease or condition. diphenyl-1,2,4-triazine, atrazine, atrazine amino deriva- [0033] According to a specific embodiment, the com- tive, arsenate, aluminium fluoride, beryllium fluoride, 10 position provided herein prevents or delays the progres- scandium fluoride, vanadate, magnesium fluoride, sion of a senescence-related disease or condition. sulfite, thiophosphate, azide, ANPP, phenylglyoxal, bu- [0034] According to a specific embodiment, the com- tanedione, dansyl chloride, 1-fluoro-2,4-dinitrobenzene, position provided herein promotes the regression of a dicarbopolyborate, almitrine, 5-hydroxy-1,2-naphtalene senescence-related disease or condition. dicarboxylic anhydride, R207910, spegazzinine, n-buta- 15 [0035] Specifically, the senescence-related disease or nol, terachlorosalicylanilide, dihydrostreptomycin, condition is selected from cardiovascular diseases, suramin, Bz-423, DMSO, hypochlorous acid, DDT, dia- atherosclerosis, cancer, osteoporosis, osteoarthritis, zoxide, HNB, N-sulfonyl or N-alkyl substituted tetrahyd- neurological disorders, dementia, cataract, kidney dis- robenzodiazepine derivatives, 4-(N-arylimidazole)-sub- eases, retinopathy, diabetes, lung fibrosis, vertebral skin stituted benzopyran derivatives, N-[1-aryl-2-(1-imidazo- 20 degeneration, age-related muscular atrophy, hair loss lo)ethyl]-cyanoguanidine derivatives, N-[1-aryl-2-(1-imi- and skin aging. dazolo)ethyl]-acylguanidine derivatives, O-[1-aryl-2-(1- [0036] According to a specific embodiment, the com- imidazolo)ethyl]-thiourethane derivatives, dio-9 com- position provided herein improves the performance of plex, ethanol and zinc. transplants. [0030] Specifically, the additional compound capable 25 [0037] According to a specific embodiment, the com- of manipulating intracellular ATP levels is an inhibitor of position provided herein prevents or attenuates senes- ADP/ATP translocases, preferably selected from the cence-associated scar formation and fibrosis. group consisting of clodronic acid, ibipinabant, atracty- [0038] According to a specific embodiment, the com- loside, carboxyatractyloside, bongkrekic acid, isobong- position ameliorates side effects of chemotherapy and krekic acid, MT-21, closantel, CD437, leelamine, 30 prevents or delays tumor relapse. L923-0673, IMD 0354, PI32-0333, S899542, nonactin [0039] Further provided herein is an in vitro method of and S838462. identifying senescent cells in a subject, comprising the [0031] Specifically, the additional compound capable steps of of manipulating intracellular ATP levels is an inhibitor of glycolysis, preferably selected from the group consisting 35 a) providing a sample of said subject, of 2-deoxy-D-glucose, lonidamine, bromopyruvic acid, b) determining the levels of at least one of lysophos- phloretin, quercetin, STF-31, WZB117, 3PO, 3- phatidylcholine, arachidonic acid and/or phospholi- bromopyruvate, dichloroacetate, oxamic acid, NHI-1, ox- pase A2 activity in said sample, ythiamine, imatinib, glucosamine, 6-aminonicotinamide, c) comparing the levels of b) to a reference level, genistein, 5-thioglucose, mannoheptulose, α-chlorohy- 40 wherein the reference level is the level of at least drin, ornidazole, oxalate, glufosfamide, N-(phospho- one of lysophosphatidylcholine, arachidonic acid nacetyl)-L-aspartate, 6-methylmercaptopurine riboside, and/or phospholipase A2 activity in non-senescent CGP 3466B maleate, sodium monofluorophosphate, cells, DASA-58, DL-serine, dichloroacetic acid, sodium dichlo- roacetate, nitrofural, 6-AN, fasentin, benserazide, astra- 45 and wherein an increase of at least 2-fold is indicative of glin, resveratrol, chrysin, GEN-27, apigenin, bis-2-(5- the presence of senescent cells in said sample. phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, CB- [0040] Specifically, an increase in the level of at least 839, azaserine, acivicin, 6-diazo-5-ox-L-norleucine, thi- one of lysophosphatidylcholine, arachidonic acid and azolidine-2,4-dione derivatives, compound 968, R-lipoic phospholipase A2 activity compared to a reference level acid, 1,3,4-thiadiazole compounds, 2-chloropropionate, 50 of at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold or 5- Nov3r, AZD7545, Pfz3, radicicol, mitaplatin, mito-DCA, fold is indicative of the presence of senescent cells in phenylbutyrate, 4,5-diarylisoxazoles, VER-246608, bet- said sample. ulinic acid, pyruvate analogs containing phosphinate or [0041] Even more specifically, an increase in the level phosphonate group, CPI-613, M77976, aromatic DCA of at least one of lysophosphatidylcholine, arachidonic derivatives, furan and thiophene carboxylic acids, riton- 55 acid and phospholipase A2 activity compared to a refer- avir, FX11, oxamate, D-fructose-6-phosphate, 6-phos- ence level, which is at least two-times, three-times, four- phogluconic acid, N-bromoacetyl-aminoethyl phos- times or five-times as high as the respective standard phate, 2-carboxyethylphosphonic acid, N-hydroxy-4- deviation is indicative of the presence of senescent cells

5 9 EP 3 643 305 A1 10 in said sample. Preferably, an increase in the level of at denoted as: *P < 0.05, **P < 0.01 and ***P < 0.001. least one of lysophosphatidylcholine, arachidonic acid Fig. 2: Phospholipases with PLA2 activity and secre- and phospholipase A2 activity compared to a reference tory phospholipase A2 receptor are increased in level, which is at least twice as high as as the respective SIPS HDFs. The transcription of several genes that 5 standard deviation is indicative of the presence of senes- either encode for enzymes with PLA2 activity or are cent cells in the sample of a subject. capable of increasing the intracellular PLA2 activity [0042] Further provided herein is a method of screen- by other means is elevated in stress-induced prema- ing for candidate compounds for eliminating senescent ture senescent HDF, indicating that enzymatic hy- cells, comprising the steps of drolysis is a likely source for the high levels of lyso 10 PC in senescent cells. PLA2G4A, PLA2G4C and a) bringing at least one test compound in contact PLA2R1 are all known to produce arachidonic acid with a sample of senescent cells, as product besides the lyso PCs during this process. b) measuring the level of arachidonic acid and/or ap- Data represent the average of three experiments. optosis and/or measuring cell viability, and Significance levels were denoted as: *P < 0.05, **P c) selecting the compounds which cause intracellular 15 < 0.01 and ***P < 0.001. accumulation of arachidonic acid, apoptosis or re- Fig. 3: Overview of the pathway exploited by the se- duced cell viability in the senescent cells treated in nolytic compositions described herein: In senescent a) compared with untreated senescent cells. cells the lipid metabolism, specifically the metabo- lism of arachidonic acid, is altered. Senescent cells [0043] According to a specific embodiment, the com- 20 comprise increased levels of arachidonic acid, which pound used in the composition described herein is iden- is metabolized into eicosanoids by e.g. COX and tified according to the screening method described here- ALOX. Inhibition of COX and ALOX leads to accu- in. mulation of arachidonic acid in senescent cells, [0044] According to a specific embodiment, the com- which in turn leads to induction of apoptosis. position for use in selectively eliminating senescent cells 25 Fig. 4: Senolytic effect of the COX-1/2 inhibitor ASA comprises at least one cyclooxygenase inhibitor inhibit- and ALOX-inhibitor MK886 alone versus prior-art se- ing or eliminating COX-1 and COX-2 activity. Specifically, nolytics Quercetin and Navitoclax. MK886, ASA and the composition provided herein comprises at least one reported senolytics Quercetin and Navitoclax were cyclooxygenase inhibitor and at least one lipoxygenase tested on H2O2-induced premature senescent hu- inhibitor, wherein the subject suffering from or being at 30 man dermal fibroblasts (SIPS) as compared to con- risk of developing a senescence-related disease or con- trol quiescent cells (Q). MK886 and ASA showed a dition is female. significant senolytic effect in SIPS HDFs, whereas [0045] According to a specific embodiment, the com- this was not the case for Quercetin or Navitoclax. position for use in selectively eliminating senescent cells Error bars are presented as mean 6 standard devi- comprises at least one cyclooxygenase inhibitor inhibit- 35 ation. Data represent the average of six experiments. ing COX-1 and COX-2. Specifically, the composition pro- Significance levels were denoted as: *P < 0.05, **P vided herein comprises at least one cyclooxygenase in- < 0.01 and ***P < 0.001. hibitor inhibiting COX-1 and COX-2, wherein the subject Fig. 5: Senolytic effect of the COX-2/ALOX dual in- suffering from or being at risk of developing a senes- hibitor Licofelone and the COX-1/2 inhibitor ASA cence-related disease or condition is male. 40 alone versus prior-art senolytics. Licofelone, ASA and reported senolytics Quercetin and Navitoclax FIGURES were tested on doxorubicin-induced premature se- nescent HUVECs (SIPS) as compared to control qui- [0046] escent cells (Q). Licofelone, ASA as well as Navito- 45 clax showed a significant senolytic effect in SIPS HU- Fig. 1: Lysophosphatidylcholines are elevated in se- VECs, whereas this was not the case for Quercetin. nescent cells. (a) Intracellular levels of lyso PPC Error bars are presented as mean 6 standard devi- (16:0 lyso PC) and lyso SPC (18:0 lyso PC) are in- ation. Data represent the average of six experiments. creasing during the replicative lifespan of HDF from Significance levels were denoted as: *P < 0.05, **P three different donors, mirroring the increased per- 50 < 0.01 and ***P < 0.001. centage of replicative senescent cells in HDF cul- Fig. 6: Synergistic effect of combined inhibition of tures at high population doublings (PD). (b) Intrac- cyclooxygenase and lipoxygenase in HDF161. Dox- ellular levels of lyso PPC (16:0 lyso PC) and lyso orubicin-induced premature senescent HDF161 SPC (18:0 lyso PC) are increasing in HDF after in- (SIPS) and quiescent control cells (Q) were either duction of telomere-independent stress-induced 55 treated with the ALOX-5 inhibitor MK886, the COX- premature senescence. Error bars are presented as 1/2 inhibitors ASA and Diclofenac or the COX-2 spe- mean 6 standard deviation. Data represent the av- cific inhibitor Celecoxib alone or in combination using erage of three experiments. Significance levels were either 0.4 mM ASA, 50 mM Diclofenac or 2 mM

6 11 EP 3 643 305 A1 12

Celecoxib combined with increasing concentrations mycin A showed a synergistic effect by reducing the of MK886.The inhibition of a single AA-metabolizing EC50 value of senescent cells and increasing the fold enzyme was not sufficient to decrease the cell via- change between the EC50 of senescent versus qui- bility in senescent cells as seen with Celecoxib escent cells. Error bars are presented as mean 6 alone. The combination of MK886 with any cycloox- 5 standard deviation. Data represent the average of ygenase inhibitor (COX-1/2 or COX-2-specific) three experiments. Significance levels were denoted showed a synergistic effect, demonstrated by the re- as: *P < 0.05, **P < 0.01 and ***P < 0.001. duction in the EC50 value of senescent cells and the Fig. 10: Synergistic effect of combined inhibition of increase in the fold change between the EC50 of se- cyclooxygenase and lipoxygenase in fHDF166 can nescent versus quiescent cells. Error bars are pre- 10 be further enhanced by addition of the calcineurin sented as mean 6 standard deviation. Data repre- inhibitor Cyclosporin A. Doxorubicin-induced prema- sent the average of three experiments. Significance ture senescent fHDF166 (SIPS) and quiescent con- levels were denoted as: *P < 0.05, **P < 0.01 and trol cells (Q) were treated with 0.4 mM ASA combined ***P < 0.001. with increasing concentrations of MK886 alone or in Fig. 7: Synergistic effect of combined inhibition of 15 combination with 2 mM Cyclosporin A. The combi- cyclooxygenase and lipoxygenase in HDF164. Dox- nation of the ALOX-5 inhibitor MK886 and the COX- orubicin-induced premature senescent HDF164 1/2 inhibitor ASA with the calcineurin inhibitor Cy- (SIPS) and quiescent control cells (Q) were either closporin A, which is known to inhibit COX-2 expres- treated with the ALOX-5 inhibitor MK886 and the sion, showed a synergistic effect by reducing the 20 COX-1/2 inhibitor Diclofenac alone or in combination EC50 value of senescent cells and increasing the fold using either 0.4 mM ASA or 50, 100 or 200 mM Di- change between the EC50 of senescent versus qui- clofenac combined with increasing concentrations escent cells. Error bars are presented as mean 6 of MK886. The combination of MK886 with a cycloox- standard deviation. Data represent the average of ygenase inhibitor showed a synergistic effect by re- three experiments. Significance levels were denoted 25 ducing the EC50 value of senescent cells and in- as: *P < 0.05, **P < 0.01 and ***P < 0.001. creasing the fold change between the EC50 of se- Fig. 11: Combinatorial treatment of SIPS HDFs with nescent versus quiescent cells. Error bars are pre- Cyclosporin A enhances the senolytic effect of sented as mean 6 standard deviation. Data repre- COX/ALOX-inhibitors. Microscopic imaging reveals sent the average of three experiments. Significance that combination treatment using MK886 and ASA levels were denoted as: *P < 0.05, **P < 0.01 and 30 induces lysis of senescent cells ("SIPS") but not in ***P < 0.001. control cells ("Q"). The senolytic effect of MK886 and Fig. 8: Synergistic effect of combined inhibition of ASA in SIPS is further increased upon addition of cyclooxygenase and lipoxygenase in fHDF166. Dox- cyclosporine A. Such effect is not observed in control orubicin-induced premature senescent fHDF166 cells. Microscopic pictures at 100 x magnification. (SIPS) and quiescent control cells (Q) were either 35 Scale bar, 100 mm. treated with the ALOX-5 inhibitor MK886 and the Fig. 12: Intracellular levels of AA are elevated in se- COX-1/2 inhibitors ASA and Diclofenac alone or in nescent HDFs (SIPS) compared to quiescent HDFs combination using either 0.4 mM ASA or 50 mM Di- (Q) and blocking of eicosanoid synthesis increases clofenac combined with increasing concentrations the intracellular levels of AA. (a) Intracellular levels of MK886. The combination of MK886 with a cycloox- 40 of AA of doxorubicin-induced senescent HDF161 ygenase inhibitor showed a synergistic effect by re- compared to quiescent HDF161. (b) Intracellular lev- ducing the EC50 value of senescent cells and in- els of AA of HDF161 treated for 6 hrs with the COX- creasing the fold change between the EC50 of se- 1/2 inhibitor Diclofenac (100 mM) and the ALOX-5 nescent versus quiescent cells. Error bars are pre- inhibitor MK886 (10 mM) or with the solvent only (1% sented as mean 6 standard deviation. Data repre- 45 DMSO). Data represent one experiment. sent the average of three experiments. Significance levels were denoted as: *P < 0.05, **P < 0.01 and DETAILED DESCRIPTION ***P < 0.001. Fig. 9: Synergistic effect of combined inhibition of [0047] Unless indicated or defined otherwise, all terms cyclooxygenase and lipoxygenase in HDF164 can 50 used herein have their usual meaning in the art, which be further enhanced by addition of the ATP synthase will be clear to the skilled person. Reference is for exam- inhibitor Oligomycin A. Doxorubicin-induced prema- ple made to the standard handbooks, such as Sambrook ture senescent HDF164 (SIPS) and quiescent con- et al, "Molecular Cloning: A Laboratory Manual" (2nd trol cells (Q) were treated with 0.4 mM ASA combined Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press with increasing concentrations of MK886 or in com- 55 (1989); Lewin, "Genes IV", Oxford University Press, New bination with 5 mM Oligomycin A. The combination York, (1990), and Janeway et al, "Immunobiology" (5th of the ALOX-5 inhibitor MK886 and the COX-1/2 in- Ed., or more recent editions), Garland Science, New hibitor ASA with the ATP synthase inhibitor Oligo- York, 2001.

7 13 EP 3 643 305 A1 14

[0048] Specific terms as used throughout the specifi- of p16 or p21 or any combination thereof; or a cell or cation have the following meaning. subject shows a process that causes those described [0049] The terms "comprise", "contain", "have" and "in- above. clude" as used herein can be used synonymously and [0057] The senescent cell may further show a de- shall be understood as an open definition, allowing fur- 5 crease in autophagy activity or a decrease in mitochon- ther members or parts or elements. "Consisting" is con- drial membrane potential, or shows a process that caus- sidered as a closest definition without further elements es those described above. Compared to a cell or subject of the consisting definition feature. Thus "comprising" is such as a known senescent cell or subject, a non-senes- broader and contains the "consisting" definition. cent cell or subject may show an increase in cell prolif- [0050] The term "about" as used herein refers to the 10 eration ability, a decrease in lipofuscin accumulation, a same value or a value differing by +/-5 % of the given decrease in β-galactosidase activity, or a combination value. thereof. Specifically, in the case of a human, a cell or [0051] Singular and plural forms can be used inter- sample that is taken from a person about 30 years or changeably herein if not otherwise indicated. older, about 40 years or older, about 50 years or older, [0052] As used herein, the term "subject" or "individual" 15 about 60 years or older, about 70 years or older, about or "patient" shall refer to a warm-blooded mammalian, 80 years or older, about 90 years or older, may be defined particularly a human being. Alternatively, it may also be as a senescent cell or sample. In certain cases a cell or an animal, for example mouse, rat, dog, cat, swine, bo- sample that is taken from a person about 30 years or vine, or a non-human primate. younger, 20 years or younger, 10 years or younger or [0053] The term "patient" includes human and other 20 even at the embryonic stage, may be defined as a se- mammalian subjects that receive either prophylactic or nescent cell or sample. Specifically, a cell or sample that therapeutic treatment for a senescence-associated con- is taken from a human child suffering from or at risk of dition or disease or are diagnosed of cellular senescence developing a senescence-related disease or condition, or a senescence-associated condition or disease. such as for example diabetes, specifically type I diabetes, [0054] The term "sample" generally refers to tissue or 25 may be defined as a senescent cell or sample. organ sample, blood, cell-free blood such as serum and [0058] The terms "cellular senescence" and "senes- plasma, platelet-poor plasma, lymph, urine, saliva and cent cells" thus refer to the essentially irreversible growth biopsy probes. arrest that occurs when cells that can divide encounter [0055] Senescent cells were found to accumulate in critically short telomeres, oncogenic stress or DNA dam- tissues and organs in response to internal or external 30 age or experience strong mitogenic signals, such as but stress as well as during the aging process. They play a not limited to oncogenes or highly expressed pro-prolif- critical role in the development and progression of se- erative genes and a senescent cell, which is a potentially nescence-related diseases and disorders. Several phar- persisting cell that is metabolically active and has under- macological compounds able to eliminate senescent gone widespread changes in protein expression. cells have been identified; however, most senolytics 35 [0059] The term "senescent cells" specifically refers to cause serious side effects, exhibit senolytic activity only cells that express a marker or combination of markers in specific cell types or lack sufficient effectiveness. Ac- that are characteristic of senescence. According to a spe- cordingly, there is an urgent need for improved senolyt- cific embodiment, such marker is any one or more of ics. lysophosphatidylcholine, specifically lyso PPC and/or [0056] Senescence refers to the gradual deterioration 40 lyso SPC, arachidonic acid and phospholipase A2 activ- of functional characteristics. Cellular senescence occurs ity. Specifically, such marker is an increase in the level in culture and in vivo as a response to extracellular or of any one or more of lysophosphatidylcholines, prefer- intracellular stress and/or due to aging. The senescence ably lyso PPC and/or lyso SPC, arachidonic acid and response locks cells into a cell-cycle arrest that prevents phospholipase A2 activity. In some embodiments, a se- the propagation of damaged cells and precludes potential 45 nescent cell expresses other markers including but not malignant transformation. Senescence refers to an array limited to increased expression relative to a reference, of changes that occurs over time. Compared to a refer- such as a non-senescent cell, in the levels of DNA-dam- ence cell (e.g., a cell or sample of the same type or age age response (DDR) markers, co-localization of DNA known to be non-senescent), a senescent cell is defined damage proteins like 53BP1 or gammaH2AX with telom- as a cell that shows one, two, three, four, five, six or more 50 eres, as well as the cell cycle inhibitors p16INK4A, or all of any of the following features: a decrease in cell p15INK4B, p21CIP1, and p53. DEC1, DCR2, and PAI1 can proliferation ability; an accumulation of lipofuscin (e.g., also be used as senescence biomarkers. In one embod- increase in lipofuscin accumulation); an increase in beta- iment, senescent cells express SA-beta-Gal (senes- galactosidase activity; an increase in the secretion of cence-associated beta galactosidase) to an extent that members of the senescence-associated secretory phe- 55 staining with X-Gal at pH=6 results in a blue color. notype (SASP), an increase of mitochondrial-derived re- [0060] Stress, the natural and in vivo equivalent of active oxygen species; an increase in nuclear DNA dam- which are unknown, causes a senescence arrest without age foci; a shortening of telomeres; increased expression significant telomere erosion. These stresses may include

8 15 EP 3 643 305 A1 16 inappropriate substrate, e.g., tissue culture plastic, se- normal cells or circulating angiotensin II, have also been rum (most cells experience plasma, not serum, in vivo), shown to induce cellular senescence. All somatic cells serum withdrawal, DNA-damage inducing substances or that have the ability to divide can undergo senescence. oxidative stress, e.g., culture in atmospheric O2, which Regardless of the disparate mechanisms of senescence- is hyperphysiological or exposure to substances that pro- 5 inducing stresses, the senescence program is activated duce reactive oxygene species, such as hydrogenperox- once a cell has sensed a critical level of damage or dys- ide, paraquat or tert-Butyl hydroperoxide. Cells also enter function. So far, the senescence growth arrest has been senescence upon loss of the PTEN tumor suppressor, a shown to depend on the activities of the major tumor- phosphatase that counteracts pro-proliferative/pro-sur- suppressor pathways controlled by p16INK4a and pRB vival kinases. Additionally, ectopic expression of the cy- 10 (retinoblastoma protein), as well as by p53. Some of the clin-dependent kinase inhibitors (CDKis) that normally molecules involved in pathways upstream and down- enforce the senescence growth arrest, notably p21WAF1 stream of the senescence-associated phenotype have and/or p16INK4a, may cause senescence. been used as markers to detect senescent cells in culture [0061] Aging is a combination of processes of deteri- and in vivo. oration that follow the period of development of an or- 15 [0064] As described herein, senescent cells have an ganism. Aging is generally characterized by a declining altered lipid metabolism. Specifically, phospholipase A2 adaptability to stress, increased homeostatic imbalance, activity is upregulated. According to a specific example, increase in senescent cells, and increased risk of dis- the secretory phospholipase A2 receptor (PLA2R1) is ease. Because of this, death is the ultimate consequence upregulated, which induces the production of lyso PCs, of aging. Environmental factors may affect aging, for ex- 20 leading to a concurrent release of arachidonic acid (AA). ample, overexposure to ultraviolet radiation accelerates Specifically, senescent cells thus have an increased AA skin aging. Different parts of the body may age at different formation rate. According to a further specific example, rates. Two organisms of the same species can also age lysophosphatidylcholine is upregulated. In particular, 1- at different rates, making biological aging and chrono- stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 logical aging distinct concepts. 25 lyso PC) and 1-palmitoyl-2-hydroxy-sn-glycero-3-phos- [0062] Acceleration of the rate of aging may be induced phocholine (16:0 lyso PC) are upregulated independent by stress conditions including, but not limited to chemical, of whether the senescence inducer is telomere depend- physical, and biological stresses. For example, acceler- ent (replicative senescence, aging) or independent ated aging can be induced by stresses caused by UV (stress-induced premature senescence). and IR irradiation, drugs and other chemicals, chemo- 30 [0065] Surprisingly, targeting said altered lipid metab- therapy, intoxicants, such as but not limited to DNA in- olism of senescent cells using a composition capable of tercalating and/or damaging agents, oxidative stressors inhibiting at least two of COX-1, COX-2 and lipoxygen- etc; mitogenic stimuli, oncogenic stimuli, toxic com- ase, efficiently and selectively eliminates senescent cells pounds, hypoxia, oxidants, exposure to environmental of different cell types. Advantageously, numerous cy- pollutants, for example, silica, exposure to an occupa- 35 clooxygenase and lipoxygenase inhibitors exist which tional pollutant, for example, dust, smoke, asbestos, or are well-studied pharmacological compounds and gen- fumes. All these stressors alone or in combination can erally have only mild side effects. also cause cellular senescence. Specifically, senes- [0066] The term "phospholipase A2" or "PLA2" as used cence is induced by combinations of stresses, e.g., two herein refers to a superfamily of lipases with PLA2 activ- or more chemical and physical stresses; two or more 40 ity. PLA2 activity refers to the ability of PLA2 enzymes chemical and biological stresses; two or more physical to hydrolyze the fatty acid from the sn-2 position of mem- and biological stresses; chemical, physical, and biologi- brane phospholipids, such as for example phosphatidyl- cal stresses in combination, etc. choline. The PLA2 superfamily comprises four main [0063] Cellular senescence may also be caused by te- types of enzymes including the secreted PLA2 (sPLA2), lomeric dysfunction (telomere uncapping) resulting from 45 cytosolic PLA2 (cPLA2), calcium-independent PLA2 repeated cell division (termed replicative senescence), (iPLA2), and platelet activating factor (PAF) acetyl hy- mitochondrial deterioration, oxidative stress, severe or drolase/oxidized lipid lipoprotein associated PLA2 irreparable DNA damage and chromatin disruption (ge- ((Lp)PLA2). Specifically, PLA2s, in particular sPLA2s notoxic stress), and the expression of certain oncogenes and cPLA2s, are known to hydrolyze phosphatidylcho- (oncogene-induced senescence). Stresses that cause 50 lines, which produces lysophosphatidylcholines and con- cellular senescence can be induced by external or inter- currently releases arachidonic acid. nal chemical and physical insults encountered during the [0067] The term "lysophosphatidylcholine" or "lyso course of the life span, during therapeutic interventions PC" as used herein refers to a chemical compound de- (for example, X-irradiation or chemotherapy), or as a con- rived from phosphatidylcholine, which is normally located sequence of endogenous processes such as oxidative 55 in the cell membrane. Lyso PC can be generated from respiration and mitogenic signals. External mitogenic sig- phosphatidylcholines (PC) either non-enzymatically nals, for example growth-related oncogene alpha through oxidation processes or enzymatically through (GROα) secretion by tumor cells in close proximity to conversion by phospholipases with PLA2 activity. Spe-

9 17 EP 3 643 305 A1 18 cifically, Lyso PC can be generated by cytosolic phos- tion destroys or kills a senescent cell in a biologically, pholipase A2 (PLA2G4A), cytosolic phospholipase A2 clinically, and/or statistically significant manner com- gamma (PLA2G4C) and/or group XV phospholipase A2 pared with its capability to destroy or kill a non-senescent (PLA2G15). Lyso PCs can have different combinations cell. Specifically, the senolytic composition described of fatty acids of varying lengths and saturation attached 5 herein alters the lipid metabolism of senescent cells by at the C-1 (sn-1) position. Fatty acids containing 16, 18 targeting cyclooxygenases and lipoxygenases in a man- and 20 carbons are the most common. 18:0 lyso PC, also ner that induces (initiates, stimulates, triggers, activates, called lyso SPC, in particular, consists of one chain of promotes) and results in (i.e., causes, leads to) death of stearic acid at the C-1 position. 16:0 lyso PC, also called the senescent cell. Additionally, the senolytic composi- lyso PPC, in particular, consists of one chain of palmitic 10 tion described herein may alter, for example, a cell sur- acid at the C-1 position. vival signaling pathway (e.g., Akt pathway) or an inflam- [0068] The term "arachidonic acid" or "AA" as used matory pathway, for example, by antagonizing a protein herein refers to a polyunsaturated omega-6 fatty acid. within the cell survival and/or inflammatory pathway in a Specifically, AA is present in phospholipids, especially senescent cell. phosphatidylcholine, of membranes of the body’s cells. 15 [0072] The term "cyclooxygenase inhibitor" or "COX In addition to being involved in cellular signaling as a lipid inhibitor" as used herein refers to any compound able to second messenger involved in the regulation of signaling bind to and inhibit, prevent or reduce cyclooxygenase enzymes, arachidonic acid is a key inflammatory inter- activity or to selectively inhibit, prevent or reduce COX- mediate and can also act as a vasodilator. Specifically, 1 or COX-2 activity, or any combination thereof. Specif- AA is released from phosphatidylcholines upon hydroly- 20 ically, cyclooxygenase (COX), officially known as pros- sis of phosphatidylcholines into lysophosphatidylcho- taglandin-endoperoxide synthase (PTGS), is an enzyme lines by lipases with PLA2 activity. that is responsible for formation of prostanoids, including [0069] AA is the precursor that is metabolized by var- thromboxane and prostaglandins such as prostacyclin, ious enzymes to a wide range of eicosanoids and me- from arachidonic acid. There are two isozymes of COX tabolites of eicosanoids. Specifically, AA is further me- 25 encoded by distinct gene products: a constitutive COX- tabolized to eicosanoids by cyclooxygenases and lipox- 1 and an inducible COX-2. ygenases. For example, the enzymes cyclooxygenase- [0073] Classical COX inhibitors are not selective and 1 and -2 (COX-1 and COX-2) metabolize arachidonic ac- inhibit all types of COX. The most frequent adverse effect id to prostaglandin G2 and prostaglandin H2, 5-lipoxy- is irritation of the gastric mucosa as prostaglandins nor- genase metabolizes arachidonic acid to 5-hydroperoxy- 30 mally have a protective role in the gastrointestinal tract. icosatetraenoic acid (5-HPETE), which in turn is metab- Newer COX inhibitors exhibit selectivity for COX-2 or for olized to various leukotrienes, 15-lipoxygenase-1 COX-1 or preferentially inhibit one of COX-2 or COX-1 (ALOX15) and 15-lipoxygenase-2 (ALOX15B) metabo- over the other. Because COX-2 is usually specific to in- lize arachidonic acid to 15-hydroperoxyicosatetraenoic flamed tissue, there is much less gastric irritation asso- acid (15-HPETE) and 12-lipoxygenase (ALOX12) me- 35 ciated with COX-2 inhibitors, with a decreased risk of tabolizes arachidonic acid to 12-hydroperoxyeicosa- peptic ulceration. tetraenoic acid (12-HPETE). [0074] Specifically, examples of non-selective COX in- [0070] Conversion of AA into eicosanoids or metabo- hibitors include acetylsalicylic acid or derivatives thereof lites of eicosanoids is essential, because intracellular ac- such as NO-aspirin (nitro derivative of acetylsalicylic ac- cumulation of AA has a toxic effect on cells. For example, 40 id), and salicylic acid, or derivatives thereof such as mag- AA released by phospholipase A2-activity can trigger ap- nesium salicylate, choline magnesium trisalicylate, optosis through the mitochondrial apoptotic pathway. As trolamine salicylate, phenyl salicylate, glycol salicylate, described herein, senescent cells have an increased AA menthyl salicylate, bismuth subsalicylate, and O-acetyl- formation rate, because PLA2 and lyso PC are upregu- L-serine, diclofenac and derivatives thereof such as e.g. lated. Therefore, inhibition of the metabolism of AA into 45 diclofenac diethylamine, ibuprofen, suprofen, ketopro- eicosanoids increases AA levels in senescent cells to a fen, fluriprofen, fluriprofen methyl ester, fenoprofen, car- greater extent than in non-senescent cells, which leads profen, dexibuprofen, loxoprofen, zaltoprofen, pranopro- to selective elimination of senescent cells. Specifically, fen, indomethacin, ketorolac, drugs of the oxicam class senescent cells are selectively eliminated through cell such as tenoxicam, piroxicam, lornoxicam, droxicam, death triggered by increased AA levels. 50 and tolmetin, naproxen, diflunisal, salsalate, metamizole [0071] The term "selectively eliminating" refers to the (dipyrone), oxaprozin, tiaprofenic acid, diethylcar- exposure of the cells or subjects to a senolytic composi- bamazine, phenylbutazone, nepafenac (prodrug of am- tion comprising at least one cyclooxygenase inhibitor and fenac), antrafenine, acemetacin, tolfenamic acid, dexke- at least one lipoxygenase inhibitor, which induces lysis toprofen trometamol, talniflumate, propacetamol, bufex- of senescent cells. Senolytic compositions described 55 amac, chlorphenesin, clodronic acid, amfenac sodium herein are compositions that selectively eliminate senes- monohydrate, ampiroxicam, salicin, fenbufen, xanthohu- cent cells, preferably by killing senescent cells e.g. by mol, sulindac, dihomo-gamma-linolenic acid and flunixin inducing apoptosis. In other words, a senolytic composi- meglumin.

10 19 EP 3 643 305 A1 20

[0075] Specifically, examples of selective COX-2 in- ased. Leukotriene formation is under control of the hor- hibitors include cyclosporine A, celecoxib, cimicoxib, lu- mone testosterone, which regulates the subcellular lo- miracoxib, rofecoxib, valdecoxib, parecoxib, firocoxib, calization of ALOX-5, the key enzyme in the biosynthesis etoricoxib, robenacoxib, deracoxib, acetaminophen (pa- of pro-inflammatory eicosanoids. In individuals with in- racetamol), nabumetone, etodolac, bromfenac, icosap- 5 creased testosterone levels, such as males, testosterone ent, meloxicam, flufenamic acid, mefenamic acid, triflu- inhibits the translocation of ALOX-5 from the cytoplasm sal, niflumic acid, aceclofenac, specifically, as prodrug to the cell membrane, thus inhibiting its arachidonic acid for diclofenac, Tacrolimus (also known as fujimycin or metabolic activity. FK-506), lenalidomide, roscovitine (seliciclib), dros- [0081] According to a specific embodiment of the in- pirenone, triamcinolone, pomalidomide, adapalene, tha- 10 vention, the composition provided herein comprises a lidomide, asaraldehyde, dexamethasone, NS-398, ru- cyclooxygenase inhibitor and a lipoxygenase inhibitor, taecarpine and nimesulide. except for only those specific uses for which it comprises [0076] Specifically, examples of inhibitors selective for only one or more cyclooxygenase inhibitors. For exam- COX-1 or its isoform COX-3 include phenacetin, antipy- ple, one of such specific use for which the composition rine, aminopyrine, SC-560 and mofezolac. 15 provided herein comprises only cyclooxygenase inhibi- [0077] Specifically, many natural compounds also ex- tors is treatment of a sex-biased senescence-related dis- hibit COX-inhibitory effects. Culinary mushrooms, like ease or condition. Specifically, wherein the subject suf- maitake, are able to partially inhibit COX-1 and COX-2, fering from or at risk of developing a senescence related a variety of flavonoids have been found to inhibit COX- disease or condition is male, the composition for use in 2, hyperforin has been shown to inhibit COX-1 around 20 selectively eliminating senescent cells comprises at least 3-18 times as much as aspirin, calcitriol (vitamin D) sig- one COX-1/COX2 inhibitor or a COX-1 inhibitor and a nificantly inhibits the expression of the COX-2 gene and COX-2 inhibitor. However, treatment of male subjects fish oils provide alternative fatty acids to arachidonic acid. with a composition comprising both, cyclooxygenase in- These acids can be turned into some anti-inflammatory hibitor and lipoxygenase inhibitor, is not excluded. prostacyclins by COX instead of pro-inflammatory pros- 25 [0082] Examples of lipoxygenase and/or FLAP inhibi- taglandins. tors include but are not limited to MK886, zileuton, maso- [0078] The term "lipoxygenase inhibitor" as used here- procol, diethylcarbamazine, azelastine, benoxaprofen, in, refers to compounds that bind to and inhibit, prevent nordihydroguaiaretic acid, abietic acid, esculetin, mon- or decrease the activity of any lipoxygenase. Lipoxyge- telukast, minocycline, MLN-977, rhein, diacerein, nabixi- nases are a family of iron-containing enzymes most of 30 mols, fostamatinib, AM103, DG031, fiboflapon, AA-861 which catalyze the dioxygenation of polyunsaturated fat- and atreleuton. ty acids in lipids containing a cis,cis-1,4- pentadiene into [0083] According to a specific embodiment, the com- cell signaling agents. Specifically, the polyunsaturated position described herein comprises at least one dual fatty acid arachidonic acid serves as substrate for lipox- cyclooxygenase/lipoxygenase inhibitor. Specifically, the ygenases. Specifically, arachidonate 5-lipoxygenase (or 35 composition for use in selectively eliminating senescent ALOX-5, or 5-LOX) is capable to metabolize and thereby cells comprises such dual inhibitor when the subject is transform arachidonic acid into eicosanoids, particularly suffering from or is at risk of developing a senescence- 5-hydroxyeicosatetraenoic acid and 5-oxo-eicosatetrae- related disease or condition is a female subject. noic acid. Leukotrienes, a family of eicosanoid inflamma- [0084] Exemplary dual inhibitors include but are not tory mediators, are produced by the oxidation of AA and 40 limited to licofelone, darbufelone, CI-987, S-2474, KME- the essential fatty acid eicosapentaenoic acid (EPA) by 4, Chebulagic acid, balsalazide, mesalazine, sulfasala- ALOX-5. zine, aminosalicylic acid, meclofenamic acid, morniflu- [0079] Arachidonate 5-Lipoxygenase Activating Pro- mate diarylpyrazole derivatives, thieno[2,3-b]pyridine tein (ALOX5AP or FLAP) is a protein which, with 5-lipox- derivatives, N-substituted 5-aminosalicylicylamides, fla- ygenase, is required for leukotriene synthesis. Leukot- 45 vocoxid, indolizine derivatives, LQFM-091, hyperforin, rienes are arachidonic acid metabolites, which have been celastrol, BW755C, tepoxalin,b-boswellic acid, D-002 (a implicated in various types of inflammatory responses. mixture of six higher aliphatic beeswax alcohols), 2,3- This protein localizes to the plasma membrane. Inhibitors diarylxanthones, phenidone and ER-34122. of its function impede translocation of 5-lipoxygenase [0085] According to a specific embodiment, the com- from the cytoplasm to the cell membrane and inhibit 5- 50 position described herein comprises at least one cycloox- lipoxygenase activation. An example of an ALOX5AP in- ygenase and at least one lipoxygenase inhibitor and an hibitor is MK886. MK-886, or L-663536, is a leukotriene additional compound capable of inhibiting intracellular antagonist, which exerts its action by blocking the 5-lipox- conversion of arachidonic acid. Specifically, such addi- ygenase activating protein, thus inhibiting 5-lipoxygen- tional compound is any one or more or all of a natural ase. Specifically, inhibitors of FLAP (or ALOX5AP) are 55 compound, an inhibitor of cytochrome P450, an inhibitor lipoxygenase inhibitors, which inhibit the enzymatic ac- of long-chain-fatty-acid CoA ligase 4 (ACSL4), an inhib- tivity of lipoxygenase indirectly. itor of a lysophosphatidylcholine acyltransferase and/or [0080] Specifically, leukotriene formation is sex-bi- an inhibitor of fatty acid elongase.

11 21 EP 3 643 305 A1 22

[0086] Specifically, an inhibitor of cytochrome P450 cular atrophy (SMA), Werdnig-Hoffman Disease (SMA1), can be an inhibitor of any one or more or all of cytochrome SMA2, Kugelberg-Welander Disease (SM3), Kennedy’s P450 2J2 (CYP2J2), cytochrome P450 2C (CYP2C), cy- disease, post-polio syndrome, hereditary spastic para- tochrome P450 4A (CYP4A) and/or cytochrome P450 4F plegia, age-related memory decline, and depression and (CYP4F). 5 mood disorders. [0087] Specifically, an inhibitor of lysophosphatidyl- [0093] The senescence-related disease or condition choline acyltransferase (LPCAT) can be an inhibitor of can be an inflammatory condition. Inflammatory condi- any one or more or all of LPCAT1, LPCAT2, LPCAT3, tions include, but are not limited to, musculoskeletal dis- LPCAT4, lysophospholipid acyltransferase 2 (MBOAT2) eases, osteoarthritis, osteoporosis, sarcopenia, lupus, and Membrane Bound O-Acyltransferase Domain Con- 10 interstitial cystitis, scleroderma, alopecia, oral mucositis, taining 7 (lysophospholipid acyltransferase 7 and/or rheumatoid arthritis, inflammatory bowel disease, kypho- MBOAT7). sis, herniated intervertebral disc, ulcerative colitis, [0088] Specifically, an inhibitor of fatty acid elongase Crohn’s disease, ulcerative asthma, renal fibrosis includ- can be an inhibitor of any one or more or all of Elongation ing post-transplant renal fibrosis, liver fibrosis, pancreatic of very long chain fatty acids protein 2 (ELOVL2), Elon- 15 fibrosis, cardiac fibrosis, skin wound healing including gation of very long chain fatty acids protein 4 (ELOVL4) diabetes related wound healing, and oral submucosa fi- and/or Elongation of very long chain fatty acids protein brosis. 5 (ELOVL5). [0094] The senescence-related disease or condition [0089] According to a specific embodiment, the com- can be a metabolic disorder. Metabolic disorders include, position described herein comprises at least one cycloox- 20 but are not limited to type I and type II diabetes mellitus. ygenase and at least one lipoxygenase inhibitor and an [0095] The senescence-related disease or condition additional compound capable of manipulating ATP lev- can be a macular degeneration, dry (non-neovascular) els, specifically increasing and/or decreasing intracellu- or wet (neovascular) macular degeneration. lar ATP levels. Specifically, such additional compound is [0096] The senescent cell-associated condition can be any one or more or all of an inhibitor of ATP synthase, 25 a pulmonary condition. Pulmonary conditions include, an inhibitor or ADP/ATP translocases and/or an inhibitor but are not limited to, idiopathic pulmonary fibrosis (TPF), of glycolysis. chronic obstructive pulmonary disease (COPD), asthma, [0090] As described herein, the compositions provided cystic fibrosis, bronchiectasis, emphysema, age-related herein can be used for the treatment of a senescence- loss of pulmonary function, and age-associated sleep ap- related disease or condition. Specifically, the composi- 30 nea. tions provided herein are used for the prevention or delay [0097] The senescence-related disease or condition of the onset of a senescence-related disease or condi- can be a dermatological condition. Dermatological con- tion, for the prevention or delay of the progression of a ditions include, but are not limited to, psoriasis, eczema, senescence-related disease or condition or to promote rhytides, pruritis, dysesthesia, papulosquamous disor- the regression of a senescence-related disease or con- 35 ders, erythroderma, lichen planus, lichenoid dermatosis, dition. atopic dermatitis, eczematous eruptions, eosinophilic [0091] The senescence-related disease or condition dermatosis, rashes, photosensitivity and photoaging re- can be a cardiovascular disease or condition, including lated diseases and disorders, reactive neutrophilic der- but not limited to angina, arrhythmia, atherosclerosis, matosis, pemphigus, pemphigoid, immunobullous der- cardiomyopathy, congestive heart failure, coronary ar- 40 matosis, fibrohistocytic proliferations of skin, skin nevi, tery disease (CAD), carotid artery disease, endocarditis, urticaria, hyperpigmentation, cutaneous lymphomas, coronary thrombosis, carotid thrombosis, myocardial in- psoriasis, and cutaneous lupus. farction (MI), high blood pressure/hypertension, aortic [0098] According to a further specific embodiment, the aneurysm, brain aneurysm, cardiac fibrosis, cardiac di- composition provided herein is used to improve the per- astolic dysfunction, hypercholesterolemia/hyperlipi- 45 formance of transplants. Specifically, presence of an in- demia, mitral valve prolapse, peripheral vascular dis- creased number of senescent cells in the donor organ ease, peripheral artery disease (PAD), cardiac stress re- and/or in the recipient negatively influences the perform- sistance, and stroke. ance of a transplant. According to a specific embodiment, [0092] The senescence-related disease or condition the composition described herein is used to selectively can be a neurological condition. Neurological conditions 50 eliminate senescent cells in the donor organ and/or the include, but are not limited to, Parkinson’s disease, recipient to improve the performance of the transplant. Alzheimer’s disease, dementia, amyotrophic lateral scle- [0099] According to a further specific embodiment, the rosis (ALS), bulbar palsy, pseudobulbar palsy, primary composition provided herein is used to prevent or atten- lateral sclerosis, motor neuron dysfunction (MND), mild uate senescence-associated scar formation and fibrosis. cognitive impairment (MCI), Huntington’s disease, ocular 55 [0100] According to a further specific embodiment, the diseases, macular degeneration (wet and dry), glauco- composition provided herein is used to ameliorate side ma, vision loss, presbyopia, cataracts, progressive mus- effects of chemotherapy. According to yet a further spe- cular atrophy, lower motor neuron disease, spinal mus- cific embodiment, the composition provided herein is

12 23 EP 3 643 305 A1 24 used to prevent or delay tumor relapse and the occur- ELISA Kit supplied by Novus Biologicals (NBP2-66372) rence of senescence-related diseases and conditions. or any other equivalent ELISA assay. According to further Therapy-induced senescence (TIS) is a common side specific examples, the level of arachidonic assay can be effect of cancer therapy and causes premature aging in measured by flow-injection electrospray ionization mass patients surviving the therapy. In addition, TIS induces 5 spectrometry as described by (Gruber et al. 2015), with senescence-associated stemness (SAS) in tumors, for high performance liquid chromatography as described example in Bcl2 lymphomas, thereby causing an in- by (Nishikiori et al. 2015) or with high performance liquid creased tumor-initiation capacity and an increased risk chromatography coupled to tandem mass spectrometry for cancer relapse. in the negative ion mode. [0101] Further provided herein is a method of identify- 10 [0107] For example, PLA2 activity can be measured ing senescent cells in a subject. Specifically, altered lev- by an assay that comprises a PLA2 substrate that on els of members of the altered lipid metabolism of senes- cleavage by the phospholipase A2 activity yields a cent cells described herein can be detected to identify flourescent product that can be quantified with a fluorim- senescent cells in a subject. Specifically, the method of eter, e.g. bis-BODIPY®-FL-C11-PC (Thermo Fisher), or identifying senescent cells in a subject comprises deter- 15 with the EnzChek™ Phospholipase A2 Assay Kit (Ther- mining the level of at least one of the biomarkers lyso- mo Fisher) or any other equivalent assay. phosphatidylcholine, arachidonic acid and phospholi- [0108] According to a specific embodiment, an in- pase A2 activity. Preferably, the biomarker is lyso SPC crease by more than two or three standard deviations of or lyso PPC or arachidonic acid. the level of at least one of the biomarkers lysophospati- [0102] The method of identifying senescent cells in a 20 dylcholine, arachidonic acid or phospholipase A2 com- subject can be specifically used for determining the pres- pared to the level of said biomarkers in a reference is ence of senescent cells in context of diagnosing senes- indicative of the presence of senescent cells or cellular cence-related diseases or conditions, diagnosing the risk senescence. of senescence-related diseases or conditions in a sub- [0109] Specifically, a more than 2.0, 2.5, 3.0, 3.1, 3.2, ject, monitoring senescence-related diseases or condi- 25 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, tions or monitoring or predicting treatment-response, or 4.6, 4.7, 4.8, 4.9 or 5.0 fold increase between the refer- monitoring exposure to senescence inducing agents (in- ence level of any one or more of said biomarkers obtained toxicants, including oxidative stressors, DNA damaging from a healthy subject or a group of healthy subjects com- agents, etc.). pared to the level of one or more of said biomarkers ob- [0103] A "control", "control sample", or "reference val- 30 tained from a sample of a subject is indicative of the pres- ue" or "reference level" are terms which can be used ence of senescent cells in said sample. interchangeably herein, and are to be understood as a [0110] Specifically a more than 2.0, 2.5, 3.0, 3.1, 3.2, sample or standard used for comparison with the exper- 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, imental sample. The control may include a sample ob- 4.6, 4.7, 4.8, 4.9 or 5.0 fold decrease between the refer- tained from a healthy subject or a subject, which is not 35 ence level of any one or more of said biomarkers obtained at risk of or suffering from senescence or is not exposed from a sample of a subject during or after senolytic treat- to stress conditions inducing senescence or which is ex- ment compared with a sample of the same subject from posed to treatment with a senolytic. Reference level spe- an earlier timepoint (intra-individual comparison or intra- cifically refers to the level of lysophosphatidylcholine, individual difference) is indicative of positive response to arachidonic acid and/or PLA2 activity quantified in a sam- 40 senolytic treatment. ple from a healthy subject, from a subject, which is not [0111] Additionally, a control may also be a standard at risk of or suffering from senescence or is not exposed reference value or range of values. The reference level to stress conditions inducing senescence or, in case of can also be determined as the average level of any one senolytic treatment monitoring, said level could also be or more of lysophosphatidylcholine, arachidonic acid and derived from a sample of a subject before starting seno- 45 phospholipase A2 activity in a sample of a healthy subject lytic treatment or during the course of senolytic treatment. and/or in a subject prior to a pharmacologic, dietary or [0104] Specifically, levels of lysophosphatidylcholine, life-style intervention. As an alternative, also a pool of arachidonic acid and/or PLA2 activity are measured us- samples from one or more subjects may be used or a ing immunoassays such as ELISA, mass spectrometry reference disclosed in literature. or high-performance liquid chromatography (HPLC). 50 [0112] The method for the detection of senescent cells [0105] For example, the levels of lysophosphatidylcho- as described herein specifically provides a diagnostic lines can be measured by high performance liquid chro- and predictive tool using a diagnostic signature or ex- matography coupled to tandem mass spectrometry in the pression pattern as described herein, comprised of an positive ion mode as described by (Gruber et al. 2015) increase in the level of any one or more or all of lyso- or by HPLC as described by (Jeschek et al. 2016). 55 phosphatidylcholine, arachidonic acid and/or phosphol- [0106] For example, the level of arachidonic acid can ipase A2, applicable over a broad range of senescence be measured by an enzyme linked immunosorbent as- of various cell types including but not limited to fibrob- say, such as for example the universal arachidonic acid lasts, endothelial cells, kidney epithelial cells, liver cells,

13 25 EP 3 643 305 A1 26 neuronal cells, skin cells, lung epithelial cells, or colon capable of inhibiting at least two of cyclooxygenase- epithelial cells. In particular, detection of senescent cells 1 (COX-1), cyclooxygenase-2 (COX-2) and lipoxy- in the tissue, blood or serum of young subjects or subjects genase, for use in selectively eliminating senescent not being exposed to senescence inducing stress con- cells. ditions and old subjects or subjects being exposed to 5 2. The composition for use according to item 1, senescence inducing stress conditions provides a diag- wherein the senescent cells are characterized by in- nostic and predictive tool that has a higher significance creased intracellular levels of at least one of lyso- for early diagnosis, long-term prognosis, and screening phosphatidylcholine, arachidonic acid and phos- of patients with cellular senescence. The method as de- pholipase A2 activity. scribed herein also provides a diagnostic tool for moni- 10 3. The composition for use according to item 2, toring treatment with senolytics. wherein the lysophosphatidylcholine is 1-steraroyl- [0113] Specifically, the method of identifying senes- 2-hydroxy-sn-glycero-3-phosphocholine (LysoSPC) cent cells as described herein can be performed as single or 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho- measurement but may also be performed by repetitive choline (LysoPPC). determinations. 15 4. The composition for use according to any one of [0114] Specifically, use of the method for detecting se- items 1 to 3, wherein one or more of the inhibitors nescent cells as described herein also encompasses pre- are COX-1 and/or COX-2 inhibitors, selected from dicting transplant organ function or predicting organ the group consisting of acetylsalicylic acid, di- transplant failure. clofenac, celecoxib, cyclosporine A, ibuprofen, [0115] According to a specific embodiment, the herein 20 acetaminophen, indomethacin, nabumetone, described method can also be used for detecting a de- ketorolac, tenoxicam, tolmetin, piroxicam, fenopro- cline of senescent cells or reduction of cellular senes- fen, etodolac, naproxen, diflunisal, suprofen, brom- cence, wherein the level of at least one of the biomarkers fenac, ketoprofen, dihomo-gamma-linolenic acid, lysophosphatidylcholine, arachidonic acid and/or PLA2 icosapent, fluriprofen, mefenamic acid, salsalate, activity is compared with the level of the corresponding 25 sulindac, salicylic acid, lumiracoxib, O-acetyl-L-ser- lysophosphatidylcholine, arachidonic acid and/or PLA2 ine, phenacetin, fluriprofen methyl ester, metami- activity prior to a treatment with senolytics, anti-aging zole, nitroaspirin, meloxicam, flufenamic acid, oxa- agents or any anti-aging intervention. Specifically, a de- prozin, tiaprofenic acid, magnesium salicylate, di- crease in the level of at least one of said biomarkers can ethylcarbamazine, lornoxicam, carprofen, phenylb- indicate the removal of senescent cells e.g. during use 30 utazone, nepafenac, antipyrine, antrafenine, choline of senolytics, anti-aging agents (e.g. rapamycin, spermi- magnesium trisalicylate, triflusal, niflumic acid, dex- dine, metformin), or any other anti-aging intervention like ibuprofen, aceclofenac, acemetacin, droxicam, loxo- diet, exercise, etc. This signature can also be used to profen, tolfenamic acid, dexketoprofen trometamol, identify subjects that would benefit from any senolytic talniflumate, propacetamol, trolamine salicylate, intervention. 35 phenyl salicylate, bufexamac, glycol salicylate, men- [0116] Specifically, the method is useful for monitoring thyl salicylate, FK-506, lenalidomide, rofecoxib, val- a subject, specifically for measuring the response of a decoxib, cimicoxib, chlorphenesin, clodronic acid, subject to senolytic treatments. Therefore, the biomark- seliciclib, drospirenone, triamcinolone, pomalido- ers may be also used for indication of efficient drug doses mide, parecoxib, firocoxib, aclofenac, adapalene, (dose finding, e.g. during development of any type of in- 40 thalidomide, etoricoxib, robenacoxib, asaraldehyde, tervention, or in terms of identifying personalized treat- zaltoprofen, deracoxib, dexamethasone, pranopro- ment options or dosing). fen, amfenac sodium monohydrate, ampiroxicam, [0117] Further provided herein is a method of screen- NS-398, bismuth subsalicylate, diclofenac diethyl- ing for senolytic compounds, which are capable of selec- amine, trometamol, rutaecarpine, salicin, fenbufen, tively eliminating senescent cells. According to a specific 45 xanthohumol, flunixin meglumin and nimesulide. embodiment, the level of arachidonic acid is measured 5. The composition for according to any one of items in senescent cells upon exposure to a compound, using 1 to 4, wherein one or more inhibitors are lipoxyge- for example enzyme-based immunoassays such as ELI- nase and/or FLAP (ALOX5AP) inhibitors selected SA, and compared to the level of arachidonic acid in a from the group consisting of MK886, zileuton, maso- non-senescent cell also exposed to the compound or to 50 procol, diethylcarbamazine, azelastine, benoxapro- a senescent cell not exposed to the compound or both. fen, nordihydroguaiaretic acid, abietic acid, escule- Specifically, compounds identified by this screening tin, montelukast, minocycline, MLN-977, rhein, diac- method are capable of selectively eliminating senescent erein, nabiximols, fostamatinib, AM103, DG031, fi- cells. boflapon, AA-861 and atreleuton. [0118] The present invention further comprises the fol- 55 6. The composition for use according to any one of lowing items: items 1 to 5, wherein one or more inhibitors are dual cyclooxygenase and lipoxygenase inhibitors, prefer- 1. A composition comprising one or more inhibitors ably selected from the group consisting of licofelone,

14 27 EP 3 643 305 A1 28 darbufelone, CI-987, S-2474, KME-4, Chebulagic terone, omeprazole, valsartan, bortezomib, nevirap- acid, balsalazide, mesalazine, sulfasalazine, ami- ine, azelastine, lornoxicam, phenylbutazone, etravir- nosalicylic acid, meclofenamic acid, morniflumate di- ine, leflunomide, sitaxentan, aminophenazone, ver- arylpyrazole derivatives, thieno[2,3-b]pyridine deriv- apamil, etoricoxib, propofol, sulfamoxole, dicou- atives, N-substituted 5-aminosalicylicylamides, flav- 5 marol, diltiazem, histamine, moclobemide, sele- ocoxid, indolizine derivatives, LQFM-091, hyper- giline, parecoxib, doconexent, acetyl sulfisoxazole, forin, celastrol, BW755C, tepoxalin,b-boswellic acid, fluconazole, pantoprazole, desloratadine, micona- D-002, 2,3-diarylxanthones, phenidone and ER- zole, amiodarone, gemfibrozil, probenecid, tenipo- 34122. side, sulfadiazine, capecitabine, fluorouracil, tranyl- 7. The composition for use according to any one of 10 cypromine, anastrozole, atovaquone, cyclizine, items 1 to 6, wherein the composition comprises an dexfenfluramine, disulfiram, epinephrine, eprosar- additional compound capable of inhibiting intracel- tan, flecainide, indinavir, methazolamide, nelfinavir, lular conversion of arachidonic acid. olanzapine, pranlukast, promethazine, sulfadimeth- 8. The composition for use according to item 7, oxine, sulfamethizole, sulfanilamide, sulfapyridine, wherein the additional compound is a natural com- 15 methimazole, tolcapone, bicalutamide, armodafinil, pound, preferably selected from the group consisting agomelatine, noscapine, clevidipine, sulconazole, of turmeric, rosemary, ginger, oregano, resveratrol, gefitinib, ticagrelor, ceritinib, floxuridine, lifitegrast, curcumin, cannabinoids, gingseng, saponins, terpe- rhein, diacerein, zucapsaicin, stiripentol, lobeglita- noids, flavonoids, polyphenols, ginkgo biloba, cap- zone, dosulepin, manidipine, cimicifuga racemose, saicin, genistein, kaempferol and quercetin. 20 curcumin, felbamate, piperine, safinamide, irponi- 9. The composition for use according to item 7, azid, oritavancin, masorpocol and pegvisomant. wherein the additional compound is an inhibitor of 10. The composition for use according to item 7, cytochrome P450, preferably an inhibitor of CYP2J, wherein the additional compound is an inhibitor of CYP2C, CYP4A or CYP4F, preferably selected from long-chain-fatty-acid-CoA ligase 4 (ACSL4), prefer- the group consisting of sulfaphenazole, avasimibe, 25 ably selected from the group consisting of triascin A, benzbromarone, rosiglitazone, troglitazone, cervis- triascin B, triascin C, triascin D, troglitazone, ciglita- tatin, warfarin, pioglitazone, lapatinib, trimethoprim, zone, pioglitazone and rosiglitazone. zafirlukast, amodiaquine, nicardipine, simvastatin, 11. The composition for use according to item 7, fluvastatin , loratadine, ethinylestradiol, irbesartan, wherein the additional compound is an inhibitor of a quinine, sorafenib, eltrombopag, losartan, 30 lysophosphatidylcholine acyltransferase, specifical- licofelone, amitriptyline, atorvastatin, mefenamic ac- ly an inhibitor of LPCAT1, LPCAT2, LPCAT3, id, meloxicam, piroxicam, erlotinib, pazopanib, di- LPCAT4, MBOAT2 and/or MBOAT7, and wherein ethylstilbestrol, enzalutamide, ponatinib, dabraf- the inhibitor of a lysophosphatidylcholine acyltrans- enib, enasidenib, lovastatin, montekulast, ketocona- ferase is preferably selected from the group consist- zole, felodipine, candesartan cilexetil, clotrimazole, 35 ing of N-phenylmaleimide derivatives, TSI-01 and mometasone, salmeterol, raloxifene, fenofibrate, thimerosal. levothyroxine, tamoxifen, quercetin, oxybutynin, me- 12. The composition for use according to item 7, droxyprogesterone acetate, nifedipine, liotrix, am- wherein the additional compound is an inhibitor of a lodipine, bezafibrate, chloramphenicol, cy- fatty acid elongase, specifically an inhibitor of closporine, cimetidine, clopidogrel, cholecalciferol, 40 ELOVL2, ELOVL4 and/or ELOVL5, and wherein the delavirdine, dextropropoxyphene, etoposide, isoni- inhibitor of a fatty acid elongase is preferably select- azid, ketoprofen, metronidazole, nilutamide, nilvad- ed from the group consisting of cycloate, adenosine ipine, paroxetine, phenelzine, pravastatin, propaf- 5’-hexadecylphosphate, endo-1k, (S)-1y and com- enone, pyrimethamine, rofecoxib, rutin, saquinavir, pound 37, 5,5-dimethyl-3-(5-methyl-3-oxo-2-phe- sulfamethoxazole, sulfinpyrazone, tegaserod, ter- 45 nyl-2,3-dihydro-1H-pyrazol-4-yl)-1-phenyl-3-(trif- fenadine, thioridazine, ticlopidine, tioconazole, tria- luoromethyl-3,5,6,7-tetrahydro-1H-indole-2,4-di- zolam, troleandomycin, valproic acid, abiraterone, one) and (3-endo)-3-(phenylsulfonyl)-N-[4-(trifluor- vismodegib, regorafenib, trametinib, idelalisib, lopi- omethyl)phenyl]-8-azabicyclo[3.2.1]octane-8-car- navir, celecoxib, efavirenz, rabeprazole, terifluno- boxamide. mide, crisaborole, belinostat, topiroxostat, cande- 50 13. The composition for use according to any one of sartan, letermovir, rucaparib, opicapone, nabilone, items 1 to 7, wherein the composition comprises an fluvoxamine, fluticasone, fluticasone furoate, flutica- additional compound capable of manipulating intra- sone propionate, bosutinib, cabozantinib, genistein, cellular ATP levels. lenvatinib, atazanavir, bexarotene, deferasirox, qui- 14. The composition for use according to item 13, nidine, mifepristone, vemurafenib, sildenafil, di- 55 wherein the additional compound is an inhibitor of clofenac, fluoxetine, valdecoxib, voriconazole, ATP synthases, preferably selected from the group etodolac, sertraline, glyburide, acenocoumarol, ro- consisting of oligomycin A, inositol nicotinate, suvastatin, imatinib, clozapine, diazepam, proges- bedaquiline, efrapeptins, leucinostatins, tentoxin,

15 29 EP 3 643 305 A1 30 tnetoxin derivates, angiostatin, enterostatin, melittin, wherein the additional compound is an inhibitor of IF1, Syn-A2, Syn-C, resveratrol, piceatannol, diethyl- glycolysis, preferably selected from the group con- stilbestrol, 4-acetoamido-4’-isothiocyanostilbene- sisting of 2-deoxy-D-glucose, lonidamine, 2,2’-disulfonate, 4,4’-D-isothiocyanatostilbene-2,2- bromopyruvic acid, phloretin, quercetin, STF-31, disulfonic acid, quercetin, kaempferol, morin, api- 5 WZB117, 3PO, 3-bromopyruvate, dichloroacetate, genin, genistein, biochanin A, daidzein, epicatechin oxamic acid, NHI-1, oxythiamine, imatinib, glu- gallate, epigallocatechin gallate, proanthocyanidin, cosamine, 6-aminonicotinamide, genistein, 5-thi- curcumin, phloretin, theaflavin, tannic acid, 4-hy- oglucose, mannoheptulose, α-chlorohydrin, ornida- droxy-estradiol, 2-hydroxy-estradiol, 17α-estradiol, zole, oxalate, glufosfamide, N-(phosphonacetyl)-L- 17β-estradiol, α-zearalenol, β-zearalenol, oligomy- 10 aspartate, 6-methylmercaptopurine riboside, CGP cin, venturicidin, apoptolidin, ossamycin, cyto- 3466B maleate, sodium monofluorophosphate, DA- varicin, peliomycin, tributyltin chloride, tricyclohexy- SA-58, DL-serine, dichloroacetic acid, sodium ltin hydroxide, triethyltin sulfate, triphenyltin chloride, dichloroacetate, nitrofural, 6-AN, fasentin, benser- dimethyltin 3-hydroxyflavone chloride, diethyltin 3- azide, astraglin, resveratrol, chrysin, GEN-27, api- hydroxyflavone chloride, dibuthyltin 3-hydroxyfla- 15 genin, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2- vone bromide, dioctyltin 3-hydroxyflavone chloride, yl)ethyl sulfide, CB-839, azaserine, acivicin, 6-diazo- diphenyltin 3-hydroxyflavone chloride, diethyltin 5-ox-L-norleucine, thiazolidine-2,4-dione deriva- 3,5,7,2’,4’-pentahydroxyflavone chloride, dibutyltin tives, compound 968, R-lipoic acid, 1,3,4-thiadiazole 3,5,7,2’,4’-pentahydroxyflavone bromide, diphenyl- compounds, 2-chloropropionate, Nov3r, AZD7545, tin 3,5,7,2’,4’-pentahydroxyflavone chloride, tributyl- 20 Pfz3, radicicol, mitaplatin, mito-DCA, phenylbu- tin 3-hydroxyflavone, triethyllead, aurovertin, citreo- tyrate, 4,5-diarylisoxazoles, VER-246608, betulinic viridin, asteltoxin, rhodamine B, rhodamine 123, acid, pyruvate analogs containing phosphinate or rhodamine 6G, rosaniline, malachite green, brilliant phosphonate group, CPI-613, M77976, aromatic green, quinacrine, quinacrine mustard, acridine or- DCA derivatives, furan and thiophene carboxylic ac- ange, coriphosphine, pyronin Y, dequalinium, sa- 25 ids, ritonavir, FX11, oxamate, D-fructose-6-phos- franin O, nile blue A, ethidium bromide, tetracaine, phate, 6-phosphogluconic acid, N-bromoacetyl-ami- dibucaine, procaine, lidocaine, chlorpromazine, trif- noethyl phosphate, 2-carboxyethylphosphonic acid, luoperazine, procainamide, propranolol, octyl gua- N-hydroxy-4-phosphono-butanamide, 2-phos- nidine, 1-dansyl amido-3-dimethypropylamine com- phoglyceric acid, iodoacetate, gossypol, bisphos- pounds, cetyltrimethylammonium, spermine, sper- 30 phonate analogs of 1,3-bisphosphoglyceric acid, midine, bathophenan throline-metal chelate, 4,4- benzene hexacarboxylic acid, 3-phosphoglyceric diphenyl-2,2-bipyridine, 3-(2-pyridyl)-5,6-diphenyl- acid, phosphonoacetohydroxamic acid, 2-phospho- 1,2,4-triazine, atrazine, atrazine amino derivative, D-glyceric acid, TLN-232 and CAP-232. arsenate, aluminium fluoride, beryllium fluoride, 17. The composition for use according to any one of scandium fluoride, vanadate, magnesium fluoride, 35 items 1 to 16, wherein the composition prevents or sulfite, thiophosphate, azide, ANPP, phenylglyoxal, delays the onset of a senescence-related disease or butanedione, dansyl chloride, 1-fluoro-2,4-dini- condition. trobenzene, dicarbopolyborate, almitrine, 5-hy- 18. The composition for use according to any one of droxy-1,2-naphtalene dicarboxylic anhydride, items 1 to 16, wherein the composition prevents or R207910, spegazzinine, n-butanol, terachlorosali- 40 delays the progression of a senescence-related dis- cylanilide, dihydrostreptomycin, suramin, Bz-423, ease or condition. DMSO, hypochlorous acid, DDT, diazoxide, HNB, 19. The composition for use according to any one of N-sulfonyl or N-alkyl substituted tetrahydrobenzodi- items 1 to 16, wherein the composition promotes the azepine derivatives, 4-(N-arylimidazole)-substituted regression of a senescence-related disease or con- benzopyran derivatives, N-[1-aryl-2-(1-imidazo- 45 dition. lo)ethyl]-cyanoguanidine derivatives, N-[1-aryl-2-(1- 20. The composition for use according to any one of imidazolo)ethyl]-acylguanidine derivatives, O-[1-ar- items 17 to 19, wherein the senescence-related dis- yl-2-(1-imidazolo)ethyl]-thiourethane derivatives, ease or condition is selected from cardiovascular dis- dio-9 complex, ethanol and zinc. ease, atherosclerosis, cancer, osteoporosis, oste- 15. The composition for use according to item 13, 50 oarthritis, neurological disorders, dementia, cata- wherein the additional compound is an inhibitor of ract, kidney disease, retinopathy, diabetes, lung fi- ADP/ATP translocases, preferably selected from the brosis, vertebral skin degeneration, age-related group consisting of clodronic acid, ibipinabant, at- muscular atrophy, hair loss and skin aging. ractyloside, carboxyatractyloside, bongkrekic acid, 21. The composition for use according to any one of isobongkrekic acid, MT-21, closantel, CD437, leela- 55 items 1 to 16, wherein the composition improves the mine, L923-0673, IMD 0354, PI32-0333, S899542, performance of transplants. nonactin and S838462. 22. The composition for use according to any one of 16. The composition for use according to item 13, items 1 to 16, wherein the composition prevents or

16 31 EP 3 643 305 A1 32

attenuates senescence-associated scar formation ed otherwise. and fibrosis. 23. The composition for use according to any one of Cell isolation items 1 to 16, wherein the composition ameliorates side effects of chemotherapy and prevents or delays 5 [0121] Human dermal fibroblasts (HDFs) were isolated tumor relapse. from skin biopsies of healthy female adult donors and 24. A method of identifying senescent cells in a sub- obtained from Evercyte (Vienna, Austria). Foreskin hu- ject, comprising the steps of man dermal fibroblasts (fHDFs) were isolated from fore- skin and obtained from Evercyte (Vienna, Austria). Male a) providing a sample of said subject, 10 human umbilical vein endothelial cells (HUVECs) were b) determining the levels of at least one of lys- isolated from human umbilical cords and obtained from ophosphatidylcholine, arachidonic acid and/or Evercyte (Vienna, Austria). All cell strains were tested phospholipase A2 activity in said sample, for mycoplasma at regular intervals. c) comparing the levels of b) to a reference level, wherein the reference level is the level of at least 15 Cell culture one of lysophosphatidylcholine, arachidonic ac- id and/or phospholipase A2 activity in non-se- [0122] HDFs and fHDFs were cultured with nescent cells, DMEM/Ham’s F-12 (1:1 mixture) (F4815, Biochrome) supplemented with 10% FCS (F7524, Sigma-Aldrich) and wherein an increase of at least 2-fold is indicative 20 and 4 mM L-Glutamine (G7513, Sigma-Aldrich) under of the presence of senescent cells in said sample. ambient oxygen, 7% CO2 and 37 °C. HUVECs were cul- 25. A method of screening for candidate compounds tured with Endopan 3 Kit (except for Gentamicin and FBS; for eliminating senescent cells, comprising the steps P04-0010K, Pan Biotech) supplemented with 10% FCS of (F7524, Sigma-Aldrich) under ambient oxygen, 7% CO2 25 and 37 °C. Cells were detached by incubation with 0.1% a) bringing at least one test compound in contact trypsin and 0.02% EDTA at 37 °C for 3-5 min and split with a sample of senescent cells, at ratios between 1:2 and 1:8 depending on cell type and b) measuring the level of arachidonic acid and/or growth rate. Cells were counted using a Vi-CELL XR apoptosis and/or measuring cell viability, and (Beckman Coulter) automated cell counter. c) selecting the compounds which cause intra- 30 cellular accumulation of arachidonic acid, apop- SIPS tosis or reduced cell viability in the senescent cells treated in a) compared with untreated se- [0123] Cells were seeded at a cell density of 2,800 nescent cells. cells/cm2 one day prior to the treatment. The cells were 35 treated nine times over a period of 11 days with 60-80 26. Use of a compound for eliminating senescent mM H2O2 supplemented to the media for one hour fol- cells in a subject, wherein said compound is identi- lowed by a media change. Alternatively, cells were seed- fied according to the method of item 25. ed at a cell density of 3,500 cells/cm2 one day prior to 27. A composition comprising at least one cycloox- the treatment and SIPS was induced with 100-150 nM ygenase inhibitor and at least one lipoxygenase in- 40 doxorubicin (D1515, Sigma-Aldrich) supplemented to the hibitor for use in selectively eliminating senescent medium for 6 days. Induction of senescence was verified cells. by SA-β-gal staining, p21 expression, and absence of BrdU incorporation. AnnexinV/PI staining was performed [0119] The examples described herein are illustrative to assure that the treatment was non-lethal and SIPS of the present invention and are not intended to be limi- 45 HDFs were cultured and monitored for over 50 days to tations thereon. Different embodiments of the present assure that the induced growth arrest was permanent invention have been described according to the present (Terlecki-Zaniewicz et al. 2018). invention. Many modifications and variations may be made to the techniques described and illustrated herein Lipid analysis without departing from the spirit and scope of the inven- 50 tion. Accordingly, it should be understood that the exam- [0124] Cells were washed once with PBS containing ples are illustrative only and are not limiting upon the 0.5 mM diethylenetriaminepentaacetic acid and after scope of the invention. adding 2.2 ml methanol containing 3% acetic acid and 0.01% butylated hydroxytoluene, cells were detached EXAMPLES 55 with a cell scraper. Lipid samples were transferred to a glass vial, air was evacuated with inert gas and samples [0120] The following materials and methods were used were stored at -20 °C after vials were sealed with para- throughout the examples provided herein unless indicat- film. Finally, samples were analyzed with flow-injection

17 33 EP 3 643 305 A1 34 electrospray ionization mass spectrometry as described [0129] Figure 1 shows that Lysophosphatidylcholines by (Gruber et al. 2015) using the version 1.6 of the Analyst are elevated in senescent cells. Figure 1a shows lipid software (Applied Biosystems). Levels of lyso PPC and analysis of human dermal fibroblasts (HDF) of three dif- lyso SPC were normalized to DPPC levels. ferent donors at early, middle and late population dou- 5 bling (PD), wherein levels of lyso PCs were normalized Next generation sequencing and data analysis to Dipalmitoylphosphatidylcholine (DPPC) levels. Figure 1b shows lipid analysis of stress-induced premature se- [0125] Library preparation and sequencing were per- nescent (SIPS) HDF, four days after SIPS treatment. formed on an Illumina HighSeq 2000 Platform (GATC SIPS was induced with chronic oxidative stress treatment 10 Biotech AG; Konstanz, Germany). All analysis steps (9 doses of H2O2 treatment over the period of 11 days). were done according to the Tuxedo Suite Pipeline (Trap- As a control for the SIPS treatment, cells were cultivated nell et al. 2012). Briefly, Illumina Casava 1.8.2 software with normal growth medium, grown to confluence during was used for base calling. RNA-seq reads were aligned the treatment and entered a quiescent state (Q). For to hg19 genome assembly using TOPHAT Version Fig.1a significance was calculated with a one-way ANO- 2.0.13 with default parameters. Transcripts were assem- 15 VA followed by a post hoc Tukey’s test. For Fig.1b sig- bled in Cufflinks Version 2.1.1 and differentially ex- nificance was calculated with a two-tailed student’s t-test. pressed genes were predicted by Cuffdiff. [0130] Lyso PC can be generated from phosphatidyl- cholines (PC) either non-enzymatically through oxidation Viability assay processes (Choi et al. 2011) or enzymatically through 20 conversion by phospholipases with PLA2 activity (Six and [0126] Alamar blue (DAL1100, Thermo Fisher Scien- Dennis 2000). Comparing RNA-seq expression data tific) assay was performed according to the manufactur- from SIPS HDF with quiescent control cells (raw data er’s instructions. Cells were treated with the respective available with GEO accession number: GSE93535), we substances for 9 days with a media change on day 0, 3 identified cytosolic phospholipase A2 (PLA2G4A), cy- and 6. The control was treated with the respective con- 25 tosolic phospholipase A2 gamma (PLA2G4C) and group centration of the solvent (DMSO). XV phospholipase A2 (PLA2G15) as possible candidates for the observed effects (Fig. 2). Quantification of arachidonic acid [0131] Figure 2 shows that phospholipases with PLA2 activity and secretory phospholipase A2 receptor are in- [0127] The cells were harvested by trypsinization, cen- 30 creased in SIPS HDFs. Premature senescence was in- trifuged at 1000 g and 4°C for 5 min and washed three duced in HDFs with chronic oxidative stress treatment (9 times with cold PBS. The Cells were counted using a Vi- doses of H2O2 treatment over the period of 11 days). As CELL XR (Beckman Coulter) automated cell counter and a control for the SIPS treatment, cells were cultivated the cell pellets were resuspended in cold PBS to obtain with normal growth medium, grew to confluency during a cell suspension with 5000 cells/ml. Subsequently the 35 the treatment and entered a quiescent state (Q). Four cells were lysed by 30 cycles of sonication (30 s on, 30 days after the SIPS treatment, RNA samples were pre- s off) in a Bioruptor (Diagenode). Cell debris was re- pared and RNA-seq was performed. Expression levels moved by 10 min centrifugation at 1500 g and 4°C and are displayed as Fragments Per Kilobase of transcript the supernatant was analyzed with an ELISA assay (No- per Million mapped reads (FPKM). The p-values were vus Biologicals; NBP2-66372) according to the manufac- 40 corrected with the false discovery rate for multiple com- turer’s instructions. parisons using the Benjamini-Hochberg method and er- ror bars indicate confidence intervals (95 %). Example 1: Altered Lipid Metabolism in Senescent Cells Example 1B: Increased production of arachidonic 45 acid (AA) in senescent cells Example 1A: Identification of increased levels of lyso PC in senescent cells [0132] The secretory phospholipase A2 receptor (PLA2R1), which induces cellular senescence (Augert et [0128] As described herein an altered lipid metabolism al. 2009) and is upregulated in our dataset (Fig. 2), is in senescent cells was discovered and two species of 50 reported to induce the production of lyso PCs and the lyso PC as novel biomarkers for senescence were iden- concurrent release of arachidonic acid (AA) by activation tified using mass spectrometry. 1-stearoyl-2-hydroxy-sn- of PLA2G4A through phosphorylation by MAPKs (Fonteh glycero-3-phosphocholine (18:0 lyso PC) and 1-palmi- et al. 2000; Pan et al. 2015; Scott et al. 2006). toyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso [0133] Given the fact that the increase of lyso PC in PC) were upregulated during senescence in several pri- 55 senescence was robust in all examined cell strains inde- mary human cell strains regardless of the inducer being pendent of the senescence inducer indicates that the al- telomere dependent (replicative senescence) or inde- tered lipid metabolism may be used as a target for the pendent (stress-induced premature senescence; Fig. 1). development of a cytotoxic treatment specific for senes-

18 35 EP 3 643 305 A1 36 cent cells and thereby for the prevention or therapy of with chronic oxidative stress treatment (9 doses of H2O2 senescence-associated diseases and disorders. treatment over the period of 11 days). Quiescent cells [0134] Increased formation of eicosanoids has been were used as control. Treatment with senolytic substanc- reported to be associated with cellular senescence and es started 11 days after completion of the SIPS treatment aging (Currais et al. 2016; Kabir et al. 2016; Li et al. 2015; 5 to allow for the establishment of a fully established se- Wang et al. 2016b). As shown herein, the generation of nescent phenotype. Growth media were supplemented lyso PC is increased and increased generation of lyso with senolytic substances for 9 days with media changes PC leads to increased generation of AA as a byproduct every 3 days. All samples, including the control, con- (Fig. 1 and Fig. 12). AA in turn can be further metabolized tained the same concentration of solvent (DMSO). Sub- to eicosanoids by enzymes such as cyclooxygenase and 10 sequently, the viability was assessed using an alamar lipoxygenase. It has been reported that accumulation of blue assay (Thermo Fisher Scientific). intracellular AA could induce apoptosis at high concen- [0140] Indeed, MK886 and the COX inhibitor ASA re- trations of AA (Penzo et al. 2004). duced the viability of senescent cells compared to qui- [0135] As described herein for the first time, cell death escent cells to a greater extent than the prior-art seno- caused by accumulation of intracellular levels of AA is 15 lytics Quercetin and Navitoclax. observed predominantly in senescent cells as these cells have an altered lipid metabolism with a higher AA forma- Example 2B: Identification of novel senolytics in HU- tion rate (Fig. 3). Indeed, as surprisingly shown in the VECs examples below, novel senolytics were identified based on the senescent cell-specific alterations in the arachi- 20 [0141] The senolytic effect of either COX or ALOX-in- donic acid metabolism and the altered arachidonic acid hibitors as well as of a dual inhibitor was assessed on metabolism could be exploited to selectively eliminate human umbilical vein endothelial cells (HUVECs). Figure senescent cells. 5 shows the effect of the COX-1/2 inhibitor ASA and of the dual COX/LOX inhibitor Licofelone compared to prior- Example 2: Identification of novel senolytics 25 art senolytics Quercetin and Navitoclax. [0142] Premature senescence was induced in HU- [0136] In accordance with the hypothesis established VECs with doxorubicin (Sigma-Aldrich), which was sup- in Example 1B, inhibition of cyclooxygenases or lipoxy- plemented to the growth media for 6 days. Treatment genases led to a cytotoxic effect, which was in general with senolytic substances started 8 days after completion more severe in senescent cells (Fig. 4-11), indicating 30 of the SIPS treatment to allow for cells to enter senes- availability of good therapeutic windows. There was cence. Quiescent cells were used as control. Growth me- some difference between the two examined cell types dia was supplemented with senolytic substances for 9 (HDFs and HUVECs) but inhibition of cyclooxygenases days with media changes every 3 days. All samples, in- and/or lipoxygenases lead to the elimination of senescent cluding the control, contained the same concentration of cells in both cell lines. Differences between different in- 35 solvent (DMSO). Subsequently, the viability was as- ducers of senescence and different donors were less pro- sessed using an alamar blue assay (Thermo Fisher Sci- nounced. This is in line with previous reports, showing a entific) (Fig. 5). cell type dependent senolytic effect by other senolytic [0143] HUVECs were more susceptible to an inhibition strategies (Fuhrmann-Stroissnigg et al. 2017; Schafer et of cyclooxygenases alone than HDFs. As expected and al. 2017; Zhu et al. 2017; Zhu et al. 2016; Zhu et al. 2015). 40 in line with a previous report (Zhu et al. 2016), Navitoclax [0137] The two reported prior-art senolytics Navitoclax did show considerable senolytic activity against HU- (ABT263; Selleck Chemicals) and Quercetin showed no VECs. In addition, the dual COX/LOX inhibitor Licofelone senolytic effects in HDFs (Fig. 4). Only in HUVECs, did also show a significant senolytic effect in HUVECs. Navitoclax was able to selectively eliminate senescent [0144] This confirmed the recent findings that different cells but not Quercetin (Fig. 5). 45 cell types react differently on senolytic compounds and indicates that cells from embryonic and neonatal origin Example 2A: Identification of novel senolytics in hu- might be more susceptible to senolytics in general man dermal fibroblasts (Hwang et al. 2018; Schafer et al. 2017). Surprisingly, the inhibition of AA metabolizing enzymes using cycloox- [0138] First, the senolytic effect of either COX or 50 ygenase and lipoxygenase inhibitors was senolytic in ALOX-inhibitors alone was assessed. Figure 4 shows both cell types, HDFs and HUVECs, and, importantly, the significant senolytic effects of the ALOX-5 inhibitor they are the only effective senolytic compounds reported MK886 (Santa Cruz Biotechnology) and the COX inhib- for adult human dermal fibroblasts so far. itor acetylsalicylic acid (ASA; Sigma-Aldrich) on human fibroblasts alone. These 2 drugs were also compared to 55 Example 3: Combined inhibition of cyclooxygenases prior-art senolytics Quercetin (Sigma-Aldrich) and and lipoxygenases in HDFs Navitoclax. [0139] Premature senescence was induced in HDF [0145] In order to increase the senolytic effects ob-

19 37 EP 3 643 305 A1 38 served, inhibition of both cyclooxygenases and lipoxyge- EC50 of MK886 as single treatment was 34 mM for se- nases using a combinatorial treatment was tested and nescent cells and dropped to 29.6 mM and 12.4 mM re- indeed showed synergistic effects. Co-inhibition of cy- spectively. In line with the results obtained with adult hu- clooxygenases and lipoxygenases with the combination man dermal fibroblasts as outlined above, the fold chang- 5 of acetylsalicylic acid (ASA) or Diclofenac (Sigma- es between the EC50 of senescent versus quiescent cells Aldrich) together with MK886 generated a potent seno- increased from 1.49 to 1.51 and 2.52 respectively. Again, lytic effect in adult HDFs as well as in foreskin HDFs (Fig. this shows the synergistic effect of the combined inhibi- 6-8). tion of cyclooxygenase and lipoxygenase compared to [0146] Premature senescence was induced in adult inhibition of cyclooxygenase and lipoxygenase alone, al- and foreskin HDFs with doxorubicin, which was supple- 10 so in foreskin derived HDFs. mented to the growth media for 6 days. Treatment with senolytic substances started 8 days after completion of Example 4: Combining cyclooxygenase/lipoxygen- the SIPS treatment to allow for cells to enter senescence. ase inhibition with ATP depletion Quiescent cells were used as control. Growth media was supplemented with senolytic substances for 9 days with 15 [0150] Lowering ATP levels by inhibition of ATP syn- media changes every 3 days. All samples, including the thase, ADP/ATP translocase or glycolysis potentiated control, contained the same concentration of solvent AA-induced mitochondrial membrane permeability in (DMSO). Subsequently, the viability was assessed using heart mitochondria indicating that AA-induced apoptosis an alamar blue assay (Thermo Fisher Scientific). can be further enhanced by depleting ATP levels. [0147] Figure 6 shows the effect of single treatment on 20 [0151] Premature senescence was induced in adult adult human dermal fibroblasts of the cell strain HDF161 HDFs with doxorubicin, which was supplemented to the using the ALOX-5 inhibitor MK886 and the COX-1/2 in- growth media for 6 days. Treatment with senolytic sub- hibitors ASA and Diclofenac or the COX-2 specific inhib- stances started 8 days after completion of the SIPS treat- itor Celecoxib (Sigma-Aldrich) compared to dual inhibi- ment to allow cells to enter senescence. Quiescent cells tion using either 0.4 mM ASA, 50 mM Diclofenac or 2 mM 25 were used as control. Growth media was supplemented Celecoxib combined with increasing concentrations of with senolytic substances for 9 days with media changes MK886. Inhibition of COX-2 alone by Celecoxib did not every 3 days. All samples, including the control, con- show any senolytic effect, indicating that the inhibition of tained the same concentration of solvent (DMSO). Sub- a single AA-metabolizing enzyme is not sufficient to re- sequently, the viability was assessed using an alamar 30 duce the cell viability in senescent cells. The EC50 of blue assay (Thermo Fisher Scientific). MK886 as single treatment was 26.4 mM for senescent [0152] Indeed, addition of 5 mM Oligomycin A (OmA; cells, whereas, combined with ASA, Diclofenac or Cayman Chemicals), an inhibitor of ATP synthase, to the Celecoxib, the EC50 dropped to 18.8 mM, 9 mM and 17 combination treatment of 0.4 mM ASA and MK886 further mM respectively. More importantly, the fold changes be- increased the senolytic effect by lowering the EC50 for 35 tween the EC50 of senescent versus quiescent cells in- senescent cells from 30.4 mM to 9.9 mM and increased creased from 1.58 to 1.95, 2.53 and 2.55 respectively. the fold changes between the EC50 of senescent versus This shows a synergistic effect of the combined inhibition quiescent cells from 1.46 to 4.51 (Fig. 9). This indicates of cyclooxygenase and lipoxygenase compared to inhi- that the AA-induced apoptosis in senescent cells is ATP bition of cyclooxygenase and lipoxygenase alone. sensitive and can thus be further enhanced by ATP de- [0148] Figure 7 shows the effect of single treatment on 40 pletion. adult human dermal fibroblasts of the cell strain HDF164 using the COX inhibitor Diclofenac and the ALOX-5 in- Example 5: Combining cyclooxygenase/lipoxygen- hibitor MK886 compared to dual inhibition using 0.4 mM ase inhibition with inhibition of the calcineurin-NFAT ASA or 50, 100 or 200 mM Diclofenac combined with pathway 45 increasing concentrations of MK886. The EC50 of MK886 as single treatment was 34.7 mM for senescent cells and [0153] Lowering the expression levels of enzymes, dropped to 30.4 mM, 17.3 mM, 11.2 mM and 8.39 mM which are metabolizing AA is also expected to have a respectively. In addition, the fold changes between the senolytic effect and can be used as a combinatorial se- EC50 of senescent versus quiescent cells increased from nolytic treatment together with the inhibition of the 1.31 to 1.46, 1.61, 1.88 and 1.64 respectively, confirming 50 COX/ALOX enzymatic activity, increasing the apoptotic the results of Fig. 7 in a cell strain derived of a different pressure even further in senescent cells. Pathway anal- donor. ysis of our RNA-seq dataset (Lämmermann et al. 2018) [0149] Figure 8 shows the effect of single treatment on indicated that the calcineurin-NFAT pathway is highly up- foreskin human dermal fibroblasts of the cell line regulated in SIPS HDFs compared to quiescent cells. fHDF166 using the COX inhibitors ASA and Diclofenac 55 Cyclosporin A (CsA) is a potent inhibitor of the cal- as well as the ALOX-5 inhibitor MK886 compared to dual cineurin-NFAT pathway and was previously shown to al- inhibition using 0.4 mM ASA or 50 mM Diclofenac com- so downregulate COX-2 expression (Hernandez et al. bined with increasing concentrations of MK886. The 2001; Lötzer et al. 2007; Yiu and Toker 2006).

20 39 EP 3 643 305 A1 40

[0154] In order to test this, premature senescence was methods such as ELISA, mass spectrometry or HPLC. induced in foreskin HDFs with doxorubicin, which was [0159] In order to verify, whether novel senolytics can supplemented to the growth media for 6 days. Treatment be identified in an efficient screen measuring the intrac- with senolytic substances started 8 days after completion ellular levels of AA, an ELISA test (Novus Biologicals; of the SIPS treatment to allow for cells to enter senes- 5 NBP2-66372) was used according to the manufacturer’s cence. Quiescent cells were used as control. Growth me- instructions to first, confirm that novel senolytics identi- dia was supplemented with senolytic substances for 9 fied in the examples above indeed lead to an accumula- days with media changes every 3 days. All samples, in- tion of intracellular AA, and second, to screen for com- cluding the control, contained the same concentration of pounds, which cause an increase of intracellular levels solvent (DMSO). Subsequently, the viability was as- 10 of AA in senescent HDFs. sessed using an alamar blue assay (Thermo Fisher Sci- [0160] As expected, the intracellular concentration of entific). AA was higher in senescent cells compared to quiescent [0155] Interestingly, adding 2 mM of CsA (Santa Cruz control cells and by blocking the production of eicosa- Biotechnology) to the combination treatment of 0.4mM noids for 6 hrs with the COX-1/2 inhibitor Diclofenac and 15 ASA and MK886 lowered the EC50 for senescent cells the ALOX-5 inhibitor MK886 the intracellular AA levels from 29.6 mM to 14.9 mM and increased the fold changes could be increased (Fig. 12). between the EC50 of senescent versus quiescent cells [0161] The senolytic effect of compounds identified in from 1.51 to 2.81 (Fig. 10 and 11). the ELISA screen is then tested using a dose response [0156] Taken together, methods and compositions tar- assay in adult and foreskin HDFs as described for COX geting the lipid metabolism therefore represent a prom- 20 and ALOX inhibitors above. Such compounds will also ising way to eliminate senescent cells and treat senes- be used as senolytic drugs that target the AA metabolism. cence-associated diseases and disorders. Our results demonstrate that the inhibition of AA utilizing enzymes REFERENCES results in a senolytic effect in adult HDFs and in foreskin HDFs as well as in neonatal HUVECs. The two reported 25 [0162] senolytics Quercetin and Navitoclax did not show con- siderable senolytic activity, except for Navitoclax in dox- Acosta JC, Banito A, Wuestefeld T, Georgilis A, Jan- orubicin-induced senescent HUVECs. In contrast to ich P, Morton JP, et al. A complex secretory program Navitoclax, there are no serious side effects reported for orchestrated by the inflammasome controls para- the inhibition of cyclooxygenases and/or lipoxygenases. 30 crine senescence. Nat. Cell Biol. 2013;15(8):978-90 In addition, the dual inhibition of cyclooxygenases and Augert A, Payré C, de Launoit Y, Gil J, Lambeau G, lipoxygenases showed a synergistic effect in senescent Bernard D. The M-type receptor PLA2R regulates HDFs of adult origin making them uniquely qualified to senescence through the p53 pathway. EMBO Rep. treat and prevent senescence-associated diseases and European Molecular Biology Organization; disorders. 35 2009;10(3):271-7 [0157] As shown herein, the senolytic effect of the in- Baar MP, Brandt RMC, Putavet DA, Klein JDD, hibition of cyclooxygenases and/or lipoxygenases could Derks KWJ, Bourgeois BRM, et al. Targeted Apop- additionally be enhanced by simultaneous inhibition of tosis of Senescent Cells Restores Tissue Homeos- ATP synthase and calcineurin. Inhibiting further AA-uti- tasis in Response to Chemotoxicity and Aging. Cell. lizing or AA-level-manipulating enzymes and pathways 40 2017;169(1):132-147.e16 such as cytochrome P450, long-chain-fatty-acid-CoA Baker DJ, Childs BG, Durik M, Wijers ME, Sieben ligase 4(ACSL4), lysophosphatidylcholine acyltrans- CJ, Zhong J, et al. Naturally occurring p16 Ink4a ferases or fatty acid elongases further potentiates the -positive cells shorten healthy lifespan. Nature. Na- senolytic effect of cyclooxygenase and/or lipoxygenase ture Publishing Group; 2016;1-20 inhibitors. 45 Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, et al. Clearance of Example 6: Quantitative Analysis of AA p16lnk4a-positive senescent cells delays ageing-as- sociated disorders. Nature. Nature Publishing [0158] As described herein, the senolytic effect is Group; 2011;479(7372):232-6 caused by increasing the intracellular levels of AA above 50 Braun H, Schmidt BMW, Raiss M, Baisantry A, Mir- a certain threshold and thereby inducing cell death. This cea-Constantin D, Wang S, et al. Cellular Senes- can be verified by measuring the intracellular AA levels cence Limits Regenerative Capacity and Allograft of quiescent and senescent cells before and after inhib- Survival. J. Am. Soc. Nephrol. 2012;23(9):1467-73 iting AA utilizing metabolic pathways. This is expected Bussian TJ, Aziz A, Meyer CF, Swenson BL, van to increase the intracellular AA levels of senescent cells 55 Deursen JM, Baker DJ. Clearance of senescent glial above a critical threshold that will not be reached by qui- cells prevents tau-dependent pathology and cogni- escent cells under the same conditions. Intracellular AA tive decline. Nature. 2018; levels can be quantified using quantitative analytical Campisi J, d’Adda di Fagagna F. Cellular senes-

21 41 EP 3 643 305 A1 42 cence: when bad things happen to good cells. Nat. senescent cells by mitochondrial targeting is regu- Rev. Mol. Cell Biol. 2007;8(9):729-40 lated by ANT2. Cell Death Differ. 2018; Chang J, Wang Y, Shao L, Laberge R-M, Demaria Hwang H V., Tran DT, Rebuffatti MN, Li C-S, Knowl- M, Campisi J, et al. Clearance of senescent cells by ton AA. Investigation of quercetin and hyperoside as ABT263 rejuvenates aged hematopoietic stem cells 5 senolytics in adult human endothelial cells. Minami- in mice. Nat. Med. Nature Publishing Group; no T, editor. PLoS One. 2018;13(1):e0190374 2016;22(1):78-83 Jeon OH, Kim C, Laberge R-M, Demaria M, Rathod Childs BG, Baker DJ, Wijshake T, Conover CA, S, Vasserot AP, et al. Local clearance of senescent Campisi J, van Deursen JM. Senescent intimal foam cells attenuates the development of post-traumatic cells are deleterious at all stages of atherosclerosis. 10 osteoarthritis and creates a pro-regenerative envi- Science. 2016;354(6311):472-7 ronment. Nat. Med. 2017; Choi J, Zhang W, Gu X, Chen X, Hong L, Laird JM, Jeschek D, Lhota G, Wallner J, Vorauer-Uhl K. A et al. Lysophosphatidylcholine is generated by spon- versatile, quantitative analytical method for pharma- taneous deacylation of oxidized phospholipids. ceutical relevant lipids in drug delivery systems. J. Chem. Res. Toxicol. 2011:24(1):111-8 15 Pharm. Biomed. Anal. 2016;119:37-44 Coppé J-P, Desprez P-Y, Krtolica A, Campisi J. The Kabir T, Leigh R, Tasena H, Mellone M, Coletta R, Senescence-Associated Secretory Phenotype: The Parkinson E, et al. A miR-335/COX-2/PTEN axis reg- Dark Side of Tumor Suppression. Annu Rev Pathol. ulates the secretory phenotype of senescent cancer- 2010;5:99-118 associated fibroblasts. Aging (Albany. NY). Currais A, Quehenberger O, M Armando A, Daugh- 20 2016;8(8):1608-35 erty D, Maher P, Schubert D. Amyloid proteotoxicity Krtolica A, Parrinello S, Lockett S, Desprez PY, initiates an inflammatory response blocked by can- Campisi J. Senescent fibroblasts promote epithelial nabinoids. npj Aging Mech. Dis. 2016;2(1):16012 cell growth and tumorigenesis: a link between cancer Demaria M, Ohtani N, Youssef S, Rodier F, Tous- and aging. Proc. Natl. Acad. Sci. U. S. A. saint W, Mitchell J, et al. An essential role for senes- 25 2001;98(21):12072-7 cent cells in optimal wound healing through secretion Lämmermann I, Terlecki-Zaniewicz L, Weinmüllner of PDGF-AA. Dev. Cell. 2014;31(6):722-33 R, Schosserer M, Dellago H, de Matos Branco AD, Dörr JR, Yu Y, Milanovic M, Beuster G, Zasada C, et al. Blocking negative effects of senescence in hu- Däbritz JHM, et al. Synthetic lethal metabolic target- man skin fibroblasts with a plant extract. npj Aging ing of cellular senescence in cancer therapy. Nature. 30 Mech. Dis. 2018;4(1):4 2013;501(7467):421-5 Lehmann M, Korfei M, Mutze K, Klee S, Skronska- Farr JN, Xu M, Weivoda MM, Monroe DG, Fraser Wasek W, Alsafadi HN, et al. Senolytic drugs target DG, Onken JL, et al. Targeting cellular senescence alveolar epithelial cell function and attenuate exper- prevents age-related bone loss in mice. Nat. Med. imental lung fibrosis ex vivo. Eur. Respir. J. 2017;23(9):1072-9 35 2017;50(2):1602367 Fonteh AN, Atsumi G, LaPorte T, Chilton FH. Secre- Lewis DA, Travers JB, Machado C, Somani A-K, tory phospholipase A2 receptor-mediated activation Spandau DF. Reversing the aging stromal pheno- of cytosolic phospholipase A2 in murine bone mar- type prevents carcinoma initiation. Aging (Albany. row-derived mast cells. J. Immunol. NY). 2011;3(4):407-16 2000;165(5):2773-82 40 Li Y, Lei D, Swindell WR, Xia W, Weng S, Fu J, et Fuhrmann-Stroissnigg H, Ling YY, Zhao J, McGow- al. Age-Associated Increase in Skin Fibroblast-De- an SJ, Zhu Y, Brooks RW, et al. Identification of rived Prostaglandin E2 Contributes to Reduced Col- HSP90 inhibitors as a novel class of senolytics. Nat. lagen Levels in Elderly Human Skin. J. Invest. Der- Commun. 2017;8(1):422 matol. 2015;135(9):2181-8 Gruber F, Ornelas CM, Karner S, Narzt M-S, Nagel- 45 Lötzer K, Jahn S, Kramer C, Hildner M, Nüsing R, reiter IM, Gschwandtner M, et al. Nrf2 deficiency Funk CD, et al. 5-Lipoxygenase/cyclooxygenase-2 causes lipid oxidation, inflammation, and matrix-pro- cross-talk through cysteinyl leukotriene receptor 2 in tease expression in DHA-supplemented and UVA- endothelial cells. Prostaglandins Other Lipid Mediat. irradiated skin fibroblasts. Free Radic. Biol. Med. 2007;84(3-4):108-15 2015;88(Pt B):439-51 50 Milanovic M, Fan DNY, Belenki D, Däbritz JHM, Hernández GL, Volpert O V, Iñiguez MA, Lorenzo Zhao Z, Yu Y, et al. Senescence-associated repro- E, Martinez-Martinez S, Grau R, et al. Selective in- gramming promotes cancer stemness. Nature. hibition of vascular endothelial growth factor-medi- 2017;553(7686):96-100 ated angiogenesis by cyclosporin A: roles of the nu- Muñoz-Espín D, Cañamero M, Maraver A, Gómez- clear factor of activated T cells and cyclooxygenase 55 López G, Contreras J, Murillo-Cuesta S, et al. Pro- 2. J. Exp. Med. 2001;193(5):607-20 grammed Cell Senescence during Mammalian Em- Hubackova S, Davidova E, Rohlenova K, Stursa J, bryonic Development. Cell. Cell Press; Werner L, Andera L, et al. Selective elimination of 2013;155(5):1104-18

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Nishikiori M, lizuka H, Ichiba H, Sadamoto K, Fuku- X, et al. Discovery of piperlongumine as a potential shima T. Determination of Free Fatty Acids in Human novel lead for the development of senolytic agents. Serum by HPLC with Fluorescence Detection. J. Aging (Albany. NY). 2016a;8(11):2915-26 Chromatogr. Sci. Oxford University Press; Wang X-F, Huang Y-F, Wang L, Xu L-Q, Yu X-T, Liu 2015;53(4):537-41 5 Y-H, et al. Photo-protective activity of pogostone Pan J, Li D, Xu Y, Zhang J, Wang Y, Chen M, et al. against UV-induced skin premature aging in mice. Inhibition of Bcl-2/xl With ABT-263 Selectively Kills Exp. Gerontol. 2016b;77:76-86 Senescent Type II Pneumocytes and Reverses Per- Xu M, Palmer AK, Ding H, Weivoda MM, Pirtskhal- sistent Pulmonary Fibrosis Induced by Ionizing Ra- ava T, White TA, et al. Targeting senescent cells diation in Mice. Int. J. Radiat. Oncol. Biol. Phys. 10 enhances adipogenesis and metabolic function in 2017;99(2) old age. Elife. 2015;4(December):e12997 Pan Y, Wan J, Liu Y, Yang Q, Liang W, Singhal PC, Yiu GK, Toker A. NFAT induces breast cancer cell et al. sPLA2 IB induces human podocyte apoptosis invasion by promoting the induction of cyclooxyge- via the M-type phospholipase A2 receptor. Sci. Rep. nase-2. J. Biol. Chem. 2006;281(18):12210-7 2015;4(1):6660 15 Yosef R, Pilpel N, Tokarsky-Amiel R, Biran A, Penzo D, Petronilli V, Angelin A, Cusan C, Colonna Ovadya Y, Cohen S, et al. Directed elimination of R, Scorrano L, et al. Arachidonic acid released by senescent cells by inhibition of BCL-W and BCL-XL. phospholipase A(2) activation triggers Ca(2+)-de- Nat. Commun. 2016;7:11190 pendent apoptosis through the mitochondrial path- Zhang X, Zhang S, Liu X, Wang Y, Chang J, Zhang way. J. Biol. Chem. American Society for Biochem- 20 X, et al. Oxidation resistance 1 is a novel senolytic istry and Molecular Biology; 2004;279(24):25219-25 target. Aging Cell. 2018;e12780 Ressler S, Bartkova J, Niederegger H, Bartek J, Zhu Y, Doornebal EJ, Pirtskhalava T, Giorgadze N, Scharffetter-Kochanek K, Jansen-Dürr P, et al. Wentworth M, Fuhrmann-Stroissnigg H, et al. New p16INK4A is a robust in vivo biomarker of cellular agents that target senescent cells: the flavone, fise- aging in human skin. Aging Cell. 2006;5(5):379-89 25 tin, and the BCL-XL inhibitors, A1331852 and Roos CM, Zhang B, Palmer AK, Ogrodnik MB, Pirt- A1155463. Aging (Albany. NY). 2017;9(3):955-63 skhalava T, Thalji NM, et al. Chronic senolytic treat- Zhu Y, Tchkonia T, Fuhrmann-Stroissnigg H, Dai ment alleviates established vasomotor dysfunction HM, Ling YY, Stout MB, et al. Identification of a novel in aged or atherosclerotic mice. Aging Cell. senolytic agent, navitoclax, targeting the Bcl-2 family 2016;15(5):973-7 30 of anti-apoptotic factors. Aging Cell. Schafer MJ, White TA, lijima K, Haak AJ, Ligresti G, 2016;15(3):428-35 Atkinson EJ, et al. Cellular senescence mediates fi- Zhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding brotic pulmonary disease. Nat. Commun. 2017;8 H, Giorgadze N, et al. The Achilles’ heel of senescent Schmitt R, Melk A. Molecular mechanisms of renal cells: From transcriptome to senolytic drugs. Aging aging. Kidney Int. 2017;92(3):569-79 35 Cell. 2015;14(4):644-58 Scott GA, Jacobs SE, Pentland AP. sPLA2-X Stim- ulates Cutaneous Melanocyte Dendricity and Pig- mentation Through a Lysophosphatidylcholine-De- Claims pendent Mechanism. J. Invest. Dermatol. 2006;126(4):855-61 40 1. A composition comprising one or more inhibitors ca- Six DA, Dennis EA. The expanding superfamily of pable of inhibiting at least two of cyclooxygenase-1 phospholipase A(2) enzymes: classification and (COX-1), cyclooxygenase-2 (COX-2) and lipoxyge- characterization. Biochim. Biophys. Acta. nase, for use in selectively eliminating senescent 2000;1488(1-2):1-19 cells. Terlecki-Zaniewicz L, Lämmermann I, Latreille J, 45 Bobbili MR, Pils V, Schosserer M, et al. Small extra- 2. The composition for use according to claim 1, where- cellular vesicles and their miRNA cargo are anti-ap- in the senescent cells are characterized by in- optotic members of the senescence-associated se- creased intracellular levels of at least one of lyso- cretory phenotype. Aging (Albany. NY). 2018 phosphatidylcholine, arachidonic acid and phos- Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kel- 50 pholipase A2 activity. ley DR, et al. Differential gene and transcript expres- sion analysis of RNA-seq experiments with TopHat 3. The composition for use according to claim 2, where- and Cufflinks. Nat. Protoc. 2012;7(3):562-78 in the lysophosphatidylcholine is 1-steraroyl-2-hy- Valentijn FA, Falke LL, Nguyen TQ, Goldschmeding droxy-sn-glycero-3-phosphocholine (LysoSPC) or R. Cellular senescence in the aging and diseased 55 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho- kidney. J. Cell Commun. Signal. Springer; choline (LysoPPC). 2018;12(1):69-82 Wang Y, Chang J, Liu X, Zhang X, Zhang S, Zhang 4. The composition for use according to any one of

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claims 1 to 3, wherein one or more inhibitors are lular conversion of arachidonic acid, wherein said COX-1 and/or COX-2 inhibitors selected from the additional compound is preferably selected from the group consisting of acetylsalicylic acid, diclofenac, group consisting of celecoxib, cyclosporin A, ibuprofen, acetaminophen, indomethacin, nabumetone, ketorolac, tenoxicam, 5 a) a natural compound, preferably selected from tolmetin, piroxicam, fenoprofen, etodolac, naproxen, the group consisting of turmeric, rosemary, gin- diflunisal, suprofen, bromfenac, ketoprofen, dihomo- ger, oregano, resveratrol, curcumin, cannabi- gamma-linolenic acid, icosapent, fluriprofen, noids, ginseng, saponins, terpenoids, flavo- mefenamic acid, salsalate, sulindac, salicylic acid, noids, polyphenols, ginkgo biloba, capsaicin, lumiracoxib, O-acetyl-L-serine, phenacetin, fluripro- 10 genistein, kaempferol and quercetin; fen methyl ester, metamizole, nitroaspirin, meloxi- b) an inhibitor of cytochrome P450, preferably cam, flufenamic acid, oxaprozin, tiaprofenic acid, selected from the group consisting of sulfaphen- magnesium salicylate, diethylcarbamazine, lornoxi- azole, avasimibe, benzbromarone, rosiglita- cam, carprofen, phenylbutazone, nepafenac, antipy- zone, troglitazone, cervistatin, warfarin, piogli- rine, antrafenine, choline magnesium trisalicylate, 15 tazone, lapatinib, trimethoprim, zafirlukast, triflusal, niflumic acid, dexibuprofen, aceclofenac, amodiaquine, nicardipine, simvastatin, acemetacin, droxicam, loxoprofen, tolfenamic acid, fluvastatin , loratadine, ethinylestradiol, irbe- dexketoprofen trometamol, talniflumate, propaceta- sartan, quinine, sorafenib, eltrombopag, losar- mol, trolamine salicylate, phenyl salicylate, bufex- tan, licofelone, amitriptyline, atorvastatin, amac, glycol salicylate, menthyl salicylate, FK-506, 20 mefenamic acid, meloxicam, piroxicam, erlo- lenalidomide, rofecoxib, valdecoxib, cimicoxib, chlo- tinib, pazopanib, diethylstilbestrol, enzaluta- rphenesin, clodronic acid, seliciclib, drospirenone, mide, ponatinib, dabrafenib, enasidenib, lovas- triamcinolone, pomalidomide, parecoxib, firocoxib, tatin, montekulast, ketoconazole, felodipine, aclofenac, adapalene, thalidomide, etoricoxib, robe- candesartan cilexetil, clotrimazole, mometa- nacoxib, asaraldehyde, zaltoprofen, deracoxib, dex- 25 sone, salmeterol, raloxifene, fenofibrate, lev- amethasone, pranoprofen, amfenac sodium mono- othyroxine, tamoxifen, quercetin, oxybutynin, hydrate, ampiroxicam, NS-398, bismuth subsali- medroxyprogesterone acetate, nifedipine, liot- cylate, diclofenac diethylamine, trometamol, rutae- rix, amlodipine, bezafibrate, chloramphenicol, carpine, salicin, fenbufen, xanthohumol, flunixin cyclosporin, cimetidine, clopidogrel, cholecalcif- meglumin and nimesulide. 30 erol, delavirdine, dextropropoxyphene, etopo- side, isoniazid, ketoprofen, metronidazole, ni- 5. The composition for use according to any one of lutamide, nilvadipine, paroxetine, phenelzine, claims 1 to 4, wherein one or more inhibitors are pravastatin, propafenone, pyrimethamine, ro- lipoxygenase and/or FLAP (ALOX5AP) inhibitors se- fecoxib, rutin, saquinavir, sulfamethoxazole, lected from the group consisting of MK886, zileuton, 35 sulfinpyrazone, tegaserod, terfenadine, thiori- masoprocol, diethylcarbamazine, azelastine, dazine, ticlopidine, tioconazole, triazolam, trole- benoxaprofen, nordihydroguaiaretic acid, abietic ac- andomycin, valproic acid, abiraterone, vismo- id, esculetin, montelukast, minocycline, MLN-977, degib, regorafenib, trametinib, idelalisib, lopina- rhein, diacerein, nabiximols, fostamatinib, AM103, vir, celecoxib, efavirenz, rabeprazole, terifluno- DG031, fiboflapon, AA-861 and atreleuton. 40 mide, crisaborole, belinostat, topiroxostat, can- desartan, letermovir, rucaparib, opicapone, na- 6. The composition for use according to any one of bilone, fluvoxamine, fluticasone, fluticasone claims 1 to 5, wherein one or more inhibitors are dual furoate, fluticasone propionate, bosutinib, cabo- cyclooxygenase and lipoxygenase inhibitors, prefer- zantinib, genistein, lenvatinib, atazanavir, bex- ably selected from the group consisting of licofelone, 45 arotene, deferasirox, quinidine, mifepristone, darbufelone, CI-987, S-2474, KME-4, Chebulagic vemurafenib, sildenafil, diclofenac, fluoxetine, acid, balsalazide, mesalazine, sulfasalazine, ami- valdecoxib, voriconazole, etodolac, sertraline, nosalicylic acid, meclofenamic acid, morniflumate di- glyburide, acenocoumarol, rosuvastatin, imat- arylpyrazole derivatives, thieno[2,3-b]pyridine deriv- inib, clozapine, diazepam, progesterone, ome- atives, N-substituted 5-aminosalicylicylamides, flav- 50 prazole, valsartan, bortezomib, nevirapine, aze- ocoxid, indolizine derivatives, LQFM-091, hyper- lastine, lornoxicam, phenylbutazone, etravirine, forin, celastrol, BW755C, tepoxalin, b-boswellic acid, leflunomide, sitaxentan, aminophenazone, ver- D-002, 2,3-diarylxanthones, phenidone and ER- apamil, etoricoxib, propofol, sulfamoxole, dicou- 34122. marol, diltiazem, histamine, moclobemide, sel- 55 egiline, parecoxib, doconexent, acetyl sulfisox- 7. The composition for use according to any one of azole, fluconazole, pantoprazole, deslorata- claims 1 to 6, wherein the composition comprises an dine, miconazole, amiodarone, gemfibrozil, additional compound capable of inhibiting intracel- probenecid, teniposide, sulfadiazine, capecitab-

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ine, fluorouracil, tranylcypromine, anastrozole, hydroxy-estradiol, 17a-estradiol, 17β-estradiol, atovaquone, cyclizine, dexfenfluramine, di- α-zearalenol, β-zearalenol, oligomycin, venturi- sulfiram, epinephrine, eprosartan, flecainide, cidin, apoptolidin, ossamycin, cytovaricin, peli- indinavir, methazolamide, nelfinavir, olanzap- omycin, tributyltin chloride, tricyclohexyltin hy- ine, pranlukast, promethazine, sulfadimethox- 5 droxide, triethyltin sulfate, triphenyltin chloride, ine, sulfamethizole, sulfanilamide, sulfapyrid- dimethyltin 3-hydroxyflavone chloride, diethyltin ine, methimazole, tolcapone, bicalutamide, ar- 3-hydroxyflavone chloride, dibuthyltin 3-hydrox- modafinil, agomelatine, noscapine, clevidipine, yflavone bromide, dioctyltin 3-hydroxyflavone sulconazole, gefitinib, ticagrelor, ceritinib, floxu- chloride, diphenyltin 3-hydroxyflavone chloride, ridine, lifitegrast, rhein, diacerein, zucapsaicin, 10 diethyltin 3,5,7,2’,4’-pentahydroxyflavone chlo- stiripentol, lobeglitazone, dosulepin, ma- ride, dibutyltin 3,5,7,2’,4’-pentahydroxyflavone nidipine, cimicifuga racemose, curcumin, fel- bromide, diphenyltin 3,5,7,2’,4’-pentahydroxy- bamate, piperine, safinamide, irponiazid, orita- flavone chloride, tributyltin 3-hydroxyflavone, tri- vancin, masorpocol and pegvisomant; ethyllead, aurovertin, citreoviridin, asteltoxin, c) an inhibitor of long-chain-fatty-acid-CoA 15 rhodamine B, rhodamine 123, rhodamine 6G, ligase 4 (ACSL4), preferably selected from the rosaniline, malachite green, brilliant green, group consisting of triascin A, triascin B, triascin quinacrine, quinacrine mustard, acridine or- C, triascin D, troglitazone, ciglitazone, pioglita- ange, coriphosphine, pyronin Y, dequalinium, zone and rosiglitazone; safranin O, nile blue A, ethidium bromide, tet- d) an inhibitor of a lysophosphatidylcholine acyl- 20 racaine, dibucaine, procaine, lidocaine, chlo- transferase, specifically an inhibitor of LPCAT1, rpromazine, trifluoperazine, procainamide, pro- LPCAT2, LPCAT3, LPCAT4, MBOAT2 and/or pranolol, octyl guanidine, 1-dansyl amido-3- MBOAT7, preferably selected from the group dimethypropylamine compounds, cetyltrimethy- consisting of N-phenylmaleimide derivatives, lammonium, spermine, spermidine, bathophen- TSI-01 and thimerosal; and/or 25 an throline-metal chelate, 4,4-diphenyl-2,2-bi- e) an inhibitor of a fatty acid elongase, specifi- pyridine, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-tri- cally an inhibitor of ELOVL2, ELOVL4 and/or azine, atrazine, atrazine amino derivative, arse- ELOVL5, and wherein the inhibitor of a fatty acid nate, aluminium fluoride, beryllium fluoride, elongase is preferably selected from the group scandium fluoride, vanadate, magnesium fluo- consisting of cycloate, adenosine 5’-hexadecyl- 30 ride, sulfite, thiophosphate, azide, ANPP, phe- phosphate, endo-1k, (S)-1y and compound 37, nylglyoxal, butanedione, dansyl chloride, 1- 5,5-dimethyl-3-(5-methyl-3-oxo-2-phenyl-2,3- fluoro-2,4-dinitrobenzene, dicarbopolyborate, dihydro-1H-pyrazol-4-yl)-1-phenyl-3-(trifluor- almitrine, 5-hydroxy-1,2-naphtalene dicarboxy- omethyl-3,5,6,7-tetrahydro-1H-indole-2,4-di- lic anhydride, R207910, spegazzinine, n-buta- one) and (3-endo)-3-(phenylsulfonyl)-N-[4-(trif- 35 nol, terachlorosalicylanilide, dihydrostreptomy- luoromethyl)phenyl]-8-azabicyclo[3.2.1]oc- cin, suramin, Bz-423, DMSO, hypochlorous ac- tane-8-carboxamide. id, DDT, diazoxide, HNB, N-sulfonyl or N-alkyl substituted tetrahydrobenzodiazepine deriva- 8. The composition for use according to any one of tives, 4-(N-arylimidazole)-substituted benzo- claims 1 to 7, wherein the composition comprises an 40 pyran derivatives, N-[1-aryl-2-(1-imidazo- additional compound capable of manipulating intra- lo)ethyl]-cyanoguanidine derivatives, N-[1-aryl- cellular ATP levels, wherein said additional com- 2-(1-imidazolo)ethyl]-acylguanidine deriva- pound is preferably selected from the group consist- tives, O-[1-aryl-2-(1-imidazo- ing of lo)ethyl]-thiourethane derivatives, dio-9 com- 45 plex, ethanol and zinc; a) an inhibitor of ATP synthases, preferably se- b) an inhibitor of ADP/ATP translocases, pref- lected from the group consisting of oligomycin erably selected from the group consisting of A, inositol nicotinate, bedaquiline, efrapeptins, clodronic acid, ibipinabant, atractyloside, car- leucinostatins, tentoxin, tnetoxin derivates, an- boxyatractyloside, bongkrekic acid, isobongkre- 50 giostatin, enterostatin, melittin, IF1, Syn-A2, kic acid, MT-21, closantel, CD437, leelamine, Syn-C, resveratrol, piceatannol, diethyl- L923-0673, IMD 0354, PI32-0333, S899542, stilbestrol, 4-acetoamido-4’-isothiocyanostil- nonactin and S838462; bene-2,2’-disulfonate, 4,4’-D-isothiocyanato- c) an inhibitor of glycolysis, preferably selected stilbene-2,2-disulfonic acid, quercetin, kaemp- from the group consisting of 2-deoxy-D-glucose, ferol, morin, apigenin, genistein, biochanin A, 55 lonidamine, bromopyruvic acid, phloretin, quer- daidzein, epicatechin gallate, epigallocatechin cetin, STF-31, WZB117, 3PO, 3-bromopyru- gallate, proanthocyanidin, curcumin, phloretin, vate, dichloroacetate, oxamic acid, NHI-1, ox- theaflavin, tannic acid, 4-hydroxy-estradiol, 2- ythiamine, imatinib, glucosamine, 6-aminonico-

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tinamide, genistein, 5-thioglucose, mannohep- side effects of chemotherapy and prevents or delays tulose, α-chlorohydrin, ornidazole, oxalate, glu- tumor relapse. fosfamide, N-(phosphonacetyl)-L-aspartate, 6- methylmercaptopurine riboside, CGP 3466B 14. An in vitro method of identifying senescent cells in a maleate, sodium monofluorophosphate, DASA- 5 subject, comprising the steps of 58, DL-serine, dichloroacetic acid, sodium dichloroacetate, nitrofural, 6-AN, fasentin, a) providing a sample of said subject, benserazide, astraglin, resveratrol, chrysin, b) determining the levels of at least one of lys- GEN-27, apigenin, bis-2-(5-phenylacetamido- ophosphatidylcholine, arachidonic acid and/or 1,3,4-thiadiazol-2-yl)ethyl sulfide, CB-839, aza- 10 phospholipase A2 activity in said sample, serine, acivicin, 6-diazo-5-ox-L-norleucine, thi- c) comparing the levels of b) to a reference level, azolidine-2,4-dione derivatives, compound 968, wherein the reference level is the level of at least R-lipoic acid, 1,3,4-thiadiazole compounds, 2- one of lysophosphatidylcholine, arachidonic ac- chloropropionate, Nov3r, AZD7545, Pfz3, id and/or phospholipase A2 activity in non-se- radicicol, mitaplatin, mito-DCA, phenylbutyrate, 15 nescent cells, 4,5-diarylisoxazoles, VER-246608, betulinic ac- id, pyruvate analogs containing phosphinate or and wherein an increase of at least 3-fold is indicative phosphonate group, CPI-613, M77976, aromat- of the presence of senescent cells in said sample. ic DCA derivatives, furan and thiophene carbox- ylic acids, ritonavir, FX11, oxamate, D-fructose- 20 15. A method of screening for candidate compounds for 6-phosphate, 6-phosphogluconic acid, N-bro- eliminating senescent cells, comprising the steps of moacetyl-aminoethyl phosphate, 2-carbox- yethylphosphonic acid, N-hydroxy-4-phospho- a) bringing at least one test compound in contact no-butanamide, 2-phosphoglyceric acid, iodoa- with a sample of senescent cells, cetate, gossypol, bisphosphonate analogs of 25 b) measuring the level of arachidonic acid and/or 1,3-bisphosphoglyceric acid, benzene hexacar- apoptosis and/or measuring cell viability, and boxylic acid, 3-phosphoglyceric acid, phospho- c) selecting the compounds which cause intra- noacetohydroxamic acid, 2-phospho-D-glyceric cellular accumulation of arachidonic acid, apop- acid, TLN-232 and CAP-232. tosis or reduced cell viability in the senescent 30 cells treated in a) compared with untreated se- 9. The composition for use according to any one of nescent cells. claims 1 to 8, wherein the composition 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 pro- 35 motes the regression of a senescence-related dis- ease or condition.

10. The composition for use according to claim 9, where- in the senescence-related disease or condition is se- 40 lected from cardiovascular disease, atherosclerosis, cancer, osteoporosis, osteoarthritis, neurological disorders, dementia, cataract, kidney disease, retin- opathy, diabetes, lung fibrosis, vertebral skin degen- eration, age-related muscular atrophy, hair loss and 45 skin aging.

11. The composition for use according to any one of claims 1 to 8, wherein the composition improves the performance of transplants. 50

12. The composition for use according to any one of claims 1 to 8, wherein the composition prevents or attenuates senescence-associated scar formation and fibrosis. 55

13. The composition for use according to any one of claims 1 to 8, wherein the composition ameliorates

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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