Use of carotenoids in the treatment of age-related diseases

Abstract

The present invention relates to a combination comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or ester or salt thereof, and a second component selected from the following: an inhibitor of one or more BCL-2 anti-apoptotic protein family members, an activator of a pro-apoptotic BCL-2 family member, a senescent cell lysing agent and an inhibitor of a senescent phenotype. The present invention also relates to a cosmeceutical, a cosmetic or a pharmaceutical composition comprising the combination of the present invention, and its use in a cosmetic method and its medical use.

Description

Technical Field

[0001] The present invention is in the field of medicine. More specifically, in the field of treatment of diseases or conditions in which removal of senescent cells is beneficial. Background Art

[0002] Cellular senescence is characterized by the irreversible loss of proliferative potential and specific changes in cell morphology and gene expression that ultimately lead to impaired cellular function.

[0003] Given that senescent cells have been causally implicated in some aspects of age-related health decline and can contribute to certain diseases, and are also induced by necessary life-sustaining chemotherapy and radiation treatments, the presence of senescent cells can have deleterious effects on millions of patients worldwide.

[0004] Document US2016339019 discloses inhibitors of the BCL-2 anti-apoptotic protein family and document EP3247375A2 discloses inhibitors of SYK and inhibitors of ASK1 as agents suitable for removing senescent cells in humans.

[0005] There remains a need in the art to provide new and existing compounds that therapeutically target senescent cells and can be used to treat diseases associated with cellular senescence. SUMMARY OF THE INVENTION

[0007] The authors of the present invention have found that carotenoids act synergistically with inhibitors of BLC2 anti-apoptotic family members, activators of BCL-2 pro-apoptotic family members, senolytic agents and senomorphic agents in alleviating aging and in cancer treatment.

[0008] Therefore, in a first aspect, the present invention relates to a combination comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or an ester or a salt thereof, and a second component selected from the following: an inhibitor of one or more BCL-2 anti-apoptotic protein family members, an activator of a BCL-2 pro-apoptotic family member, a senescent cell lytic agent and a senescent phenotype inhibitor.

[0009] In a second aspect, the present invention relates to a cosmeceutical, cosmetic or pharmaceutical composition comprising the combination of the present invention.

[0010] In a third aspect, the present invention relates to a cosmetic method for preventing and / or reducing skin aging and / or for improving the cosmetic adverse effects of aging, comprising administering to a subject in need thereof the combination according to any one of claims 1 to 4 or the cosmeceutical or cosmetic composition according to claim 5.

[0011] In a fourth aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0012] In a fifth aspect, the invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in the treatment of cancer.

[0013] In a sixth aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a senescence-related disease or disorder.

[0014] In a seventh aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in enhancing the effectiveness of an anti-tumor compound.

[0015] In an eighth aspect, the present invention relates to a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or an ester or salt thereof for use in treating a senescence-related disease or disorder.

[0016] In a ninth aspect, the present invention relates to a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or an ester or salt thereof for enhancing the effectiveness of an anti-tumor compound.

[0017] In a tenth aspect, the present invention relates to carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for use in reducing adverse effects of anti-tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 IL secretion of SK-Mel-103 cells after treatment with palbociclib. Error bars indicate standard deviation from 3 technical replicates. SC, senescent cells; F, fucoxanthin.

[0020] Figure 2 IL secretion of SK-Mel-103 cells after treatment with doxorubicin. Error bars indicate standard deviation from 3 technical replicates. SC, senescent cells; F, fucoxanthin.

[0021] Figure 3 Fold change in α-SMA expression in HSCs from cirrhotic rats when incubated with 10 μM amarouciaxanthin A. Values ​​are normalized to vehicle control and error bars indicate standard deviation.

[0022] Figure 4Fold change in COL1A1 expression in LX2 cells when incubated with 10 μM amarouciaxanthin A. Values ​​are normalized to vehicle control and error bars indicate standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0024] combination

[0025] The authors of the present invention have found that carotenoids and BCl-2 inhibitors exhibit synergistic cytotoxic effects in certain cancer cell lines. Therefore, in a first aspect, the present invention relates to a combination comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or an ester or salt thereof, and a second component selected from the group consisting of: an inhibitor of one or more BCL-2 anti-apoptotic protein family members, an activator of a BCL-2 pro-apoptotic family member, a senescent cell lytic agent, and a senescent phenotype inhibitor.

[0026] As used herein, "combination" or "composition" indicates that carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof and one or more BCL-2 anti-apoptotic protein family members, activators of BCL-2 pro-apoptotic family members, senescent cell lytic agents and senescence phenotype inhibitors can be formulated in the same pharmaceutical preparation, or they can also be formulated in different pharmaceutical preparations, but the different pharmaceutical preparations should be contained in the same drug by combined packaging and should be used simultaneously, sequentially or one after the other.

[0027] As used herein, the term "carotenoid" refers to a group of naturally occurring pigments produced primarily by plants, yeast, and algae that have a common polyisoprenoid-based structure, a long polyene chain forming a chromophore, and approximate symmetry around a central double bond. 20 Tail-to-tail ligation of geranylgeranyl diphosphate molecules produces the parent C 40 The polyene chain may also have a ring group at one or both ends of the molecule.

[0028] Carotenoids are divided into two classes, xanthophylls (which contain oxygen atoms) and carotenes (which do not).

[0029] In a preferred embodiment, the carotenoid of the present invention is a xanthophyll. In a more preferred embodiment, the xanthophyll is selected from the group consisting of α-cryptoxanthin, β-cryptoxanthin, adonirubin, adonixanthin, alloxanthin, amarouchiaxanthin (particularly amarouchiaxanthin), A), antheraxanthin, astaxanthin, auroxanthin, caloxanthin, cantaxanthin, capsanthin, capsanthin-5-6-epoxide, capsorubin, crocoxanthin, diadinoxanthin, diatoxanthin, echinenone, fucoxanthin, fucoxanthinol, isofucoxanthin, isofucoxanthin, lutein, luteoxanthin, mutatoxanthin, neoxanthin, nostoxanthin, violaxanthin, zeaxanthin, and combinations or derivatives thereof. More preferably, the xanthophylls are selected from amarouciaxanthin A, capsaicin, fucoxanthin, fucoxanthol, neoxanthin, lutein, zeaxanthin and combinations thereof. More preferably, the xanthophylls are selected from amarouciaxanthin A, fucoxanthin, fucoxanthol, neoxanthin, lutein and combinations thereof. More preferably, the xanthophylls are selected from amarouciaxanthin A, fucoxanthin, fucoxanthol, neoxanthin and combinations thereof. More preferably, the xanthophylls are selected from amarouciaxanthin A, fucoxanthin, fucoxanthol, neoxanthin and combinations thereof. More preferably, the xanthophylls are selected from amarouciaxanthin A, fucoxanthin, fucoxanthol and combinations thereof. Even more preferably, the xanthophylls are fucoxanthin or amarouciaxanthin A.

[0030] In one embodiment, the above xanthophylls refer to their all-trans forms.

[0031] In another preferred embodiment, the carotenoid is fucoxanthin. In another preferred embodiment, the carotenoid is fucoxanthin. In another preferred embodiment, the carotenoid is amarouciaxanthin. In a more preferred embodiment, the carotenoid is amarouciaxanthin A. In another preferred embodiment, the carotenoid is neoxanthin. In another preferred embodiment, the carotenoid is astaxanthin. In another preferred embodiment, the carotenoid is zeaxanthin. In another preferred embodiment, the carotenoid is lutein.

[0032] The carotenoids of the present invention may be provided from algae, fungi or plant extracts. When the carotenoids used in the present invention (e.g., the above-mentioned xanthophylls) are from plant extracts or algae extracts, the product is obtained by an extraction process performed on any organism or plant, the extraction process being performed by soaking the organism or plant with a solvent or by extraction involving more complex techniques using pressure or supercritical fluids.

[0033] Examples of suitable algae for providing carotenoids in the combination of the present invention include microalgae from Cyanophyta, Chlorophyta, Rhodophyta, Heterokontophyta and Haptophyta. The algae can be green microalgae, such as Chlorella, Scenedesmus, Dunaliella (for beta-carotene), Haematococcus (for astaxanthin) and Bracteacoccus; haptophyte microalgae, such as Isochrysis (for fucoxanthin and lutein); and microalgae from Heterokontophyta, such as Phaeodactylum, Ochromonas and Odontella. Examples of suitable macroalgae include all brown algae, and in particular Fucus vesiculosus, Fucus evanescens, Laminaria sp., and Sargassum sp. (all for fucoxanthin). Certain fungi are known to produce xanthophylls, such as Xanthophyllomyces dendrorhous. Additionally, carotenoids can also be obtained from animal sources, such as egg yolk. Plants and plant parts suitable for producing carotenoids, such as xanthophylls for use in the present invention, include, but are not limited to, marigold flower, corn, kiwi fruit, red seedless grapes, zucchini, pumpkin, spinach, orange pepper, yellow squash, cucumber, peas, green pepper, red grape, butternut, orange, honeydew, celery, green grape, Brussels sprout, scallion, green bean, broccoli, apple, mango, green lettuce, tomato, peach, yellow pepper, nectarine, red pepper, carrot, cantaloupe, apricot, bell pepper, and green kidney bean.

[0034] As used herein, "carotenoid metabolites" refer to carotenoids modified under different conditions by, for example, hydrogenation, dehydrogenation, double bond migration, chain shortening or elongation, rearrangement, isomerization, oxidation or a combination of these processes. In addition, carotenoid metabolites can also be formed due to the presence of enzymes monooxygenases, cyclooxygenases and dioxygenases.

[0035] Examples of β-carotene metabolites are retinol, retinal and retinoic acid, β-ionone, β-apo-14′-carotenal, β-apo-10′-carotenal, β-apo-8′-carotenal and β-carotene 5,8-endoperoxide-2,3-dihydro-β-apocarotene-13-one, 5,6-monoepoxide, retinoyl β-D-glucuronide, β-apo-12′-carotenoic acid, β-apo-12′-carotenoic acid, β-apo-14′-carotenoic acid, β-apo-14′-carotenal, β-apo-14′-carotenoic acid and β-apo-13-carotene.

[0036] Lycopene metabolites are, for example, acyclo retinoic acid, 2,7,11-trimethyl-tetradecahexaene-1,14-dial, (E,E,E)-4-methyl-8-oxo-2,4,6-nonatrienal (MON), apo-6-lycopenal, apo-8′-lycopenal, apo-10′-lycopenal, apo-12′-lycopenal and apo-14′-lycopenal and apo-10′-lycopene acid. Examples of lutein metabolites are 3'-epilutein, 3'-oxolutein, 3'-dehydrolutein, meso-zeaxanthin, methoxy-zeaxanthin, 3-hydroxy-β-ionone and the oxime derivative of 3-hydroxy-14-apocarotene, 3-hydroxy-3',4'-didehydro-β,γ-carotene and 3-hydroxy-2',3'-didehydro-β,ε-carotene.

[0037] Astaxanthin metabolites are for example 3-hydroxy-4-oxo-β-ionone and 3-hydroxy-4-oxo-7,8-dihydro-β-ionone. An example of a canthaxanthin metabolite is 4-oxoretinoic acid.

[0038] "Derivatives of carotenoids" are also included in the context of the present invention. In a specific embodiment, the derivatives include xanthophylls and carotenes containing one or more cis double bonds, including but not limited to 9-cis derivatives, 9'-cis derivatives, 13-cis derivatives, 13'-cis derivatives, 15-cis derivatives, 15'-cis derivatives and any combination thereof.

[0039] In another embodiment, synthetic derivatives are used, wherein xanthophylls and carotenes are used as the starting scaffolds of the carotenoid derivatives. In some embodiments, carotenoid derivatives include compounds having a structure comprising a polyene chain (i.e., the backbone of the molecule). The polyene chain may include about 5 to about 15 unsaturated bonds, more particularly 7 or more conjugated double bonds.

[0040] Combinations of the present invention may also include carotenoid analogs. As used herein, the term "carotenoid analogs" may be generally defined as carotenoids and their biologically active structural analogs. Typical analogs include molecules that exhibit equivalent or improved biologically useful and relevant functions but are structurally different from the parent compound. Parent carotenoids are selected from more than 600 naturally occurring carotenoids described in the literature, and stereo and geometric isomers thereof. Such analogs may include, but are not limited to, esters, ethers, carbonates / esters, amides, carbamates / esters, phosphates and ethers, sulfates / esters, glycoside ethers, with or without spacers (joints).

[0041] The combinations of the invention may also comprise esters of carotenoids (eg acetate, formate and benzoate derivatives).

[0042] The combination of the present invention may also contain salts of carotenoids, carotenoid metabolites, carotenoid derivatives or carotenoid analogs. The term "salt" is understood to mean any form of the active compound according to the invention: wherein it is in ionic form or is charged and coupled to a counterion (cation or anion) or is in solution. This is also understood to be a complex of the active compound with other molecules and ions, in particular a complex complexed by ionic interactions. This definition particularly includes physiologically acceptable salts; the term must be understood to be equivalent to "pharmacologically acceptable salts" or "pharmaceutically acceptable salts".

[0043] According to the present invention, the second component is selected from a senescent cell lysing agent, a senescent phenotype inhibitor, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, and an activator of a BCL-2 pro-apoptotic family member. In the present invention, the second component is not troglitazone and / or punicic acid.

[0044] As used herein, "senescent cell lysing agents" refer to compounds that (preferentially or to a greater extent) destroy, kill, remove senescent cells, or promote the selective destruction of senescent cells. In other words, compared with its ability to destroy or kill non-senescent cells, senescent cell lysing agents destroy or kill senescent cells in a biologically, clinically, and / or statistically significant manner. The amount and time of use of the senescent cell lysing agent are sufficient to selectively kill determined senescent cells in a clinically significant or biologically significant manner but are not sufficient to kill non-senescent cells (destroy non-senescent cells, cause non-senescent cell death). In certain embodiments, the senescent cell lysing agents described herein change at least one signal transduction pathway in a manner that induces (triggers, stimulates, triggers, activates, promotes) and causes (i.e., causes, causes) senescent cell death. Senescent cell lysing agents can change, for example, one or both of cell survival signal transduction pathways (e.g., Akt pathways) or inflammatory pathways, for example by antagonizing proteins within cell survival and / or inflammatory pathways in senescent cells. Senescent cell lysing agents can perform their functions by inducing (activating, stimulating, eliminating inhibition of) apoptotic pathways that cause cell death.

[0045] Those skilled in the art are aware of methods for identifying and selecting senescent cell lysing agents, such as by determining that the agent reduces senescent cells. The level of senescent cells can be determined according to any in vitro assay or technique known in the art. For example, senescent cells can be detected by: morphology (e.g., observed by microscopy); the production of senescent-related markers, such as senescent-related β-galactosidase (SA-β-gal), p16INK4a, p21, PAI-I, or any one or more SASP factors (e.g., IL-6, MMP3). Therefore, those skilled in the art will readily recognize that by comparing the activity of the test agent with an appropriate negative control (e.g., only a carrier or diluent and / or a composition or compound known in the art that does not kill senescent cells) and an appropriate positive control, the agent can be characterized as a senescent cell lysing agent and the killing level of the agent can be determined. In vitro cell-based assays for characterizing senescent cell lysing agents also include controls for determining the effects of agents on non-senescent cells (e.g., quiescent cells or proliferative cells). A senescent cell lysing agent decreases (ie, reduces) the survival percentage of a plurality of senescent cells (ie, somehow decreases the amount of viable senescent cells in an animal or in a cell-based assay) as compared to one or more negative controls.

[0046] In certain embodiments, the senescent cell lytic agent can be a polypeptide, a peptide, an antibody, an antigen binding fragment (i.e., peptides and polypeptides comprising at least one complementarity determining region (CDR)), a peptibody, a recombinant viral vector, or a nucleic acid. In certain embodiments, the senescent cell lytic agent is an antisense oligonucleotide, siRNA, shRNA, or a peptide. In a specific embodiment, a polynucleotide or oligonucleotide (e.g., including shRNA) can be delivered by a recombinant vector into which a polynucleotide or oligonucleotide of interest has been incorporated. In another preferred embodiment, the senescent cell lytic agent is a polyclonal or monoclonal antibody.

[0047] In a preferred embodiment, the senescent cell lysing agent is selected from Nutlin-3a, RG-7112, ABT-263 (navitoclax), ABT-199 (venetoclax), ABT-737, WEHI-539A-1155463, MK-2206. In a more preferred embodiment, the senescent cell lysing agent is selected from navitoclax and venetoclax. In a more preferred embodiment, the senescent cell lysing agent is navitoclax. In another preferred embodiment, the senescent cell lysing agent is venetoclax.

[0048] In another preferred embodiment, the senescent cell lytic agent is a flavonoid.

[0049] As used herein, "flavonoid" refers to compounds having a general structure of a 15-carbon backbone consisting of two benzene rings (A and B) and a heterocyclic ring (C). This carbon structure can be abbreviated as C6-C3-C6.

[0050] In a more preferred embodiment, the flavonoid is selected from the group consisting of fisetin, curcumin, alvocidib and quercetin.

[0051] In another preferred embodiment, the combination of the present invention may comprise an inhibitor of the senescence phenotype.

[0052] As used herein, "senescence phenotype inhibitors" relate to small molecules that inhibit the senescence phenotype in the absence of cell killing.

[0053] In a preferred embodiment, the senescence phenotype inhibitor is selected from rapamycin, fluspirilene, cycloheximide, NVP-BEZ235, loperamide, Timosaponin A-III, iguldipine and nordihydroguaiaretic acid.

[0054] Screening methods for identifying inhibitors of the senescence phenotype and distinguishing from senescence cell lytic agents are known in the art by way of illustrative example in Fuhrmann-Stroissnigg H. et al., Nat Commun. 2017 Sep 4; 8(1):422.

[0055] In another preferred embodiment, the combination of the present invention may comprise an inhibitor of the BCL-2 anti-apoptotic protein family. In the present invention, the inhibitor is not troglitazone and / or punicic acid.

[0056] "Inhibitors of one or more members of the BCL-2 anti-apoptotic protein family" relate to inhibitors of a class of key regulators of caspase activation consisting of anti-apoptotic (pro-survival) proteins with a BHI-BH4 domain (BCL-2 (i.e., BCL-2 protein members of the BCL-2 anti-apoptotic protein family), BCL-xL, BCL-w, Al, MCL-I and BCL-B).

[0057] In certain embodiments, the BCL2 inhibitor is a selective inhibitor, meaning that it preferentially binds to BCL2 family members (e.g., BCL-2, MCL-1, BCL-w, BCL-b, and BFL-1 / A1) relative to other proteins.

[0058] Methods for measuring the binding affinity of inhibitors of BCL-2 family proteins are known in the art. For example, the binding affinity of the inhibitor can be determined using a competition fluorescence polarization assay, in which a fluorescent BAK BH3 domain peptide is incubated with a BCL-xL protein (or other BCL-2 family protein) in the presence or absence of increasing concentrations of the inhibitor, as previously described, for example, in U.S. Patent Publication No. 20140005190.

[0059] Additionally, any method known in the art can be used to determine whether a compound is a BCL2 inhibitor, by way of example and not limitation, by determining the ability to inhibit anti-apoptotic effects, such as performing an MTT assay, an Annexin V binding assay, a caspase activity assay, a mitochondrial membrane potential assay, or analyzing DNA fragmentation and morphology.

[0060] In certain embodiments, the BCL2 inhibitor of one or more BCL-2 anti-apoptotic protein family members is a small molecule, a polypeptide, a peptide, an antibody antigen-binding fragment (i.e., a peptide and a polypeptide comprising at least one complementary determining region (CDR)), a peptibody, a recombinant viral vector, or a nucleic acid. In certain embodiments, the BCL2 inhibitor is an antisense oligonucleotide, siRNA, shRNA, or a peptide. For example and in certain embodiments, the BCL-xL selective peptide inhibitor is a BH3 peptide mimetic. In another preferred embodiment, the BCL2 inhibitor is a polynucleotide or oligonucleotide that specifically hybridizes with a portion of an mRNA encoding a target protein (e.g., BCL-2, MCL-1, BCL-w, BCL-b, and BFL-1 / A1, BCL-x). Antisense polynucleotides bind to nucleic acids (e.g., mRNA or DNA) in a sequence-specific manner. Identifying oligonucleotides and ribozymes used as antisense agents and identifying DNA encoding a target gene for targeted delivery involves methods well known in the art. For example, the desired properties, length, and other characteristics of such oligonucleotides are well known.

[0061] In a preferred embodiment, the BCL2 inhibitor is selected from the group consisting of compounds of formula (I), Disarib, S55746, A1331852, WEHI-S39, Obatoclax, TW-37, AT101, Sabutoclax, A1210477, Umi-77, BTSA1, BAM7, MIM1, AMG-176, MIK665 (S64315), APG-1252, BXI-61 and BXI-72.

[0062] The compound of formula (I) and its synthesis have been described in US 8,546,399 B2. The compound of formula (I) has the structure shown below, or a pharmaceutically acceptable salt thereof:

[0063]

[0064] in

[0065] R 1 It is H;

[0066] R 2 Selected from NO2, SO2R 7 ,H,CN,F,Cl,Br,I,CF3,R 7 , OR7 , SR 7 and C(O)NH2.

[0067] R 3 Selected from NHR 8 、NHC(O)R 8 , OR 8 , R 8 , F, Br, I and Cl;

[0068] R 4 and R 5 independently selected from H, F, Br, I and Cl;

[0069] R 6 Selected from H, OR 9 , R 9 , SR 9 、S(O)R 9 、SO2R 9 、C(O)R 9 、CO(O)R 9 、OC(O)R 9 、NHR 9 、N(R 9 )2. NHC(O)R 9 NR 9 C(O)R 9 NHSO2R 9 NR 9 S02R 9 、NHC(O)OR 9 and NR 9 C(O)OR 9 ;

[0070] R 7 is a linear or branched C1-C6 alkyl group, which is optionally substituted by one or more substituents independently selected from F, Cl, Br and I;

[0071] R 8 is a linear or branched C1-C6 alkyl group, which is optionally substituted by one or more substituents selected from the following: SR 10 , OR 10 and R 10 , and a 5- to 10-membered monocyclic or bicyclic heterocyclic group containing one or more heteroatoms selected from N, O and S;

[0072] R 9 is selected from 5- to 10-membered monocyclic or bicyclic heteroaryl groups containing one or more heteroatoms selected from N, O and S;

[0073] R 10 It is C6-C 10Monocyclic or bicyclic aryl.

[0074] In one embodiment, in the compound of formula (I), R 1 It's H.

[0075] In another specific embodiment, in the compounds of formula (I), R 2 Selected from NO2, SO2R 7 , where R 7 is a linear or branched C1-C3 alkyl group, which is optionally substituted by one or more F atoms.

[0076] In another specific embodiment, in the compounds of formula (I), R 3 NHR 8 , where R 8 It is a straight chain or branched C1-C6 alkyl group, which is optionally substituted by one or more substituents selected from the following: -S-phenyl, and a 5- to 6-membered monocyclic heterocyclic group containing one or two heteroatoms selected from N and O; preferably a C1-C6 alkyl group, which is optionally substituted by one or more substituents selected from -S-phenyl, morpholinyl and tetrahydropyranyl.

[0077] In another specific embodiment, in the compounds of formula (I), R 4 It's H.

[0078] In another specific embodiment, in the compounds of formula (I), R 5 It's H.

[0079] In another specific embodiment, in the compounds of formula (I), R 6 Selected from: H and OR 9 , where R 9 is selected from 8- to 10-membered bicyclic heteroaryl groups containing one or more nitrogen atoms; preferably H and OR 9 , where R 9 is pyrrolopyridinyl; more preferably H and OR 9 , where R 9 It is 7-azaindole.

[0080] In another specific embodiment, in the compounds of formula (I), R 8 is a straight chain or branched C1-C6 alkyl group, which is optionally substituted by one or more substituents selected from: -S-phenyl, and a 5- to 6-membered monocyclic heterocyclic group containing one or two heteroatoms selected from N and O (preferably morpholinyl or tetrahydropyranyl).

[0081] In one embodiment, in the compound of formula (I), R 1 , R 4 and R 5It is H; R 2 Selected from NO2, SO2R 7 ; R 3 NHR 8 ; R 6 Selected from H and OR 9 ; R 7 is a linear or branched C1-C3 alkyl group, which is optionally substituted by one or more F atoms; R 8 is a linear or branched C1-C6 alkyl group, which is optionally substituted by one or more substituents selected from: -S-phenyl, and a 5- to 6-membered monocyclic heterocyclic group containing one or two heteroatoms selected from N and O (preferably morpholinyl or tetrahydropyranyl); and R 9 is selected from an 8- to 10-membered bicyclic heteroaryl group containing one or more nitrogen atoms; or a pharmaceutically acceptable salt thereof (preferably pyrrolopyridinyl, more preferably 7-azaindole); or a pharmaceutically acceptable salt thereof.

[0082] As used herein, some of the atoms, groups, chains or rings present in the general structures of the present invention are "optionally substituted". This means that these atoms, groups, chains or rings can be unsubstituted or substituted at any position by one or more, for example 1, 2, 3 or 4 substituents, 1, 2 or 3 substituents or 1 or 2 substituents, whereby hydrogen atoms bound to the unsubstituted atoms, groups, chains or rings are replaced by the indicated substituents. When two or more substituents are present, each substituent may be the same or different.

[0083] "Alkyl" groups can be branched or unbranched, and preferably have 1 to 6 carbon atoms. A more preferred class of alkyl groups has 1 to 4 carbon atoms or 1 to 3 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, pent-3-yl, and n-hexyl.

[0084] As used herein, "heterocyclyl" refers to a 5 to 10-membered monocyclic or bicyclic ring system containing one or more heteroatoms (e.g., 1, 2 or 3 heteroatoms, preferably 1 or 2 heteroatoms) selected from N, O and S. In a bicyclic ring system, the heteroatoms may be in one or both rings forming the heterocyclyl ring system. Examples of heterocyclyl rings are morpholinyl, tetrahydropyranyl, piperidinyl, thiomorpholinyl, tetrahydrofuranyl and piperazinyl. Preferably, the heterocyclyl ring is fully saturated. Preferably, the heterocyclyl ring is a 5 to 6-membered monocyclic ring containing 1 or 2 heteroatoms selected from N and O.

[0085] As used herein, "heteroaryl" refers to an aromatic 5- to 10-membered monocyclic or bicyclic ring system containing one or more heteroatoms (e.g., 1, 2 or 3 heteroatoms, preferably 1 or 2 heteroatoms) selected from N, O and S. In a bicyclic ring system, the heteroatoms can be in one or both rings forming the heteroaryl ring system. Examples of heteroaryl rings are pyrrolopyridinyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzo benzoxazolyl, naphthyridinyl, pyridinyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isothiazolyl, Azolyl, Azolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, oxadiazolyl, thiadiazolyl, pyridazinyl and triazinyl. Preferably, the heteroaryl ring is an 8 to 10 membered bicyclic ring containing 1 or 2 heteroatoms, preferably 1 or 2 nitrogen atoms, in the ring.

[0086] As used herein, "aryl" refers to an aromatic 6-monocyclic or bicyclic ring system consisting of hydrogen and 6 to 10 carbon atoms. Examples of aryl rings are phenyl and naphthyl, preferably phenyl.

[0087] The term "pharmaceutically acceptable salt" refers to any salt that can (directly or indirectly) provide a compound described herein after being applied to a recipient. For example, the pharmaceutically acceptable salt of the compound provided herein can be an acid addition salt, a base addition salt or a metal salt, and it can be synthesized by a parent compound comprising an alkaline or acidic part by conventional chemical methods. Typically, such salts are prepared, for example, by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent or in a mixture of the two. Typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of acid addition salts include inorganic acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, mesylate and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as ammonium, and organic base salts such as ethylenediamine, ethanolamine, N,N-dialkyleneethanolamine, triethanolamine, glucosamine and basic amino acid salts. Examples of metal salts include, for example, sodium, potassium, calcium, magnesium, aluminum and lithium salts.

[0088] In a more preferred embodiment, the BCL2 inhibitor is selected from venetoclax, naviclax, Disarib, S55746, A1331852, WEHI-S39, Obatoclax, TW-37, AT101, Sabutoclax, A1210477, Umi-77, BTSA1, BAM7, MIM1, AMG-176, MIK665 (S64315), APG-1252, BXI-61 and BXI-72. In a preferred embodiment, the BCL2 inhibitor is selected from venetoclax and naviclax. In a preferred embodiment, the BCL2 inhibitor is venetoclax, also known as GDC-0199, ABT-199, RG7601, CAS No. 12557044-40-8, and its chemical formula is shown below (Compound Ia). In another preferred embodiment, the BCL2 inhibitor is naviridavir, also known as ABT-263, CAS No. 923564-51-6, and its chemical formula is shown below (Compound Ib).

[0089]

[0090] In a preferred embodiment, the combination of the invention comprises an inhibitor of one or more BCL-2 anti-apoptotic protein family members selected from naviclat or venetoclax, and a carotenoid selected from fucoxanthin, fucoxanthinol, amarouciaxanthin A and neoxanthin.

[0091] Thus, in a preferred embodiment, the combination of the invention comprises navicrata and fucoxanthin, navicrata and fucoxanthinol, navicrata and amarouciaxanthin A, navicrata and neoxanthin, venetoclax and fucoxanthin, venetoclax and fucoxanthinol, venetoclax and amarouciaxanthin A, or venetoclax and neoxanthin.

[0092] In a more preferred embodiment, the combination of the present invention comprises an inhibitor of one or more BCL-2 anti-apoptotic protein family members selected from naviclat or venetoclax, and a carotenoid selected from fucoxanthin, fucoxanthinol and amarouciaxanthin A.

[0093] Therefore, in another more preferred embodiment, the combination of the present invention comprises naviclat and fucoxanthin, naviclat and fucoxanthinol, naviclat and amarouciaxanthin A, venetoclax and fucoxanthin, venetoclax and fucoxanthinol, or venetoclax and amarouciaxanthin A.

[0094] In another preferred embodiment, the combination of the invention comprises an activator of a BCL-2 pro-apoptotic family member.

[0095] As used herein, "activators of pro-apoptotic family members of BCL-2" refer to activators of a class of pro-apoptotic proteins (BAX, BAK, and BOK) having BH1, BH2, and BH3 domains, and pro-apoptotic BH3-only proteins (BIK, BAD, BID, BIM, BMFHRK, NOXA, and PUMA). In a preferred embodiment, the activator of the pro-apoptotic family member of BCL-2 is a synthetic peptide. In another preferred embodiment, the activator is any compound that interacts with the surface pocket of BCL-2, such as HA14-1 or compound 106 as disclosed in Zhao G. et al. (Mol Cell Biol. 2014 Apr; 34 (7): 1198-1207).

[0096] Any method known in the art can be used to determine whether a compound is an activator of a pro-apoptotic family member of BCL-2. The in vitro binding affinity of an organic compound to a Bcl-2 protein can be determined by, for example, a competitive binding assay based on fluorescence polarization. In addition, any method previously disclosed for the ability to inhibit anti-apoptotic effects can be used to determine the ability of a compound to activate pro-apoptosis.

[0097] In another preferred embodiment, the combination of the invention comprises an activator of a BCL-2 pro-apoptotic family member and a carotenoid selected from the group consisting of fucoxanthin, fucoxanthinol, amarouciaxanthin A and neoxanthin.

[0098] The combination of the present invention shows a synergistic effect, for example, in producing a cytotoxic effect or reducing the number of senescent cells, so that the same cytotoxic effect or senolytic effect or senomorphic effect is obtained with a lower dose of an inhibitor of the BCL-2 anti-apoptotic protein family, an activator of a BCL-2 pro-apoptotic family member, a senescent cell lytic agent or a senescent phenotype inhibitor, respectively.

[0099] In a specific embodiment, the molar ratio of the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof to the second component selected from the group consisting of inhibitors of one or more BCL-2 anti-apoptotic protein family members, activators of BCL-2 pro-apoptotic family members, senescent cell lytic agents and senescence phenotype inhibitors in the combination is 0.1:100 to 100:0.01, preferably 0.2:100 to 100:0.05, more preferably 0.5:100 to 100:0.1. In some other embodiments, the molar ratio of the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof to the second component selected from one or more inhibitors of BCL-2 anti-apoptotic protein family members, activators of BCL-2 pro-apoptotic family members, senescent cell lytic agents and senescence phenotype inhibitors in the combination is 1:100 to 10:0.01, 1:10 to 1:0.01, 1:1 to 0.1:0.01, or 0.1:1 to 1:1.

[0100] In the combination of the present invention, the second component is not troglitazone and / or punicic acid.

[0101] Food, cosmeceuticals, nutritional supplements, cosmetics or pharmaceutical compositions

[0102] In another aspect, the present invention relates to a cosmeceutical, cosmetic or pharmaceutical composition comprising the combination of the present invention.

[0103] As used herein, the term "foodstuff" refers to any substance or product, natural or processed, of any nature, solid or liquid, which, by reason of its properties, application, composition, preparation and state of preservation, is usually or desirably usable for some of the following purposes: a) as normal nutrition for humans or animals or as a pleasurable food; or b) as a dietary product in the special case of human or animal food (feed). The term "feedstuff" includes all natural materials and manufactured products of any origin, either separately from one another or conveniently mixed together, suitable for use as animal food.

[0104] Ready-to-eat food is a food that does not need to be diluted, for example, by an aqueous solution suitable for consumption. In principle, as those skilled in the art would consider, the ingredients present in the ready-to-eat food are balanced, and it is not necessary to add other ingredients to the food to make it ready-to-eat. Concentrated food is a food in which one or more ingredients exist in a higher concentration than in the ready-to-eat food, so when used, it must be diluted, for example, by an aqueous solution suitable for consumption. The non-limiting illustrative examples of food provided by the invention include dairy products and derivatives such as fermented milk, yogurt, kefir (kephir), curd, cheese, butter, ice cream, milk-based desserts, and non-dairy products such as baked products, cakes and cakes, cereals, chocolate, jam, fruit juice, other fruit derivatives, oils and margarine, dishes made, etc.

[0105] As used herein, the term "cosmeceutical product" refers to a product suitable for use in the body or animal body, which contains one or more cosmeceutical products (functional cosmetics, dermaceuticals or active cosmetics), i.e., a topical hybrid product with cosmetic-pharmaceutical properties, which contains active ingredients that have an effect on the user's skin, hair and / or nails at a higher or more effective concentration, and is therefore at an intermediate level between cosmetics and drugs. Illustrative examples of cosmeceutical products include essential oils, ceramides, enzymes, minerals, peptides, vitamins, etc.

[0106] As used herein, the term "nutraceutical" refers to a product suitable for use in humans or animals that contains one or more therapeutic natural products that provide health benefits or are associated with disease prevention or alleviation, and includes dietary supplements that are present in non-food matrices (e.g., capsules, powders, etc.) of concentrated natural bioactive products that are normally present (or not present) in foods, and that, when taken in higher doses than those present in those foods, exert a beneficial effect on health with a greater effect than normal foods can have. Thus, the term "nutraceutical" includes isolated or purified foods and additives or food supplements that are usually present in dosage forms that are usually used orally (e.g., capsules, tablets, sachets, drinkable phials, etc.); such products provide physiological benefits or protection against disease, usually against chronic diseases. If desired, the nutritional products provided by the present invention may also contain one or more nutritional products (products or substances related to disease prevention or alleviation) in addition to luteins, such as flavonoids, ω-3 fatty acids, etc., and / or one or more prebiotics (non-digestible food ingredients that stimulate the activity and / or growth of probiotics), such as oligofructose, pectin, inulin, galacto-oligosaccharides, lactulose, human milk oligosaccharides, dietary fiber, etc.

[0107] As used herein, the term "nutritional composition" of the present invention relates to a food that beneficially affects one or more functions of the body to provide a better health condition. Thus, such a nutritional composition can be used to prevent and / or treat a disease or a factor causing a disease. Thus, the term "nutritional composition" of the present invention can be used as a synonym for a functional food or a food for a specific nutritional purpose or a medical food. A nutritional composition is similar to a conventional food and is consumed as part of a normal diet appearance.

[0108] As used herein, the term "cosmetic composition" or "personal care composition" refers to a composition suitable for personal hygiene of humans or animals or for enhancing natural beauty or changing the appearance of the body without affecting the structure or function of the human or animal body, which comprises one or more products that provide such an effect. If desired, the cosmetic composition provided by the present invention may also contain one or more cosmetics or beauty products in addition to the combination of the present invention, that is, such substances or mixtures, which are intended to contact with the external parts of the human or animal body (such as epidermis, hair system, finger / toe nails, lips, etc.) or with teeth and buccal mucosa, with the sole or main purpose of cleaning it, perfuming it, changing its appearance, protecting it, keeping it in good condition or correcting body odor. Illustrative examples of cosmetically acceptable carriers include products included in the INCI (International Nomenclature of Cosmetic Ingredients) list. Cosmetic or personal care compositions include products such as balms, pads, pomades, creams, etc., oils, surfactants, moisturizers, botanical extracts, vitamins, antioxidants, sunscreens, fragrances, preservatives, etc. Illustrative examples are moisturizers, botanical extracts, vitamins, antioxidants, and sunscreens.

[0109] The ingredients as described above are preferably provided in cosmetic compositions that can be formulated into creams, gels, lotions, oils, ointments, powders, sticks, cakes or other topically applied forms. The resulting cosmetic composition can be in the form of a liquid, solid, semisolid, dispersion, suspension, solution or emulsion, and it can be water-based or anhydrous. The cosmetic composition of the present invention can also be in the form of a color cosmetic composition, such as foundation makeup, mascara, lip gloss, blush, eye shadow, etc. Certain other derivatives are lipophilic in nature and will be more likely to be present in the oil phase of the emulsion. The combination of the present invention is preferably present in the aqueous phase of the emulsion or encapsulated in the aqueous phase in the liposome.

[0110] As used herein, the term "cosmetically effective amount" relates to a sufficient amount of a compound (i.e., a combination of the present invention) to provide the desired effect, and is generally determined, among other reasons, by the properties of the compound itself and the cosmetic effect to be achieved. The dosage for obtaining a cosmetically effective amount also depends on a range of factors, such as the age, weight, sex or tolerance of the animal (preferably a mammal, and more preferably a human).

[0111] In a specific and preferred embodiment of the present invention, the cosmetic composition of the present invention is administered by topical route. Suitable formulations for topical administration of the composition of the present invention are detailed in the context of the pharmaceutical composition of the present invention and are equally applicable to the cosmetic composition of the present invention.

[0112] If desired, the cosmetic composition of the present invention is incorporated into a fabric, nonwoven fabric, or medical device. Illustrative examples of the fabric, nonwoven fabric, or medical device include, but are not limited to, bandages, gauze, T-shirts, pantyhose, socks, underwear, belts, gloves, diapers, sanitary napkins, dressings, bed covers, wipes, adhesive patches, non-adhesive patches, occlusive patches, microelectric patches, and face masks.

[0113] Furthermore, the present invention also relates to pharmaceutical compositions comprising the combination according to the invention.

[0114] As used herein, the term "pharmaceutical composition" relates to a composition comprising at least the combination provided by the present invention and a pharmaceutically acceptable carrier.

[0115] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable carrier," "pharmaceutically acceptable diluent," or "pharmaceutically acceptable excipient," used interchangeably herein, refer to any conventional type of nontoxic solid, semisolid or liquid filler, diluent, encapsulating material, or formulation aid. Pharmaceutically acceptable carriers are substantially nontoxic to recipients at the dosages and concentrations employed and are compatible with the other ingredients of the formulation. The amount and nature of the pharmaceutically acceptable carrier depends on the desired form of administration. Pharmaceutically acceptable carriers are known and may be prepared by methods well known in the art. They participate in carrying or transporting the subject chemical from one organ or part of the body to another without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (a) sugars (e.g., lactose, glucose, and sucrose), (b) starches (e.g., corn starch and potato starch), (c) cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate), (d) powdered tragacanth, (e) malt, (f) gelatin, (g) talc, (h) excipients (e.g., cocoa butter and suppository waxes), (i) oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), (j) glycols (e.g., propylene glycol), (k) polyols (e.g., glycerol, sorbitol, mannitol, and polyethylene glycol), (l) esters (e.g., ethyl oleate and ethyl laurate), (m) agar, (n) buffers (e.g., magnesium hydroxide and aluminum hydroxide), (o) alginic acid, (p) pyrogen-free water, (q) isotonic saline, (r) Ringer's solution Solution), (s) ethanol, (t) phosphate buffer and (u) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents and flavor enhancers, preservatives and antioxidants may also be present in the composition. Examples of pharmaceutically acceptable antioxidants include: (a) water-soluble antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite or sodium sulfite), (b) oil-soluble antioxidants (such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate or α-tocopherol), and (c) metal chelators (such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid or phosphoric acid).

[0116] More preferably, the pharmaceutical product comprises a carrier or vehicle suitable for topical or oral administration.

[0117] Depending on the specific mode of administration, the pharmaceutical product can be formulated into tablets, pills, capsules, sachets, granules, powders, suspensions, emulsions, anhydrous or aqueous topical preparations and solutions.

[0118] Pharmaceutically acceptable carriers or vehicles are well known to those skilled in the art and are readily available to the public. Preferably, a pharmaceutically acceptable carrier or vehicle is one that is chemically inert to the active formulation and each of its components, and one that has no deleterious side effects or toxicity under the conditions of use.

[0119] In some embodiments, the drug product is suitable as a delivery system for oral, parenteral, or intravenous transport of a therapeutic agent into the circulatory system of a subject.

[0120] In addition to intravenous administration, the composition may include a small amount of wetting agent or emulsifier or pH buffer. The composition may be a liquid solution, suspension, emulsion, gel, polymer or sustained release formulation. As known in the art, the composition can be formulated with traditional adhesives and carriers. The preparation may include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers with recognized functions in drug manufacturing. A variety of delivery systems are known and can be used to administer the therapeutic agent of the present invention, including encapsulation in liposomes, microparticles, microcapsules, etc.

[0121] If necessary, the combination or pharmaceutical composition of the present invention is included in a composition that also includes a solubilizing agent and a local anesthetic to alleviate any pain at the injection site. Typically, the components are provided separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or bag indicating the amount of the active agent. In the case where the composition is to be administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case where the combination is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0122] Formulations suitable for oral administration include liquid solutions dissolved in a diluent such as water or saline; capsules, sachets, tablets, lozenges, each containing a predetermined amount of the combination of the present invention; powders; suspensions in a suitable liquid; and emulsions. Liquid dosage forms for oral administration may include emulsions, solutions, suspensions, syrups, and elixirs containing pharmaceutically acceptable inert diluents commonly used in the technology, such as water. These compositions may also contain excipients such as wetting agents, emulsifiers and suspending agents, as well as sweeteners, flavoring agents, and flavoring agents.

[0123] Solid dosage forms for oral administration can include conventional capsules, sustained release capsules, conventional tablets, sustained release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. Under these solid dosage forms, the active compound can be mixed with at least one inert excipient such as sucrose, lactose or starch. In normal practice, such dosage forms can also include other substances except inert diluents, such as lubricants, such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, dosage forms can also include buffers. Tablets and pills can be prepared with enteric coatings.

[0124] Parenteral formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as water for injection, immediately prior to use.

[0125] The pharmaceutical composition of the present invention can be applied by surface, transdermal or subcutaneous route. Illustrative examples of surface or transdermal application include but are not limited to iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive dressing, microinjection, needleless injection by pressure, microelectric patch and any combination thereof. In any case, excipients will be selected according to the selected pharmaceutical dosage form.

[0126] Injectable preparations (e.g., sterile injectable aqueous or oleaginous suspensions) can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Acceptable carriers and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile oils are also commonly used as solvents or suspending media.

[0127] In a specific and preferred embodiment of the present invention, the pharmaceutical composition of the present invention is administered by topical routes. For topical administration, the pharmaceutical composition of the present invention can be formulated into creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid bars, emulsions, microemulsions and the like, which can be formulated according to conventional methods using suitable excipients (e.g., emulsifiers, surfactants, thickeners, colorants and combinations of two or more thereof).

[0128] In addition, the pharmaceutical composition of the present invention can be administered in the form of a transdermal patch or an iontophoresis device. Suitable transdermal patches are well known to those skilled in the art.

[0129] Several drug delivery systems are known and can be used to administer the combination of the present invention, including, for example, being encapsulated in liposomes, microvesicles, emulsions, microgranules, microcapsules, nanoparticles, nanocapsules and the like. The desired dosage can be administered in a single unit or sustained release form. In a specific and preferred embodiment of the present invention, the pharmaceutical composition is encapsulated in liposomes.

[0130] Sustained release forms and suitable materials and methods for their preparation are well known in the prior art. In one embodiment of the invention, the oral administration form of the combination according to the present invention is a sustained release form, which also comprises at least one coating or matrix. Coating or sustained release matrix include but are not limited to natural polymers, semi-synthetic or synthetic water-insoluble modified waxes, fats, fatty alcohols, fatty acids, natural semi-synthetic or synthetic plasticizers, or a combination of two or more thereof. Enteric coatings can be applied using conventional methods known to experts in the art.

[0131] All terms and embodiments previously described with respect to the combination of the present invention are equally applicable to this aspect of the invention.

[0132] Beauty Methods

[0133] The authors of the present invention have found that the combination of carotenoids and senescent cell lytic agents (e.g., Bcl-2 inhibitors) produces a synergistic senescent cell lytic effect. Therefore, the present invention also provides methods for treating diseases in which there is an undesirable accumulation of senescent cells, as well as cosmetic methods in which it is desired to eliminate senescent cells.

[0134] Therefore, in a further aspect, the present invention relates to a cosmetic method for preventing and / or reducing skin aging and / or for improving the cosmetically adverse effects of aging, which comprises administering to a subject in need thereof a combination according to the invention or a cosmeceutical or cosmetic composition according to the invention.

[0135] As used herein, the term "cosmetic method" relates to a method for enhancing the appearance of skin in a subject. Cosmetic compositions for use in the cosmetic methods of the present invention include skin creams, lotions, powders, lipsticks, eye and facial foundations, wipes, gels, deodorants, hand soaps, baby products, bath oils, bubble baths, bath salts, butters, and many other types of products.

[0136] Skin aging is a multifactorial process that affects almost every aspect of its biology and function; it is driven by both intrinsic (e.g., time, genetic factors, hormones) and extrinsic (e.g., UV exposure, pollution, cigarette smoke) factors. Skin aging also results from aging.

[0137] Cellular senescence is a growth arrest that occurs as a result of different damaging stimuli, including DNA damage, telomere shortening and dysfunction or oncogenic stress. Senescent cells exert pleiotropic effects on development, tissue aging and regeneration, inflammation, wound healing and tumor suppression. Senescent cells are characterized by their inability to proliferate, resistance to apoptosis and secretion of factors that promote inflammation and tissue degradation.

[0138] Senescent keratinocytes and fibroblasts have been shown to accumulate in human skin with age. Furthermore, senescent cells express genes for degradative enzymes, growth factors, and inflammatory cytokines with long-term pleiotropic effects.

[0139] As used herein, "cosmetic adverse effects of aging" refers to the characteristics of aging that are inherent or occur over time, and include, by way of illustration and not limitation, visible signs such as thin and dry skin, fine wrinkles, decreased elasticity, abnormal pigmentation, graying of hair, and hair loss.

[0140] As used herein, "skin aging" refers to a state of aging and, in particular, damage to the epidermal cells of human skin caused by partial damage or complete destruction of the cells, conversion of imide bonds to amide bonds in collagen and / or elastin caused by toxic byproducts of oxygen metabolism, free radical pathology, as well as photodamage and general aging.

[0141] The cosmetic methods of the present invention are intended to reduce epidermal cells and thereby reduce skin aging in humans by reducing or inhibiting aging, including through one or more of the following effects: reversing photodamage or other regenerative effects, such as increasing underlying skin vascularity, increasing the rate of cell replication and desquamation to produce a more youthful appearance, increasing collagen synthesis and uniformity, delaying skin atrophy and thinning of the epidermis and dermis, etc.

[0142] The combination of the present invention can be applied in a cosmetically effective amount. As used herein, the term "cosmetically effective amount" refers to a sufficient amount of a compound (i.e., a combination of the present invention) to provide a desired effect, and is generally determined by the properties of the compound itself and the cosmetic effect to be achieved, among other reasons. The dosage for obtaining a cosmetically effective amount also depends on a series of factors, such as the age, weight, sex or tolerance of the animal (preferably a mammal, and more preferably a human).

[0143] In a specific and preferred embodiment of the cosmetic method according to the invention, the cosmetic composition according to the invention is applied by topical route. Suitable formulations for topical application of the combination according to the invention have already been detailed in the context of the cosmetic composition according to the invention and are equally applicable to the cosmetic method according to the invention.

[0144] If desired, the cosmetic composition of the present invention is incorporated into a fabric, nonwoven fabric, or medical device. Illustrative examples of the fabric, nonwoven fabric, or medical device include, but are not limited to, bandages, gauze, T-shirts, pantyhose, socks, underwear, belts, gloves, diapers, sanitary napkins, dressings, bed covers, wipes, adhesive patches, non-adhesive patches, occlusive patches, microelectric patches, and face masks.

[0145] All terms and embodiments described previously are equally applicable to this aspect of the invention.

[0146] Medical Uses

[0147] In a further aspect the invention relates to a combination according to the invention or to a pharmaceutical composition according to the invention for use in medicine.

[0148] When used in medicine, the composition according to the present invention can be used by simultaneous administration or by separate or sequential administration of therapeutically effective amounts of (i) carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof, and (ii) inhibitors of one or more BCL-2 anti-apoptotic protein family members, activators of BCL-2 pro-apoptotic family members, senescent cell lytic agents or senescent phenotype inhibitors. The term "combination" also represents multiple combinations of compounds (i) and (ii), such as in a single composition, in a combined mixture (e.g., "tank-mix") consisting of separate preparations of a single active compound, and in the combined use of single active ingredients when applied in a sequential manner (i.e., one after another in a reasonably short time (e.g., hours or days)) or in simultaneous administration.

[0149] Preferably, if administration is not simultaneous, the compounds are administered at close times to each other. In addition, it is not important whether the compounds are administered in the same dosage form or by the same route, for example, one compound may be administered topically and the other compound may be administered orally. Suitably, both compounds are administered orally.

[0150] The combination of the present invention can be formulated for simultaneous, separate or sequential administration thereof. This means that the combination of the two compounds can be administered as follows:

[0151] - Combinations as part of the same pharmaceutical preparation, then both compounds are always administered simultaneously.

[0152] - As a combination of two units, each unit having one substance so that simultaneous, sequential or separate administration is possible.

[0153] In a specific embodiment, the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof and the senescent cell lytic agent, senescent phenotype inhibitor, inhibitor of one or more BCL-2 anti-apoptotic protein family members, or activator of BCL-2 pro-apoptotic family members are administered independently (i.e., in two units) but simultaneously.

[0154] In another specific embodiment, the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof is administered first, and the senescent cell lytic agent, senescent phenotype inhibitor, inhibitor of one or more BCL-2 anti-apoptotic protein family members, or activator of BCL-2 pro-apoptotic family members is administered separately or sequentially thereafter.

[0155] In yet another specific embodiment, a senescent cell lytic agent, a senescent phenotype inhibitor, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, or an activator of a BCL-2 pro-apoptotic family member is administered first, and the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof is then administered separately or sequentially as defined.

[0156] In a further aspect, the invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in the treatment of cancer.

[0157] Alternatively, the present invention relates to a method for preventing and / or treating cancer, which comprises administering the combination according to the present invention or the pharmaceutical composition according to the present invention to a subject in need thereof.

[0158] Alternatively, the present invention relates to the use of a combination according to the invention or a pharmaceutical composition according to the invention for the preparation of a medicament for preventing and / or treating cancer in a subject in need thereof.

[0159] As used herein, the term "prevention" and variations thereof relate to the administration of a combination according to the invention or a medicament comprising the combination to a subject who has not yet been diagnosed as being likely to suffer from, for example, cancer, but who is generally expected to develop the disease or be at an increased risk of developing the disease. Prevention is intended to avoid the occurrence of the disease. Prevention may be complete (e.g., the complete absence of the disease). Prevention may also be partial, such that, for example, the occurrence of the disease in the subject is lower than the occurrence of the disease without the administration of the composition of the invention. Prevention also refers to reducing susceptibility to clinical conditions.

[0160] As used herein, the term "treatment" refers to any type of treatment intended to terminate, prevent, improve, or reduce susceptibility to a clinical condition described herein. In a preferred embodiment, the term treatment refers to a preventive treatment of a disease or condition as defined herein (i.e., a treatment that reduces susceptibility to a clinical condition). Thus, "treatment" and its equivalents refer to obtaining a desired pharmacological or physiological effect, covering any treatment of a pathological condition or disease in a mammal (including a human). The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects attributable to the disease. That is, "treatment" includes (1) preventing the occurrence or recurrence of a disease in a subject, (2) inhibiting a disease, such as preventing its development, (3) stopping or terminating a disease or at least symptoms associated therewith, so that the host no longer suffers from the disease or its symptoms, for example, such as causing regression of the disease or its symptoms by restoring or repairing a lost, missing or defective function or stimulating an ineffective process, or (4) alleviating, alleviating, or improving a disease or symptoms associated therewith, wherein improvement is used in a broad sense to refer to at least reducing the magnitude of a parameter.

[0161] As used herein, the term "cancer" refers to a disease characterized by uncontrolled cell division (or increased resistance to survival or apoptosis), and the ability of the cells to invade other adjacent tissues (invasion) and spread through lymphatics and blood vessels to other areas of the body where the cells are not normally located (metastasis), circulate through the bloodstream, and then invade normal tissues in other parts of the body. Tumors are classified as benign or malignant depending on whether they can spread by invasion and metastasis: benign tumors are tumors that cannot spread by invasion or metastasis, i.e., they grow only locally; while malignant tumors are tumors that can spread by invasion and metastasis. As used herein, the term cancer includes, but is not limited to, the following types of cancer: breast cancer; biliary tract cancer; bladder cancer; brain cancer, including glioblastomas, particularly glioblastoma multiforme and medulloblastomas; cervical cancer; head and neck cancer; choriocarcinoma; colon cancer, colorectal cancer; endometrial cancer; esophageal cancer; gastric cancer; blood neoplasms, including acute lymphoblastic and myeloid leukemias; T-cell acute lymphoblastic leukemia / lymphoma; hairy cell leukemia; chronic myeloid leukemia, multiple myeloma; AIDS-related leukemia and adult T-cell leukemia / lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer, hepatoma; lung cancer, pleural mesothelioma; lymphomas, including Hodgkin's disease (Hodgkin's Disease) and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; parotid cancer; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; kidney cancer, adrenal cancer; rectal cancer; sarcoma, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma; cervical cancer, endometrial cancer; testicular cancer, including reproductive tumors, such as seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumor and germ cell tumor; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancer, including adenocarcinoma and Wilms tumor. Other cancers will be known to the ordinary technician.

[0162] In a preferred embodiment, the cancer is a blood cancer or hematological malignancy. The term "blood cancer or hematological malignancy" refers to a type of cancer that affects the blood, bone marrow, and lymph nodes. Hematological malignancies can be derived from one of the two major blood cell lineages, the myeloid cell lineage and the lymphoid cell lineage. The myeloid cell lineage typically produces granulocytes, red blood cells, platelets, macrophages, and mast cells; the lymphoid cell lineage produces B, T, NK, and plasma cells. Lymphomas, lymphocytic leukemias, and myelomas are from the lymphoid lineage, while acute and chronic myeloid leukemias, myelodysplastic syndromes (MDS), and myeloproliferative diseases all originate from the bone marrow. Non-limiting illustrative examples of hematologic malignancies are acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), and myeloma such as multiple myeloma (MM). In a more preferred embodiment, the hematologic malignancy is a leukemia. In a more preferred embodiment, the leukemia is from a bone marrow source. In a more preferred embodiment, the leukemia is acute myeloid leukemia. In another preferred embodiment, the hematologic cancer is AML, NHL, MDS, MM, CLL, or myelofibrosis.

[0163] In another preferred embodiment, the cancer is breast cancer, lung cancer, melanoma, pancreatic cancer, colon cancer, kidney cancer, basal cell carcinoma, squamous cell carcinoma, recurrent or refractory malignancies, prostate cancer, neuroblastoma, glioblastoma, astrocytoma or ovarian cancer. In a preferred embodiment, the cancer is skin cancer, more preferably melanoma.

[0164] The authors of the present invention have shown that the combined use of carotenoids and senescent cell lytic agents results in a synergistic senescent cell lytic effect. Therefore, the present invention also provides the use of these compositions for the treatment of diseases requiring the selective destruction of senescent cells.

[0165] In another aspect, the invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of a senescence-related disease or disorder.

[0166] Alternatively, the present invention relates to a method for preventing and / or treating a senescence-related disease or disorder, which comprises administering a combination or pharmaceutical composition according to the present invention.

[0167] Alternatively, the present invention relates to a combination or pharmaceutical composition of the present invention for use in the preparation of a medicament for preventing and / or treating a disease or disorder associated with aging.

[0168] As used herein, a "senescence-associated disease or disorder" relates to a clinical disorder in which the presence and effects of senescent cells significantly contribute to the pathophysiology of the disorder.

[0169] In another preferred embodiment, the senescence-associated disease or disorder is fibrosis.

[0170] As used herein, "fibrosis" refers to the formation of excess fibrous connective tissue in an organ or tissue during a repair or reaction process. Although fibrosis can be a benign state, the present invention preferably relates to pathological fibrosis. In a preferred embodiment, fibrosis is characterized by having a large number of senescent cells.

[0171] In another preferred embodiment, the fibrosis having a high number of senescent cells is pulmonary fibrosis, chronic obstructive pulmonary disease, myocardial fibrosis, renal fibrosis, myelofibrosis, or liver fibrosis.

[0172] As used herein, "a large number of senescent cells" refers to an increase in the number of senescent cells in a tissue with fibrosis relative to the number of cells in a normal tissue. In a preferred embodiment, the number of senescent cells in a tissue with fibrosis is at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or even more compared to the number of senescent cells in a normal tissue. Normal tissue means tissue that does not have fibrosis.

[0173] Senescent cells may exhibit any one or more of the following characteristics. (1) Senescent growth arrest is essentially permanent and cannot be reversed by known physiological stimuli. (2) Senescent cells increase in size, sometimes more than twofold relative to their non-senescent counterparts. (3) Senescent cells express senescence-associated β-galactosidase (SAP-gal), which in part reflects an increase in lysosomal mass. (4) Most senescent cells express p16INK4a, which is not normally expressed by quiescent or terminally differentiated cells. (5) Senescent cells with persistent DDR signaling have persistent nuclear foci, called DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). These foci contain activated DDR proteins and can be distinguished from transient damage foci. DNA-SCARS include dysfunctional telomeres or telomere dysfunction-induced foci (TIFs). (6) Senescent cells express and can secrete molecules referred to herein as senescent cell-associated molecules, which in some cases can be observed in the presence of persistent DDR signaling.

[0174] The presence of senescent cells in a tissue can be detected by the presence of senescent cell-associated molecules, including growth factors, proteases, cytokines (e.g., inflammatory cytokines), chemokines, cell-associated metabolites, reactive oxygen species (e.g., H2O2), and other molecules that stimulate inflammation and / or other biological effects or responses that can promote or exacerbate the subject's underlying disease. In a preferred embodiment, senescent cells are detected by detecting the senescence-associated secretory phenotype (SASP, i.e., it includes secreted factors that can constitute the pro-inflammatory phenotype of senescent cells), the senescent-messaging secretome, and the DNA damage secretory program (DDSP).

[0175] Senescent cells and senescent cell-related molecules can be detected by techniques and procedures described in the art. For example, the presence of senescent cells in tissues can be analyzed by histochemistry or immunohistochemistry techniques that detect the senescence marker SA-β galactosidase (SA-Pgal) (see, for example, Dimri et al, Proc. Natl. Acad. Sci. USA 92: 9363-9367 (1995)). The presence of senescent cell-related polypeptide p16 can be determined by any of a variety of immunochemical methods practiced in the art (e.g., immunoblot analysis). The expression of p16 mRNA in cells can be measured by a variety of techniques practiced in the art (including quantitative PCR). The presence and level of senescent cell-related polypeptides (e.g., SASP polypeptides) can be determined by using automation and high-throughput assays, such as the automatic Luminex array assay described in the art (see, for example, Coppe et al, PLoS Biol 6: 2853-68 (2008)). The presence of senescent cells can also be determined by detecting senescent cell-associated molecules, including growth factors, proteases, cytokines (e.g., inflammatory cytokines, such as determining the level of IL-1β as in Example 4), chemokines, cell-associated metabolites, reactive oxygen species (e.g., H2O2), and other molecules that stimulate inflammation and / or other biological effects or responses that may promote or exacerbate the subject's underlying disease.

[0176] Aging-associated diseases and disorders include, for example, cardiovascular diseases and disorders, inflammatory diseases and disorders, autoimmune diseases and disorders, pulmonary diseases and disorders, ocular diseases and disorders, metabolic diseases and disorders, nervous system diseases and disorders (e.g., neurodegenerative diseases and disorders); age-related diseases and disorders arising from senescence; skin disorders; age-related diseases; skin diseases and disorders; and transplantation-related diseases and disorders.

[0177] In another specific embodiment, the senescence-related disease or disorder is a cardiovascular disease selected from the group consisting of atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, cerebral aneurysm and stroke. In another specific embodiment, the senescence-related disease or disorder is an inflammatory or autoimmune disease or disorder selected from the group consisting of osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis and intervertebral disc herniation. In another specific embodiment, the senescence-related disease or disorder is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment and motor neuron dysfunction. In another specific embodiment, the aging-related disease or condition is a metabolic disease selected from diabetes, diabetic ulcers, metabolic syndrome and obesity. In another specific embodiment, the aging-related disease or condition is a pulmonary disease selected from pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis and age-related loss of lung function. In another specific embodiment, the aging-related disease or condition is an eye disease or condition selected from macular degeneration, glaucoma, cataracts, presbyopia and decreased vision. In another specific embodiment, the aging-related disease or condition is an age-related condition selected from kidney disease, renal failure, weakness, hearing loss, muscle fatigue, skin disorders, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucous fibrosis and sarcopenia. In another specific embodiment, the senescence-related disease or disorder is a skin disease or disorder selected from the group consisting of eczema, psoriasis, pigmentation, moles, rashes, atopic dermatitis, urticaria, diseases and disorders associated with photosensitivity or photoaging; rhytides; pruritus; insensitivity; eczematous eruptions; eosinophilic dermatosis; reactive neutrophilic dermatosis; pemphigus; pemphigoid; immunobullous dermatosis; fibrohistocytic proliferations of skin; cutaneous lymphoma; and cutaneous lupus. In another specific embodiment, the senescence-related disease or disorder is atherosclerosis; osteoarthritis; pulmonary fibrosis; hypertension, chronic obstructive pulmonary disease; myelofibrosis; or liver fibrosis.

[0178] In a preferred embodiment, the senescence-associated disease or disorder is selected from cancer, fibrosis, and chemotherapy- or radiotherapy-induced senescence.

[0179] In a preferred embodiment, the senescence-associated disease or disorder is cancer, more preferably a blood cancer or a skin cancer.

[0180] In another preferred embodiment, the fibrosis having a high number of senescent cells is pulmonary fibrosis, chronic obstructive pulmonary disease, myocardial fibrosis, renal fibrosis, myelofibrosis, or liver fibrosis.

[0181] In another preferred embodiment, the aging disorder is therapy-induced aging, preferably chemotherapy- or radiotherapy-induced aging. In a more preferred embodiment, the therapy-induced aging is in cancer. Some illustrative, non-limiting examples of cancer have been described previously.

[0182] As used herein, "therapy-induced senescence" or TIS refers to senescence induced by treatment. DNA damaging radiation therapy and chemotherapy can induce SASP in vivo (see, e.g., Coppe et al, 2008, PLoS Biol. 6: 2853-2868), which can have deleterious systemic effects and the ability to stimulate the regeneration of tumor cells that have not been eradicated by anticancer therapy. Unlike apoptotic cell death, whose remnants are rapidly cleared by phagocytosis without much inflammatory response, senescent cells may persist at the tumor site for varying periods of time.

[0183] Several assays can be used to determine the presence of treatment-induced senescence, such as the SA-β-gal assay, Ki67 and / or H3K9me3 staining, or determining an increase in IL-1β.

[0184] In a preferred embodiment, the treatment is radiation therapy or chemotherapy. In a preferred embodiment, the radiation therapy is gamma irradiation.

[0185] "Chemotherapy" refers to treatment using chemotherapeutic agents. The term "chemotherapeutic agent" includes standard chemotherapy drugs (which generally attack any rapidly dividing cells), targeted therapeutic agents, and immunomodulators. Illustrative, non-limiting examples of cancer chemotherapeutic agents that may be used according to the present invention include alkylating agents, antimetabolite drugs, anthracycline antibiotics, antibodies targeting angiogenic factors, topoisomerase inhibitors, anti-microtubule agents, cyclin-dependent kinase inhibitors, low molecular weight tyrosine kinase inhibitors of angiogenic growth factors, and matrix metalloproteinase inhibitors.

[0186] In a more preferred embodiment, the chemotherapeutic agent is selected from Aphidocolin, Bleomycin, Camptothecin, Carboplatin, Docetaxel, Cisplatin, Cyclophosphamide, Daunorubicin, Doxorubicin, Cytarabine, Palbociclib, 5-Fluorouracil, Diaziquone / AZQ, Epigallocatechin gallate, Etoposide, Hydroxyurea, K858, Lovastatin, Mitoxantrone, MLN4924, MLN8054, Pyrithione, Resveratrol, TPA, PEP005, PEP008 and VO-OHpic.

[0187] In a more preferred embodiment, the chemotherapeutic agent is doxorubicin. In another preferred embodiment, the chemotherapeutic agent is palbociclib.

[0188] For the medical use of the present invention, the combination of the present invention or a pharmaceutical composition comprising the combination may be administered in a therapeutically effective amount.

[0189] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of a compound (i.e., a combination of the present invention) to provide the desired effect, and is generally determined by, among other reasons, the properties of the compound itself and the therapeutic effect to be achieved. It also depends on the subject to be treated, the severity of the disease suffered by the subject, the selected dosage form, the route of administration, etc. Therefore, those skilled in the art must adjust the dosage according to the aforementioned variables.

[0190] For medical use according to the present invention, the combination or pharmaceutical composition of the present invention can be administered by any route of administration, for example by systemic (e.g. intravenous, subcutaneous, intramuscular injection), oral, parenteral (intranasal, sublingual) or topical administration.

[0191] There are numerous reports across several cancer types indicating that aging is associated with a poor therapeutic index for cancer therapeutics.

[0192] It is well known in the art that many tumor cells become resistant to chemotherapy by entering senescence, and once the chemotherapy is removed, the cells leave senescence and resume proliferation or induce proliferation of neighboring cells (see, for example, Gordin and Nelson, Drug Resist Updat. 2012, 15: 123-131). Therefore, in view of its senescent cell lytic effect, the composition according to the present invention can be used to destroy cells that have entered senescence in response to chemotherapy treatment, thereby resulting in an increase in the efficacy of chemotherapy treatment. Therefore, in another aspect, the present invention relates to a combination according to the present invention or a pharmaceutical composition according to the present invention, which is used to enhance the effectiveness of an anti-tumor compound.

[0193] As used herein, "enhancing effectiveness" includes expanding, strengthening, increasing, amplifying and enhancing the effects produced by the anti-tumor compound.

[0194] Since the combination of the present invention reduces senescence induced by the treatment, this results in an improvement or enhancement of the therapeutic and / or prophylactic benefit of the drug treatment compared to the benefit observed in the absence of administration of the agent. Thus, a specific dose of an anti-tumor compound together with the combination of the present invention results in a higher effect than the same dose of the anti-tumor compound in the absence of administration of the combination of the present invention to the same subject.

[0195] All terms and embodiments previously described with respect to the combinations of the present invention are equally applicable to these aspects of the present invention.

[0196] Medical uses of carotenoids

[0197] The authors of the present invention have shown that carotenoids are able to restore the senescent phenotype induced by cytotoxic agents (see Example 3). This therefore allows carotenoids to be used to treat diseases characterized by an undesirable number of senescent cells by promoting cells to abandon the senescent phenotype. Thus, in another aspect, the present invention relates to carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for use in treating senescence-related diseases or disorders.

[0198] Alternatively, the present invention relates to the use of carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for the preparation of a medicament for treating aging-related diseases or disorders.

[0199] Alternatively, the present invention relates to a method for treating a disease or condition associated with aging, comprising administering a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog thereof, or an ester or salt thereof to a subject in need thereof. The term "disease or condition associated with aging" and its embodiments have been described previously and are equally applicable to this aspect of the present invention.

[0200] In a preferred embodiment, the senescence-associated disease or disorder is cancer, more preferably a blood cancer or a skin cancer.

[0201] In another preferred embodiment, the senescence-related disease or disorder is fibrosis. In a preferred embodiment, fibrosis is characterized by having a large number of senescent cells.

[0202] In another preferred embodiment, the fibrosis having a high number of senescent cells is pulmonary fibrosis, chronic obstructive pulmonary disease, myocardial fibrosis, or renal fibrosis.

[0203] In another preferred embodiment, the aging disorder is therapy-induced senescence, more preferably chemotherapy- or radiotherapy-induced senescence.

[0204] In a preferred embodiment, the carotenoid is selected from the group consisting of fucoxanthin, fucoxanthinol, amarouciaxanthin A, neoxanthin, astaxanthin, zeaxanthin and lutein. In a preferred embodiment, the carotenoid is fucoxanthin. In another preferred embodiment, the carotenoid is amarouciaxanthin A. In another preferred embodiment, the carotenoid is not fucoxanthin. In another preferred embodiment, the carotenoid is not fucoxanthinol. In another preferred embodiment, the carotenoid is not amarouciaxanthin A. In another preferred embodiment, the carotenoid is not neoxanthin. In another preferred embodiment, the carotenoid is not astaxanthin. In another preferred embodiment, the carotenoid is not zeaxanthin. In another preferred embodiment, the carotenoid is not lutein.

[0205] In another aspect, the present invention relates to carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for use in enhancing the effectiveness of anti-tumor compounds.

[0206] In addition, it is well known that certain anti-tumor agents cause secondary effects due to the induction of senescence in non-tumor cells (see Demaria, Mol. Cell Oncol. 2017; 4(3): e1299666.). Therefore, the ability of carotenoids to promote cells to leave the senescent phenotype allows carotenoids to be used to reduce the adverse effects of anti-tumor therapy. Therefore, in another aspect, the present invention relates to carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof, which are used to reduce the adverse effects of anti-tumor therapy.

[0207] As used herein, "adverse effects", side effects, toxic side effects or deleterious side effects relate to undesirable harmful effects caused by a drug with an anti-tumor compound. Preferably, the adverse effect is gastrointestinal toxicity, peripheral neuropathy, fatigue, weakness, low physical activity, blood toxicity, liver toxicity, alopecia, pain, mucositis, fluid retention, skin toxicity, fatigue or cardiotoxicity.

[0208] As used herein, "reducing an adverse effect" relates to a lower likelihood of the adverse effect occurring, or a lower intensity of the adverse effect, compared to a subject not receiving the carotenoid, carotenoid metabolite, carotenoid derivative, analog, or ester or salt thereof.

[0209] In a preferred embodiment, the likelihood of an adverse effect in a subject treated with an anti-tumor compound is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%.

[0210] Reducing the likelihood of suffering an adverse effect can be measured by methods known in the art depending on the adverse effect to be determined.

[0211] In a preferred embodiment, the adverse effect is due to chemotherapeutic treatment. In another preferred embodiment, the adverse effect is due to radiotherapeutic treatment.

[0212] All terms and embodiments previously described with respect to the combinations of the present invention are equally applicable to these aspects of the present invention.

[0213] Methods for inhibiting aging

[0214] In another aspect, the present invention relates to a method for inhibiting senescence in a cell population or a subject, comprising administering an effective amount of a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or an ester or salt thereof to a cell population or a subject in need thereof.

[0215] As used herein, "inhibit" refers to reducing or lowering aging. As used herein, "reducing" refers to reducing aging by more than at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% compared to the level of aging in the absence of administration of carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof.

[0216] Methods for determining senescence have been described previously. In a preferred embodiment, the level of senescence is determined by detecting the number of senescent cells. In another preferred embodiment, the level of senescence is determined by quantifying the number of senescence markers.

[0217] The method for inhibiting aging in a cell population of the present invention comprises a first step of contacting the cell population with a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog thereof, or an ester or salt thereof. In the case where the cell population forms part of an individual, the contacting step can be performed by administering a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog thereof, or an ester or salt thereof to the individual. Any method previously described for administering a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog thereof, or an ester or salt thereof can be used. The level of senescent cells can be determined according to any in vitro assay or technique known in the art. For example, senescent cells can be detected by: morphology (e.g., by microscopy); the production of senescence-related markers, such as the production of senescence-related β-galactosidase (SA-β-gal), p16INK4a, p21, PAI-I, or any one or more SASP factors (e.g., IL-6, MMP3).

[0218] In addition, the method for inhibiting senescence includes a second step of measuring the senescence level and comparing it with a reference value.

[0219] As used herein, "reference value" refers to a laboratory value used as a reference for the value / data obtained from a sample. A reference value (or reference level) can be an absolute value, a relative value, a value with an upper and / or lower limit, a series of values, an average value, a median value, a mean value, or a value represented by a control or reference value as a reference. A reference value can be based on a value obtained from a single sample, such as a value obtained from a research sample but obtained at a previous time point. A reference value can be based on a large number of samples, such as a value obtained in a sample group, or based on a sample amalgamation containing or not containing a sample to be tested.

[0220] In a specific embodiment, the reference value is the level of senescence in a cell or subject in the absence of the same carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof.

[0221] All terms and embodiments previously described with respect to the combinations of the present invention are equally applicable to these aspects of the present invention.

[0222] The following examples illustrate the invention and must not be considered as limiting the invention. Example

[0223] Example 1 - Synergistic effects of carotenoids and BCL2 inhibitors in cancer

[0224] Materials and methods

[0225] Cell culture

[0226] Human MV-4-11 and PL-21 cell lines were used to evaluate the effects of fucoxanthin and amarouciaxanthin A in combination with the BCL-2 family inhibitors navicla (ABT-263) and venetoclax (ABT-199). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere in RPMI medium supplemented with 10% FCS and were always mycoplasma free as assessed by PCR. All passages were performed at 80% to 90% confluence and viability greater than 90%.

[0227] MTT assay

[0228] For cytotoxicity assays, MV-4-11 cells and PL-21 cells were cultured at 10 4 Cells / well were plated in 96-well microplates at a density of 100 cells / well. After 24 hours, different concentrations of navicla or venetoclax and fucoxanthin or amarouciaxanthin A were added. The plate was incubated for 72 hours at 37°C and 5% CO2. DMSO was used as a vehicle control. After this period, 50 μL of 10% SDS was added to the negative control wells (0% survival rate, 100% mortality rate). Then, the culture medium was replaced with fresh culture medium with 5mg / ml MTT in 10 μL of PBS in each well. The plate was incubated for 4 hours at 37°C and 5% CO2. Next, the culture medium was removed and 100 μL of extraction buffer (SDS, DSA, acetic acid 2%, pH 4.7) was added to each well. After the plate was incubated for 1 hour at 37°C and 5% CO2, the culture was homogenized. Finally, the plate was read at 570nm with a spectrophotometer Multiskan Ascent (MTX Lab systems). The potency of each product was determined by calculating the IC50 (half maximal inhibitory concentration).

[0229] result

[0230] To test the cytotoxic effects of fucoxanthin and amarouciaxanthin A in combination with different drugs (BCL2 inhibitors) in cancer cells, a series of concentrations of fucoxanthin were co-incubated with the following compounds:

[0231] - Navikla (ABT-263)

[0232] -Venetoclax (ABT-199).

[0233] The cytotoxic potency of each compound and its combination potency with fucoxanthin were obtained in the MTT assay in MV-4-11 and PL-21 cells. This parameter is expressed as IC50 in Table 1. Different synergistic combination scores were also obtained and are shown in Table 2 for MV-4-11 cells and in Table 3 for PL-21 cells.

[0234] Table 1. IC50 of different drugs in two hematological cancer cell lines (6 replicates).

[0235]

[0236] SD, standard deviation.

[0237] Table 2. Synergy scores of different drug combinations with fucoxanthin in MV-4-11 cells.

[0238]

[0239] The combination index was calculated according to the Chou-Talalay method (Cancer Res. 2010 Jan 15; 70(2): 440-6.) The values ​​are interpreted as follows: <1 synergy, >1 antagonism, and =1 additivity. Conc, concentration; CI, combination index.

[0240] Table 3. Synergy scores of different drug combinations with fucoxanthin in PL-21 cells.

[0241]

[0242] The combination index was calculated according to the Chou-Talalay method cited above. Values ​​are interpreted as follows: <1 synergy, >1 antagonism, and =1 additivity. Conc, concentration; CI, combination index.

[0243] As seen in Tables 2 and 3, synergistic reductions in IC50 were observed for fucoxanthin and naviclaz (in both MV-4-11 and PL-21 cells), amarouciaxanthin A and naviclaz, and fucoxanthin and venetoclax (in MV-4-11).

[0244] Example 2 - Role of carotenoids and BCL2 inhibitors in aging

[0245] Materials and methods

[0246] Cell culture

[0247] The Sk-Mel-103 cell line (human skin melanoma) was cultured in high glucose DMEM supplemented with 10% FCS. All cells were cultured at 37°C in a humidified 5% CO2 atmosphere and were always mycoplasma free as assessed by PCR. All passages were performed at 80% to 90% confluence and viability greater than 90%.

[0248] Treatments and experimental conditions

[0249] 10 6 Sk-Mel-103 cells were seeded into 24-well plates. The cultures were incubated with Palbociclib (as a senescence inducer), fucoxanthin, or a combination thereof, such as Error! No seencuentraelorigendelareferencia. After 7 days, the cultures were treated with different concentrations of the BCL2 inhibitor naviclat. The cytotoxicity of each condition was determined by calculating the IC50 (half maximal inhibitory concentration) by MTT assay.

[0250] Table 4. Experimental conditions used to determine the effect of fucoxanthin pretreatment on the sensitivity of cells to BCL2 inhibitors.

[0251]

[0252] MTT assay

[0253] For cytotoxicity assays, cells were plated at 10 4 The density of cells / well was plated in 96-well microplates. After the above initial treatment, different concentrations of navicla were added. The plate was then incubated for 72 hours at 37°C and 5% CO2. DMSO was used as a vehicle control. After this period, 50 μL of 10% SDS was added to the negative control wells (0% survival rate, 100% mortality rate). Then, the culture medium was replaced with fresh culture medium, with 5mg / mlMTT in 10 μL of PBS in each well. The plate was incubated for 4 hours at 37°C and 5% CO2. Next, the culture medium was removed, and 100 μL of extraction buffer (SDS, DSA, acetic acid 2%, pH 4.7) was added to each well. After the plate was incubated for 1 hour at 37°C and 5% CO2, the culture was homogenized. Finally, the plate was read at 570nm with a spectrophotometer Multiskan Ascent (MTX Lab systems). The efficacy of each product was determined by calculating IC50 (half maximum inhibition concentration).

[0254] result

[0255] To investigate the effect of pretreatment with fucoxanthin on the sensitivity of cells to BCL2 inhibitors, incubations with palbociclib (senescence inducer) alone or in combination with fucoxanthin were performed for 7 days (as in Error! Noseencuentraelorigendelareferenca seen). Then, the cytotoxicity of navicla (BCL2 inhibitor) was evaluated by MTT assay. The data were compared with control cells (non-senescent) in basal state or treated with fucoxanthin.

[0256] Table 5. IC50 of navicla in Sk-Mel-103 cells treated with different conditions (6 replicates).

[0257] IC50 (μM) (mean ± SD) Control cells 8.017±1.306 Senescent cells 0.136±0.012 Control cells + fucoxanthin 4.785±0.911 Senescent Cells + Fucoxanthin 0.055±0.005

[0258] SD, standard deviation.

[0259] As seen in Table 5, the IC50 of Navitra in control cells and senescent cells decreased when cells were pretreated with fucoxanthin. Thus, pretreatment with fucoxanthin increased the senescent cell lytic activity of Navitra.

[0260] Example 3 - Role of carotenoids in aging

[0261] Materials and methods

[0262] Cell culture

[0263] BJ-11 cell line (human primary skin fibroblasts) was cultured in MEM supplemented with 10% FCS plus 2 mM L-glutamine. All cells were cultured at 37°C in a humidified 5% CO2 atmosphere and were always mycoplasma free, as assessed by PCR. All passages were performed at 80% to 90% confluence and viability greater than 90%.

[0264] Treatments and experimental conditions

[0265] In order to evaluate the effect of fucoxanthin on aging, 10 4 BJ-11 cells were seeded in 24-well plates. The cultures were incubated with different concentrations of Palbociclib (Palbo) or doxorubicin (Doxo) (as senescence inducers) and fucoxanthin (Fuco) as described in Table 6.

[0266] Table 6. Experimental conditions used to determine the effects of fucoxanthin on aging.

[0267]

[0268] Doxorubicin treatment was performed for only 24 hours. Thereafter, doxorubicin was removed and cells were cultured in complete medium for the periods indicated below. On the other hand, palbociclib was continuously present in the culture medium and fresh was added regularly.

[0269] Two different approaches are followed:

[0270] Method 1: The role of fucoxanthin in preventing aging

[0271] Cell cultures were treated with fucoxanthin and senescence inducers as indicated below. After 7 days, β-galactosidase activity was measured.

[0272] Solution 1. Experimental procedures to test the effects of fucoxanthin on aging prevention

[0273]

[0274] Method 2: Fucoxanthin’s Effect on Reversing Aging

[0275] As shown below, the cell cultures were first treated with a senescence inducer for 7 days. Then, the treatment with fucoxanthin was performed for 7 days. At the end of this period, the β-galactosidase activity was determined.

[0276] Solution 2. Experimental procedures to test the effects of fucoxanthin on aging reversal.

[0277]

[0278] Senescence-associated β-galactosidase (SA-β-gal) staining

[0279] After treatment, SA-β-galactosidase staining was used to determine the expression of senescence-related β-galactosidase in BJ-11 cells. β-galactosidase staining was performed according to the manufacturer's recommendations (9860S, Cell Signaling Technology). Growth medium was removed from the cells and the plates were washed once with PBS. Then, a fixative was added to each well and the cells were allowed to fix at room temperature for 10 to 15 minutes. The plate was washed twice with PBS and then β-galactosidase staining solution was added to each well. Finally, the plate was incubated at 37°C for 24 hours in a dry incubator (no CO2) to prevent pH changes, which can affect the staining results. The amount of cells (dyed blue) positive for β-galactosidase activity was evaluated by manual staining cell counting.

[0280] result

[0281] To investigate the effects of fucoxanthin on the prevention and reversal of senescence, incubation with the cytostatic drugs doxorubicin and palbociclib was performed in BJ-11 cells as previously described. The activity of β-galactosidase was determined and used as a senescence marker. After fucoxanthin treatment, the percentage of senescent cells positive for β-galactosidase activity decreased, as can be seen in Tables 7 and 8.

[0282] Table 7. Percentage of cells positive for β-galactosidase activity in Method 1 (Evaluation of the effect of fucoxanthin on the prevention of aging).

[0283] Pabocini Doxorubicin Control cells (basal state) 0％ 0％ Senescent cells >98％ >80％ Senescent Cells + Fucoxanthin <5％ <5％

[0284] Table 8. Percentage of cells positive for β-galactosidase activity in Method 2 (Evaluation of the effect of fucoxanthin on senescence reversal).

[0285] Pabocini Doxorubicin Control cells (basal state) <2％ <2％ Senescent cells >95％ >80％ Senescent Cells + Fucoxanthin <5％ <5％

[0286] Example 4 - Role of carotenoids in reducing the senescence-associated secretory phenotype (SASP)

[0287] Materials and methods

[0288] Cell culture

[0289] The Sk-Mel-103 cell line (human skin melanoma) was cultured in high glucose DMEM supplemented with 10% FCS. All cells were cultured at 37°C in a humidified 5% CO2 atmosphere and were always mycoplasma free as assessed by PCR. All passages were performed at 80% to 90% confluence and viability greater than 90%.

[0290] Treatments and experimental conditions

[0291] 10 6 Sk-Mel-103 cells were seeded into 24-well plates. The cultures were incubated with palbociclib or doxorubicin (as senescence inducers), fucoxanthin, or a combination thereof, as described in Table 9. After treatment, cells and supernatants were collected by centrifugation and stored at -20°C.

[0292] Table 9. Experimental conditions used to determine the effect of fucoxanthin on SASP

[0293]

[0294] Determination of secreted interleukins (IL)

[0295] The secretion of IL-1β, IL-6 and IL-8 in the supernatant was quantified by commercial ELISA kits (DY201, DY206, DY208, R&D Sytems) according to the manufacturer's instructions. The values ​​obtained were normalized by the total cellular protein content in each well quantified by Bradford assay (B6916, Sigma-Aldrich) at the end of the experiment. A second normalization was performed by dividing the interleukins secreted after fucoxanthin incubation by their corresponding senescent cell controls.

[0296] result

[0297] IL-1β, IL-6 and IL-8 are upregulated in senescent cells and constitute part of the SASP. To investigate the effect of fucoxanthin on SASP factors, secreted ILs were quantified in the supernatants of SK-Mel-103 cells in which senescence was induced with doxorubicin or palbociclib.

[0298] like Figure 1 and Figure 2 As shown in , for both pro-senescence treatments, the secretion of IL-1β, IL-6, and IL-8 was significantly reduced after incubation with fucoxanthin (p-values ​​< 0.01).

[0299] Example 5 - Effect of amarouciaxanthin A on liver fibrosis

[0300] Hepatic stellate cells (HSCs) are found in the perisinusoidal spaces of the liver. They constitute the major cell type involved in liver fibrosis, responsible for scar tissue formation in response to liver injury.

[0301] Materials and methods

[0302] Experimental conditions

[0303] In order to evaluate the effect of amarouciaxanthin A on HSC activation, immortalized human cell line LX2 was used. In addition, primary HSC was extracted from cirrhotic rats that had been subjected to carbon tetrachloride inhalation. In both cases, cells were cultured for 24 hours before adding the compound. Then, amarouciaxanthin A (10 μM) or vehicle (DMSO) was added to the culture. Cells were harvested 24 hours and 72 hours after treatment, and lysed to analyze fibrosis biomarkers. Gene transcription was measured by quantitative PCR at 24 and 72 hours, and protein expression was measured by western blot at 72 hours. α-smooth muscle actin (α-SMA) was analyzed in rat HSC. Collagen I a1 (COL1A1), a major component of the extracellular matrix, was analyzed in LX2.

[0304] result

[0305] α-SMA is a structural protein that forms microfilaments and a common activation biomarker of HSC. Under pathological conditions, HSC increases the synthesis of COL1A1, thereby promoting the occurrence of liver fibrosis.

[0306] like Figure 3 As shown in , amarouciaxanthin A inhibited α-SMA transcription in cirrhotic rat HSCs by about 80% after 24 and 72 hours. Protein expression was also reduced by 95%. Luteins also inhibited COL1A1 transcription in human LX2 cells by about 50% (after 24 hours) and 80% (after 72 hours) ( Figure 4 As quantified by western blot, collagen expression was reduced by approximately 95% ( Figure 4 ).

[0307] In summary, amarouciaxanthin A promotes HSC inactivation and inhibits the enhanced synthesis of extracellular matrix, as demonstrated by the downregulation of fibrotic markers at both the transcriptional and translational levels.

Claims

1. A composition comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or a salt thereof, and a second component, wherein the second component is an inhibitor of one or more BCL-2 anti-apoptotic protein family members, the one or more inhibitors of BCL-2 anti-apoptotic protein family members are selected from naviclat or venetoclax, and the carotenoid, carotenoid metabolite, carotenoid derivative, an analog or a salt thereof is selected from fucoxanthin, fucoxanthinol and amarouciaxanthin A.

2. A cosmeceutical, cosmetic or pharmaceutical composition comprising the composition of claim 1.

3. Use of the composition according to claim 1 or the pharmaceutical composition according to claim 2 in the manufacture of a medicament for treating acute myeloid leukemia (AML), cutaneous melanoma or skin aging.

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