CN112804996A

CN112804996A - Use of carotenoids in the treatment of age-related disorders

Info

Publication number: CN112804996A
Application number: CN202080005597.2A
Authority: CN
Inventors: 欧亨尼娅·鲁伊斯卡诺瓦斯; 阿里亚德娜·埃梅里克卡斯特拉; 哈维尔·阿尔瓦雷斯米科; 豪梅·梅卡德罗加; 诺埃利亚·赫斯泰拉佩雷斯
Original assignee: Garter Treatment Ltd
Current assignee: Garter Treatment Ltd
Priority date: 2019-03-05
Filing date: 2020-03-04
Publication date: 2021-05-14

Abstract

The present invention relates to a combination comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof, and a second component selected from the group consisting of: one or more inhibitors of BCL-2 anti-apoptotic protein family members, activators of pro-apoptotic BCL-2 family members, senescent cell lytic agents, and senescent phenotype inhibitors. The invention also relates to a food product, cosmeceutical, nutraceutical, cosmetic or pharmaceutical composition comprising the combination of the invention, and to its use in a cosmetic method and to its medical use.

Description

Use of carotenoids in the treatment of age-related disorders

Technical Field

The present invention is in the medical field. More particularly in the field of treatment of diseases or conditions in which the removal of senescent cells is beneficial.

Background

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

Given that certain aspects of aging cells that have been associated with age-related health decline are causally related and can cause certain diseases, and are also induced by necessary life-sustaining chemotherapy and radiation therapy, the presence of aging cells can have deleterious effects on millions of patients worldwide.

Document US2016339019 discloses inhibitors of the BCL-2 family of anti-apoptotic proteins and document EP3247375a2 discloses inhibitors of SYK and inhibitors of ASK1 as agents suitable for the removal of senescent cells in humans.

There remains a need in the art to provide new and existing compounds that are therapeutically targeted to aging cells and that are useful in the treatment of diseases associated with cellular aging.

Summary of The Invention

The authors of the present invention have found that carotenoids act synergistically with inhibitors of the BLC2 anti-apoptotic family member, activators of BCL-2 pro-apoptotic family members, senolytic agents (senolytic agents) and senomorphic agents (senomorphic agents) in reducing senescence and in the treatment of cancer.

Accordingly, in a first aspect, the present invention relates to a combination comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof, and a second component selected from the group consisting of: 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 senescent phenotype inhibitors.

In a second aspect, the present invention relates to a food product, cosmeceutical, nutraceutical, cosmetic or pharmaceutical composition comprising the combination of the invention.

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

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

In a fifth 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 cancer.

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 disease or disorder associated with aging.

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

In an eighth aspect, the present invention relates to a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or an ester or a salt thereof for use in the treatment of an aging-related disease or disorder.

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

In a tenth aspect, the present invention relates to a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof for use in reducing the adverse effects of an anti-tumor therapy.

Brief Description of Drawings

FIG. 1 IL secretion from SK-Mel-103 cells following treatment with Palbociclib (palbociclib). Error bars indicate standard deviation from 3 technical replicates. SC, senescent cells; f, fucoxanthin (fucoxanthin).

FIG. 2 IL secretion from SK-Mel-103 cells following treatment with doxorubicin (doxorubicin). Error bars indicate standard deviation from 3 technical replicates. SC, senescent cells; f, fucoxanthin.

FIG. 3 fold change in α -SMA expression in HSCs from liver-cirrhosis rats when incubated with 10 μ M amarocixanthin A. Values were normalized to vehicle control and error bars indicate standard deviation.

FIG. 4 fold change in COL1A1 expression in LX2 cells when incubated with 10. mu.M amarocixanthin A. Values were normalized to vehicle control and error bars indicate standard deviation.

Detailed Description

Combination of

The authors of the present invention have found that carotenoids and BCl-2 inhibitors exhibit synergistic cytotoxic effects in certain cancer cell lines. Thus, in a first aspect, the present invention relates to a combination comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof, and a second component selected from the group consisting of: 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 senescent phenotype inhibitors.

As used herein, "combination" or "composition" indicates that the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt 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 may be formulated in the same pharmaceutical formulation, or they may also be formulated in different pharmaceutical formulations, but the different pharmaceutical formulations should be contained in the same medicament by combined packaging and should be used simultaneously, sequentially or sequentially.

As used herein, the term "carotenoid" refers to a group of naturally occurring pigments produced primarily by plants, yeasts and algae, which have a common polyisoprenoid-based structure, long polyene chains forming chromophores, and approximate symmetry around a central double bond. Two C₂₀Mother body C is generated by tail-to-tail ligation of geranylgeranyl diphosphate molecules₄₀A carbon skeleton. The polyene chain may also have a cyclic group at one or both ends of the molecule.

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

In a preferred embodiment, the carotenoids of the invention are xanthophylls. In a more preferred embodiment, the xanthophyll is selected from the group consisting of α -cryptoxanthin (α -cryptoxanthin), β -cryptoxanthin (β -cryptoxanthin), adonirubin (adonirubin), adonixanthin (adonixanthin), isoxanthin (alloxanthin), amarocoxanthin (particularly amarocoxanthin A), epoxyzeaxanthin (atheraxanthin), astaxanthin (astaxantine), aureoxanthin (auroxanthin), xanthophyll (caloxanthin), gallicin (canthaxanthin), capsanthin (capsanthin), capsanthin-5-6-epoxide, capsorubin (capsorubin), carthamin (crocoxanthin), diadinoxanthin (diadinoxanthin), phycoerythrin (phycoerythrin), fucoxanthin (neoxanthin (fucoxanthin), fucoxanthin (neoxanthin), fucoxanthin (neoxanthin), xanthophyll (neoxanthin), xanthophyll (neoxanthin, neoxanthin (neoxanthin), xanthophyll (neoxanthin, or a, neoxanthin, or a, neoxanthin, or a, Violaxanthin (violaxanthin), zeaxanthin (zeaxanthin) and combinations or derivatives thereof. More preferably, the xanthophylls are selected from the group consisting of amarocixanthin a, capsaicinoids, fucoxanthin, neoxanthin, lutein, zeaxanthin, and combinations thereof. More preferably, the xanthophylls are selected from the group consisting of amarocixanthin a, fucoxanthin alcohol, neoxanthin, lutein, and combinations thereof. More preferably, the xanthophylls are selected from the group consisting of amarocixanthin a, fucoxanthin alcohol, neoxanthin, and combinations thereof. More preferably, the xanthophyll is selected from the group consisting of amarocixanthin a, fucoxanthin alcohol, and combinations thereof. Even more preferably, the xanthophyll is fucoxanthin or amarocixanthin a.

In one embodiment, the above mentioned xanthophylls refer to all-trans forms thereof.

In another preferred embodiment, the carotenoid is fucoxanthin. In another preferred embodiment, the carotenoid is fucoxanthin. In another preferred embodiment, the carotenoid is amarocixanthin. In a more preferred embodiment, the carotenoid is amarocixanthin 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.

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

Examples of suitable algae for providing carotenoids in the combination of the invention include microalgae from the phylum cyanobacteria (Cyanophyta), the phylum Chlorophyta (Chlorophyta), the phylum Rhodophyta (Rhodophyta), the phylum anisodinophyta (Heterokontophyta) and the phylum dinoflagellata (Haptophyta). The algae may be green microalgae such as Chlorella (Chlorella), Scenedesmus (Scenedesmus), dunaliella (dunaliella) (for beta-carotene), Haematococcus (Haematococcus) (for astaxanthin) and bracteacococcus; microalgae (hypophosphites) such as Isochrysis (Isochrysis) (for fucoxanthin and lutein); and heterodinoflagellates, such as Phaeodactylum (Phaeodactylum), phaeoflagellate (Ochromonas) and odotella. Examples of suitable macroalgae include all brown algae, and in particular, Fucus vesiculosus (Fucus vesiculosus), Fucus evanescens (Fucus evanescens), Laminaria (Laminaria sp.) and Sargassum (Sargassum sp.) (all for fucoxanthin). Certain fungi are known to produce xanthophylls, such as Phaffia rhodozyma (Xanthophyllomyces dendrorhous). Additionally, carotenoids may also be obtained from animal sources, such as egg yolk. Plants and plant parts suitable for producing carotenoids (e.g., xanthophylls used in the present invention) include, but are not limited to, calendula (marigold flower), corn, kiwi, red seedless grape, pumpkin, squash, spinach, orange pepper (orange peel), yellow squash (yellow squash), cucumber, pea, green pepper, red grape, grey walnut, orange, honeydew melon (honeydew), celery, green grape, Brussels sprouts (Brussels sprouts), shallot (onion), mung bean (green bean), broccoli, apple, mango, green lettuce (green bean), tomato, peach, yellow pepper, nectarine, red pepper, carrot, cantaloupe, apricot, bell pepper (bell pepper), and green kidney bean (green kidney bean).

As used herein, "carotenoid metabolite" relates to a carotenoid that is modified under different conditions by, for example, hydrogenation, dehydrogenation, double bond migration, chain shortening or extension, rearrangement, isomerization, oxidation, or a combination of these methods. Besides, carotenoid metabolites can also be formed by the presence of the enzymes monooxygenase, cyclooxygenase and dioxygenase.

Examples of metabolites of beta-carotene are retinol, retinal and retinoic acid, beta-ionone, beta-apo-l 4 ' -carotenal, beta-apo-10 ' -carotenal, beta-apo-8 ' -carotenal and beta-carotene 5, 8-endoperoxide-2, 3-dihydro-beta-apocarotenan-13-one, 5, 6-monoepoxide, retinoyl beta-D-glucuronide (retinoyl beta-D-glucuronide), beta-apo-12 ' -carotenoic acid (beta-apo-12 ' -carotenoic acid), beta-apo-14 ' -carotenoic acid, beta-apo-14 ' -carotenal, Beta-apo-14' -carotenoic acid and beta-apo-13-caronone.

Lycopene metabolites are, for example, acyclic retinoic acid (acyclo retinoic acid), 2, 7, 11-trimethyl-tetradecahexaene-1, 14-dialdehyde (2, 7, 11-trimethyl-tetradecahexaene-1, 14-diol), (E, E, E) -4-methyl-8-oxo-2, 4, 6-nonanetrialdehyde ((E, E, E) -4-methyl-8-oxo-2, 4, 6-nonanarial) (MON), apo-6-lycopene aldehyde (apo-6-lycopenial), apo-8 '-lycopene aldehyde, apo-10' -lycopene aldehyde, apo-12 '-lycopene aldehyde and apo-14' -lycopene aldehyde, and apo-10 '-lycopene acid (apo-10' -lycopenoic acid). Examples of lutein metabolites are 3 '-epi-lutein (3' -epilutein), 3 '-oxo-lutein, 3' -dehydrolutein, meso-zeaxanthin, methoxy-zeaxanthin, oxime derivatives of 3-hydroxy-beta-ionone and 3-hydroxy-14-apocarotenal, 3-hydroxy-3 ', 4' -didehydro-beta, gamma-carotene and 3-hydroxy-2 ', 3' -didehydro-beta, epsilon-carotene.

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

"derivatives of carotenoids" are also included in the context of the present invention. In a particular embodiment, the derivatives include xanthophylls and carotenes that contain one or more cis double bonds, including but not limited to 9 cis derivatives, 9 ' cis derivatives, 13 ' cis derivatives, 15 ' cis derivatives, and any combination thereof.

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

The combination of the invention may also comprise carotenoid analogs. As used herein, the term "carotenoid analog" can 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. The parent carotenoid is selected from the group consisting of more than 600 naturally occurring carotenoids described in the literature, and the stereo-and geometric isomers thereof. Such analogs may include, but are not limited to, esters, ethers, carbonates, amides, carbamates, phosphates and ethers, sulfates, glycoside ethers, with or without a spacer (linker).

The combination of the invention may also comprise esters of carotenoids (e.g. acetate, formate and benzoate derivatives).

The combination of the invention may also comprise a carotenoid, a carotenoid metabolite, a carotenoid derivative or a salt of a carotenoid analogue. The term "salt" is understood to mean any of the following forms of the active compounds according to the invention: wherein it is in ionic form or is charged and coupled to a counter ion (cation or anion) or is in solution. This is also to be understood as meaning complexes of the active compounds with other molecules and ions, in particular complexes which complex through ionic interactions. This definition includes in particular physiologically acceptable salts; the term must be understood as equivalent to "pharmacologically acceptable salts" or "pharmaceutically acceptable salts".

According to the invention, the second component is selected from the group consisting of a senescent cell lysis 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 (troglitazone) and/or punicic acid (punicic acid).

As used herein, a "senescent cell lytic agent" refers to a compound that destroys, kills, removes or promotes selective destruction of senescent cells (preferentially or to a greater extent). In other words, the senescent cell lysing agent destroys or kills senescent cells in a biologically, clinically and/or statistically significant manner as compared to its ability to destroy or kill non-senescent cells. The senescent cell lysing agent is used in an amount and for a time sufficient to selectively kill certain senescent cells in a clinically significant or biologically significant manner but insufficient to kill non-senescent cells (destroy non-senescent cells, cause non-senescent cell death). In certain embodiments, a senescent cell lytic agent described herein alters at least one signaling pathway in a manner that induces (initiates, stimulates, triggers, activates, promotes) and causes (i.e., causes) senescent cell death. Senescent cell lytic agents can alter, for example, one or both of a cell survival signaling pathway (e.g., the Akt pathway) or an inflammatory pathway, for example, by antagonizing proteins within the cell survival and/or inflammatory pathways in senescent cells. Senescent cell lytic agents are capable of performing their function by inducing (activating, stimulating, abrogating) apoptotic pathways that lead to cell death.

The person skilled in the art is aware of methods for identifying and selecting a lytic agent of senescent cells, for example by determining the reduction of senescent cells by an agent. 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 microscopic observation); production of senescence-associated markers, such as senescence-associated β -galactosidase (SA- β -gal), p16INK4a, p21, PAI-I, or any one or more of SASP factors (e.g., IL-6, MMP 3). Thus, one skilled in the art will readily recognize that characterization of an agent as a senescent cell lytic agent and determination of the level of killing of the agent can be achieved by comparing the activity of the test agent to an appropriate negative control (e.g., vehicle or diluent only and/or a composition or compound known in the art that does not kill senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing senescent cell lytic agents also include controls for determining the effect of an agent on non-senescent cells (e.g., resting cells or proliferating cells). The senescent cell lytic agent reduces (i.e., decreases) the percent survival of a plurality of senescent cells (i.e., reduces the amount of viable senescent cells in an animal or in a cell-based assay in some manner) as compared to one or more negative controls.

In certain embodiments, the senescent cell lysing 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 peptide. In a particular embodiment, the polynucleotide or oligonucleotide (e.g., including shRNA) can be delivered by a recombinant vector into which the polynucleotide or oligonucleotide of interest has been incorporated. In another preferred embodiment, the senescent cell lysing agent is a polyclonal or monoclonal antibody.

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 the group consisting of nevira and tenettog. In a more preferred embodiment, the senescent cell lysing agent is nevira. In another preferred embodiment, the senescent cell lysing agent is tenentog.

In another preferred embodiment, the senescent cell lysing agent is a flavonoid.

As used herein, "flavonoid" relates to a compound having the general structure of a 15-carbon skeleton consisting of two benzene rings (A and B) and a heterocycle (C). The carbon structure may be abbreviated as C6-C3-C6.

In a more preferred embodiment, the flavonoid is selected from fisetin, curcumin (curcumin), alvocidib and quercetin (quercetin).

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

As used herein, "senescence phenotype inhibitor" relates to a small molecule that inhibits the senescence phenotype in the absence of cell killing.

In a preferred embodiment, the senescence phenotype inhibitor is selected from rapamycin (rapamycin), fluspirilene (fluspirilene), cycloheximide (cycloheximide), NVP-BEZ235, Loperamide (Loperamide), Timosaponin A-III (Timosaponin A-III), niguldipine (igudiline), and nordihydroguaiaretic acid (nordihydroguaiaretic acid).

Screening methods for identifying senescence phenotype inhibitors and distinguishing them from senescent cell lysing agents are described in Fuhrmann-Stroissnigg h.et al, Nat commun.2017 Sep 4; 8(1): the manner of the illustrative example in 422 is known in the art.

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

"inhibitors of one or more BCL-2 anti-apoptotic protein family members" relates to inhibitors of the key class of caspase-activated modulators (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) consisting of anti-apoptotic (pro-survival) proteins with a BHI-BH4 domain.

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

Methods for measuring the binding affinity of inhibitors of BCL-2 family proteins are known in the art. For example, the binding affinity of an inhibitor can be determined using a competitive fluorescence polarization assay, in which a fluorescent BAK BH3 domain peptide is incubated with BCL-xL protein (or other BCL-2 family protein) in the presence or absence of an increasing concentration of the inhibitor, as previously described, for example, in U.S. patent publication 20140005190.

Additionally, any method known in the art can be used to determine whether a compound is a BCL2 inhibitor, illustratively and without limitation by determining the ability to inhibit anti-apoptotic effects, e.g., performing MTT assays, annexin V binding assays, caspase activity assays, mitochondrial membrane potential assays, or analyzing DNA fragmentation and morphology.

In certain embodiments, the BCL2 inhibitor of one or more BCL-2 anti-apoptotic protein family members is a small molecule, polypeptide, peptide, antibody antigen-binding fragment (i.e., peptides and polypeptides comprising at least one Complementarity Determining Region (CDR)), peptibody, recombinant viral vector, or nucleic acid. In certain embodiments, the BCL2 inhibitor is an antisense oligonucleotide, siRNA, shRNA, or peptide. For example and in certain embodiments, the BCL-xL selective peptide inhibitor is a BH3 peptide mimetic. In another preferred embodiment, the inhibitor of BCL2 is a polynucleotide or oligonucleotide that specifically hybridizes to a portion of the mRNA encoding the 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 for use 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.

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.

Compounds of formula (I) and their synthesis have been described in US 8,546,399B 2. The compound of formula (1) has the structure shown below, or a pharmaceutically acceptable salt thereof:

wherein

R¹Is H;

R²selected from NO₂、SO₂R⁷、H、CN、F、Cl、Br、I、CF₃、R⁷、OR⁷、SR⁷And C (O) NH₂。

R³Selected from NHR⁸、NHC(O)R⁸、OR⁸、R⁸F, Br, I and Cl;

R⁴and R⁵Independently selected from H, F, Br, I and Cl;

R⁶selected from H, OR⁹、R⁹、SR⁹、S(O)R⁹、SO₂R⁹、C(O)R⁹、CO(O)R⁹、OC(O)R⁹、NHR⁹、N(R⁹)₂、NHC(O)R⁹、NR⁹C(O)R⁹、NHSO₂R⁹、NR⁹SO₂R⁹、NHC(O)OR⁹And NR⁹C(O)OR⁹；

R⁷Is straight or branched C₁-C₆Alkyl optionally substituted with one or more substituents independently selected from F, Cl, Br and I;

R⁸is straight or branched C₁-C₆Alkyl, optionally substituted with one or more substituents selected from: SR¹⁰、OR¹⁰And R¹⁰And a 5 to 10 membered monocyclic or bicyclic heterocyclic group containing one or more heteroatoms selected from N, O and S;

R⁹selected from 5 to 10 membered monocyclic or bicyclic heteroaryl comprising one or more heteroatoms selected from N, O and S;

R¹⁰is C₆-C₁₀Monocyclic or bicyclic aryl.

In a particular embodiment, in the compounds of formula (I), R¹Is H.

In another embodiment, in the compounds of formula (I), R²Selected from NO₂、SO₂R⁷Wherein R is⁷Is straight or branched C₁-C₃Alkyl, optionally substituted with one or more F atoms.

In another embodiment, in the compounds of formula (I), R³Is NHR⁸Wherein R is⁸Is straight or branched C₁-C₆Alkyl, optionally substituted with one or more substituents selected from: -S-phenyl, and a 5 to 6 membered monocyclic heterocyclyl comprising one or two heteroatoms selected from N and O; preferably C₁-C₆Alkyl, optionally substituted with one or more substituents selected from-S-phenyl, morpholinyl and tetrahydropyranyl.

In another embodiment, in the compounds of formula (I), R⁴Is H.

In another embodiment, in the compounds of formula (I), R⁵Is H.

In another embodiment, in the compounds of formula (I), R⁶Selected from: h and OR⁹Wherein R is⁹Selected from 8 to 10 membered bicyclic heteroaryl comprising one or more nitrogen atoms; preferably H and OR⁹Wherein R is⁹Is a pyrrolopyridinyl group; more preferably H and OR⁹Wherein R is⁹Is a 7-azaindole.

In another embodiment, in the compounds of formula (I), R⁸Is straight or branched C¹-C₆Alkyl, optionally substituted with one or more substituents selected from: -S-phenyl, and 5 to 6 membered monocyclic heterocyclyl (preferably morpholinyl or tetrahydropyranyl) containing one or two heteroatoms selected from N and O.

In a particular embodiment, in the compounds of formula (I), R¹、R⁴And R⁵Is H; r²Selected from NO₂、SO₂R⁷；R³Is NHR⁸；R⁶Selected from H and OR⁹；R⁷Is straight or branched C₁-C₃Alkyl optionally substituted with one or more F atoms; r⁸Is straight or branched C₁-C₆Alkyl, optionally substituted with one or more substituents selected from: -S-phenyl, and 5 to 6 membered monocyclic heterocyclyl (preferably morpholinyl or tetrahydropyranyl) containing one or two heteroatoms selected from N and O; and R is⁹Selected from 8 to 10 membered bicyclic heteroaryl comprising one or more nitrogen atoms; or a pharmaceutically acceptable salt thereof (preferably pyrrolopyridinyl, more preferably 7-azaindole); or a pharmaceutically acceptable salt thereof.

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 may be unsubstituted or substituted in 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 the hydrogen atom bound to the unsubstituted atom, group, chain or ring is replaced by the indicated substituent. When two or more substituents are present, each substituent may be the same or different.

The "alkyl" group may be branched or unbranched and preferably has 1 to 6 carbon atoms. One 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-dimethylpropyl, pent-3-yl and n-hexyl.

As used herein, "heterocyclyl" refers to a 5 to 10 membered monocyclic or bicyclic ring system comprising one or more heteroatoms (e.g., 1, 2 or 3 heteroatoms, preferably 1 or 2 heteroatoms) selected from N, O and S. In bicyclic ring systems, the heteroatom may be in one or both rings forming a 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 comprising 1 or 2 heteroatoms selected from N and O.

As used herein, "heteroaryl" refers to an aromatic 5-to 10-membered monocyclic or bicyclic ring system comprising one or more heteroatoms (e.g., 1, 2, or 3 heteroatoms, preferably 1 or 2 heteroatoms) selected from N, O and S. In bicyclic ring systems, the heteroatom may be in one or both rings forming a heteroaryl ring system. Examples of heteroaryl rings are pyrrolopyridyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzofurazanyl

Oxazolyl (benzoxazolyl), naphthyridinyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isothiazolyl

Azolyl group,

Oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, oxazolyl,

oxadiazolyl, thiadiazolyl, pyridazinyl and triazinyl groups. 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.

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.

The term "pharmaceutically acceptable salt" refers to any salt that, upon administration to a recipient, is capable of providing (directly or indirectly) a compound described herein. For example, a pharmaceutically acceptable salt of a compound provided herein can be an acid addition salt, a base addition salt, or a metal salt, and it can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Typically, such salts are prepared, for example, by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Examples of the 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, methanesulfonate, and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as ammonium, and organic base salts such as ethylenediamine, ethanolamine, N-dialkylenethanolamine, triethanolamine, glucosamine, and basic amino acid salts. Examples of the metal salt include, for example, sodium, potassium, calcium, magnesium, aluminum, and lithium salts.

In a more preferred embodiment, the BCL2 inhibitor is selected from the group consisting of Venetok, Navigila, 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 the group consisting of tenectetocel and neviral. In a preferred embodiment, the BCL2 inhibitor is Venetok, also known as GDC-0199, ABT-199, RG7601, CAS number 12557044-40-8, and is of the formula shown below (Compound Ia). In another preferred embodiment, the BCL2 inhibitor is Navigilant, also known as ABT-263, CAS number 923564-51-6, and is of the formula shown below (Compound Ib).

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 nevira or vinatok, and a carotenoid selected from fucoxanthin, fucoxanthin alcohol, amarocixanthin a and neoxanthin.

Thus, in a preferred embodiment, the combination of the invention comprises nevira and fucoxanthin, nevira and amarocixanthin a, nevira and neoxanthin, vinatork and fucoxanthin, vinatork and amarocixanthin a, or vinatork and neoxanthin.

In a more preferred embodiment, the combination of the invention comprises an inhibitor of one or more BCL-2 anti-apoptotic protein family members selected from nevira or vinatork, and a carotenoid selected from fucoxanthin, fucoxanthin and amarocixanthin a.

Thus, in another more preferred embodiment, the combination of the invention comprises nevira and fucoxanthin, nevira and amarocixanthin a, vinatok and fucoxanthin, or vinatok and amarocixanthin a.

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

As used herein, "activators of BCL-2 pro-apoptotic family members" relates to a class of pro-apoptotic proteins having BH1, BH2, and BH3 domains (BAX, BAK, and BOK) and pro-apoptotic BH 3-only proteins (BIK, BAD, BID, BIM, BMF HRK, NOXA, and PUMA). In a preferred embodiment, the activator of a BCL-2 pro-apoptotic family member is a synthetic peptide. In another preferred embodiment, the activator is any compound that interacts with the surface pocket (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).

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

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, amarocixanthin A and neoxanthin.

The combinations of the invention show a synergistic effect, for example in producing a cytotoxic effect or in reducing the number of senescent cells, so that the same cytotoxic effect or senescent cell lysis (senolytic effect) or senescent phenotype inhibition (senomorphic effect) is obtained with lower doses of an inhibitor of the BCL-2 anti-apoptotic protein family, an activator of a BCL-2 pro-apoptotic family member, a senescent cytolytic agent or a senescent phenotype inhibitor, respectively.

In a particular embodiment, the molar ratio of the carotenoid, carotenoid metabolite, carotenoid derivative, analogue or ester or salt thereof in the combination 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 senescent phenotype inhibitors is from 0.1: 100 to 100: 0.01, preferably from 0.2: 100 to 100: 0.05, more preferably from 0.5: 100 to 100: 0.1. In still other embodiments, the molar ratio of the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof in the combination 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 senescent phenotype inhibitors is from 1: 100 to 10: 0.01, from 1: 10 to 1: 0.01, from 1: 1 to 0.1: 0.01, or from 0.1: 1 to 1: 1.

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

Food, cosmeceutical, nutraceutical, cosmetic or pharmaceutical composition

In another aspect, the present invention relates to a food product, cosmeceutical, nutraceutical, cosmetic or pharmaceutical composition comprising the combination of the invention.

As used herein, the term "food product" refers to any substance or product, natural or processed, of any nature, solid or liquid, which may be used for some of the following purposes, either generally or ideally, due to its nature, application, composition, preparation and state of preservation: a) as a normal nutrition for humans or animals or as a pleasant food; or b) as a dietary product in the special case of human or animal food (feed). The term "feed" includes all natural materials and manufactured products of any origin that are separate from one another or conveniently mixed together and are suitable for use as animal food.

Ready-to-eat food is food that does not require dilution, for example, by an aqueous solution suitable for consumption. In principle, as one skilled in the art would recognize, the ingredients present in a ready-to-eat food product are balanced and no other ingredients need to be added to the food product in order to make it ready-to-eat. A concentrated food product is a food product in which one or more ingredients are present in a higher concentration than in a ready-to-eat food product and therefore, at the time of use, it must be diluted by, for example, an aqueous solution suitable for consumption. Non-limiting illustrative examples of food products provided by the present invention include dairy products and derivatives such as fermented milks, yogurts, kephirs, curds, cheeses, butter, ice creams, milk based desserts, and the like, as well as non-dairy products such as baked products, cakes and pastries, cereals, chocolates, jams, juices, other fruit derivatives, oils and margarines, prepared dishes, and the like.

As used herein, the term "cosmeceutical" refers to a product suitable for use in the body or animal body comprising one or more cosmeceutical products (functional cosmetics, dermopharmaceuticals or active cosmetics), i.e. surface (topical) mixed products with cosmetic-pharmaceutical properties, comprising active ingredients that have an effect on the skin, hair and/or nails of the user at higher or more effective concentrations, and therefore at intermediate levels between cosmetics and pharmaceuticals. Illustrative examples of cosmeceutical products include essential oils, ceramides, enzymes, minerals, peptides, vitamins, and the like.

As used herein, the term "nutritional product" refers to a product suitable for use in humans or animals that comprises one or more natural products having a therapeutic effect, which provides health benefits or is associated with disease prevention or mitigation, and which includes dietary supplements that exist in non-food matrices (e.g., capsules, powders, etc.) that are concentrated in natural bioactive products that are typically present (or not present) in foods, and which exert a beneficial effect on health at greater effects than normal foods may have when ingested at higher doses than are present in those foods. Thus, the term "nutritional product" includes isolated or purified food products as well as additives or food supplements that are typically present in dosage forms that are commonly used orally (e.g., capsules, tablets, sachets, drinkable vials, etc.); such products provide a physiological benefit or protection against disease, typically chronic disease. If desired, the present invention provides nutritional products which may contain in addition to xanthophylls one or more nutraceuticals (products or substances associated with disease prevention or reduction), such as flavonoids, omega-3 fatty acids and the like, and/or one or more prebiotics (non-digestible food ingredients that stimulate probiotic activity and/or growth), such as fructooligosaccharides, pectins, inulin, galactooligosaccharides, lactulose, human milk oligosaccharides, dietary fibers and the like.

As used herein, the term "nutritional composition" of the present invention relates to a food product that beneficially affects one or more functions of the body to provide a better health condition. Such nutritional compositions are therefore useful for the prevention and/or treatment of disease or disease-causing factors. Thus, the term "nutritional composition" according to the present invention may be used as a synonym for functional food or food for specific nutritional purposes or medical food. The nutritional composition is similar to conventional food and is consumed as part of the normal dietary appearance.

As used herein, the term "cosmetic composition" or "personal care composition" refers to a composition suitable for use in personal hygiene of humans or animals or for enhancing the natural beauty or altering the physical appearance without affecting the structure or function of the human or animal body, comprising one or more products that provide such an effect. If desired, the cosmetic compositions provided by the present invention may comprise, in addition to the combination of the invention, one or more cosmetic or cosmetical products, i.e. substances or mixtures intended to come into contact with external parts of the human or animal body (such as the epidermis, the hair system, the nails, the lips, etc.) or with the teeth and buccal mucosa, with the sole or primary purpose of cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odor. Illustrative examples of cosmetically acceptable carriers include the products contained in the INCI (International Nomenclature of Cosmetic Ingredients) list. Cosmetic or personal care compositions include products such as: balm, pad, hair oil, cream, etc., oil, surfactant, humectant, plant extract (botanic extract), vitamin, antioxidant, sunscreen agent, perfume, antiseptic, etc. Illustrative examples of moisturizers, plant extracts, vitamins, antioxidants, and sunscreens.

The ingredients as described above are preferably provided in a cosmetic composition which may be formulated as a cream, gel, lotion, oil, ointment, powder, stick, cake or other topically applicable form. The resulting cosmetic composition may be in the form of a liquid, solid, semi-solid, dispersion, suspension, solution or emulsion, and it may be water-based or anhydrous. The cosmetic composition of the present invention may also be in the form of a color cosmetic composition, such as foundation (foundation makeup), mascara, lip gloss, blush (blush), eye shadow, and the like. Certain other derivatives are lipophilic in nature and will more likely be present in the oil phase of the emulsion. The combination of the invention is preferably present in the aqueous phase of the emulsion or in the aqueous phase encapsulated within liposomes.

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

In a particular and preferred embodiment of the invention, the cosmetic composition of the invention is applied by topical route. Suitable formulations for topical application of the compositions of the invention are detailed in the context of the pharmaceutical compositions of the invention and are equally suitable for use in the cosmetic compositions of the invention.

If desired, the cosmetic compositions of the present invention are incorporated into fabrics, nonwovens or medical devices. Illustrative examples of such fabrics, nonwovens, or medical devices include, but are not limited to, bandages, gauzes, T-shirts, pantyhose, socks, undergarments, waistbands, gloves, diapers, sanitary napkins, dressings, bed covers, wet wipes, adhesive patches, non-adhesive patches, occlusive patches, micro-electrical patches, and face masks.

In addition, the present invention also relates to pharmaceutical compositions comprising the combinations of the present invention.

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

The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable diluent," or "pharmaceutically acceptable excipient," used interchangeably herein, refer to any conventional type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Pharmaceutically acceptable carriers are substantially non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The number and nature of the pharmaceutically acceptable carriers depends on the desired form of administration. Pharmaceutically acceptable carriers are known and can be prepared by methods well known in the art. Which is involved in carrying or transporting the subject chemical substance from one organ or body part to another without undue toxicity, irritation, allergic response, or other problem or complication, 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 injurious 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, ethylcellulose and cellulose acetate), (d) powdered tragacanth, (e) malt, (f) gelatin, (g) talc, (h) excipients (e.g., cocoa butter and suppository wax), (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) raw water, (p) pyrogen-free, (q) isotonic saline, (r) Ringer's solution,(s) ethanol, (t) phosphate buffer and (u) other non-toxic compatible substances used in pharmaceutical formulations. Wetting, emulsifying and lubricating agents (such as sodium lauryl sulfate and magnesium stearate), as well as coloring, mold release, coating, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (a) water-soluble antioxidants (e.g., ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, or sodium sulfite), (b) oil-soluble antioxidants (e.g., ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, or alpha-tocopherol), and (c) metal chelators (e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, or phosphoric acid).

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

The pharmaceutical products can be formulated as tablets, pills, capsules, sachets, granules, powders, suspensions, emulsions, anhydrous or aqueous surface preparations and solutions, depending on the particular mode of administration.

Pharmaceutically acceptable carriers or vehicles are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier or vehicle is one that is chemically inert to the active agent and each of its components, and one that does not have deleterious side effects or toxicity under the conditions of use.

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

In cases other than intravenous administration, the composition may contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition may be a liquid solution, suspension, emulsion, gel, polymer or sustained release formulation. The composition may be formulated with conventional binders and carriers, as is known in the art. The formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium sugar (sodium saccharate), cellulose, magnesium carbonate and the like inert carriers having recognized functions in the manufacture of medicaments. A variety of delivery systems are known and can be used to administer the therapeutic agents of the present invention, including encapsulation in liposomes, microparticles, microcapsules, and the like.

If desired, the combination or pharmaceutical composition of the invention is included in a composition that also includes a solubilizing agent and a local anesthetic to reduce any pain at the site of injection. Typically, the ingredients are provided separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container (e.g., ampoule or bag) that indicates the amount of active agent. Where the composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administration of the combination by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

Formulations suitable for oral administration include liquid solutions dissolved in a diluent (e.g., water or saline); capsules, sachets, tablets, lozenges, each comprising a predetermined amount of a combination of the invention; powder; suspensions in suitable liquids; 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 art, such as water. These compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

Solid dosage forms for oral administration may include conventional capsules, sustained release capsules, conventional tablets, sustained release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. In these solid dosage forms, the active compound may be mixed with at least one inert excipient such as sucrose, lactose or starch. In normal practice, such dosage forms may also contain other substances in addition to inert diluents, for example lubricating agents, such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills may be prepared with an enteric coating.

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

The pharmaceutical compositions of the invention may be administered by topical, transdermal or subcutaneous routes. Illustrative examples of topical or transdermal administration include, but are not limited to, iontophoresis (ionophoresis), sonophoresis (sonophoresis), electroporation, mechanical pressure, osmotic pressure gradients, occlusive dressings (occlusive dressing), microinjection, needle-free injection with pressure, micro-electrical patches, and any combination thereof. In any case, the excipients will be selected according to the selected pharmaceutical dosage form.

Injectable formulations (e.g., sterile injectable aqueous or oleaginous suspensions) may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. Acceptable carriers and solvents that may be used include water, ringer's solution and isotonic sodium chloride solution. Sterile oils are also conventionally employed as a solvent or suspending medium.

In a particular and preferred embodiment of the invention, the pharmaceutical composition of the invention is administered by topical route. For topical administration, the pharmaceutical compositions of the present invention may be formulated as creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid strips, emulsions, microemulsions and the like, which may be formulated according to conventional methods using suitable excipients (e.g., emulsifiers, surfactants, thickeners, colorants and combinations of two or more thereof).

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

Several drug delivery systems are known and can be used to administer the combinations of the present invention, including for example encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules, nanoparticles, nanocapsules and the like. The desired dosage may be administered in a single unit or in a sustained release form. In a specific and preferred embodiment of the invention, the pharmaceutical composition is encapsulated in liposomes.

Sustained release forms and suitable materials and methods for their preparation are well known in the art. In one embodiment of the invention, the oral administration form of the combination according to the invention is a sustained release form further comprising at least one coating or matrix. The coating or sustained release matrix includes, but is 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 combinations of two or more thereof. The enteric coating may be applied using conventional methods known to those skilled in the art.

All terms and embodiments previously described in relation to the combination of the invention are equally applicable to this aspect of the invention.

Cosmetic method

The authors of the present invention have found that the combination of a carotenoid and a senescent cell lytic agent (e.g., a Bcl-2 inhibitor) produces synergistic senescent cell lysis. Thus, the invention also provides methods for treating diseases in which there is an undesirable accumulation of senescent cells, and cosmetic methods in which it is desirable to eliminate senescent cells.

Thus, in a further aspect, the present invention relates to a cosmetic method for preventing and/or reducing skin ageing and/or for ameliorating the cosmetic adverse effects of ageing, comprising administering to a subject in need thereof a combination according to the invention or a food, cosmeceutical, nutraceutical or cosmetic composition according to the invention.

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 method of the invention include skin creams, lotions, powders, lipsticks, eye and face foundations, wipes, gels, deodorants, hand washes, baby products, bath oils, bubble baths, bath salts, butters and many other types of products.

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.

Cellular senescence is a growth arrest that occurs as a result of various destructive 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, resist apoptosis, and secrete factors that promote inflammation and tissue degradation.

Aged keratinocytes and fibroblasts appear to accumulate in human skin with age. In addition, senescent cells express genes for degradative enzymes, growth factors and inflammatory cytokines with long-term pleiotropic effects.

As used herein, "cosmetic adverse effects of aging" relate to the intrinsic or chronological aging characteristics and include, by way of illustration and not limitation, visible signs such as thin and dry skin, fine wrinkles, decreased elasticity, abnormal pigmentation, hair whitening and hair loss.

As used herein, "skin aging" relates to a state that means the aging and in particular the damage of epidermal cells of human skin due to: partial or complete destruction of cells, conversion of imide bonds in collagen and/or elastin to amide bonds caused by toxic byproducts of oxygen metabolism, radical pathological mechanisms, and photodamage and general aging.

The cosmetic method of the invention is intended to reduce epidermal cells and thus skin aging in humans by reducing or inhibiting aging, including by one or more of the following effects, for example: reversing photodamage or other regenerative effects, such as increasing underlying skin vessels, increasing the rate of cell replication and desquamation to produce a younger appearance, increasing collagen synthesis and uniformity, delaying skin atrophy and thinning of the epidermis and dermis, and the like.

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

In a particular 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 of the invention have been detailed in the context of the cosmetic composition of the invention and are equally suitable for the cosmetic method of the invention.

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

Medical use

In a further aspect, the present invention relates to a combination according to the invention or a pharmaceutical composition according to the invention for use in medicine.

When used in medicine, the compositions according to the invention may be used by administering simultaneously or by any means separately or sequentially a therapeutically effective amount of: (i) a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or ester or salt thereof, and (ii) an inhibitor of one or more BCL-2 anti-apoptotic protein family members, an activator of BCL-2 pro-apoptotic family members, a senescent cell lytic agent, or a senescent phenotype inhibitor. The term "combination" also denotes multiple combinations of compounds (i) and (ii), for example in a single composition, in a combined mixture consisting of separate preparations of a single active compound (e.g. "tank-mix"), and in combined use of the single active ingredients when applied in a sequential manner, i.e. one after the other in a reasonably short time (e.g. hours or days), or in a simultaneous application.

Preferably, if the administration is not simultaneous, the compounds are administered within a time period close to each other. Furthermore, it is not important whether the compounds are administered in the same dosage form or by the same route, e.g., one compound may be administered topically and another compound may be administered orally. Suitably, both compounds are administered orally.

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

combinations as part of the same pharmaceutical preparation, both compounds then always being administered simultaneously.

As a combination of two units, each unit having one substance so that administration can be simultaneous, sequential or separate.

In a particular embodiment, the carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof is administered independently (i.e., in two units) but simultaneously with a senescent cytolytic agent, a senescent phenotype inhibitor, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, or an activator of BCL-2 pro-apoptotic family members.

In another embodiment, a carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof is administered first, and then a senescent cell lysing agent, a senescent phenotype inhibitor, an inhibitor of one or more BCL-2 anti-apoptotic protein family members, or an activator of BCL-2 pro-apoptotic family members is administered separately or sequentially.

In yet another embodiment, the senescent cell lytic agent, the senescent phenotype inhibitor, the inhibitor of one or more BCL-2 anti-apoptotic protein family members, or the 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 subsequently administered separately or sequentially as defined.

In another 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 cancer.

Alternatively, the present invention relates to a method for the prevention and/or treatment of cancer comprising administering to a subject in need thereof a combination according to the present invention or a pharmaceutical composition according to the present invention.

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 the prevention and/or treatment of cancer in a subject in need thereof.

As used herein, the term "prevention" and variations thereof relates to the administration of a combination according to the invention or a medicament comprising said combination to a subject who has not been diagnosed as likely to suffer from e.g. cancer but who is generally expected to suffer from said disease or to have an increased risk of developing said disease. Prevention aims to avoid the occurrence of said diseases. Prevention may be complete (e.g., complete absence of disease). Prevention can also be partial, such that, for example, the occurrence of disease in a subject is lower than the occurrence of disease in the absence of administration of a composition of the invention. Prevention also refers to reducing susceptibility to clinical conditions.

As used herein, the term "treatment" relates to any type of treatment intended to terminate, prevent, ameliorate, or reduce susceptibility to a clinical condition described herein. In a preferred embodiment, the term treatment relates to prophylactic treatment (i.e. treatment that reduces susceptibility to a clinical condition) of a disease or condition as defined herein. Thus, "treatment" and its equivalents refer to obtaining a desired pharmacological or physiological effect and encompass any treatment of a pathological condition or disease in a mammal, including a human. The effect may be prophylactic in terms of completely or partially preventing the disease or a symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects attributable to the disease. That is, "treating" includes (1) preventing the occurrence or recurrence of a disease in a subject, (2) inhibiting the disease, e.g., arresting its development, (3) halting or terminating the disease or at least symptoms associated therewith such that the host no longer suffers from the disease or symptoms thereof, e.g., causing regression of the disease or symptoms thereof, e.g., as by restoring or repairing lost, absent, or defective function or otherwise stimulating ineffective processes, or (4) alleviating, relieving, or ameliorating the disease or symptoms associated therewith, wherein amelioration is used in a broad sense to refer to at least reducing the size of the parameter.

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 cell to invade other adjacent tissues (invasion) and spread through lymph and blood vessels to other areas of the body where cells are not normally located (metastasis), circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Tumors are classified as benign or malignant, depending on whether they can spread by invasion and metastasis: a benign tumor is a tumor that cannot spread by invasion or metastasis, i.e. it grows 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 cancers: breast cancer; biliary tract cancer; bladder cancer; brain cancer, including glioblastoma, particularly glioblastoma multiforme and medulloblastoma; cervical cancer; head and neck cancer; choriocarcinoma; colon cancer, colorectal cancer; endometrial cancer; esophageal cancer; gastric cancer; blood neoplasms, including acute lymphocytic and myelocytic leukemias; t cell acute lymphocytic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous 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 and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; parotid cancer; ovarian cancer, including those arising from epithelial, stromal, germ, and mesenchymal cells; pancreatic cancer; prostate cancer; kidney, adrenal gland cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers including melanoma, Merkel cell carcinoma (Merkel cell carcinoma), Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; cervical cancer, endometrial cancer; testicular cancer, including germ cell tumors, such as seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumors, and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; and renal cancers, including adenocarcinoma and Wilms tumor. Other cancers will be known to the ordinarily skilled artisan.

In a preferred embodiment, the cancer is a hematologic cancer or hematologic malignancy. The term "hematologic cancer or hematologic malignancy" refers to the type of cancer that affects the blood, bone marrow, and lymph nodes. Hematological malignancies can be derived from one of two major blood cell lineages myeloid and lymphoid cell lines. Myeloid cell lines typically produce granulocytes, erythrocytes, platelets, macrophages and mast cells; lymphoid cell lines give rise to B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias and myelomas are from lymphoid lineages, while acute and chronic myelogenous leukemias, myelodysplastic syndromes (MDS) and myeloproliferative diseases all originate in the bone marrow. Non-limiting illustrative examples of hematological malignancies are Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL) and myelomas such as Multiple Myeloma (MM). In a more preferred embodiment, the hematologic malignancy is 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.

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 malignancy, prostate cancer, neuroblastoma, glioblastoma, astrocytoma, or ovarian cancer. In a preferred embodiment, the cancer is a skin cancer, more preferably melanoma.

The authors of the present invention have shown that the combined use of carotenoids with senescent cell lytic agents results in synergistic senescent cell lysis. Thus, the invention also provides the use of these compositions for the treatment of diseases requiring the selective destruction of senescent cells.

In a further aspect, the present 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 disease or disorder associated with aging.

Alternatively, the present invention relates to a method for the prevention and/or treatment of a disease or disorder associated with aging, comprising administering a combination or pharmaceutical composition according to the invention.

Alternatively, the present invention relates to a combination or pharmaceutical composition of the invention for use in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with aging.

As used herein, a "senescence-associated disease or disorder" relates to a pathophysiological clinical condition in which the presence and action of senescent cells significantly contributes to the disorder.

In another preferred embodiment, the disease or disorder associated with aging is fibrosis.

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

In another preferred embodiment, the fibrosis with a high number of senescent cells is pulmonary fibrosis, chronic obstructive pulmonary disease, cardiac fibrosis, renal fibrosis, myelofibrosis or hepatic fibrosis.

As used herein, "a plurality 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-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or even more compared to the number of senescent cells in a normal tissue. Normal tissue means tissue without fibrosis.

The senescent cells can exhibit any one or more of the following characteristics. (1) The senescence growth arrest is essentially permanent and cannot be reversed by known physiological stimuli. (2) The size of senescent cells increases, sometimes more than two-fold relative to the size of non-senescent counterparts. (3) The senescent cells express senescence-associated P-galactosidase (SAP-gal), which reflects, in part, an increase in lysosomal weight. (4) Most senescent cells express p 16. sup. INK4a, which is not normally expressed by quiescent or terminally differentiated cells. (5) Senescent cells with persistent DDR signaling have persistent foci (nuclear foci) known as DNA fragments with chromatin alterations that enhance senescence (DNA-SCARS). These foci contain activated DDR proteins and can be distinguished from transient lesion foci. DNA-SCARS include dysfunctional telomeres or telomere dysfunction-inducing foci (TIF). (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.

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 reactions that may promote or exacerbate the underlying disease in a subject. In a preferred embodiment, senescent cells are detected by detecting a senescence-associated secretory phenotype (SASP, i.e., which includes secretory factors that can constitute a pro-inflammatory phenotype of senescent cells), a senescence-information secretory set (senescent-inducing secretion), and a DNA Damage Secretory Program (DDSP).

Senescent cells and senescent cell-associated molecules can be detected by techniques and procedures described in the art. For example, tissues can be analyzed for the presence of senescent cells by histochemical or immunohistochemical techniques that detect the senescence marker SA- β galactosidase (SA-Pgal) (see, e.g., Dimri et al, Proc. Natl. Acad. Sci. USA 92: 9363-9367 (1995)). The presence of senescent cell-associated polypeptide p16 can be determined by any of a variety of immunochemical methods practiced in the art, such as immunoblot analysis. Expression of pl6 mRNA in cells can be measured by a variety of techniques practiced in the art, including quantitative PCR. The presence and levels of senescent cell-associated polypeptides (e.g., SASP polypeptides) can be determined by using automated and high-throughput assays, such as the automated Luminex array assay described in the art (see, e.g., 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, e.g., determining the level of IL-1. beta. as in example 4), chemokines, cell-associated metabolites, reactive oxygen species (e.g., H)₂O₂) And other molecules that stimulate inflammation and/or other biological effects or responses that may promote or exacerbate the underlying disease in the subject.

Aging-related 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, neurological diseases and disorders (e.g., neurodegenerative diseases and disorders); age-related diseases and disorders caused by aging; skin disorders; age-related diseases; skin diseases and disorders; and transplantation-related diseases and disorders.

In another specific embodiment, the senescence-associated disease or disorder is a cardiovascular disease (cardiovascular disease) selected from the group consisting of: atherosclerosis, angina pectoris, 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 stress resistance), cardiac fibrosis, cerebral aneurysm, and stroke. In another specific embodiment, the senescence-associated disease or disorder is an inflammatory or autoimmune disease or disorder selected from osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis, and herniated intervertebral disc. In another specific embodiment, the senescence-associated disease or disorder is a neurodegenerative disease selected from 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 disorder is a metabolic disease selected from diabetes, diabetic ulcers, metabolic syndrome, and obesity. In another specific embodiment, the senescence-associated disease or disorder 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 embodiment, the senescence-associated disease or disorder is an ocular disease or disorder selected from macular degeneration, glaucoma, cataracts, presbyopia, and vision loss. In another specific embodiment, the aging-related disease or disorder is an age-related disorder selected from the group consisting of kidney disease, renal failure, frailty, hearing loss, muscle fatigue, a skin disorder, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosal fibrosis, and sarcopenia. In another specific embodiment, the aging-related disease or condition is a skin disease or condition selected from the group consisting of: eczema, psoriasis, pigmentation, nevi, rash, atopic dermatitis, urticaria, diseases and disorders associated with photosensitivity or photoaging; wrinkles (rhytides); itching; dysesthesia; eczema eruptions (eczematous eruptions); an eosinophilic skin disease; reactive neutrophilic dermatoses; pemphigus; pemphigoid; immune bullous skin disease (immunobullous dermatitis); dermal fibroblastic proliferations (skin); cutaneous lymphomas; and cutaneous lupus. In another embodiment, the disease or disorder associated with aging is atherosclerosis; osteoarthritis; pulmonary fibrosis; hypertension, chronic obstructive pulmonary disease; myelofibrosis; or liver fibrosis.

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

In a preferred embodiment, the senescence-associated disease or disorder is cancer, more preferably hematological cancer or skin cancer.

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

As used herein, "treatment-induced senescence" or TIS relates 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 as well as the ability to stimulate tumor cell regeneration that is not eradicated by anti-cancer therapy. Unlike apoptotic cell death, where the remainder is rapidly cleared by phagocytosis without too much inflammatory response, senescent cells may persist at the tumor site for different periods of time.

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-1 β.

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

"chemotherapy" relates to treatment with chemotherapeutic agents. The term "chemotherapeutic agent" includes standard chemotherapeutic drugs (which typically attack any rapidly dividing cells), targeted therapeutic agents, and immunomodulators. Illustrative, non-limiting examples of cancer chemotherapeutic agents that can be in accordance with the present invention include alkylating agents, antimetabolite drugs, anthracyclines, antibodies targeting pro-angiogenic factors, topoisomerase inhibitors, antimicrotubule agents, cyclin-dependent kinase inhibitors, low molecular weight tyrosine kinase inhibitors of pro-angiogenic growth factors, and matrix metalloproteinase inhibitors.

In a more preferred embodiment, the chemotherapeutic agent is selected from the group consisting of aphidicolin (Aphidocolin), bleomycin, camptothecin, carboplatin, docetaxel, cisplatin, cyclophosphamide, daunorubicin, doxorubicin, cytarabine, palbociclib, 5-fluorouracil, diazaquinone (diaziquuone)/AZQ, Epigallocatechin gallate (Epigallocatechin gate), etoposide, hydroxyurea, K858, lovastatin, mitoxantrone, MLN4924, MLN8054, Pyrithione (Pyrrithione), resveratrol, TPA, PEP005, PEP008 and VO-Ohpic.

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

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

As used herein, the term "therapeutically effective amount" refers to a sufficient amount of a compound (i.e., a combination of the invention) to provide the desired effect, and is generally determined by the identity of the compound itself and the therapeutic effect to be achieved, among other reasons. 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 and the like. Therefore, the skilled person must adjust the dosage according to the aforementioned variables.

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

There are many reports throughout several cancer types that suggest that aging is associated with a poor therapeutic index for cancer therapeutics.

It is well known in the art that many tumor cells become resistant to chemotherapy by entering senescence, and once chemotherapy is removed, the cells leave senescence and resume proliferation or induce proliferation of neighboring cells (see, e.g., Gordin and Nelson, Drug resistance update.2012, 15: 123-131). Thus, in view of its senescent cell lysis, the composition according to the invention can be used to destroy cells that have entered senescence in response to chemotherapy treatment, resulting in an increase in the efficacy of the chemotherapy treatment. Thus, in a further aspect, the present invention relates to a combination according to the invention or a food, nutraceutical or pharmaceutical composition according to the invention for use in enhancing the effectiveness of an anti-tumor compound.

As used herein, "enhancing effectiveness" includes augmenting, potentiating, augmenting, amplifying and enhancing the effect produced by an anti-tumor compound.

Since the combination of the invention reduces the ageing 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, the same dose of the anti-tumor compound together with the combination of the invention results in a higher effect than a specific dose of the anti-tumor compound in the absence of administration of the combination of the invention to the same subject.

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.

Medical use of carotenoids

The authors of the present invention have shown that carotenoids are able to restore the senescence phenotype induced by cytotoxic agents (see example 3). This therefore allows the use of carotenoids in the treatment of diseases characterized by an undesirable number of senescent cells by promoting the cells to abandon the senescent phenotype. Thus, in another aspect, the present invention relates to a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof, for use in the treatment of an age-related disease or disorder.

Alternatively, the present invention relates to the use of a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof for the manufacture of a medicament for the treatment of an age-related disease or disorder.

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

In another preferred embodiment, the disease or disorder associated with aging is fibrosis. In a preferred embodiment, fibrosis is characterized by having a high number of senescent cells.

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

In another preferred embodiment, the aging disorder is treatment-induced aging, more preferably chemotherapy-or radiotherapy-induced aging.

In a preferred embodiment, the carotenoid is selected from the group consisting of fucoxanthin, fucoxanthin alcohol, amarocixanthin a, neoxanthin, astaxanthin, zeaxanthin and lutein. In a preferred embodiment, the carotenoid is fucoxanthin. In another preferred embodiment, the carotenoid is amarocixanthin a. In another preferred embodiment, the carotenoid is not fucoxanthin. In another preferred embodiment, the carotenoid is not fucoxanthin. In another preferred embodiment, the carotenoid is not amarocixanthin 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.

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.

In addition, it is well known that certain antineoplastic agents cause secondary effects due to the induction of senescence in non-neoplastic cells (see Demaria, mol. cell Oncol.2017; 4 (3): e 1299666.). Thus, the ability of carotenoids to promote cell departure from the aging phenotype allows carotenoids to be used to reduce the adverse effects of anti-tumor therapy. Thus, in another aspect, the present invention relates to carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof, for use in reducing the adverse effects of anti-tumor therapy.

As used herein, "adverse effect", side effect, toxic side effect or harmful side effect relates to an undesired harmful effect caused by a drug having an anti-tumor compound. Preferably, the adverse effect is gastrointestinal toxicity, peripheral neuropathy, fatigue, weakness, low physical activity, hematologic toxicity, hepatotoxicity, alopecia, pain, mucositis, fluid retention, skin toxicity, fatigue or cardiotoxicity.

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

In a preferred embodiment, the likelihood of an adverse effect 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% in a subject treated with an anti-neoplastic compound.

Reducing the likelihood of an adverse effect suffering from an adverse effect can be measured by methods known in the art based on the adverse effect to be determined.

In a preferred embodiment, the adverse effect is due to chemotherapy treatment. In another preferred embodiment, the adverse effect is due to radiation therapy treatment.

Method for inhibiting aging

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

As used herein, "inhibit" relates to reducing or decreasing aging. As used herein, "reducing" relates to a reduction in 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% as compared to the level of aging in the absence of administration of a carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof.

Methods for determining aging have been previously described. 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.

The method of the present invention for inhibiting senescence in a cell population comprises a first step of contacting the cell population with a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or ester or salt thereof. In the case where the population of cells forms part of the individual, then the contacting step may be carried out by administering to the individual a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analog or ester or salt thereof. Any of the methods previously described for administering carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof may 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 microscopic observation); production of senescence-associated markers, e.g., senescence-associated β -galactosidase (SA- β -gal), p16INK4a, p21, PAI-I, or production of any one or more of SASP factors (e.g., IL-6, MMP 3).

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

As used herein, "reference value" relates to a laboratory value used as a reference for values/data obtained from a sample. The reference value (or reference level) may be an absolute value, a relative value, a value having an upper and/or lower limit, a series of values, an average value, a median value, an average value, or a value represented by reference to a control or reference value. The reference value may be based on a value obtained from a single sample, e.g. a value obtained from a study sample but obtained at a previous point in time. The reference value may be based on a large number of samples, e.g. values obtained in a sample population, or on a sample pool comprising or not comprising the sample to be tested.

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

The following examples illustrate the invention and must not be considered as limiting it.

Examples

Example 1-synergistic Effect of carotenoids and BCL2 inhibitor in cancer

Materials and methods

Cell culture

Human MV-4-11 and PL-21 cell lines were used to evaluate the effects of fucoxanthin and amarocixanthin A in combination with the BCL-2 family inhibitors nevira (ABT-263) and vinatork (ABT-199). Cells were humidified at 37 ℃ with 5% CO₂Cultured in RPMI medium supplemented with 10% FCS under atmosphere and consistently free of mycoplasma as assessed by PCR. All passages were performed at 80% to 90% confluence and at a survival rate (viatility) of greater than 90%.

MTT assay

For cytotoxicity assays, MV-4-11 cells and PL-21 cells were counted at10⁴Density of individual cells/well plated in 96-well microplates. After 24 hours, different concentrations of nevira or vinatork in combination with fucoxanthin or amarocixanthin a were added. The plates were incubated at 37 ℃ and 5% CO₂Incubate for 72 hours. DMSO was used as vehicle control. After this period, 50 μ L of 10% SDS was added to the negative control wells (0% survival, 100% mortality). The medium was then changed to fresh medium with 10. mu.L of 5mg/ml MTT in PBS per well. The plates were incubated at 37 ℃ and 5% CO₂Incubate for 4 hours. Next, the medium was removed, and 100 μ L of extraction buffer (SDS, DSA, acetic acid 2%, pH 4.7) was added to each well. The plates were incubated at 37 ℃ and 5% CO₂After 1 hour of incubation, the culture was homogenized. Finally, the plate was read with a spectrophotometer Multiskan Ascent (MTX Lab systems) at 570 nm. By calculating IC50 (half maximal inhibitory concentration)The efficacy of each product was determined.

Results

To test the cytotoxic effect of fucoxanthin and amarocixanthin a in combination with different drugs (BCL2 inhibitors) in cancer cells, a range of concentrations of fucoxanthin were co-incubated with the following compounds:

-Navkela (ABT-263)

-Venetork (ABT-199).

The cytotoxic potency of each compound and its combined potency with fucoxanthin were obtained in MTT assays in MV-4-11 and PL-21 cells. This parameter is indicated in table 1 as IC 50. Different synergistic combination scores were also obtained and are shown in Table 2 for MV-4-11 cells and Table 3 for PL-21 cells.

TABLE 1 IC50(6 replicates) for different drugs in two blood cancer cell lines.

SD, standard deviation.

Table 2 synergy scores for different drug combinations with fucoxanthin in MV-4-11 cells.

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

Table 3 synergy scores for different drug combinations with fucoxanthin in PL-21 cells.

The combination index was calculated according to the Chou-Talalay method cited above. The values are explained as follows: < 1 synergy, > 1 antagonism, and ═ 1 additivity. Conc, concentration; CI, combination index.

As seen in tables 2 and 3, a synergistic reduction in IC50 was observed for fucoxanthin and nevira (in both MV-4-11 and PL-21 cells), amarouracixanthin A and nevira, and fucoxanthin and vinatork (in MV-4-11).

Example 2 Carotenoid and BCL2 inhibitor Effect in aging

Materials and methods

Cell culture

The Sk-Mel-103 cell line (human skin melanoma) was cultured in high glucose DMEM supplemented with 10% FCS. All cells were humidified at 37 ℃ with 5% CO₂Cultured under atmosphere and consistently free of mycoplasma as assessed by PCR. All passages were performed at 80% to 90% confluence and at greater than 90% survival.

Treatment and Experimental conditions

Will 10⁶One Sk-Mel-103 cell was seeded in a 24-well plate. Incubating the culture with palbociclib (Palbo) (as senescence inducer), fucoxanthin, or a combination thereof, e.g.

No se encuentra el origen de la preferencia. After 7 days, the cultures were treated with different concentrations of the BCL2 inhibitor neviras. Cytotoxicity was determined for each condition by calculating IC50 (half maximal inhibitory concentration) by MTT assay.

Table 4 experimental conditions used to determine the effect of fucoxanthin pretreatment on cell sensitivity to BCL2 inhibitors.

MTT assay

For cytotoxicity assayThen, the cells are treated with 10⁴Density of individual cells/well plated in 96-well microplates. After the initial treatment described above, different concentrations of nevira were added. The plates were then incubated at 37 ℃ and 5% CO₂Incubate for 72 hours. DMSO was used as vehicle control. After this period, 50 μ L of 10% SDS was added to the negative control wells (0% survival, 100% mortality). The medium was then changed to fresh medium with 10. mu.L of 5mg/ml MTT in PBS per well. The plates were incubated at 37 ℃ and 5% CO₂Incubate for 4 hours. Next, the medium was removed, and 100 μ L of extraction buffer (SDS, DSA, acetic acid 2%, pH 4.7) was added to each well. The plates were incubated at 37 ℃ and 5% CO₂After 1 hour of incubation, the culture was homogenized. Finally, the plate was read with a spectrophotometer Multiskan Ascent (MTX Lab systems) at 570 nm. The efficacy of each product was determined by calculating the IC50 (half maximal inhibitory concentration).

Results

To investigate the effect of pretreatment with fucoxanthin on the sensitivity of cells to BCL2 inhibitors, incubation of palbociclib (senescence inducer), alone or in combination with fucoxanthin, was performed for 7 days (e.g., at

No se encuentra el origen de la preferencia). Then, the cytotoxicity of nevira (BCL2 inhibitor) was evaluated by MTT assay. The data were compared to control cells (non-senescent) in the basal state or treated with fucoxanthin.

TABLE 5 IC50(6 replicates) of neviras in Sk-Mel-103 cells treated with different conditions.

	IC50(μ M) (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

SD, standard deviation.

As seen in table 5, the IC50 of nevira was decreased in control and senescent cells when the cells were pretreated with fucoxanthin. Thus, pretreatment with fucoxanthin increased the senescent cell lytic activity of nevira.

Example 3 Carotenoid Effect in aging

Materials and methods

Cell culture

BJ-11 cell line (human primary skin fibroblasts) was cultured in MEM supplemented with 10% FCS plus 2mM L-glutamine. All cells were humidified at 37 ℃ with 5% CO₂Cultured under atmosphere and consistently free of mycoplasma as assessed by PCR. All passages were performed at 80% to 90% confluence and at greater than 90% survival.

Treatment and Experimental conditions

To evaluate the effect of fucoxanthin on aging, 10 was used⁴Individual BJ-11 cells were seeded in 24-well plates. As described in table 6, cultures were incubated with varying concentrations of palbociclib (Palbo) or doxorubicin (Doxo) as senescence inducer and fucoxanthin (Fuco).

TABLE 6 Experimental conditions for determining the effect of fucoxanthin on aging.

Doxorubicin treatment was only performed for 24 hours. Thereafter, doxorubicin was removed and the cells were cultured in complete medium for the period indicated below. On the other hand, palbociclib is continuously present in the medium and is periodically added with fresh.

Two different approaches were followed:

the method comprises the following steps: fucosan yellow for preventing aging

Cell cultures were treated with fucoxanthin and a senescence-inducing agent simultaneously as shown below. After 7 days, the beta-galactosidase activity was determined.

Scheme 1.Experimental procedure for testing the Effect of fucoxanthin on aging prevention

The method 2 comprises the following steps: fucosan yellow effect on aging reversal

Cell cultures were first treated with senescence-inducing agents for 7 days as shown below. Then, treatment with fucoxanthin was performed for 7 days. At the end of this period, β -galactosidase activity was measured.

Scheme 2.Experimental procedure to test the effect of fucoxanthin on senescence reversal.

Senescence-associated beta-galactosidase (SA-beta-gal) staining

Following treatment, SA- β -galactosidase staining was used to determine senescence-associated β -galactosidase expression in BJ-11 cells. Beta-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, fixative is added to each well and the cells are allowed to fix at room temperature for 10 to 15 minutes. Plates were washed twice with PBS and then β -galactosidase staining solution was added to each well. Finally, the plates were placed in a dry incubator (CO free)₂) At 37 ℃ for 24 hours to prevent pH changes, which may affect the staining results. The amount of cells positive for β -galactosidase activity (blue staining) was assessed by manual staining cell counting.

Results

To investigate the effect of fucoxanthin on the prevention and reversal of aging, incubation with the cytostatic drugs doxorubicin and palbociclib was performed in BJ-11 cells as previously described. The activity of beta-galactosidase was determined and used as a marker for senescence. The percentage of senescent cells positive for β -galactosidase activity decreased after fucoxanthin treatment, as seen in tables 7 and 8.

TABLE 7. percentage of cells positive for beta-galactosidase activity in method 1 (evaluation of the effect of fucoxanthin on senescence prevention).

	Pabociclib	Doxorubicin
			Control cells (basal status))	0％	0％
Senescent cells	＞98％	＞80％
			Senescent cells + fucoxanthin	＜5％	＜5％

Table 8. percentage of cells positive for β -galactosidase activity in method 2 (evaluation of the effect of fucoxanthin on senescence reversal).

	Pabociclib	Doxorubicin
			Control cells (basal state)	＜2％	＜2％
Senescent cells	＞95％	＞80％
			Senescent cells + fucoxanthin	＜5％	＜5％

Example 4 Effect of carotenoids in reducing the senescence-associated secretory phenotype (SASP)

Materials and methods

Cell culture

Treatment and Experimental conditions

Will 10⁶One Sk-Mel-103 cell was seeded in a 24-well plate. The cultures were incubated with palbociclib or doxorubicin (as a senescence inducer), fucoxanthin, or a combination thereof, as described in table 9. After treatment, cells and supernatant were collected by centrifugation and stored at-20 ℃.

TABLE 9 Experimental conditions for determining the Effect of fucoxanthin on SASP

Determination of secreted Interleukin (IL)

The secretion of IL-1 β, IL-6 and IL-8 in the supernatant was quantified by a commercial ELISA kit (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 the Bradford assay (B6916, Sigma-Aldrich) at the end of the experiment. A second normalization was performed by dividing the secreted interleukins after fucoxanthin incubation by their corresponding senescent cell controls.

Results

IL-1 β, IL-6 and IL-8 are up-regulated in senescent cells and form part of SASP. To investigate the effect of fucoxanthin on SASP factor, secreted IL was quantified in the supernatant of SK-Mel-103 cells in which senescence was induced with doxorubicin or palbociclib.

As shown in FIGS. 1 and 2, for both pro-aging treatments, secretion of IL-1 β, IL-6 and IL-8 was significantly reduced after incubation with fucoxanthin (< 0.01 in p-value).

Example 5 Effect of amarocixanthin A on hepatic fibrosis

Hepatic Stellate Cells (HSCs) are found in the periantral space of the liver. It constitutes the main cell type involved in liver fibrosis, responsible for scar tissue formation in response to liver injury.

Materials and methods

Conditions of the experiment

To evaluate the effect of amarocixanthin a on HSC activation, the immortalized human cell line LX2 was used. In addition, primary HSCs were extracted from cirrhosis rats that had received carbon tetrachloride inhalation. In both cases, cells were cultured for 24 hours prior to addition of the compound. Then, amaurociaxanthin a (10 μ M) or vehicle (DMSO) was added to the culture. Cells were harvested 24 and 72 hours after treatment and lysed to analyze fibrosis biomarkers. Gene transcription was determined by quantitative PCR at 24 and 72 hours, and protein expression was determined by western blot at 72 hours. Alpha-smooth muscle actin (alpha-SMA) was analyzed in rat HSC. Collagen I a1(COL1a1), a major component of extracellular matrix, was analyzed in LX 2.

Results

alpha-SMA is a common activation biomarker for microfilament-forming structural proteins and HSCs. Under pathological conditions, HSCs increase the synthesis of COL1a1, thereby promoting the development of liver fibrosis (apoptosis).

As shown in figure 3, amaurociaxanthin a inhibited alpha-SMA transcription by about 80% in the cirrhosis rat HSCs after 24 hours and 72 hours. Protein level expression was also reduced by 95%. Xanthophylls also inhibited COL1a1 transcription in human LX2 cells by about 50% (after 24 hours) and 80% (after 72 hours) (fig. 4). Collagen expression was reduced by about 95% as quantified by western blot (figure 4).

Taken together, amaurociaxanthin a promotes HSC inactivation and inhibits the exacerbated synthesis of extracellular matrix as evidenced by down-regulation of fibrotic markers at both the transcriptional and translational levels.

Claims

1. A combination, comprising: a carotenoid, a carotenoid metabolite, a carotenoid derivative, an analogue or ester or salt thereof, and a second component selected from the group consisting of: 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 senescent phenotype inhibitors.

2. The combination according to claim 1, wherein the carotenoid is selected from the group consisting of fucoxanthin, fucoxanthin alcohol, amarocixanthin a, neoxanthin, astaxanthin, zeaxanthin and lutein.

3. The combination according to any one of claims 1 or 2, wherein the second component is an inhibitor of one or more BCL-2 anti-apoptotic protein family members.

4. The combination according to claim 3, wherein the inhibitor of one or more BCL-2 anti-apoptotic protein family members is nevira or vinatork and the carotenoid is selected from fucoxanthin, amarocixanthin A and neoxanthin.

5. A food product, cosmeceutical, nutraceutical, cosmetic or pharmaceutical composition comprising a combination according to any one of claims 1 to 4.

6. Cosmetic method for preventing and/or reducing skin ageing and/or for improving the cosmetic adverse effects of ageing, comprising the administration of a combination according to any one of claims 1 to 4 or a food, cosmeceutical, nutraceutical or cosmetic composition according to claim 5 to a subject in need thereof.

7. A combination according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5 for use in medicine.

8. The combination according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 for use in the treatment of cancer.

9. The combination or pharmaceutical composition according to claim 8, wherein the cancer is a skin cancer or a hematological cancer, in particular AML, NHL, MDS; MM, CLL or myelofibrosis.

10. The combination according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 for use in the treatment of an aging-related disease or disorder.

11. The combination according to any one of claims 1 to 4 or the food, nutraceutical or pharmaceutical composition according to claim 5 for use in enhancing the effectiveness of an anti-tumor compound.

12. Carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for use in the treatment of aging-related diseases or disorders.

13. A carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof for use according to claim 12, wherein said senescence-associated disease is cancer.

14. A carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof for use according to claim 13, wherein said cancer is a hematological cancer or a skin cancer.

15. A combination for use according to claim 10 or a carotenoid, carotenoid metabolite, carotenoid derivative, analogue or ester or salt thereof for use according to claim 12, wherein said senescence-associated disorder is fibrosis.

16. A combination for use or a carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof, according to claim 15 wherein said fibrosis is characterized by having a high number of senescent cells.

17. A carotenoid, carotenoid metabolite, carotenoid derivative, analogue or ester or salt thereof, for use or a combination for use according to any one of claims 15 or 16, wherein said fibrosis is pulmonary fibrosis, chronic obstructive pulmonary disease, cardiac fibrosis, renal fibrosis, myelofibrosis or liver fibrosis.

18. A carotenoid, carotenoid metabolite, carotenoid derivative, analogue or ester or salt thereof, for use or a combination for use according to any one of claims 15 or 16, wherein said fibrosis is liver fibrosis.

19. A combination for use according to claim 10 or a carotenoid, carotenoid metabolite, carotenoid derivative, analogue or ester or salt thereof for use according to claim 12, wherein the ageing disorder is treatment-induced ageing.

20. Carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for use in enhancing the effectiveness of an anti-neoplastic compound.

21. Carotenoids, carotenoid metabolites, carotenoid derivatives, analogs or esters or salts thereof for use in reducing the adverse effects of anti-tumor therapy.

22. A carotenoid, carotenoid metabolite, carotenoid derivative, analog or ester or salt thereof, for use according to claim 21, wherein said antineoplastic therapy is chemotherapy or radiation therapy.

23. A carotenoid for use according to any one of claims 12 to 22 wherein the carotenoid is selected from fucoxanthin, fucoxanthin alcohol, amarocixanthin a, neoxanthin, astaxanthin, zeaxanthin and lutein.