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WO2024254165A2 - Procédés et matériels pour utiliser et évaluer des traitements anti-sénescence chez des mammifères - Google Patents

Procédés et matériels pour utiliser et évaluer des traitements anti-sénescence chez des mammifères Download PDF

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Publication number
WO2024254165A2
WO2024254165A2 PCT/US2024/032575 US2024032575W WO2024254165A2 WO 2024254165 A2 WO2024254165 A2 WO 2024254165A2 US 2024032575 W US2024032575 W US 2024032575W WO 2024254165 A2 WO2024254165 A2 WO 2024254165A2
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Prior art keywords
polypeptides
circulating
mammal
level
treatment
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WO2024254165A3 (fr
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Marissa J. SCHAFER
Nathan K. Lebrasseur
Andrew J. HAAK
Chase M. CARVER
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This document relates to methods and materials for using and/or assessing antisenescence treatments (e.g., admini strati on(s) of one or more senotherapeutic agents) within a mammal (e.g., a human).
  • antisenescence treatments e.g., admini strati on(s) of one or more senotherapeutic agents
  • the methods and materials provided herein can be used to determine and/or classify the efficacy of an anti-senescence treatment in a mammal (e.g., a human).
  • a circulating polypeptide signature of a mammal e.g., a human having been administered an anti-senescence treatment (e.g., admini strati on(s) of one or more senotherapeutic agents) can be used to determine and/or classify the efficacy of the anti-senescence treatment in treating the mammal.
  • an anti-senescence treatment e.g., admini strati on(s) of one or more senotherapeutic agents
  • Age is the single greatest risk factor for a multitude of chronic and progressive diseases (Marengoni et al., Ageing Res. Rev, 10:430-439 (2011); and Lopez-Otin et al., Cell, 153: 1194-1217 (2013)).
  • Cellular senescence is an age-associated mechanism that contributes to pathogenesis in diverse organ systems, including immune cell dysfunction (Bianchi et al., Oncogene, 25:4110-4115 (2006); and Liu et al. , Aging Cell, 8:439-448 (2009)), metabolic and cardiovascular diseases (Gorenne et al., Cardiovasc.
  • This document provides methods and materials for assessing the efficacy of and/or for using an anti-senescence treatment (e.g., administration(s) of one or more senotherapeutic agents).
  • the methods and materials provided herein can be used to determine the efficacy of an anti-senescence treatment in a mammal (e.g., a human).
  • a circulating polypeptide signature of a sample e.g., a blood sample such as a plasma sample
  • a mammal e.g., a human
  • this document provides methods and materials for treating a mammal (e.g., a human) having a disease or disorder characterized by the presence of senescent cells or elevated cellular senescence.
  • a mammal e.g., a human
  • an anti-senescence treatment e.g., one or more senotherapeutic agents
  • the circulating polypeptide signature of a sample obtained from the mammal can be assessed to determine and/or classify whether or not the anti-senescence treatment effectively reduced cellular senescence within the mammal.
  • a circulating polypeptide signature that includes one or more of a decreased level of an interleukin 23 receptor (IL23R) polypeptide, a decreased level of a glucagon (GCG) polypeptide, a decreased level of a C-C motif chemokine ligand 5 (CCL5) polypeptide, a decreased level of an interleukinl7A(IL17A) polypeptide, and an increased level of a carbonic anhydrase 13 (CAI 3) polypeptide can indicate that the administered anti-senescence treatment effectively reduced cellular senescence within that mammal.
  • IL23R interleukin 23 receptor
  • GCG glucagon
  • CCL5 C-C motif chemokine ligand 5
  • CAI 3 carbonic anhydrase 13
  • a circulating polypeptide signature that lacks each of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide can indicate that the administered anti-senescence treatment did not effectively reduce cellular senescence within that mammal.
  • an increased level of a IL23R polypeptide, an increased level of a GCG polypeptide, an increased level of a CCL5 polypeptide, an increased level of a IL17A polypeptide, and/or a decreased level of a CAI 3 polypeptide in blood (e.g., plasma) of a mammal (e.g., a human) can be used as an indicator of the systemic signatures of senescence (e.g., senescent cell burden of that mammal).
  • treating a mammal with an anti-senescence treatment can reduce cellular senescence within the mammal such that the level of IL23R polypeptides is decreased, the level of GCG polypeptides is decreased, the level of CCL5 polypeptides is decreased, the level of IL17A polypeptides is decreased, and/or the level of CA I 3 polypeptides is increased in blood (e.g., plasma) of the mammal.
  • a circulating polypeptide signature that includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide within the mammal following a particular anti-senescence treatment, then that particular anti-senescence treatment can be continued within that mammal.
  • a circulating polypeptide signature lacks each of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA I 3 polypeptide following a particular anti-senescence treatment, then that particular antisenescence treatment can be discontinued within that mammal. In such a case, the mammal can be treated with an alternative anti-senescence treatment.
  • the same agents can be administered to the mammal but at an altered (e.g., an increased or decreased) dosing level (e.g., an altered amount of agent(s) or an altered frequency of administration of the same amount of agent(s)) or a different agent or set of agents can be administered to the mammal.
  • the circulating polypeptide signature within a mammal can be monitored during an anti-senescence treatment to assess effectiveness of the anti-senescence treatment and/or to make adjustments to an anti- senescence treatment when it is determined to be not effective.
  • one aspect of this document features methods for identifying an effective treatment within a mammal where the mammal received an anti- senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, (a) determining that a mammal that received an anti-senescence treatment including administration of one or more senotherapeutic agents can include a decreased level of circulating IL23R polypeptides and optionally one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating LL17A polypeptides, and an increased level of circulating CAI 3 polypeptides, and (b) classifying the anti-senescence treatment as being an effective anti-senescence treatment for the mammal.
  • the mammal can be a human.
  • the determining step (a) can include determining that the mammal has one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CA I 3 polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating GCG polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating CCL5 polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating IL 17 A polypeptides.
  • the determining step (a) can include determining that the mammal has an increased level of circulating CA13 polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CA 13 polypeptides.
  • the determining step (a) can include comparing a level of the circulating IL23R polypeptides within a first sample obtained from the mammal when or at a time prior to when the mammal received the anti-senescence treatment to a level of the circulating IL23R polypeptides within a second sample obtained from the mammal at a time after the time when the mammal received the anti-senescence treatment, to determine that the mammal has the decreased level of the circulating IL23R polypeptides.
  • the determining step (a) can include comparing a level of the circulating GCG polypeptides within the first sample to a level of the circulating GCG polypeptides within the second sample to determine that the mammal has the decreased level of the circulating GCG polypeptides.
  • the determining step (a) can include comparing a level of the circulating CCL5 polypeptides within the first sample to a level of the circulating CCL5 polypeptides within the second sample to determine that the mammal has the decreased level of the circulating CCL5 polypeptides.
  • the determining step (a) can include comparing a level of the circulating IL17A polypeptides within the first sample to a level of the circulating IL17A polypeptides within the second sample to determine that the mammal has the decreased level of the circulating IL17A polypeptides.
  • the determining step (a) can include comparing a level of the circulating CAI 3 polypeptides within the first sample to a level of the circulating CAI 3 polypeptides within the second sample to determine that the mammal has the increased level of the circulating CAI 3 polypeptides.
  • the first sample and the second sample can be blood samples.
  • the blood samples can be plasma samples.
  • the first sample can have been obtained from the mammal when the mammal received the antisenescence treatment.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • the method also can include administering the effective antisenescence treatment to the mammal after the determining step.
  • this document features methods for identifying an ineffective treatment within a mammal, where the mammal received an anti-senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, (a) determining that a mammal that received an anti-senescence treatment including administration of one or more senotherapeutic agents lacks a decreased level of circulating IL23R polypeptides, a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides, and (b) classifying the anti-senescence treatment as being an ineffective anti- senescence treatment for the mammal.
  • the mammal can be a human.
  • the determining step (a) can include comparing a level of the circulating IL23R polypeptides, a level of the circulating GCG polypeptides, a level of the circulating CCL5 polypeptides, a level of the circulating IL17A polypeptides, and a level of the circulating CAI 3 polypeptide within a first sample obtained from the mammal when or at a time prior to when the mammal received the anti-senescence treatment to a level of the circulating IL23R polypeptides, a level of the circulating GCG polypeptides, a level of the circulating CCL5 polypeptides, a level of the circulating IL17A polypeptides, a level of the circulating CNTN4 polypeptides, and a level of the circulating CA13 polypeptide within a second sample obtained from the mammal at a time after the time when the mammal received the anti-senescence treatment, to
  • the first sample and the second sample can be blood samples.
  • the blood samples can be plasma samples.
  • the first sample can have been obtained from the mammal when the mammal received the anti-senescence treatment.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, A1155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • the method also can include administering a revised anti-senescence treatment that is different from the anti-senescence treatment to the mammal after the determining step.
  • the revised anti-senescence treatment can include administering a higher amount of the one or more senotherapeutic agents of the anti-senescence treatment to the mammal.
  • the revised anti-senescence treatment can include administering one or more senotherapeutic agents that are different from the one or more senotherapeutic agents of the anti-senescence treatment.
  • this document features methods for treating a mammal, where the mammal received a prior anti-senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, (a) determining that a mammal that received a prior anti-senescence treatment including administration of one or more senotherapeutic agents can include a decreased level of circulating IL23R polypeptides and optionally one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides, and (b) administering the prior anti- senescence treatment or a slightly revised version of the prior anti-senescence treatment to the mammal.
  • the mammal can be a human.
  • the determining step (a) can include determining that the mammal has one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating GCG polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating CCL5 polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating IL17A polypeptides.
  • the determining step (a) can include determining that the mammal has an increased level of circulating CAI 3 polypeptides.
  • the determining step (a) can include determining that the mammal has a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CA I 3 polypeptides.
  • the determining step (a) can include comparing a level of the circulating IL23R polypeptides within a first sample obtained from the mammal when or at a time prior to when the mammal received the anti-senescence treatment to a level of the circulating IL23R polypeptides within a second sample obtained from the mammal at a time after the time when the mammal received the anti-senescence treatment, to determine that the mammal has the decreased level of the circulating IL23R polypeptides.
  • the determining step (a) can include comparing a level of the circulating GCG polypeptides within the first sample to a level of the circulating GCG polypeptides within the second sample to determine that the mammal has the decreased level of the circulating GCG polypeptides.
  • the determining step (a) can include comparing a level of the circulating CCL5 polypeptides within the first sample to a level of the circulating CCL5 polypeptides within the second sample to determine that the mammal has the decreased level of the circulating CCL5 polypeptides.
  • the determining step (a) can include comparing a level of the circulating IL17A polypeptides within the first sample to a level of the circulating IL17A polypeptides within the second sample to determine that the mammal has the decreased level of the circulating IL17A polypeptides.
  • the determining step (a) can include comparing a level of the circulating CA I 3 polypeptides within the first sample to a level of the circulating CAI 3 polypeptides within the second sample to determine that the mammal has the increased level of the circulating CAI 3 polypeptides.
  • the first sample and the second sample can be blood samples.
  • the blood samples can be plasma samples.
  • the first sample can have been obtained from the mammal when the mammal received the antisenescence treatment.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, A1155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • the method also can include administering the prior antisenescence treatment to the mammal after the determining step.
  • this document features methods for treating a mammal, where the mammal received a prior anti-senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, administering a subsequent administration of a prior anti-senescence treatment or a slightly revised version of the prior anti-senescence treatment to a mammal that received a prior anti-senescence treatment including administration of one or more senotherapeutic agents, where the mammal was identified as having a decreased level of circulating IL23R polypeptides and optionally one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CA13 polypeptides following the prior anti- senescence treatment.
  • the mammal can be a human.
  • the mammal can have been identified as having one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides.
  • the mammal can have been identified as having a decreased level of circulating GCG polypeptides.
  • the mammal can have been identified as having a decreased level of circulating CCL5 polypeptides.
  • the mammal can have been identified as having a decreased level of circulating IL17A polypeptides.
  • the mammal can have been identified as having an increased level of circulating CAI 3 polypeptides.
  • the mammal can have been identified as having a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • this document features methods for treating a mammal, where the mammal received a prior anti-senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, (a) determining that a mammal that received a prior anti-senescence treatment including administration of one or more senotherapeutic agents lacks a decreased level of circulating IL23R polypeptides, a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides, and (b) administering a revised antisenescence treatment different from the prior anti-senescence treatment to the mammal.
  • the mammal can be a human.
  • the determining step (a) can include comparing a level of the circulating IL23R polypeptides, a level of the circulating GCG polypeptides, a level of the circulating CCL5 polypeptides, a level of the circulating IL17A polypeptides, and a level of the circulating CAI 3 polypeptide within a first sample obtained from the mammal when or at a time prior to when the mammal received the anti-senescence treatment to a level of the circulating IL23R polypeptides, a level of the circulating GCG polypeptides, a level of the circulating CCL5 polypeptides, a level of the circulating IL17A polypeptides, a level of the circulating CNTN4 polypeptides, and a level of the circulating CA I 3 polypeptide within a second sample obtained from the mammal at a time after the time when the mammal received the anti-senescence treatment,
  • the first sample and the second sample can be blood samples.
  • the blood samples can be plasma samples.
  • the first sample can have been obtained from the mammal when the mammal received the anti-senescence treatment.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • the revised anti-senescence treatment can include administering a higher amount of the one or more senotherapeutic agents of the anti-senescence treatment to the mammal.
  • the revised anti-senescence treatment can include administering one or more senotherapeutic agents that are different from the one or more senotherapeutic agents of the anti- senescence treatment.
  • this document features methods for treating a mammal, where the mammal received a prior anti-senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, administering a revised anti-senescence treatment different from the prior anti-senescence treatment to a mammal that received a prior anti-senescence treatment including administration of one or more senotherapeutic agents, where the mammal was identified as lacking a decreased level of circulating IL23R polypeptides, a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides following the prior anti-senescence treatment.
  • the mammal can be a human.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, A1155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • the revised anti-senescence treatment can include administering a higher amount of the one or more senotherapeutic agents of the anti-senescence treatment to the mammal.
  • the revised anti-senescence treatment can include administering one or more senotherapeutic agents that are different from the one or more senotherapeutic agents of the anti- senescence treatment.
  • this document features methods for continuing a prior antisenescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, administering an anti-senescence treatment that is the same as a prior anti-senescence treatment, or a slightly revised version of the prior anti-senescence treatment, to a mammal identified as having a decreased level of circulating IL23R polypeptides and optionally one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides following the prior anti-senescence treatment.
  • the mammal can be a human.
  • the mammal can have been identified as having one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CA I 3 polypeptides.
  • the mammal can have been identified as having a decreased level of circulating GCG polypeptides.
  • the mammal can have been identified as having a decreased level of circulating CCL5 polypeptides.
  • the mammal can have been identified as having a decreased level of circulating IL17A polypeptides.
  • the mammal can have been identified as having an increased level of circulating CAI 3 polypeptides.
  • the mammal can have been identified as having a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, Al 331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin- 3a, ruxolitinib, metformin, and/or rapamycin.
  • this document features methods for changing an ineffective use of a prior anti-senescence treatment including administration of one or more senotherapeutic agents.
  • the methods can include, or consist essentially of, administering an anti-senescence treatment that is different from a prior anti-senescence treatment to a mammal identified as lacking a decreased level of circulating IL23R polypeptides, a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CAI 3 polypeptides following the prior anti-senescence treatment.
  • the mammal can be a human.
  • the one or more senotherapeutic agents can be dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and/or rapamycin.
  • the revised anti-senescence treatment can include administering a higher amount of the one or more senotherapeutic agents of the anti-senescence treatment to the mammal.
  • the revised antisenescence treatment can include administering one or more senotherapeutic agents that are different from the one or more senotherapeutic agents of the anti-senescence treatment.
  • FIG. 1 Study design for young and old pl6-hikAttac mice treated with a senotherapeutic or vehicle.
  • VEN venetoclax
  • NAV navitoclax
  • FIS fisetin
  • LUT luteolin
  • Figures 3A-3N Senescence- and age-associated genes were expressed across tissues and sexes in old female and male pl6-InkAttac mice.
  • Figures 3A-3F Shown are RT-PCR gene expression values from young female (open pink circle), old female (solid pink triangle), young male (open blue circle), and old male (solid blue triangle) tissues for (Figure 3 A) kidney, ( Figure 3B) liver, ( Figure 3C) spleen, (Figure 3D) cerebral cortex, ( Figure 3E) perigonadal adipose, and (Figure 3F) lung. Values are normalized relative to the expression of young control mice of each sex per tissue. Black bars indicate median values for each group.
  • Figures 3G-3L Shown are ordinal logistic regression models comparing gene expression from young (open black circle) and old (solid red triangle) kidney, liver, spleen, cortex, adipose, and lung for (Figure 3G) pl6 mk4a , (Figure 3H) pl6 total , ( Figure 31) Bcl2ala, ( Figure 3J) Il23r, ( Figure 3K) Ccl3, ( Figure 3L) Ccl5, ( Figure 3M) [lib. and ( Figure 3N) 116.
  • Figures 4A-4F Senescence- and age-associated genes, including Il23r, were coexpressed in senescent fibroblasts in vitro.
  • Figure 4A Representative lOx images of senescence-associated 0-galactosidase (SA- -gal) staining in sham and irradiated mouse embryonic fibroblasts (MEFs). Left panels are bright-field images, right panels are DAP1 fluorescent images of the same field of view.
  • Figures 4B-4C Shown are quantifications for ( Figure 4B) percentage and (Figure 4C) total number of SA-P-gal cells per image field.
  • Figure 4D Quantification of nucleus area in sham and irradiated cells as depicted in Figure 4A.
  • Figure 4E RT-PCR gene expression of senescence- and age-associated markers in sham (open circles) and irradiated (solid triangles) MEFs. Values are relative expression to sham control.
  • Figures 5A-5J Age-related changes in select plasma polypeptides were suppressed by senotherapeutic interventions. Plasma polypeptide levels are demonstrated for all biomarkers that differed by age and at least one senolytic intervention significantly reversed the age- related change in at least one sex. This criterion was met for ( Figures 5A-5B) IL23R, ( Figures 5C-5D) CCL5, ( Figures 5E-5F) GCG, ( Figures 5G-5H) IL17A, ( Figures 5I-5J) CA I 3. Figures 5 A, 5C, 5E, 5G, and 51 show female groups. Figures 5B, 5D, 5F, 5H, and 5 J show male groups.
  • Figures 6A-6L The p!6 expression in old p!6-InkAttac mice was reduced in response to transgenic clearance or senolytic treatment. RT-PCR was used to assess p!6 gene expression between old control (OLD CON) and senolytic drug treated mice, in ( Figures 6A- 6D) kidney, ( Figures 6E-6H) spleen, and ( Figures 6L6L) cortex. Values are relative expression to YNG CON per group.
  • Figures 6A, 6C, 6E, 6G, 61, and 6K show female groups.
  • Figures 6B, 6D, 6F, 6H, 6J, and 6K show male groups.
  • Figures 6A, 6B, 6E, 6F, 61, and 6J corresponds to exon 2-3, Cdkn2a transcript variant 2.
  • Figures 7A-7L Ccl5 gene expression decreased in aged tissues following p!6- InkAttac transgenic clearance or senolytic treatment. Comparison of Ccl5 gene expression measured by RT-PCR between senolytic drug intervention and OLD CON in ( Figures 7A- 7B) kidney, ( Figures 7C-7D) liver, ( Figures 7E-7F) spleen, ( Figures 7G-7H) lung, ( Figures 7I-7J) perigonadal adipose, and (Figures 7K-7L) cortex.
  • Figures 7A, 7C, 7E, 7G, 71, and 7K show female groups.
  • Figures 7B, 7D, 7F, 7H, 7J, and 7L show male groups.
  • Figures 8A-8L Il23r gene expression decreased in aged tissues following p!6- InkAttac transgenic clearance or senolytic treatment. Comparison of Il23r gene expression measured by RT-PCR between senolytic drug intervention and OLD CON in ( Figures 8A- 8B) kidney, ( Figures 8C-8D) liver, ( Figures 8E-8F) spleen, ( Figures 8G-8H) lung, ( Figures 8L8J) perigonadal adipose, and (Figures 8K-8L) cortex.
  • Figures 8A, 8C, 8E, 8G, 81, and 8K show female groups.
  • Figures 8B, 8D, 8F, 8H, 8J, and 8L show male groups.
  • FIG. 10A-10D (O) tissues from female and male mice.
  • Figures 10A-10D Figures 10A-10B) Spearman correlations between pl6 mk4a RT- PCR gene expression for each tissue and plasma polypeptides in (Figure 10A) female and ( Figure 10B) male mice.
  • Figures 10C-10D Spearman correlations between kidney gene expression of senescence factors and the plasma polypeptides CCL3, CCL5, CCN1, IL17A, IL23R, and MIA for ( Figure IOC) female and ( Figure 10D) male mice.
  • Figure 11A-1 IL RT-PCR was used to assess p!6 gene expression between old control (OLD CON) and senolytic drug treated mice in ( Figures 11 A-l ID) liver, ( Figures 11E-11H) lung, and ( Figures 111-1 IL) perigonadal adipose.
  • Figures 11 A, 11C, IE, 11G, 111, and 1 IK show female groups.
  • Figures 1 IB, 1 ID, 1 IF, 11H, 11 J, and 1 IL show male groups.
  • Figure 12A-12X RT-PCR relative gene expression from female kidney for senescence and inflammatory markers ( Figures 12A and 12M) Bcl2, ( Figures 12B and 12N) Bcl2ala, ( Figures 12C and 121) Cal3, ( Figures 12D and 12P) Ccl2, ( Figures 12E and 12Q) Ccl3, ( Figures 12F and 12R) Ccl20, ( Figures 12G and 12S) Cent, ( Figures 12H and 12T) Cd38, ( Figures 121 and 12U) Cdknla, ( Figures 12J and 12V) Illb, ( Figures 12K and 12W) 116, and ( Figures 12L and 12X) Tnf following senolytic drug intervention, compared to OLD CON.
  • Figures 12A-12L show female groups.
  • Figures 12M-12X show male groups.
  • Figures 13A-13X Relative gene expression from liver of treated groups of mice for ( Figures 13A and 13M) Bcl2, ( Figures 13B and 13N) Bcl2cila, ( Figures 13C and 131) Cal3, ( Figures 13D and 13P) Ccl2, ( Figures 13E and 13Q) Ccl3, ( Figures 13F and 13R) Ccl20, ( Figures 13G and 13S) Ccnl, ( Figures 13H and 13T) Cd38, ( Figures 131 and 13U) Cdknla, ( Figures 13 J and 13V) Illb, ( Figures 13K and 13W) 116, and ( Figures 13L and 13X) Tnf.
  • Figures 13A-13L show female groups.
  • Figures 13M-13X show male groups.
  • Figures 14A-14X Relative gene expression from spleen of treated groups of mice for ( Figures 14A and 14M) Bcl2, ( Figures 14B and 14N) Bcl2ala, ( Figures 14C and 141) Cal 3, ( Figures 14D and 14P) Ccl2, ( Figures 14E and 14Q) Ccl3, ( Figures 14F and 14R) Ccl20, ( Figures 14G and 14S) Cent, ( Figures 14H and 14T) Cd38, ( Figures 141 and 14U) Cdknla, ( Figures 14J and 14 V) Il lb, ( Figures 14K and 14W) 116, and ( Figures 14L and 14X) Tnf.
  • Figures 14A-14L show female groups.
  • Figures 14M-14X show male groups.
  • Figures 15A-15X Relative gene expression from lung of treated groups of mice for ( Figures 15A and 15M) Bcl2, ( Figures 15B and 15N) Bcl2ala, ( Figures 15C and 151) Cal 3, ( Figures 15D and 15P) Ccl2, ( Figures 15E and 15Q) Ccl3, ( Figures 15F and 15R) Ccl20, ( Figures 15G and 15S) Ccnl, ( Figures 15H and 15T) Cd38, ( Figures 151 and 15U) Cdknla, ( Figures 15J and 15 V) Il lb, ( Figures 15K and 15W) 116, and ( Figures 15L and 15X) Tnf.
  • Figures 15A-15L show female groups.
  • Figures 15M-15X show male groups.
  • Figures 16A-16X Relative gene expression from perigonadal adipose of treated groups of mice for ( Figures 16A and 16M) Bcl2, ( Figures 16B and 16N) Bcl2ala, ( Figures 16C and 161) Cal 3, ( Figures 16D and 16P) Ccl2, ( Figures 16E and 16Q) Ccl3, ( Figures 16F and 16R) Ccl20, ( Figures 16G and 16S) Ccnl, ( Figures 16H and 16T) Cd38, ( Figures 161 and 16U) Cdknla, ( Figures 16J and 16 V) Il lb, ( Figures 16K and 16W) 116, and ( Figures 16L and 16X) Tnf.
  • Figures 16A-16L show female groups.
  • Figures 16M-16X show male groups.
  • Figures 17A-17X Relative gene expression from cerebral cortex of treated groups of mice for ( Figures 17A and 17M) Bcl2, ( Figures 17B and 17N) Bcl2ala, ( Figures 17C and 171) Cal 3, ( Figures 17D and 17P) Ccl2, ( Figures 17E and 17Q) Ccl3, ( Figures 17F and 17R) Ccl20, ( Figures 17G and 17S) Ccnl, ( Figures 17H and 17T) Cd38, ( Figures 171 and 17U) Cdknla, ( Figures 17J and IN)' Il lb, ( Figures 17K and 17W) 116, and ( Figures 17L and 17X) Tnf.
  • Figures 17A-17L show female groups.
  • Figures 17M-17X show male groups.
  • Figures 19A-19N Senescence- and age-associated genes were expressed across tissues and sexes in old female and male p!6-InkAttac mice.
  • Figures 19A-19F Shown are RT-PCR gene expression values from young female (open pink circle), old female (solid pink triangle), young male (open blue circle), and old male (solid blue triangle) tissues for (Figure 19A) kidney, ( Figure 19B) liver, ( Figure 19C) spleen, (Figure 19D) cerebral cortex, ( Figure 19E) perigonadal adipose, and (Figure 19F) lung. Values were normalized relative to the expression of young control mice of each sex per tissue. Black bars indicate median values for each group.
  • Figures 19G-19L Shown are ordinal logistic regression models comparing gene expression from young (open black circle) and old (solid red triangle) kidney, liver, spleen, cortex, adipose, and lung for (Figure ( Figure 19H) Cdkn2a, ( Figure 191) Bcl2ala, ( Figure 19J) Il23r, ( Figure 19K) Ccl3, ( Figure 19L) Ccl5, (Figure 19M) Il lb, and ( Figure 19N) 116.
  • FIGs 20A-20M Age-related changes in plasma proteins were reverted by senotherapeutic interventions.
  • Figure 20A Treatment groups (top) and senotherapeutic treatment timeline (bottom) of young and old mice that received either vehicle or AP20187 (AP) by intraperitoneal (i.p) injection and either vehicle or one of four seno lytic drugs (VEN, NAV, FIS, or LUT) by oral gavage. Beginning on week one, mice were treated once daily for five days, followed by two weeks without treatment, followed by five days of further treatment, followed by one week without treatment prior to necropsy.
  • VEN seno lytic drugs
  • Figures 20B-20M Plasma protein levels were demonstrated for all biomarkers that differed by age and at least one senolytic intervention significantly reversed the age-related change in at least one sex. This criterion was met for ( Figures 20B-20C) IL23R, ( Figures 20D-20E) CCL5, ( Figures 20F-20G) GCG, ( Figures 20H-20I) IL17A, ( Figures 20J-20K) CAB, and ( Figures 20L-20M) CNTN4.
  • Figures 20B, 20D, 20F, 20H, 20J, and 20L show female groups.
  • Figures 20C, 20E, 20G, 201, 20K, and 20M show male groups.
  • Figures 21 A-21L p!6 expression in old p 16-InkAttac mice was reduced in a subset of aged tissues in response to transgenic or pharmacological senescent cell targeting.
  • RT-PCR was used to assess Cdk2na and pl6 ,nk4a gene expression between old control (OLD CON) versus senolytic drug-treated mice, in ( Figures 21A-21D) kidney, ( Figures 21E-21H) spleen, and ( Figures 21L21L) cortex. Values are expression relative to YNG CON per group.
  • Figures 21 A, 21 C, 2 IE, 21 G, 211, and 2 IK show female groups.
  • Figures 2 IB, 2 ID, 2 IF, 21H, 21J, and 21L show males groups.
  • Figures 21A, 21B, 21E, 21F, 211, 21J) Cdkn2a detected both transcript variants 1 (pl ⁇ 'f) and 2 (pl6 mk4a ), and
  • Figures 22A-22L Ccl5 gene expression decreased in a subset of aged tissues in response to transgenic or pharmacological senescent cell targeting. Comparison of Ccl5 gene expression measured by RT-PCR between senolytic drug intervention and OLD CON in ( Figures 22A-22B) kidney, ( Figures 22C-22D) liver, ( Figures 22E-22F) spleen, ( Figures 22G-22H) lung, ( Figures 22I-22J) perigonadal adipose, and (Figures 22K-22L) cortex are shown.
  • Figures 22A, 22C, 22E, 22G, 221, and 22K show female groups.
  • Figures 22B, 22D, 22F, 22H, 22J, and 22L show males groups.
  • FIGS 23 A-23L Il23r gene expression was altered in a subset of aged tissues in response to transgenic or pharmacological senescent cell targeting. Comparison of Il23r gene expression measured by RT-PCR between senolytic drug intervention and OLD CON in ( Figures 23A-23B) kidney, ( Figures 23C-23D) liver, ( Figures 23E-23F) spleen, ( Figures 23G-23H) lung, ( Figures 23L23J) perigonadal adipose, and (Figures 23K-23L) cortex are shown. Figures 23 A, 23C, 23E, 23G, 231, and 23K show female groups.
  • Figures 23B, 23D, 23F, 23H, 23 J, and 23L show males groups.
  • FIGS 27A-27J In situ hybridization (RNAscope) o H23r+ cells co-localized to p!6 ink4a and Cd3e in the renal sinus and perivascular areas of aged mouse kidney.
  • Figure 27A Representative images of p!6 mk4a and I123r in C57BL/6 aged kidney perivascular tissue (21 mo. old). An open channel was used for contrast of tissue architecture.
  • Figure 27B RNAscope of pl6‘ nk4c Il23r, and Cd3e from young (top, 2 months old) and old (bottom, 24 months old) kidney tissue from mice on p!6-InkAttac background.
  • Figures 27C-27F and old kidney ( Figures 27G-27J) tissues from RNAscope are shown.
  • Figures 27C, 27D, 27G, and 27H Interstitial and perivascular tissue exhibited co-localization of Cd3e, Il23r, and p!6 mk4a in old but not young kidney.
  • Figures 27E and 271) Aged glomeruli showed the presence of Cd3e+Il23r+ cells.
  • Figures 27F and 27J) Connective tissue showed dense accumulation of Cd3e+ cells in both young and old kidney, but only old tissue exhibited colocalization with 7/23/' and p!6 mk4a . All scale bars 100 pm.
  • Figures 28A-28G Senescence- and age-associated genes, including Il23r, were expressed in senescent fibroblasts in vitro.
  • Figure 28A Representative lOx images of senescence-associated P-galactosidase (SA- ⁇ -gal) staining in sham and irradiated (IR) mouse embryonic fibroblasts (MEFs). Left panels are bright-field images, right panels are DAPI fluorescent images of the same field of view.
  • Figures 28B-28C Shown are quantifications for percentage ( Figure 28B) and total number of SA- -gal cells (Figure 28C) per image field.
  • Figure 28D Quantification of nucleus area in sham and irradiated cells as depicted in Figure 28A.
  • Figure 28E RT-PCR gene expression of senescence- and age-associated markers in sham (open circles) and irradiated (solid triangles) MEFs. Values are expression relative to sham control.
  • Figure 28F Concentrations of senescence-related proteins in conditioned media (MEF CM) measured in pg/mL with Luminex multiplex assay.
  • FIGS 29A-29F Senolytic compounds showed differential selectivity, potency, and efficacy in apoptosis of senescent cells in vitro. Cleaved caspase-3 activity was measured in proliferating or etoposide-induced senescent primary human lung fibroblasts.
  • Figures 29A- 29D Concentration-response curves for percent change in cleaved caspase-3 intensity within senescent (diamonds) or proliferating (circles) cells and after incubation (3 days) with senolytic compounds VEN (Figure 29 A), NAV (Figure 29B), FIS (Figure 29C), or LUT (Figure 29D).
  • Figures 30A-30L RT-PCR was used to assess pl6 gene expression between old control (OLD CON) and senolytic drug treated mice, in liver ( Figures 30A-30D), lung ( Figures 30E-30H), and perigonadal adipose (Figures 3OI-3OL).
  • Figures 30A, 30C, 30E, 30G, 301, and 30K show female groups.
  • Figures 30B, 30D, 30F, 30H, 30J, and 30L show male groups.
  • Figures 31 A- 3 IL RT-PCR was used to assess p21 gene expression between old control (OLD CON) and senolytic drug treated mice, in kidney ( Figures 31 A- 3 IB), liver ( Figures 31C-31D), spleen ( Figures 31E-31F), lung ( Figures 31G-31H), perigonadal adipose ( Figures 311-31 J), and cortex ( Figures 3 IK-3 IL).
  • Figures 31 A, 31C, 3 IE, 31G, 3 II, and 3 IK show female groups.
  • Figures 3 IB, 3 ID, 3 IF, 31H, 31 J, and 3 IL show male groups.
  • YNG CON versus OLD CON reflect statistics provided in Figure 19 with exact p-values provided.
  • FIG 33 Heatmap of normalized mean relative gene expression from female and male tissue RT-PCR, comparing old-vehicle treated mice to young vehicle-treated mice.
  • F denotes female, M denotes male mice.
  • Data points of relative gene expression for each sample and median values for young and old can be found in Figures 19A-19F.
  • FIG. 34 RT-PCR relative gene expression from female kidney for markers Bcl2, Bcl2ala, Cal 3, Ccl2, Ccl3, Ccl20, Ccnl, Cd38, Cntn4, lllb, 116, and Tnf following senolytic drug intervention, compared to OLD CON.
  • A-L Female groups are shown in pink, and (M- X) males are shown in blue.
  • FIG 35 Relative gene expression from liver of treated groups of mice for Bcl2, Bcl2ala, Cal3, Ccl2, Ccl3, Ccl20, Ccnl, Cd38, Cntn4, lllb, 116, and Tnf.
  • A-L Female groups are shown in pink, and (M-X) males are shown in blue.
  • FIG. 36 Relative gene expression from spleen of treated groups of mice for Bcl2, Bcl2ala, Cal 3, Ccl2, Ccl3, Ccl20, Ccnl, Cd38, Cntn4, lllb, 116, and Tnf.
  • A-L Female groups are shown in pink, and (M-X) males are shown in blue.
  • FIG 37 Relative gene expression from lung of treated groups of mice for Bcl2, Bcl2ala, Cal 3, Ccl2, Ccl3, Ccl20, Ccnl, Cd38, Cntn4, lllb, 116, and Tnf.
  • A-L Female groups are shown in pink, and (M-X) males are shown in blue.
  • FIG 38 Relative gene expression from perigonadal adipose of treated groups of mice for Bcl2, Bcl2ala, Cal 3, Ccl2, Ccl3, Ccl20, Ccnl, Cd38, Cntn4, Illb, 116, and Tnf.
  • A- L Female groups are shown in pink, and (M-X) males are shown in blue.
  • FIG 39 Relative gene expression from cerebral cortex of treated groups of mice for Bcl2, Bcl2ala, Cal3, Ccl2, Ccl3, Ccnl, Ccn4, Cntn4, Illb, 116, and Tnf.
  • A-L Female groups are shown in pink, and (M-X) males are shown in blue.
  • Figure 40 Principal component analysis of the intersection between female (left column) and male (right column) kidney gene expression and plasma protein abundance in young vehicle-treated (YNG VEH; green), old vehicle-treated (OLD VEH; blue), and old senolytic-treated (OLD AP, OLD VEN, OLD NAV, OLD FIS, OLD LUT; red) mice.
  • Figures 41 A-41B Cleaved caspase-3 intensity in senescent lung fibroblasts. Etoposide-induced senescence primary human lung fibroblasts were cultured for 24 hours with IpM staurosporine to induce apoptosis. Cells were fixed, and immunostained for cleaved caspase-3 and DAPI. Images were collected using an automated high throughput fluorescence microscope ( Figure 41 A), and the mean pixel intensity for cleaved caspase-3 was quantified and plotted as % change in intensity ( Figure 41B).
  • this document provides methods and materials for assessing anti-senescence treatments as well as methods and materials for effectively using anti-senescence treatments within mammals. For example, this document provides methods and materials for determining whether or not a particular anti-senescence treatment administered to a mammal (e.g., a human) is effective (e.g., is effective to reduce cellular senescence within that mammal), in some cases, determining that a circulating polypeptide signature of a mammal (e.g., a human) following administration of an anti-senescence treatment includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA13 polypeptide (e.g., as compared to the level of such one or more polypeptides prior to being administered the anti-senescence treatment) can indicate that that anti-sen
  • determining that a circulating polypeptide signature of mammal (e.g., a human) following administration of an antisenescence treatment lacks each of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA I 3 polypeptide (e.g., as compared to the level of such polypeptides prior to being administered the anti-senescence treatment) can indicate that that anti-senescence treatment is not effective in that mammal and can be discontinued, altered in amount and/or frequency, or changed to a different type of antisenescence treatment in that mammal.
  • Any appropriate mammal can be administered an anti-senescence treatment and have its effectiveness assessed as described herein. Any appropriate mammal that was administered or instructed to self-administer an anti-senescence treatment can be assessed as described herein to determine if the anti-senescence treatment is effective in that mammal. Any appropriate mammal can be treated with one or more anti-senescence treatments that can be assessed as described herein to arrive at a particular anti-senescence treatment that is determined to be effective in that mammal.
  • a mammal that can be administered an anti-senescence treatment and have its effectiveness assessed as described herein can be a female mammal.
  • a mammal that can be administered an anti- senescence treatment and have its effectiveness assessed as described herein can be a male mammal.
  • the mammal can be any appropriate age.
  • the human when assessing and/or treating a human as described herein, can be an adult that is at least 60 years of age (e.g., about 60 years of age, about 65 years of age, about 68 years of age, about 70 years of age, about 72 years of age, about 75 years of age, about 78 years of age, about 80 years of age, or older).
  • 60 years of age e.g., about 60 years of age, about 65 years of age, about 68 years of age, about 70 years of age, about 72 years of age, about 75 years of age, about 78 years of age, about 80 years of age, or older.
  • a mammal e.g., a human having been administered an anti-senescence treatment (e.g., having been administered one or more senotherapeutic agents) can be assessed to determine and/or classify the efficacy of the anti-senescence treatment.
  • an anti-senescence treatment e.g., having been administered one or more senotherapeutic agents
  • a mammal e.g., a human having been administered an anti-senescence treatment (e.g., having been administered one or more senotherapeutic agents) can be assessed to determine whether or not the anti-senescence treatment is effective within that mammal by comparing a circulating polypeptide signature of a sample (e.g., a blood sample such as a plasma sample) obtained from the mammal following the anti- senescence treatment to the circulating polypeptide signature in a comparable sample obtained from the mammal prior to the antisenescence treatment.
  • a sample e.g., a blood sample such as a plasma sample
  • a circulating polypeptide signature e.g., the circulating level of one or more particular polypeptides
  • blood e.g., plasma
  • a mammal e.g., a human
  • an increased level of a IL23R polypeptide, an increased level of a GCG polypeptide, an increased level of a CCL5 polypeptide, an increased level of a IL17A polypeptide, and/or a decreased level of a CAI 3 polypeptide in blood (e.g., plasma) of a mammal (e.g., a human) can be used as an indicator of the systemic senescent cell burden of that mammal.
  • a circulating polypeptide signature can be used to determine whether cellular senescence in a mammal (e.g., a human) is increasing, decreasing, or staying the same or essentially the same, and can therefore be used to determine and/or classify whether or not an anti-senescence treatment is effective (e.g., whether or not an antisenescence treatment reduced cellular senescence within the mammal).
  • a mammal e.g., a human
  • an anti-senescence treatment e.g., whether or not an antisenescence treatment reduced cellular senescence within the mammal.
  • an anti- senescence treatment is determined to be effective, the same antisenescence treatment can be administered to the mammal (e.g., human). In some cases, if an anti-senescence treatment is determined to be effective, an anti-senescence treatment that is a slight modification of the original anti-senescence treatment can be administered to the mammal (e.g., human).
  • an anti-senescence treatment when determined to be effective within a mammal, that mammal can be administered or instructed to self-administer a slightly higher dose (e.g., l.lx or 1.2x that of the original dose) or a slightly lower dose (e.g., 0.9x or 0.8x that of the original dose) of the same agents of the effective antisenescence treatment.
  • a slightly higher dose e.g., l.lx or 1.2x that of the original dose
  • a slightly lower dose e.g., 0.9x or 0.8x that of the original dose
  • a revised anti-senescence treatment can be administered.
  • that mammal can be administered or instructed to self-administer a revised anti-senescence treatment that includes (a) anti-senescence treatment(s) that are different from the previously administered antisenescence treatment(s), (b) anti-senescence treatment(s) that are of a higher amount of the previously administered one or more senotherapeutic agents of the previously administered anti-senescence treatment(s), and/or (c) anti-senescence treatment(s) that include one or more senotherapeutic agents that are different from the one or more senotherapeutic agents of the previously administered anti-senescence treatment(s). Any of the methods described herein used to determine if an anti-senescence treatment is effective also can be used to determine if a revised anti-senescence treatment is effective.
  • an anti-senescence treatment that is different from the previously administered anti-senescence treatment can include administering one or more different senotherapeutic agents. Different anti-senescence treatments also can include administering the previously administered one or more senotherapeutic agents and an additional one or more senotherapeutic agents that were not previously administered. In some cases, an antisenescence treatment that is of a higher amount of the previously administered one or more senotherapeutic agents of the anti-senescence treatment can include an increased dosage, a more frequent dosage regime, or both. Increased dosage can include increasing the dosage by, for example, 1.3x, 1.4x, 1.5x, I.75x, or 2x compared to the previous dosage of the administered senotherapeutic agent(s).
  • a sample e.g., a blood sample such as a plasma sample
  • a mammal e.g., a human
  • an anti-senescence treatment e.g., having been administered one or more senotherapeutic agents
  • a circulating polypeptide signature of the sample obtained from a mammal after the mammal was administered an anti-senescence treatment can be compared to the circulating polypeptide signature of a comparable sample obtained from the mammal prior to the mammal being administered the anti-senescence treatment.
  • a circulating polypeptide signature of the sample obtained from a mammal five or more weeks (e g., about 5 weeks) after the mammal was administered an anti-senescence treatment can be compared to the circulating polypeptide signature of a comparable sample obtained from the mammal prior to the mammal being administered the anti-senescence treatment.
  • a circulating polypeptide signature described herein can include any appropriate number of polypeptides.
  • a circulating polypeptide signature can include any one or more (e.g., one, two, three, four, five, six, or more) polypeptides.
  • a circulating polypeptide signature that can be used as an indicator of the systemic senescent cell burden of a mammal as described herein can include an altered level (e.g., an increased level or a decreased level) of one or more polypeptides.
  • polypeptides whose presence, absence, or level can be included in a circulating polypeptide signature described herein include, without limitation, IL23R polypeptides, GCG polypeptides, CCL5 polypeptides, IL 17A polypeptides, and CA13 polypeptides.
  • IL23R polypeptides examples include, without limitation, polypeptides having an amino acid sequence set forth in the UniProt Knowledgebase (UniProtKB; see, e.g., The UniProt Consortium, Nucleic Acids Research, 51(D1):D523- D531 (2023)) at Accession Nos. Q8K4B4 and Q5VWK5.
  • GCG polypeptides that can be used as described herein (to determine the efficacy of an anti-senescence treatment in a mammal) include, without limitation, polypeptides having an amino acid sequence set forth in the UniProtKB at Accession Nos. P55095 and P01275.
  • CCL5 polypeptides that can be used as described herein (to determine the efficacy of an anti-senescence treatment in a mammal) include, without limitation, polypeptides having an amino acid sequence set forth in the UniProtKB at Accession Nos. P30882 and P13501.
  • IL17A polypeptides that can be used as described herein (to determine the efficacy of an anti-senescence treatment in a mammal) include, without limitation, polypeptides having an amino acid sequence set forth in the UniProtKB at Accession Nos. Q62386 and Q16552.
  • CAI 3 polypeptides that can be used as described herein (to determine the efficacy of an anti-senescence treatment in a mammal) include, without limitation, polypeptides having an amino acid sequence set forth in the UniProtKB at Accession Nos. Q9D6N1 and Q8N1Q1.
  • a circulating polypeptide signature of a sample obtained from a mammal after the mammal was administered an anti-senescence treatment can be indicative of an effective anti-senescence treatment in that mammal.
  • a circulating polypeptide signature can include an altered level (e.g., an increased level or a decreased level) of one or more polypeptides.
  • an altered level of a polypeptide can be an increased level.
  • the term “increased level” as used herein with respect to a level of a polypeptide in a sample refers to any level that is higher than a level of the polypeptide within a comparable sample obtained from the mammal prior to the mammal being administered the anti-senescence treatment.
  • an increased level of a polypeptide described herein can be a level present within a sample (e.g., a blood sample) obtained five or more weeks (e.g., about 5 weeks) after administration of an anti-senescence treatment that is at least 5 percent (e.g., at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 45 percent, at least 50 percent, at least 75 percent, or at least 100 percent) higher than the level of that polypeptide present within a comparable sample (e.g., a comparable blood sample) obtained prior to the administration of the anti-senescence treatment.
  • a comparable sample e.g., a comparable blood sample
  • an altered level of a polypeptide can be a decreased level.
  • the term “decreased level” as used herein with respect to a level of a polypeptide in a sample refers to any level that is lower than a level of the polypeptide within a comparable sample obtained from the mammal prior to the mammal being administered the anti-senescence treatment or, in some cases.
  • a decreased level of a polypeptide described herein can be a level present within a sample (e.g., a blood sample) obtained five or more weeks (e.g., about 5 weeks)after administration of an anti- senescence treatment that is at least 5 percent (e.g., at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 45 percent, at least 50 percent, at least 75 percent, or at least 100 percent) lower than the level of that polypeptide present within a comparable sample (e.g., a comparable blood sample) obtained prior to the administration of the anti-senescence treatment.
  • a comparable sample e.g., a comparable blood sample
  • the level of a polypeptide in a circulating polypeptide signature of a sample obtained from a mammal after (e.g., about 5 weeks after) the mammal was administered an anti-senescence treatment can be compared to a reference level of the polypeptide.
  • the term “reference level” as used herein with respect to a level of a polypeptide refers to the level of the polypeptide typically observed in a control sample. Control samples are samples obtained from mammals (e.g., humans) that do not have a disease or disorder characterized by the presence of senescent cells or elevated cellular senescence such as young, healthy humans.
  • a control sample can be sample obtained from a mammal (e.g., a human) of the same age as a mammal (e.g., a human) that was administered an anti-senescence treatment.
  • a control sample can be pooled sample including two or more control samples. It will be appreciated that levels of polypeptides from comparable samples are used when determining whether or not a particular polypeptide has an altered level.
  • the level of a polypeptide in a circulating polypeptide signature of a sample obtained from a mammal after (e.g., about 5 weeks after) the mammal was administered an anti-senescence treatment can be compared to a cutoff level of the polypeptide.
  • a sample e.g., a blood sample such as a plasma sample
  • a mammal e.g., a human
  • an antisenescence treatment e.g., one or more senotherapeutic agents
  • a sample e.g., a blood sample such as a plasma sample
  • a mammal e.g., a human
  • an anti-senescence treatment e.g., one or more senotherapeutic agents
  • a sample e.g., a blood sample such as a plasma sample
  • a mammal e.g., a human
  • a mammal e.g., a human
  • Such a determined circulating polypeptide signature within the mammal prior to being administered the anti-senescence treatment can be used to determine if the circulating polypeptide signature within the mammal determined after the mammal was administered an anti-senescence treatment includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide following the anti-senescence treatment.
  • a circulating polypeptide signature of the mammal that includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA I 3 polypeptide after the mammal was administered an anti-senescence treatment can indicate that that anti-senescence treatment is effective in that mammal.
  • a sample e.g., a blood sample such as a plasma sample
  • mammal e.g., a human
  • a sample can be obtained from a mammal (e.g., a human) at the same time (e.g., at the start of an anti-senescence treatment) that the mammal is being administered an antisenescence treatment and assessed to determine the circulating polypeptide signature within the mammal at that time.
  • Such a determined circulating polypeptide signature within the mammal at the time of administration of the anti-senescence treatment can be used to determine if the circulating polypeptide signature within the mammal determined after the mammal was exposed to the administered anti-senescence treatment for a period of time includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide following the anti-senescence treatment.
  • a circulating polypeptide signature that includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide within the mammal after the mammal was exposed to the administered anti-senescence treatment for a period of time can indicate that that an anti-senescence treatment is effective in that mammal.
  • a sample e.g., a blood sample such as a plasma sample
  • a sample can be obtained from the mammal (e.g., a human) at any appropriate time after the mammal was administered an antisenescence treatment and assessed to determine the circulating polypeptide signature within the mammal after being administered the anti-senescence treatment.
  • a sample e.g., a blood sample such as a plasma sample
  • a mammal e.g., a human
  • five or more weeks e.g., about 5 weeks
  • Such a determined circulating polypeptide signature within the mammal after being administered the antisenescence treatment can be used to determine if the circulating polypeptide signature within the mammal determined after the mammal was administered an anti- senescence treatment includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide following the anti-senescence treatment.
  • a circulating polypeptide signature that includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide within the mammal after the mammal was administered an antisenescence treatment can indicate that that an anti- senescence treatment is effective in that mammal.
  • the circulating polypeptide signature within a mammal can be detected at different time points over a course of an anti-senescence treatment (and/or a revised anti-senescence treatment) to determine the efficacy of the anti-senescence treatment (and/or revised anti-senescence treatment).
  • two or more samples e.g., two, three, four, five, six, or more
  • samples e.g., blood samples, urine samples, or saliva samples
  • the circulating polypeptide signature in the samples can be used to determine the efficacy of the anti-senescence treatment (and/or a revised anti-senescence treatment).
  • a first sample e.g., a first blood sample such as a first plasma sample
  • a mammal e.g., a human
  • an anti-senescence treatment e.g., prior to being administered one or more seno therapeutic agents
  • a second sample e.g., a second blood sample such as a second plasma sample
  • optionally subsequent samples can be obtained from the mammal after the mammal has been administered the anti-senescence treatment.
  • the circulating polypeptide signature of the second sample includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAB polypeptide as compared to the first sample, then the anti-senescence treatment (e.g., one or more senotherapeutic agents) can be determined and/or classified as being an effective anti- senescence treatment for that mammal.
  • the anti-senescence treatment e.g., one or more senotherapeutic agents
  • the anti-senescence treatment e.g., one or more senotherapeutic agents
  • the anti-senescence treatment can be determined and/or classified as not being an effective treatment for that mammal.
  • any appropriate sample from a mammal can be assessed for the presence, absence, or level of one or more (e.g., one, two, three, four, five, six, or more) polypeptides (e.g., IL23R polypeptides, GCG polypeptides, CCL5 polypeptides, IL17A polypeptides, and/or CAI 3 polypeptides).
  • a sample can be a biological sample.
  • a sample can contain one or more biological molecules (e.g., nucleic acids such as DNA and RNA, polypeptides, carbohydrates, lipids, hormones, and/or metabolites).
  • samples that can be assessed to determine the level of one or more polypeptides as described herein include, without limitation, fluid samples (e.g., urine samples, saliva samples, cerebrospinal fluid samples, and blood samples such as whole blood samples, serum samples, and plasma samples), and tissue samples (e.g., adipose tissue samples and lymph tissues such as lymph nodes).
  • a biological sample can be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample).
  • a biological sample can be a processed sample (e g., to isolate or extract one or more biological molecules).
  • a blood sample can be obtained from a mammal (e.g., a human) and processed to obtain a sample of circulating polypeptides, that can be assessed for the presence, absence, or level of one or more polypeptides as described herein.
  • a mammal e.g., a human
  • a sample of circulating polypeptides that can be assessed for the presence, absence, or level of one or more polypeptides as described herein.
  • any appropriate method can be used to detect the presence, absence, or level of one or more (e.g., one, two, three, four, five, six, or more) polypeptides (e.g., L23R polypeptides, GCG polypeptides, CCL5 polypeptides, IL 17A polypeptides, and/or CAI 3 polypeptides) within a sample (e.g., a sample obtained from a mammal such as a human) to determine the efficacy of an anti-senescence treatment.
  • a sample e.g., a sample obtained from a mammal such as a human
  • the presence, absence, or level of one or more polypeptides within a sample can be determined by detecting the presence, absence, or level of the polypeptides in the sample.
  • immunoassays e.g., immunohistochemistry (IHC) techniques, western blotting techniques, and enzyme- linked immunosorbent assays
  • mass spectrometry techniques e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays
  • proximity extension assays e.g., SOMAscan assays
  • spatial proteomics techniques can be used to determine the presence, absence, or level of one or more polypeptides in a sample.
  • the immunoassay can use any appropriate antibodies.
  • the presence, absence, or level of one or more (e.g., one, two, three, four, five, six, or more) polypeptides e.g., IL23R polypeptides, GCG polypeptides, CCL5 polypeptides, IL17A polypeptides, and/or CA I 3 polypeptides
  • a sample e.g., a sample obtained from a mammal such as a human
  • a sample obtained from a mammal such as a human can be detected as described in Example 1.
  • the level of mRNA encoding a polypeptide can be measured to indicate the level of that polypeptide within a mammal.
  • a sample containing cells can be obtained from a mammal, and the level of mRNA encoding a polypeptide (e.g., mRNA encoding a IL23R polypeptide, mRNA encoding a GCG polypeptide, mRNA encoding a CCL5 polypeptide, mRNA encoding a IL17A polypeptide, and/or mRNA encoding a CA13 polypeptide) can be measured to determine if the level of that polypeptide within the mammal was altered following administration of an anti-senescence treatment.
  • PCR polymerase chain reaction
  • RT quantitative reverse transcription
  • qRT-PCR quantitative reverse transcription
  • spatial transcriptomic techniques spatial transcriptomic techniques
  • single-cell RNA sequencing can be used to determine the presence, absence, or level of mRNA encoding a polypeptide in the sample.
  • Any appropriate anti-senescence treatment can be administered to a mammal (e.g., a human) and be assessed for effectiveness as described herein (e.g., by assessing the level of one or more polypeptides (e.g., the level of IL23R polypeptides, the level of GCG polypeptides, the level of CCL5 polypeptides, the level of IL17A polypeptides, and/or the level of CAI 3 polypeptides) after the mammal was administered the anti-senescence treatment being assessed).
  • An anti-senescence treatment can be designed to inhibit or reduce cellular senescence within a mammal.
  • an antisenescence treatment that can be administered to a mammal (e.g., a human) and/or assessed as described herein can be a treatment that includes administering one or more senotherapeutic agents to the mammal (e.g., the human).
  • a senotherapeutic agent can be a flavonoid.
  • senotherapeutic agents that can be used as at least a part of an anti-senescence treatment described herein include, without limitation, geroprotector agents such as melatonin, carnosine, metformin, nicotinamide mononucleotide (NMN) and delta sleep-inducing peptide, senescence-associated secretory phenotype (SASP) inhibitors such as glucocorticoids, statins (e.g., simvastatin), JAK1/2 inhibitors (e.g., ruxolitinib), NF-KB inhibitors, p38 inhibitors, and IL-la inhibitors, senolytic agents such as FOXO4-related peptides, inhibitors of a BCL-2 family polypeptide (e.g., BCL-2 inhibitors), Src inhibitors, USP7 inhibitors, quercetin, dasatinib, fisetin, navitoclax, piperlongumine, azithromycin
  • senotherapeutic agents that can be administered and/or assessed as described herein include, without limitation, A1331852, A1155463, luteolin, geldanamycin, tanespimycin, alvespimycin, panobinostat, nutlin-3a, ruxolitinib, and venetoclax.
  • a senotherapeutic agent that can be used as described herein can be a senotherapeutic agent as described elsewhere (see, e.g., Kirkland et al., J. Intern. Med., 288(5):518-536 (2020) at, for example, Table 1).
  • a mammal e.g., a human
  • a mammal e.g., a human
  • the anti-senescence treatment (and/or revised anti-senescence treatment) determined to be effective within that mammal can be continued as is (or with one or more slight alterations).
  • a method for treating a mammal can include administering a previously administered anti-senescence treatment (or slight modification thereof) to a mammal that received that previous anti-senescence treatment, where that previous anti-senescence treatment was determined to be effective within that mammal as described herein.
  • that previously effective anti-senescence treatment can be continued as is.
  • that previously effective anti-senescence treatment can be continued with one or more slight alterations.
  • a method for treating a mammal can include administering a first anti-senescence treatment to the mammal, determining that that first anti-senescence treatment is not effective within that mammal as described herein, and administering a second (e.g., a revised anti-senescence treatment) to the mammal (and optionally confirming the effectiveness of that revised anti-senescence treatment within that mammal) as described herein.
  • a mammal e.g., a human
  • a second e.g., a revised anti-senescence treatment
  • a method for treating a mammal can include administering an anti-senescence treatment to a mammal that received a previous anti-senescence treatment that was determined to be ineffective as described herein.
  • the administered an anti-senescence treatment can be different from that of the previous anti-senescence treatment that was determined to be ineffective.
  • the effectiveness of that administer anti-senescence treatment can be assessed as described herein.
  • a method of treating a mammal (e.g., a human) to inhibit or reduce cellular senescence can include a step of determining whether or not a circulating polypeptide signature of the mammal includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA13 polypeptide following a previous administration of an anti-senescence treatment to the mammal as compared to the level of the polypeptide(s) within a circulating polypeptide signature of the mammal prior to the previous administration.
  • the method can include administering the same anti-senescence treatment (or a slightly modified version thereof) to the mammal (e.g., human) if the circulating polypeptide signature of the mammal includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA I 3 polypeptide as compared to the level of the polypeptide(s) within a circulating polypeptide signature of the mammal prior to the previous administration of an anti-senescence treatment.
  • the mammal e.g., human
  • the method can include administering a revised anti-senescence treatment to the mammal (e.g., human) if the circulating polypeptide signature of the mammal lacks each of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA13 polypeptide as compared to the level of the polypeptide(s) within a circulating polypeptide signature of the mammal prior to the previous administration of an anti-senescence treatment.
  • a revised anti-senescence treatment to the mammal (e.g., human) if the circulating polypeptide signature of the mammal lacks each of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptid
  • the revised anti-senescence treatment can be a higher amount (e.g., 1.3x, 1.4x, 1.5x, 1.75x, or 2x) of the same senotherapeutic agent(s) of the previous administration of an anti-senescence treatment as compared to the amounts of the senotherapeutic agent(s) of the previous administration of an anti-senescence treatment.
  • the revised antisenescence treatment can be designed to include one or more senotherapeutic agents that are different from the senotherapeutic agent(s) of the previous administration of an antisenescence treatment.
  • any appropriate anti-senescence treatment, revised anti- senescence treatment, or senotherapeutic agent described herein can be administered to a mammal (e.g., a human) to inhibit or reduce cellular senescence within a mammal and/or to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease.
  • a mammal e.g., a human
  • an anti-senescence treatment e.g., one or more senotherapeutic agents
  • a composition e.g., a pharmaceutically acceptable composition
  • one or more senotherapeutic agents can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents.
  • a pharmaceutically acceptable carrier, excipient, or diluent can be a naturally occurring pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutically acceptable carrier, excipient, or diluent can be a non-naturally occurring (e.g., an artificial or synthetic) pharmaceutically acceptable carrier, excipient, or diluent.
  • Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, serum proteins (e.g., human serum albumin), water, propylene glycol, glycerol, and salts or electrolytes (e.g., phosphate salts, saline, protamine sulfate, and DMSO).
  • serum proteins e.g., human serum albumin
  • water propylene glycol
  • glycerol e.g., propylene glycol, glycerol
  • salts or electrolytes e.g., phosphate salts, saline, protamine sulfate, and DMSO.
  • An anti-senescence treatment (e.g., one or more senotherapeutic agents) can be administered to a mammal (e.g., a human) by any appropriate route (e.g., oral, intranasal, inhalation, transdermal, and parenteral).
  • a mammal e.g., a human
  • any appropriate route e.g., oral, intranasal, inhalation, transdermal, and parenteral.
  • an anti-senescence treatment can be administered to a mammal locally or systemically.
  • an anti-senescence treatment can be administered systemically by oral administration to a mammal (e.g., a human).
  • An anti-senescence treatment (e.g., one or more senotherapeutic agents) can be administered to a mammal (e.g., a human) at any appropriate frequency.
  • the frequency of administration of an anti-senescence treatment can be from about twice a day to about one every other day, from about once a day to about once a week, from about once a day to about once a month, from about once a week to about once a month, or from about twice a month to about once a month.
  • the frequency of administration can remain constant or can be variable during the duration of treatment.
  • An anti-senescence treatment (e.g., one or more senotherapeutic agents) can be administered to a mammal (e.g., a human) for any appropriate duration.
  • the duration can vary from several days to several weeks, from several weeks to several months, or from several months to several years.
  • methods for treating a mammal (e.g., a human) as described herein can include administering to the mammal one or more (e.g., one, two, three, four, or more) senotherapeutic agents as the sole active ingredient.
  • a composition containing one or more senotherapeutic agents can include the one or more senotherapeutic agents as the sole active ingredient in the composition for inhibiting or reducing cellular senescence.
  • methods for treating a mammal as described herein also can include administering to the mammal one or more (e.g., one, two, three, or more) additional agents used to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease and/or performing one or more (e.g., one, two, three, or more) therapies used to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease on the mammal.
  • additional agents used to treat a mammal having a disease or disorder characterized by excess cellular senescence
  • therapies used to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease on the mammal.
  • a combination therapy used to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease can include administering to the mammal (e.g., a human) one or more senotherapeutic agents as described herein and one or more (e.g., one, two, three, or more) additional agents used to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease.
  • the mammal e.g., a human
  • one or more senotherapeutic agents as described herein and one or more (e.g., one, two, three, or more) additional agents used to treat a mammal having a disease or disorder characterized by excess cellular senescence such as an age-related disease.
  • a course of treatment can be monitored as described herein.
  • the circulating levels of one or more (e.g., one, two, three, four, five, six, or more) polypeptides e.g., the level of IL23R polypeptides, the level of GCG polypeptides, the level of CCL5 polypeptides, the level of IL 17A polypeptides, and/or the level of CAI 3 polypeptides) within the mammal can be monitored over the course of treatment to determine whether or not the treatment is effective and/or remains effective over time.
  • Example 1 Senotherapeutic control of aging biomarkers in circulation and tissues
  • This Example describes the identification of circulating polypeptides that are secreted by senescent cells and/or that are modulated (e.g., decreased) by senescent cells.
  • circulating polypeptides can be used as markers for systemic signatures of senescence (e.g., senescent cell burden of that mammal).
  • Circulating plasma polypeptides increase and decrease in abundance in aging
  • Circulating biomarkers that are altered by aging prior to testing the influence of acute, intermittent senolytic targeting were identified.
  • discovery-based Olink Proximity Extension Assays were implemented to quantify the abundance of a diverse polypeptide panel, including SASP, metabolic, developmental, cell scaffolding, and signaling factors (Fig. 2).
  • the utility of this approach was to probe both senescence-related and generalized factors of systemic health to identify age-related factors and then assess on-target and off-target effects of senescence-targeting strategies.
  • IL23R, CCL5, and CCN1 significantly increased and CNTN4 and MIA significantly decreased in aged plasma.
  • the polypeptides that decreased in aged plasma were more likely cell membrane or cytoplasmic in origin, possibly related to DNA damage and transcriptional defects (Stegeman and Weake, J. Mol. Biol., 429:2427-2437 (2017)).
  • Most of the polypeptides that increased in aged plasma were secreted factors (Coppe et al., PLoS Biol., 6:2853-2868 (2008); and Freund et al., Trends Mol Med. , 16:238-246 (2010)). It was next sought to measure the downstream expression of these and other aging biomarkers in tissues.
  • Il23r gene expression was observed in old female kidney, liver, and adipose compared to young female mice. In old males, H23r expression also increased in kidney, liver, adipose, and lung compared to young male mice (Fig. 3J).
  • the age-dependent elevation of plasma IL23R polypeptide highly correlated with p!6 ,nk4a across tissues, with the most robust positive association identified between plasma IL23R and kidney p!6 mk4a expression in both sexes (Fig. 10A-10B).
  • 116 (Fig. 3N) was systemically increased in aging across multiple tissues including kidney, liver, spleen, cortex, and adipose, with Ccl5 more consistently increased relative to other analyzed chemokines.
  • Senescent cells express Il23r and Ccl5
  • IL23R or CCL5 could originate from senescent cells.
  • Mouse embryonic fibroblasts (MEFs) were exposed to lOGy irradiation (IR) and analyzed 10 days after IR- exposure, relative to sham cultures (Fig. 4).
  • IR-exposed MEFs were confirmed as senescent through senescence-associated P-galactosidase (SA-P-gal) staining, RT-PCR gene expression assessment, and multiplexed immunoassay SASP analysis.
  • SA-P-gal senescence-associated P-galactosidase
  • RT-PCR gene expression assessment RT-PCR gene expression assessment
  • multiplexed immunoassay SASP analysis multiplexed immunoassay SASP analysis.
  • IR induced significantly greater percentage Fig. 4B
  • Fig. 4C total number of SA-P-gal+ cells.
  • cells from IR cultures were characterized by larger nuclear areas (Fig. 4D).
  • senescent cell targeting through transgenic (AP) or pharmacological senolytic intervention reversed the direction of age-identified changes of plasma polypeptides.
  • Treatment groups were compared to old control mice using one-tailed statistical tests for polypeptides that were previously identified significant age-dependent changes (Fig. 3).
  • Factors in which a significant treatment-dependent rejuvenation effect was identified in either or both sexes were evaluated (Fig. 5).
  • AP, VEN, and LUT significantly reduced plasma abundance of IL23R
  • Fig. 5B In old male mice, VEN significantly reduced plasma abundance of IL23R (Fig. 5B).
  • AP and NAV significantly reduced plasma abundance of CCL5 in old females (Fig. 5C).
  • VEN, FIS, and LUT significantly reduced plasma abundance of GCG in old females
  • Fig. 5E In old males, VEN, NAV, FIS, and LUT significantly reduced plasma abundance of IL17A
  • Fig. 5H CA I 3 which was lower in old female plasma, increased following senolytic targeting treatment with AP, VEN, NAV, FIS, or LUT (Fig. 51).
  • senescent cell elimination reverts the abundance of age-associated circulating polypeptides to more youthful levels, including modulation of factors that are both increased and decreased in aging.
  • IL23R was most consistently regulated by age and senescent cell clearance across sexes.
  • Drugs targeting senescent cells concentrate in secretory organs
  • AP exhibited a low volume of distribution with relatively high plasma concentration compared to tissues.
  • the steadystate concentrations of AP achieved in plasma, kidney, and liver gradually decreased with an approximate half-life of 3-4 hours, but detectable levels remained at 72 hours (plasma: 2.1 - 6.6 pM, kidney: 0.2-0.8 pM, liver: 0.8-2.2 pM).
  • AP was detected in only one brain sample at one timepoint (0.07 pM, with a limit of detection of 0.04 pM).
  • maximum concentration in plasma 2.9-6.0 pM
  • kidney 2.8-4.1 pM
  • liver 18.8-29.5 pM
  • VEN was consistently detected in the brain (20-300 nM) at 20 minutes to six hours following the fourth treatment.
  • Senescence biomarkers are differentially altered across tissues by senotherapeutics
  • AP reduced Illb in females and /A/2 and Cal 3 in males (Fig. 14).
  • AP, VEN, FIS, and LUT increased expression of Cal3
  • LUT increased expression of Ccnl
  • Fig. 16 In female perigonadal adipose, NAV and FIS reduced Cal 3 (Fig. 16).
  • AP and LUT significantly reduced 116 and Ccnl expression in male perigonadal adipose; this was the only tissue where age-dependent increase in Ccnl was observed, and Ccnl remained unchanged in other tissues after senolytic treatment.
  • male cortex AP, VEN and FIS increased p21 (Fig. 17).
  • VEN exhibited the highest efficacy in mitigating senescence-related gene expression in our acute treatment paradigm, with partial phenocopy of AP-mediated senescent cell clearance in kidney.
  • Il23r and Ccl5 are senescence-associated tissue and plasma biomarkers that increase with age and are reduced by senotherapeutics
  • circulating polypeptide signature of a mammal can be used as an indicator of the systemic senescent cell burden of that mammal.
  • a mammal e.g., a human
  • an increased level of a IL23R polypeptide, an increased level of a GCG polypeptide, an increased level of a CCL5 polypeptide, an increased level of a IL17A polypeptide, and/or a decreased level of a CAI 3 polypeptide in blood (e.g., plasma) of a mammal (e.g., a human) can be used as an indicator of the systemic senescent cell burden of that mammal.
  • mice Male and female pl6-InkAttac mice (C57BL/6 background) were used in this study, and sex-specific features of age were analyzed separately. Mice were group-housed in ventilated cages with a constant temperature of 25 °C, 30-70% humidity, a 12-hour light/dark cycle, and provided standard chow diet and water ad libitum. At the time of tissue collection, young mice were 3-4 months of age and old mice were 16-20 months old. Mice were euthanized with a lethal dose of pentobarbital. Blood was acquired from the inferior vena cava with EDTA-coated syringes and stored in EDTA-coated tubes.
  • mice were transcardially perfused with ice-cold PBS. All mice were examined for gross pathology and tumor prevalence, with exclusion of mice harboring pronounced splenomegaly. Brain cortex, liver, kidney, spleen, lung, and perigonadal adipose were immediately removed and placed in Trizol® for gene expression analyses.
  • AP20187 drug dilution a 12.5 mg/mL stock solution in 100% ethanol was created and stored at -20°C.
  • One mL of AP20187 working solution (2 mg/kg) was prepared by mixing 40 pL of the stock solution with 100 pL PEG-400 and 860 pL of 2% Tween-20 in ddFEO.
  • the solution was vortexed and administered to the mice by intraperitoneal (i.p.) injection within 30 min. of diluting the stock.
  • the corresponding vehicle was composed of ethanol, PEG-400, and Tween-20 and was administered to control animals via i.p. injection.
  • a stock oral gavage vehicle was created as a solution of 60% Phosphatidylcholine in propylene glycol (Phosal 50 PG, Lipoid GmbH), 30% PEG-400, and 10% pure ethanol. Senolytic compounds were freshly vortexed into vehicle solution at room temperature and stored at 4°C the week of injections.
  • Old pl6-InkAttac mice were randomized (based on body weight and age) to receive vehicle or AP20187 (2 mg/kg, i.p. injection), NAV (50 mg/kg), VEN (50 mg/kg), FIS (50 mg/kg), or LUT (30 mg/kg) (by oral gavage). All mice received both gavage and i.p.
  • mice 18-month old mice were treated once daily with 2 mg/kg AP (IP), 50 mg/kg VEN (oral gavage), or control vehicle for four days. Mice were euthanized 20 minutes, 2 hours, 6 hours, or 72 hours following the fourth and final treatment for collection of flash-frozen tissues.
  • IP 2 mg/kg AP
  • VEN oral gavage
  • RT-PCR Real-time polymerase chain reaction
  • RNA samples collected at necropsy were immediately stored in Trizol® reagent.
  • RNA was isolated using Trizol®-based chloroform-isopropanol precipitation, followed by nanodrop concentration and purity analysis.
  • 3 pg of total RNA was used for cDNA synthesis through M-MLV reverse transcription (Invitrogen, cat# 18091200).
  • RT- PCR was performed on a QuantStudio5 RT-PCR system (ThermoFisher) with Taqman PrimeTime qPCR Primer assays from Integrated DNA Technologies (IDT).
  • the p!6 mk4a primer denotes coverage of the Cdkn2a variant 1 and pi6 total detects both Cdkn2a variant 1 and variant 2.
  • the Olink Target 96 Mouse Exploratory panel was used to analyze polypeptide biomarkers from mouse plasma by proximity extension assay (PEA) (Table 1). Targetspecific antibody pairs are linked to DNA oligonucleotides, and upon binding to the target polypeptide, the antibodies are brought into close proximity and the oligonucleotides hybridize. Polypeptide cycle threshold values were normalized by log2 scale, denoted as normalized protein expression (NPX). 25 polypeptides were excluded from age-associated and senolytic comparisons due to low or lack of detection. 1 pL plasma was used for PEA per sample.
  • PEA proximity extension assay
  • fibroblast culture model was used to validate hallmark markers of senescence ( 41 Baker et al., 2016).
  • Embryonic day 13.5 fibroblasts were harvested from C57BL/6 mice and cultured in media containing 10% fetal bovine serum (FBS) until passage 4 (P4).
  • FBS fetal bovine serum
  • P4 cells were plated in 6-well plates or T75 flasks.
  • FBS fetal bovine serum
  • lOGy RadSource Technologies RS 2000
  • Cells were harvested 24 hours after sham treatment or 10 days post irradiation. Twenty-four hours before harvest or fixing, culture media was replaced with FBS-free media.
  • Conditioned media was collected from T75 flasks, 0.2 pm filtered, and snap-frozen at -80 °C. Cells were trypsinized, resuspended in ImL TRIzol®, and snap-frozen at -80 °C prior to processing for RT-PCR.
  • DAPI 4',6-diamidino-2-phenylindole
  • HPLC high-performance liquid chromatography
  • MS mass spectrometry
  • HPLC followed by MS was performed using internal standards for AP and VEN, and measured within the plasma, brain, muscle, liver, and kidney of each animal.
  • Plasma polypeptide For PEA polypeptide data from plasma samples, we first conducted sex-stratified comparisons of young vehicle (YNG CON) versus old vehicle (OLD CON) samples using two-tailed Kruskal- Wallis rank sum and ordinal logistic regression tests. Beta-value estimates were determined, reflective of magnitude of change in aging. We considered a polypeptide was age-altered at a significance level of p ⁇ 0.05 (Fig. 2, 5, Table 1). For head-to-head senolytic drug treatments, statistical testing was performed to identify intervention of age-dependent polypeptides in plasma.
  • Each treatment group was then compared with the old vehicle group (OLD CON) by one-tailed, pairwise comparison using ordinal logistic regression.
  • the p-values were adjusted using Dunnett correction to account for multiple comparisons to the control group, and a significant drug modulation was denoted as p ⁇ 0.10 corresponding to hypothesis-directed directionality of change.
  • Sample sizes were 5-8 mice per group.
  • Tissue gene expression Similarly, for gene expression profiling, we first conducted tissue-specific, sex- stratified comparison of young vehicle and old sample data to initially assess evidence of age differences using two-tailed Kruskal-Wallis rank sum and ordinal logistic regression models that included the terms sex, tissue type, and group (young versus old) fit for each gene. Genes with an age group effect at the p ⁇ 0.05 significance level were identified for further evaluation. Using this subset of genes and considering only sample data derived from vehicle or drug-treatment of old animals, ordinal logistic models were fit that included sex, tissue type, and group variables. For genes with an overall group difference of p ⁇ 0.10, one-tailed comparisons were made between old drug-treated and the old vehicle group for each tissue type.
  • Circulating plasma proteins increased and decreased in abundance with aging
  • transcript expression of genes encoding age-increased plasma factors alongside canonical senescence markers were measured across tissues in old and young p!6-InkAttac mice.
  • the results are shown as RT-PCR gene expression data in individual sample cycle threshold (Fig. 25) and relative expression (Figs. 19A-19F), ordinal regression (Figs. 19G- 19N), and normalized relative expression heatmap (Fig. 33)plots.
  • Fig. 19A individual sample cycle threshold
  • Figs. 19A-19F relative expression
  • Figs. 19G- 19N ordinal regression
  • Fig. 33 normalized relative expression heatmap
  • p21 expression was higher in aged kidney in both sexes and in female adipose tissue (Figs. 19A and 19E).
  • the SCAP gene Bcl2 increased in old male and female spleen (Fig. 19C).
  • Bcl2ala a downstream effector and cytokine-stimulated mediator of the BCL2 cell survival pathway, was higher in aged kidney in both sexes, liver in females, and spleen in males (Fig. 191). Higher expression of Il23r was observed in old female kidney, liver, and adipose, and in old male liver, adipose, and lung (Figs. 19A, 19B, 19E, 19F, and 19J).
  • the kidney was characterized by consistent age-elevated senescence gene signatures (Fig. 19), in which pl6 mk4a , Cdkn2a, Bcl2ala, Ccl2, Ccl3, Ccl5, Illb, 116, and 1123r, and/or Tnf gene expression were positively associated with plasma IL23R, CCL2, CCL3, CCL5, and/or IL17A and negatively associated with MIA (Fig. 26). It was shown that IL23R and/or CCL5 plasma protein also increased concomitantly with the expression of p!6, SASP, and SCAP factors in kidney, liver, spleen, and adipose (Fig.
  • IR irradiation-induced senescence was implemented in mouse embryonic fibroblasts (MEFs) (Fig. 28).
  • IR induced a greater percentage (Fig. 28B) and total number (Fig. 28C) of SA-P-gal+ cells compared to sham. Cells from IR cultures were characterized by larger nucleus areas (Fig. 28D).
  • senescence and SASP genes were analyzed (Fig. 28E).
  • pl6 mk4a , Cdkn2a, p21, Gdfl5, 116, and Mmp3 expressions increased in IR MEFs.
  • 1123r expression was 2.4 ⁇ 0.3-fold greater
  • Cc/5 expression was 6.3 ⁇ 0.5-fold greater in IR MEFs, compared to the sham cultures.
  • Mia expression also increased in IR MEFs, but Ccnl expression was lower than the sham.
  • CA I 3 which was lower in old female plasma, increased following treatment with AP, VEN, NAV, FIS, or LUT (Fig. 20J).
  • CNTN4 was also lower in old female plasma and increased following treatment with NAV (Fig. 20L).
  • senotherapeutic compounds reverted the abundance of age-associated circulating proteins to more youthful levels, including modulation of factors that were both increased and decreased with aging.
  • IL23R was most consistently regulated by age and senotherapeutics across sexes. It was also tested whether any drugs exacerbated the age-dependent change in plasma protein levels.
  • FIS further increased the age-dependent change in CDH6, CPE, FSTL3, MIA, QDPR, and CCN4.
  • VEN further increased the age-dependent change in CDH6 and CNTN4.
  • AP exhibited a low volume of distribution with relatively high plasma concentration compared to tissues.
  • the steady-state concentrations of AP achieved in plasma, kidney, and liver gradually decreased with an approximate half-life of 3-4 hours, but detectable levels remained at 72 hours (plasma: 2.1 - 6.6 pM, kidney: 0.2-0.8 pM, liver: 0.8- 2.2 pM).
  • maximum concentration in plasma 2.9-6.0 pM
  • kidney 2.8-4.1 pM
  • liver 18.8-29.5 pM
  • Il23r and Ccl5 are senescence-associated tissue biomarkers that increased with age and were reduced by senotherapeutics
  • Ccl5 expression in tissues decreased in response to each intervention (Fig. 22).
  • Cel 5 exhibited widespread differential expression among aged organs and was most abundantly expressed in the aged spleen (Fig. 25).
  • VEN, FIS, and LUT decreased Cc/5 expression (Fig. 22E).
  • VEN treatment decreased Ccl5 transcript abundance in female kidney, spleen, and cortex (Figs. 22A, 22E, and 22K).
  • Significant reductions in Ccl5 in males occurred following LUT treatment in liver (Fig. 22D) and by VEN treatment in spleen (Fig. 22F).
  • FIG. 23 It was also tested whether senotherapeutics mitigated age-associated Il23r transcriptional changes (Fig. 23).
  • AP treatment reduced Il23r expression (Figs. 23 A and 23C).
  • VEN reversed the age-dependent increase o ll23r in female kidney (Fig. 23 A), liver (Fig. 23C), spleen (Fig. 23E), and adipose (Fig. 231).
  • FIS reversed the age-dependent increase Ct H23r in kidney (Fig. 23B).
  • FIS reduced Il23r expression in old female kidney and spleen, and LUT reduced Il23r in kidney, compared to controls (Figs. 23A and 23E).
  • IL23R protein increased in aged human plasma
  • IL23R enzyme-linked immunosorbent assay
  • mice Male and female heterozygous pl6-InkAttac mice (C57BL/6 background) were used in this study, and sex-specific features of age were analyzed independently. Mice were group-housed in ventilated cages with a constant temperature of 25°C, 30-70% humidity and a 12-hour light/dark cycle. Water and the 5053 PicoLab Rodent Diet 20 were provided ad libitum. At the time of tissue collection, young mice were 3-4 months of age and old mice were 16-20 months old. Mice were euthanized with a lethal dose of pentobarbital. Blood was acquired from the inferior vena cava with EDTA-coated syringes and stored in EDTA-coated tubes.
  • mice were transcardially perfused with ice-cold PBS. All mice were examined for gross pathology and tumor prevalence, with exclusion of mice harboring pronounced splenomegaly. Brain cortex, liver, kidney, spleen, lung, and perigonadal adipose were immediately removed and placed in TRIzol" for gene expression analyses.
  • AP20187 drug dilution a 12.5 mg/mL stock solution in 100% ethanol was created and stored at -20°C.
  • One mL of AP20187 working solution (2 mg/kg) was prepared by mixing 40 pL stock solution with 100 pL PEG-400 and 860 pL of 2% Tween-20 in ddFFO. The solution was vortexed and administered to mice by intraperitoneal (i.p.) injection within 30 minutes of diluting the stock.
  • the corresponding vehicle was composed of ethanol, PEG-400, and Tween-20 and was administered to control animals via i.p. injection.
  • a stock oral gavage vehicle was created as a solution of 60% Phosphatidylcholine in propylene glycol (Phosal 50 PG, Lipoid GmbH), 30% PEG-400, and 10% ethanol. Senolytic compounds were freshly vortexed into vehicle solution at room temperature and stored at 4°C the week of injections.
  • Old pl6-InkAttac mice were randomized (based on body weight and age) to receive vehicle or AP20187 (2 mg/kg, i.p. injection), NAV (50 mg/kg), VEN (50 mg/kg), FIS (50 mg/kg), or LUT (30 mg/kg) by oral gavage. All mice received both gavage and i.p.
  • NAV ABT-263, #11500
  • LUT 3',4',5,7-Tetrahydroxyflavone, #10004161
  • VEN ABT- 199, #A8194
  • FIS 3,7,3’,4’-Tetrahydroxyflavone, #T0121
  • AP20187 was acquired from WuXi AppTec. All other reagents, unless otherwise denoted, were acquired from Sigma- Aldrich/Millipore.
  • mice received 5 consecutive daily administrations for one week, two weeks off, and then another 5-day treatment. Treatments were discontinued one week prior to necropsy (Fig. 20A).
  • Fig. 20A For the pharmacokinetic study of drug accumulation/elimination in tissue, 18-month old mice were treated once daily with 2 mg/kg AP (i.p.), 50 mg/kg VEN (oral gavage), or control vehicle for four days. Mice were euthanized 20 minutes, 2 hours, 6 hours, or 72 hours following the fourth and final treatment, perfused with ice-cold PBS, and collected tissues were flash- frozen in liquid nitrogen.
  • RT-PCR Real-time polymerase chain reaction
  • RNA samples collected at necropsy were immediately stored in TRIzol® reagent.
  • RNA was isolated using TRlzol®-based chloroform-isopropanol precipitation, followed by nanodrop concentration and purity analysis.
  • 2 pg of total RNA was used for cDNA synthesis through M-MLV reverse transcription (Invitrogen, #18091200).
  • RT-PCR was performed on a QuantStudio5 RT-PCR system (ThermoFisher) with PerfeCTa FastMix II Low ROX (Quantabio) and Taqman PrimeTime qPCR Primer assays from Integrated DNA Technologies (IDT).
  • Experimental genes were normalized to Hprt or Tbp housekeeper gene expression. Relative gene expression compared to the young control group was derived from the 2’ AACT value for each tissue sample. In studies involving RT-PCR analyses in young versus old tissues, CT values were reported in addition to the relative expression results (Fig. 25). In system-wide comparisons across multiple drug treatments, both the relative expression and total abundance of the transcripts (cycle threshold values) were examined according to tissue of origin and age. The total abundance qualitative comparison was feasible since cDNA was synthesized from the same amount of input RNA per tissue sample (2 pg).
  • Kidney was collected from 4-month and 24-month old mice, fixed in 4% PFA for 24 hours, sectioned at 5 pm, and embedded in paraffin.
  • the RNAscope fluorescent multiplex assay (ACD-Bio/Biotechne) was performed on FFPE (formalin fixed, paraffin embedded) tissue, according to the manufacturer’s specifications and protocols.
  • Cdkn2a,p ' ld nk4a probe (#447491, Mm-Cdkn2a-tv2) was used in channel 1 and paired with TSA Vivid 520 fluorophore (#323271).
  • TSA Vivid fluorophores were reconstituted in 100 pL DMSO and diluted with TSA Buffer at a 1 : 1000 concentration. Tissues were quenched with TrueBlackPlus (IX in PBS, Biotium) for 10 minutes prior to adding Vectashield and coverslip.
  • Sections were imaged with a Nikon Ti2 Eclipse Inverted microscope with 10X, 20X, and 40X Plan Apo objectives, 8-channel Spectra III light engine, and using the Orca Fusion BT sCMOS camera with Nikon Elements AR software. 405, 488, 561, 647 laser lines were used with an image exposure time of 100-200 ms per channel in 16- bit readout mode.
  • the Olink Target 96 Mouse Exploratory panel was used to analyze 92 protein biomarkers from mouse plasma by proximity extension assays (PEA).
  • PEA proximity extension assays
  • Target-specific antibody pairs were linked to DNA oligonucleotides, and upon binding to the target protein, the antibodies were brought into close proximity and the oligonucleotides hybridized, enabling amplification with signal quantification.
  • Protein cycle threshold values were normalized by log2 scale, denoted as Normalized Protein Expression (NPX). Sixty seven proteins were analyzed and reported. Twenty five proteins were excluded from age- associated and senolytic comparisons due to low or lack of detection. 1 pL plasma was used for PEA per sample.
  • Samples corresponded to 40 women and 40 men between 20 and 90 years of age. Participants with a history of cancer, other than breast cancer and melanoma, before the age of 50, or autoimmune diseases (e.g., rheumatoid arthritis, lupus), and women with BMI less than 18.5 kg/m 2 or greater than 40.0 kg/m 2 and men with BMI less than 18.5 kg/m 2 or greater than 38.0 kg/m 2 were excluded. One male participant was excluded as an outlier on the basis of BMI by Grubbs outlier test. EDTA blood was collected and processed into platelet-poor plasma (PPP) immediately, and aliquots were frozen at -80°C until used.
  • PPPP platelet-poor plasma
  • EDTA PPP One milliliter of EDTA PPP was thawed on ice and spun down at 15000 x g, 4°C for 5 minutes to remove insoluble particles. The supernatant was transferred to a fresh clean tube, homogenized, and re-aliquoted into smaller aliquots to use on each day to avoid repeated freeze-thaw cycles, and these aliquots were kept at -80°C.
  • Ancillary Reagent Kit for ELISA (DY008B, R&D/Bio-Techne) was used and the antibody pair from the Human IL-23R DuoSet ELISA (DY1400-05,R&D Systems/ Bio-Techne) was used for the development of an ELISA protocol with feasible performance to quantify the amount of soluble IL-23R in EDTA plasma.
  • the optimal condition was analyzed for linear range, accuracy, precision, and parallelism. The experiments were run according to the manufacturer’s protocol, except for the blocking buffer (2% BSA, 4 mM EDTA in PBS) and sample/standard diluent (BSA 1%, EDTA 4 mM, in PBS).
  • the linear range was 94-6000 pg/mL, and the recovery ranged from 80-105%.
  • the optical density was read using iD3 Spectramax (Molecular Devices) and 5 parameter non-linear regression was used to generate the standard curve and interpolated the results using SoftMax Pro Software version 6.1 (Molecular Devices).
  • fibroblast culture model was used to validate hallmark markers of senescence (Baker et al., Nature, 530: 184-189 (2016)).
  • Embryonic day 13.5 fibroblasts were harvested from C57BL/6 mice and cultured in DMEM containing 10% fetal bovine serum (FBS) until passage 4 (P4).
  • FBS fetal bovine serum
  • P4 cells were plated in 6-well plates or T75 flasks. At 40% confluency, cells were subjected to sham conditions or irradiated at lOGy (RadSource Technologies RS 2000). Cells were harvested 24 hours after sham treatment or 10 days post irradiation. Twenty-four hours before the harvest or fixing, culture media was replaced with FBS-free media.
  • Conditioned media was collected from T75 flasks, 0.2 pm filtered, and snap-frozen at -80°C. Cells were trypsinized, resuspended in 1 mL TRIzol®, and snap-frozen at -80°C prior to processing for RT-PCR.
  • the resulting classifier was then run on the entire set of images producing binary masks.
  • Cell Profiler was used to quantify and describe the cells and SA-P-Gal stained regions within each image (Stirling et al., BMC Bioin, 22:433 (2021)).
  • Tiff files of DAPI staining were used for the cells and the tiff files produced by iLastik were used for SA-P-Gal.
  • DAPI images were run through the ColorToGray module to generate a grayscale image after which both the DAPI grayscale and the iLastik SA-P-Gal binary images were ran through the following modules (with distinct settings): IdentifyPrimaryObjects, MeasureObjectSizeShape, OverlayOutlines, Saveimages, and ExportTo Spreadsheet. The objects identified in IdentifyPrimaryObjects were overlayed onto images and saved for review. All measurements were exported into *.csv files and used for quantification.
  • conditioned media was aspirated off from cells grown in T75 flasks and the cell monolayer washed once with 5 rnL of IX PBS.
  • Two mL of TrypLE (Gibco) was added for 4 minutes at room temperature after which the flasks were tapped against the bench three times to dislodge all cells, and the TrypLE was quenched with 5 mL of MEF media.
  • the cell suspension was pipetted over the flask three times, then transferred to a 15 mL tube, spun at 188 x g for 5 minutes at 22°C, the supernatant was aspirated, the pellet resuspended in 1 mL of Trizol®, solution was transferred to a 1.5 mL tube, and stored at -80°C. RNA extraction was then performed as described above.
  • fibroblasts Primary human lung fibroblasts were purchased from ScienCell and cultured in EMEM containing 10% fetal bovine serum. Senescence was induced by treating cells with 20 pM etoposide for 48 hours, followed by 6 days in normal culture media as described elsewhere (Schafer et al., Nat Comm, 8: 14532 (2017); (Zhang et al., Aging Cell, 20:el3486 (2021)). Proliferating and senescence cells were then plated into 96-well plates and allowed to attach. After 6 hours, media was changed to EMEM containing 0% FBS and the indicated concentration of compound was added.
  • etoposide-induced senescent primary human lung fibroblasts were cultured for 24 hours with 1 pM staurosporine to induce apoptosis and determine the ceiling of cleaved caspase-3 activity (Fig. 41).
  • Cells were fixed, and immunostained for cleaved caspase-3 and DAPI, with 4 samples collected per group. Percent change in cleaved caspase-3 intensity was normalized to the greatest response, and concentration-response curves were fit by the Hill curve.
  • Human plasma IL23R data were first tested for normality by Shapiro-Wilk and Kolmogorov- Smirnov tests. Both male and female data were found to be normally distributed and best fit by Pearson linear regression and correlation R values determined.
  • Step 2 Group-level effects in old plasma.
  • Step 3 Drug-dependent effects in old plasma. Finally, pairwise comparisons were performed to test the hypothesis that drug treatment would reverse the trajectory of the previously detected age-dependent effects.
  • a one-sided test was performed comparing each treated group (OLD AP, OLD VEN, OLD NAV, OLD FIS, and OLD LUT) with OLD VEH.
  • the alternative hypothesis of interest was that the mean value of treated group was closer to the young vehicle values.
  • an estimate value was generated in comparison of treated group to OLD VEH. P-values were adjusted using Dunnett correction to account for multiple comparisons compared to the OLD VEH group.
  • a first blood sample (e.g., a first plasma sample) is obtained from a human prior to the human being administered an anti-senescence treatment (e.g., prior to being administered one or more senotherapeutic agents), and a second blood sample (e.g., a second plasma sample) is obtained from the human after the human is administered the anti-senescence treatment.
  • an anti-senescence treatment e.g., prior to being administered one or more senotherapeutic agents
  • a second blood sample e.g., a second plasma sample
  • the first and second blood samples are assessed to determine whether or not the circulating polypeptide signature in the second blood sample includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CAI 3 polypeptide as compared to the circulating polypeptide signature of the first blood sample.
  • the circulating polypeptide signature of the second blood sample includes one or more of a decreased level of a IL23R polypeptide, a decreased level of a GCG polypeptide, a decreased level of a CCL5 polypeptide, a decreased level of a IL17A polypeptide, and an increased level of a CA I 3 polypeptide as compared to the circulating polypeptide signature of the first blood sample, then the anti-senescence treatment (e.g., one or more senotherapeutic agents) can be determined to be an effective treatment for that human. In this case, the human can continue with the same anti-senescence treatment.
  • the anti-senescence treatment e.g., one or more senotherapeutic agents
  • the anti-senescence treatment e.g., one or more senotherapeutic agents
  • the human can discontinue that particular anti- senescence treatment.
  • that human also can initiate an increased dosing regimen of that particular anti-senescence treatment or can initiate a different anti-senescence treatment.

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Abstract

Ce document concerne des procédés et des matériels pour utiliser et/ou évaluer un ou plusieurs agents sénothérapeutiques. Par exemple, l'invention concerne des procédés et des matériels pour déterminer l'efficacité d'un traitement anti-sénescence chez un mammifère (par exemple, un être humain) ainsi que des procédés et des matériels pour traiter efficacement un mammifère avec un traitement anti-sénescence.
PCT/US2024/032575 2023-06-05 2024-06-05 Procédés et matériels pour utiliser et évaluer des traitements anti-sénescence chez des mammifères Pending WO2024254165A2 (fr)

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