Methods and materials for using and assessing Anti-senescence treatments within mammals

Abstract

This document relates to methods and materials for using and / or assessing one or more senotherapeutic agents. For example, methods and materials for determining the efficacy of an anti-senescence treatment in a mammal (e.g., a human) as well as methods and materials for effectively treating a mammal with an anti-senescence treatment are provided.

Description

[0001] METHODS AND MATERIALS FOR USING AND ASSESSING ANTISENESCENCE TREATMENTS WITHIN MAMMALS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 471, 193, filed on June 5, 2023. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] STATEMENT REGARDING FEDERAL FUNDING

[0005] This invention was made with government support under AG071686 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] 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). In some cases, 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). For example, 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.

[0008] BACKGROUND

[0009] 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. Res., 72:9-17 (2006); Wang et al., Circulation, 132: 1909-1919 (2015); Roos et al., Aging Cell, 15:973-977 (2016); and Schafer et al., Mol. Cell. Endocrinol. , 455:93-102 (2017)), osteoporosis (Khosla et al., Nat. Rev. Endocrinol . , 16:263-275 (2020)), kidney disease (Li et al., FASEB J., 35:e21229 (2021)), hepatic damage (Ogrodgnik et al., Nat. Commun., 8: 15691 (2017)), disc and muscle injury (Zhang et al., Nat. Aging, 2:601-615 (2022)), and cognitive decline (Chinta et al., Exp. Gerontol., 68:3-7 (2015); Bussian et al., Nature, 562:578-582 (2018); Musi et al., Aging Cell, 17:el2840 (2018); Dehkordi et al., Nat. Aging, 1 :1107-1116 (2021); and Zhang et al., Nat. Commun., 13:5671 (2022)).

[0010] SUMMARY

[0011] 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). In some cases, 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). For example, a circulating polypeptide signature of a sample (e.g., a blood sample such as a plasma sample) obtained from a mammal (e.g., a human) having been administered one or more senotherapeutic agents can be used to determine and / or classify the efficacy of the one or more senotherapeutic agents. In some cases, 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. For example, an anti-senescence treatment (e.g., one or more senotherapeutic agents) can be administered to a mammal having a disease or disorder characterized by the presence of senescent cells or elevated cellular senescence, and 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. In some cases, 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. In some cases, 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.

[0012] As demonstrated herein, 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). As also described herein, treating a mammal with an anti-senescence treatment (e.g., administering one or more senotherapeutic agents to a mammal) 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. In some cases, when 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. In some cases, when 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. For example, 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. As described herein, 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. In general, 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.

[0013] In another aspect, 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 determine that the mammal has the decreased level of the circulating IL23R polypeptides, the decreased level of the circulating GCG polypeptides, the decreased level of the circulating CCL5 polypeptides, the decreased level of the circulating IL17A polypeptides, and the increased level of the circulating CA13 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 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.

[0014] In another aspect, 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.

[0015] In another aspect, 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.

[0016] In another aspect, 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, to determine that the mammal has the decreased level of the circulating IL23R polypeptides, the decreased level of the circulating GCG polypeptides, the decreased level of the circulating CCL5 polypeptides, the decreased level of the circulating IL17A polypeptides, and the increased level of the circulating CA13 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 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. In another aspect, 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.

[0017] In another aspect, 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.

[0018] In another aspect, 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.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0020] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0021] BRIEF DESCRIPTION OF THE DR WINGS

[0022] Figure 1. Study design for young and old pl6-hikAttac mice treated with a senotherapeutic or vehicle. Treatment groups of young and old mice that received either vehicle or AP20187 (AP) by intraperitoneal (i.p.) injection and either vehicle or one of four senolytic drugs (venetoclax (VEN), navitoclax (NAV), fisetin (FIS), or luteolin (LUT)) by oral gavage. Beginning on week one, mice were treated 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.

[0023] Figures 2A-2B. Conserved and distinct plasma polypeptides were altered by age in female and male p!6-InkAttac mice. Shown are beta estimates and confidence intervals corresponding to comparison of old control (OLD CON) versus young control (YNG CON) polypeptide abundance in (Figure 2A) female and (Figure 2B) male plasma samples. Statistically significant differences between OLD CON and YNG CON are highlighted in orange (p < 0.05, two-sided Kruskal-Wallis rank sum test), with negative values indicating decrease in polypeptide abundance in aging, and positive values indicating increase in polypeptide abundance in aging samples, n = 5-8 mice per age and sex.

[0024] 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) pl6mk4a, (Figure 3H) pl6total, (Figure 31) Bcl2ala, (Figure 3J) Il23r, (Figure 3K) Ccl3, (Figure 3L) Ccl5, (Figure 3M) [lib. and (Figure 3N) 116. Symbols in G-L denote ordinal prediction mean ± standard error. * p < 0.05 significance between old and young mice of same sex. n = 5-8 mice per age and sex.

[0025] 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. Figure 4F) Concentrations of senescence analytes measured in pg / mL with Luminex multiplex assay, Bars denote mean ± s.e.m., * p < 0.05 vs sham by Mann- Whitney U test, with multiple-comparison correction for data in Figures 4E and 4F. n = 6 samples per group.

[0026] 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. Polypeptide values are reported as normalized protein expression (NPX). Bars summarize the median group values, n = 5-8 mice per treatment group and sex. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. # < 0.05 and * p < 0.10.

[0027] 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 6C, 6D, 6G, 6H, 6L, and 6L) p!6totalcorresponding to exon 3-4 and detects both Cdkn2a transcript variants 1 and 2. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. one-tailed * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex. Similar tissue analyses for liver, lung, and perigonadal adipose, and are shown in Figure 11.

[0028] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. one-tailed * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0029] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. one-tailed * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0030] Figure 9. Cycle threshold values from RT-PCR gene expression in young (Y) and old

[0031] (O) tissues from female and male mice. Figures 10A-10D. Figures 10A-10B) Spearman correlations between pl6mk4aRT- 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. R-values are provided, n = 5-8 samples per group.

[0032] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0033] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0034] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0035] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0036] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex

[0037] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0038] 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. YNG CON versus OLD CON reflect statistics provided in Figure 3 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0039] Figure 18. Conserved and distinct plasma proteins were altered by age in female and male mice. Beta estimates and confidence intervals corresponding to comparison of old control (OLD CON) versus young control (YNG CON) protein abundance as determined by Olink proximity extension assays are shown in female and male plasma samples. Statistically significant differences between OLD CON and YNG CON are in gray (p < 0.05, two-sided Kruskal-Wallis rank sum test), with negative values indicating a decrease in protein levels in aging, and positive values indicating an increase in protein abundance in aging samples, n = 5-8 mice per age and sex.

[0040] 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. Symbols in G-L denote ordinal prediction estimate ± standard error. * p < 0.05 significance between old and young mice of same sex. n = 5-8 mice per age and sex.

[0041] Figures 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. 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. Protein values were measured by Olink proximity extension assays and are reported as Normalized Protein Expression (NPX). Bars summarize the median values, n = 5-8 mice per treatment group and sex. YNG CON versus OLD CON reflect statistics provided in Figure 19 with exact p-values provided. Kruskal- Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. # p < 0.05 and * p < 0.10.

[0042] 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,nk4agene 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 (pl6mk4a), and (Figures 21C, 2 ID, 21G, 21H, 21K, and 21L) p!6mk4adetected only transcript variant 2. YNG CON versus OLD CON reflect statistics provided in Figure 19 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. One-tailed * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0043] 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. YNG CON versus OLD CON reflect statistics provided in Figure 19 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. One-tailed * p < 0. 10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0044] Figures 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. YNG CON versus OLD CON reflect statistics provided in Figure 19 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. One-tailed * p < 0. 10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0045] Figures 24A-24C. Plasma IL23R protein increased with age in humans. Measurement of IL23R (pg / mL) in human EDTA plasma in (Figure 24A) combined, (Figure 24B) female, and (Figure 24C) male study participants aged 20 to 82 years. Best fit line and 95% confidence interval (dashed lines) were determined by linear regression. Females: (F = 4.545, df = 38, p = 0.0395). Males (F = 4.837, df = 37, p = 0.0342). Pearson correlation values are provided. Figure 25. Cycle threshold values from RT-PCR gene expression in young (Y) and old (O) tissues from female (pink) and male (blue) mice. The maximum number of cycles for each experiment was 40.

[0046] Figures 26A-26H. Spearman correlations between tissue gene expression of senescence factors in (Figures 26A-26B) kidney, (Figures 26C-26D) liver, (Figures 26E-26F) spleen, and (Figures 26G-26H) perigonadal adipose, and the plasma proteins CA13, CCL2, CCL3, CCL5, CCN1, IL17A, IL23R, and MIA for (Figures 26A, 26C, 26E, 26G) female and (26B, 26D, 26F, and 26H) male mice. R-values are provided, n = 5-8 samples per group.

[0047] Figures 27A-27J. In situ hybridization (RNAscope) o H23r+ cells co-localized to p!6ink4aand Cd3e in the renal sinus and perivascular areas of aged mouse kidney. Figure 27A) Representative images of p!6mk4aand 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‘nk4cIl23r, and Cd3e from young (top, 2 months old) and old (bottom, 24 months old) kidney tissue from mice on p!6-InkAttac background. Young (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!6mk4ain 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!6mk4a. All scale bars = 100 pm.

[0048] 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. Figure 28G) Normalized protein expression (NPX) of senescence-related proteins in conditioned media measured with Olink PEA assay. Inset shows IL23R values re-scaled. Bars denote mean ± s.e.m., * p < 0.05 vs. sham by Mann- Whitney U test, with Holm-Sidak multiple-comparison correction for data in Figures 28E, 28F, & 28G. n = 6 samples per group.

[0049] Figures 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 29E-29F) Etoposide-induced senescent fibroblast (Figure 29E) or proliferating fibroblast (Figure 29F) concentration-response curves of normalized cleaved caspase intensity by application of VEN (circles), NAV (squares), FIS (triangles), or LUT (downward triangles) compounds. Symbols denote mean ± s.e.m., n = 4 samples per group.

[0050] 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. YNG CON versus OLD CON reflect statistics provided in Figure 19 with exact p- values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0051] 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. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON. One-tailed * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex. Figure 32. Schematic of data analyses.

[0052] Figure 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. Left axis: F denotes female, M denotes male mice. KID = kidney; LIV = liver; SPN = spleen; CTX = cortex; ADP = perigonadal adipose; LNG = lung, n = 5-8 mice per age and sex. Data points of relative gene expression for each sample and median values for young and old can be found in Figures 19A-19F.

[0053] Figure 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. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0. 10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0054] Figure 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. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal- Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0055] Figure 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. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal- Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0056] Figure 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. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal-Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0057] Figure 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. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal- Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0058] Figure 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. YNG CON versus OLD CON reflect statistics provided in Figure 2 with exact p-values provided. Kruskal- Wallis tests with multiple comparison correction were used for comparison of old treatment groups with OLD CON, * p < 0.10. Bars summarize the median group values, n = 5-8 mice per group and sex.

[0059] 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.

[0060] 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).

[0061] DETAILED DESCRIPTION

[0062] 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-senescence treatment is effective in that mammal and can be continued as is (or with one or more slight alterations) in that mammal. In some cases, 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.

[0063] 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. Examples of mammals that can be assessed and / or treated as described herein include, without limitation, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, naked mole rats, guinea pigs, and hamsters. In some cases, a mammal that can be administered an anti-senescence treatment and have its effectiveness assessed as described herein can be a female mammal. In some cases, a mammal that can be administered an anti- senescence treatment and have its effectiveness assessed as described herein can be a male mammal. When assessing and / or treating a mammal (e.g., a human) as described herein, the mammal can be any appropriate age. For example, when assessing and / or treating a human as described herein, the human 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).

[0064] 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. For example, 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. As described herein, a circulating polypeptide signature (e.g., the circulating level of one or more particular polypeptides) 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. For example, 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. Accordingly, 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).

[0065] If 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). For example, when an anti-senescence treatment is 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.

[0066] If an anti-senescent treatment is determined and / or classified to not be effective, a revised anti-senescence treatment can be administered. For example, when an antisenescence treatment is determined not to be effective within a mammal, 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.

[0067] In some cases, 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).

[0068] In some cases, a sample (e.g., a blood sample such as a plasma sample) can be obtained from a mammal (e.g., a human) having been administered an anti-senescence treatment (e.g., having been administered one or more senotherapeutic agents) to determine whether or not the anti-senescence treatment is effective within that mammal. In some cases, 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. For example, 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.

[0069] A circulating polypeptide signature described herein can include any appropriate number of polypeptides. For example, a circulating polypeptide signature can include any one or more (e.g., one, two, three, four, five, six, or more) polypeptides. In some cases, 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. Examples of 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.

[0070] Examples of IL23R 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 UniProt Knowledgebase (UniProtKB; see, e.g., The UniProt Consortium, Nucleic Acids Research, 51(D1):D523- D531 (2023)) at Accession Nos. Q8K4B4 and Q5VWK5.

[0071] Examples of 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.

[0072] Examples of 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.

[0073] Examples of 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.

[0074] Examples of 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.

[0075] In some cases, 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. In some cases, a circulating polypeptide signature can include an altered level (e.g., an increased level or a decreased level) of one or more polypeptides. In some cases, 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. For example, 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.

[0076] In some cases, 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. For example, 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.

[0077] In some cases, 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. For example, 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. In some cases, 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.

[0078] It will be appreciated that levels of polypeptides from comparable samples are used when determining whether or not a particular level is an altered level of a polypeptide.

[0079] In some cases, 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.

[0080] A sample (e.g., a blood sample such as a plasma sample) can be obtained from a mammal (e.g., a human) before and / or after the mammal has been administered an antisenescence treatment (e.g., one or more senotherapeutic agents). In some cases, a sample (e g., a blood sample such as a plasma sample) can be obtained from a mammal (e.g., a human) at any appropriate time before the mammal has been administered an anti-senescence treatment and assessed to determine the circulating polypeptide signature of the mammal prior to being administered the anti-senescence treatment. For example, a sample (e.g., a blood sample such as a plasma sample) can be obtained from a mammal (e.g., a human) one hour, six hours, 12 hours, one day, two days, three days, five days, one week, two weeks, three weeks, four weeks, on month, two months, three months, four months, six months, eight months, ten months, one year, two years, three years, or five years before the mammal was administered an anti-senescence treatment and assessed to determine the circulating polypeptide signature of the mammal prior to being administered the anti-senescence treatment. 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.

[0081] In some cases, a sample (e.g., a blood sample such as a plasma sample) can be obtained from mammal (e.g., a human) during the time that the mammal is administered an anti-senescence treatment and assessed to determine the circulating polypeptide signature within the mammal at that time. For example, a sample (e.g., a blood sample such as a plasma 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.

[0082] In some cases, to assess the effectiveness of an anti-senescence treatment within a mammal, a sample (e.g., a blood sample such as a plasma 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. For example, a sample (e.g., a blood sample such as a plasma sample) can be obtained from a mammal (e.g., a human) five or more weeks (e.g., about 5 weeks)after an initial administration of an antisenescence treatment and assessed to determine the circulating polypeptide signature within the mammal after being administered the anti-senescence treatment. 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.

[0083] In some cases, the circulating polypeptide signature within a mammal (e.g., within a sample obtained from 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). For example, two or more (e.g., two, three, four, five, six, or more) samples (e.g., blood samples, urine samples, or saliva samples) can be obtained from a mammal at different time points over the course of an anti-senescence treatment (and / or a revised anti-senescence treatment), and 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).

[0084] In some cases, a first sample (e.g., a first blood sample such as a first plasma sample) can be obtained from a mammal (e.g., a human) prior to the mammal being administered an anti-senescence treatment (e.g., prior to being administered one or more seno therapeutic agents), and a second sample (e.g., a second blood sample such as a second plasma sample), and optionally subsequent samples, can be obtained from the mammal after the mammal has been administered the anti-senescence treatment. When 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. When the circulating polypeptide signature of the second sample 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 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 not being an effective treatment for that mammal.

[0085] Any appropriate sample from a mammal (e.g., a human) 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). In some cases, a sample can be a biological sample. For example, 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). Examples of 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). In some cases, a biological sample can be a processed sample (e g., to isolate or extract one or more biological molecules). For example, 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.

[0086] 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. In some cases, 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. For example, 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, SOMAscan assays, and spatial proteomics techniques can be used to determine the presence, absence, or level of one or more polypeptides in a sample. When an immunoassay is used to determine the presence, absence, or level of one or more polypeptides in a sample, the immunoassay can use any appropriate antibodies. In some cases, 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) within a sample (e.g., a sample obtained from a mammal such as a human) can be detected as described in Example 1.

[0087] In some case, the level of mRNA encoding a polypeptide can be measured to indicate the level of that polypeptide within a mammal. For example, 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. Any appropriate method can be used to measure the level of mRNA encoding a polypeptide within a sample. For example, polymerase chain reaction (PCR)-based techniques such as quantitative reverse transcription (RT)-PCR (qRT-PCR) techniques, spatial transcriptomic techniques, and single-cell RNA sequencing can be used to determine the presence, absence, or level of mRNA encoding a polypeptide in the sample.

[0088] Any appropriate anti-senescence treatment (and / or revised 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. For example, 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). In some cases, a senotherapeutic agent can be a flavonoid. Examples of 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, roxithromycin, senescence-specific killing compound 1 (SSK1), GLS1 inhibitors, 25-hydroxycholesterol, Procyanidin Cl, and EF-24, and senomorphic agents such as apigenin, rapamycin, and everolimus. Additional examples of 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. In some cases, 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). This document also provides methods and materials for treating a mammal (e.g., a human) in need of inhibited or reduced cellular senescence. For example, a mammal (e.g., a human) can be treated as described herein by administering any appropriate anti-senescence treatment (and / or revised anti-senescence treatment) to the mammal and by confirming the effectiveness of that anti-senescence treatment (and / or revised anti-senescence treatment) within that mammal as described herein. In such cases, 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).

[0089] In some cases, a method for treating a mammal (e.g., a human) provided herein 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. In some cases, that previously effective anti-senescence treatment can be continued as is. In some cases, that previously effective anti-senescence treatment can be continued with one or more slight alterations.

[0090] In some cases, a method for treating a mammal (e.g., a human) provided herein 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.

[0091] In some cases, a method for treating a mammal (e.g., a human) provided herein 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. In such cases, the administered an anti-senescence treatment can be different from that of the previous anti-senescence treatment that was determined to be ineffective. In some cases, the effectiveness of that administer anti-senescence treatment can be assessed as described herein.

[0092] In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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.

[0093] 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. In some cases, an anti-senescence treatment (e.g., one or more senotherapeutic agents) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having a disease or disorder characterized by excess cellular senescence (e.g., an age-related disease). For example, one or more senotherapeutic agents can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents. In some cases, a pharmaceutically acceptable carrier, excipient, or diluent can be a naturally occurring pharmaceutically acceptable carrier, excipient, or diluent. In some cases, 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).

[0094] 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). In some cases, an anti-senescence treatment can be administered to a mammal locally or systemically. For example, an anti-senescence treatment can be administered systemically by oral administration to a mammal (e.g., a human).

[0095] 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. For example, the frequency of administration of an anti-senescence treatment (e.g., one or more senotherapeutic agents) 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.

[0096] 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. For example, the duration can vary from several days to several weeks, from several weeks to several months, or from several months to several years. In some cases, 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. For example, 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.

[0097] In some cases, methods for treating a mammal (e.g., a human) 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. For example, 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.

[0098] In some cases, a course of treatment can be monitored as described herein. For example, 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.

[0099] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0100] EXAMPLES

[0101] Example 1: Senotherapeutic control of aging biomarkers in circulation and tissues

[0102] 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. Such circulating polypeptides can be used as markers for systemic signatures of senescence (e.g., senescent cell burden of that mammal).

[0103] RESULTS

[0104] Circulating plasma polypeptides increase and decrease in abundance in aging

[0105] Circulating biomarkers that are altered by aging prior to testing the influence of acute, intermittent senolytic targeting were identified. Among young and old mice, 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. Relative to young females, in old females four plasma polypeptides were identified that were significantly increased (CCL5, CCN1, IL23R, and GCG) and three plasma polypeptides that were significantly reduced (CA13, CNTN4, and MIA) (Fig. 2A). Relative to young males, in old males four plasma polypeptides were identified that were significantly increased (CCL3, CCN1, IL17A, and IL23R) and 12 plasma polypeptides that were significantly reduced (CCN4, CDH6, CLMP, CPE, FSTL3, LPL, MIA, N0TCH3, PLIN1, TGFBR3, TNFRSF12A, and VSIG2) (Fig. 2B). Across both sexes, IL23R, CCL5, and CCN1 significantly increased and CNTN4 and MIA significantly decreased in aged plasma. According to the gene ontology terms, 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.

[0106] Genes that encode age-associated plasma polypeptides increase with senescence-related transcripts in aged mouse tissues

[0107] It was evaluated whether age-related polypeptides in circulation may originate from senescent cells. Whether expression of genes encoding age-increased plasma factors increased with canonical senescence markers across tissues in old versus youn pl6-InkAttac mice was tested by RT-PCR. Compared to young mice, significant increases in pl6,nk4aexpression were detected in old mouse kidney (Fig. 3A), liver (Fig. 3B), spleen (Fig. 3C), and brain cerebral cortex (Fig. 3D) of both sexes. p!6,nk4aincreased in perigonadal adipose of male mice (Fig. 3E). In examination of relative cycle threshold values from RT-PCR, it was observed that aged spleen harbored the greatest abundance of pl6mk4atranscript across young and old tissues (Fig. 9). However, in comparison of the fold-change of pl6mk4abetween young and old tissues, our ordinal prediction model demonstrates the largest aging change in pl6mk4ainkidney, followed by liver (Fig. 3G). Age-dependent, increases in expression of Ccl2, Ccl3, Ccl5, Illb, and 116 SASP factors accompanied increases in p!6tnk4aexpression across kidney, liver, spleen, and / or cortex (Fig. 3). The broader Cdkn2a INK4a / ARF locus (pl6totak) expression closely tracked with expression of p!6mk4aper organ (Fig. 3H). p21 expression increased in aged kidney and adipose (Fig. 3A, 3E). The SCAP gene Bcl2 increased in old male and female spleen (Fig. 3C). However, Bcl2ala, a downstream effector and cytokine-stimulated mediator of the BCL2 cell survival pathway, increased in aged kidney, liver, and spleen (Fig. 31). Old females exhibited greater expression of Cd38 in spleen and adipose compared to young controls (Fig. 3C). Significantly greater 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,nk4aacross tissues, with the most robust positive association identified between plasma IL23R and kidney p!6mk4aexpression in both sexes (Fig. 10A-10B). Expression of chemokines Ccl3 (Fig. 3K) and Ccl5 (Fig. 3L), Illb (Fig.

[0108] 3M), and 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.

[0109] In comparison of plasma polypeptides and transcript abundance in distinct tissues, expression of SASP and immune mediators such as Ccl2, Ccl3, Ccl5, and Il23r significantly correlated with their encoded polypeptide abundance in plasma. However, Cal 3, Ccnl, Ccn4, Cntn4, Mia, and Rgma gene transcripts did not consistently correlate with corresponding polypeptides, despite age-associated changes of the polypeptide in plasma. Alongside the significant increase in CCN1 in aged plasma (Fig. 2), Ccnl transcript selectively displayed age-associated increase in adipose (Fig. 3E), whereas Il23r and Ccl5 increased prominently across aged tissues. It was discovered that Il23r and / or Ccl5 expression increased concomitant with expression of p!6, SASP, and SCAP pathway factors in kidney, liver, spleen, adipose, and cortex. A general pattern was observed of senescence and SASP gene signatures increasing most reliably in the aged kidney. In kidney, gene expression of pl6mk4a, pl&°tal, Ccl2, Ccl3, Ccl5, Illb, 116, and Il23r, and Tnf showed positive correlations with plasma CCL3, CCL5, CCN1, LL17A, and IL23R, and negative correlation with MIA (Fig. 10C-10D).

[0110] Senescent cells express Il23r and Ccl5

[0111] To definitively determine whether IL23R or CCL5 could originate from senescent cells, an established cell culture model of senescence was implemented. 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. In quantification of SA-P-gal from sham and IRMEFs, IR induced significantly greater percentage (Fig. 4B) and total number (Fig. 4C) of SA-P-gal+ cells. In addition, cells from IR cultures were characterized by larger nuclear areas (Fig. 4D).

[0112] For RT-PCR gene expression profiling, established senescence and SASP genes were examined, as well as genes encoding polypeptides that displayed age-associated changes in plasma (Fig. 4E). Relative to sham, pl6mk4a, pl6total, p21, Gdfl5, 116,r\v3 Mmp3 expression significantly increased in IR MEFs. Coinciding with this senescence signature, 1123r expression increased by 2.4 ± 0.3 fold, and Ccl5 expression increased by 6.3 ± 0.5 fold in IR MEFs. Mia also increased in IR MEFs, but Ccnl significantly decreased. In measurement of SASP polypeptides in conditioned media via multiplexed LUMINEX® immunoassays, CCL3, CCL5, CCL11, GDF15, MMP2, MMP12, OPN, and TNFRSF1 A polypeptides significantly increased in IR media as compared to sham (Fig. 4F). Collectively, this demonstrated that / / 23 / ' and Ccl5 expression is induced in senescent cells, which suggests that soluble IL23R and CCL5 polypeptides in aged blood circulation could originate from senescent cells.

[0113] Age-associated changes in plasma polypeptides are suppressed by senotherapeutic senescent cell targeting

[0114] It was next tested whether age-associated plasma polypeptides that can originate from senescent cells were modulated by senescent cell clearance strategies. In old p!6-InkAttac mice, a head-to-head comparison was conducted of AP20187 (AP)-mediated transgenic clearance or senolytic compounds delivered through an intermittent strategy of five once- daily treatments, followed by two weeks without treatment, followed by five once-daily treatments, followed by a week without treatment prior to necropsy (Fig. 1).

[0115] It was evaluated whether 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). In old female mice, AP, VEN, and LUT significantly reduced plasma abundance of IL23R (Fig. 5A). 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). Thus, 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. Among these data, IL23R was most consistently regulated by age and senescent cell clearance across sexes.

[0116] To interrogate potential off-target effects of the senolytic compounds, it was also tested whether some treatments ‘exacerbate’ the age-dependent trajectory of plasma polypeptide levels. Whether the abundance of polypeptides from treated old mice shifted in the direction further away from the young control values was determined using one-sided tests (Table 1). In male plasma, FIS further augmented the age-dependent shift in CDH6, CPE, FSTL3, MIA, QDPR, and CCN4. In female plasma, VEN further augmented the agedependent shift in CDH6 and CNTN4.

[0117] Drugs targeting senescent cells concentrate in secretory organs

[0118] Pharmacokinetic analysis was performed using mass spectrometry at 20 minutes, two hours, six hours, and three days following four consecutive days of treatment with vehicle, AP (2 mg / kg) or VEN (50 mg / kg), based on the observation that these senolytic interventions reversed age-associated changes in plasma biomarker abundance. 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). In mice treated with VEN, maximum concentration in plasma (2.9-6.0 pM), kidney (2.8-4.1 pM), and liver (18.8-29.5 pM) was detected 2 hours after the final dose, and the majority of the drug was eliminated by 72 hours. VEN was consistently detected in the brain (20-300 nM) at 20 minutes to six hours following the fourth treatment. These results demonstrate that AP and VEN, drugs targeting senescent cells, concentrate and persist in kidney and liver, which harbor a significant burden of senescent cell makers (Fig. 3) and thus, may be more likely to effect on-target clearance in these tissues.

[0119] Senescence biomarkers are differentially altered across tissues by senotherapeutics

[0120] It was next examined whether agents targeting senescent cells reduced the abundance of transcripts encoding canonical senescence biomarkers in register with transcripts encoding age- and senolytic-altered plasma polypeptides. Ordinal regression models were fit with interactions for tissue*group*sex, and pairwise comparisons with multiple comparison corrections were derived from the models for females and males by tissue. It was discovered that treatment of aged mice with acute, intermittent AP treatment reduced p!6 expression in the aged kidney (Fig. 6). All senolytics reduced pl6totalin female kidney (Fig. 6C), and FIS significantly reduced p!6,otalin aged male kidney (Fig. 6D). In female spleen, VEN and NAV reduced p!6mk4a(Fig. 6E) and NAV reduced pl6'""-4(Fig. 6G). FIS reduced p!6mk4a, and AP, FIS, and LUT each significantly reduced pl6totalm cortex of male mice (Fig. 6L). Senolytics did not significantly shift pl6 in liver, lung, or perigonadal adipose (Fig. 11).

[0121] It was next comparatively assessed if the interventions reduced other senescence- related and SASP factors. In aged female kidney, AP-mediated transgenic clearance reduced Bcl2ala (Fig. 12). In addition, among all tissues, the kidney alone demonstrated a significant decrease in Cd38 expression following AP treatment in females (Fig. 12). VEN significantly reduced Ccl2, Ccl3 Ccl20, and Tnfm ' female kidney. In the liver, VEN reduced 116 in females, and reduced Bcl2 and Tnf expression in males (Fig. 13). Female spleen was sensitive to many of the senolytic drugs as indicated by reductions in Bcl2, Bcl2ala, Ccl2, Ccl3, p21, and 116 (Fig. 14). AP reduced Illb in females and / A / 2 and Cal 3 in males (Fig. 14). In aged female lung, AP, VEN, FIS, and LUT increased expression of Cal3, LUT increased expression of Ccnl, and AP reduced expression Ci H6 (Fig. 15). In female perigonadal adipose, NAV and FIS reduced Cal 3 (Fig. 16). Interestingly, 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. In male cortex AP, VEN and FIS increased p21 (Fig. 17). Overall, similarity was observed between tissues that were sensitive to pl6- / / ? / c4 / toc-directed clearance and their responsiveness to senolytic treatment. 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.

[0122] Il23r and Ccl5 are senescence-associated tissue and plasma biomarkers that increase with age and are reduced by senotherapeutics

[0123] In parallel to the tissue-specific gene expression profiling of senescence biomarkers described above, it was determined if expression of genes encoding the circulating aging biomarkers such as CCL5 (Fig. 7), decreased in response to each intervention. Although Cc / 5 exhibited widespread differential expression among organs, spleen showed highest baseline levels (Fig. 9). In aged female spleen, VEN, FIS, and LUT significantly decreased Ccl5 expression (Fig. 7E). VEN treatment significantly decreased Ccl5 transcript abundance in kidney, liver, spleen, and cortex (Fig. 7, Fig. 9). Significant reductions in Ccl5 in males occurred following LUT treatment in liver (Fig. 7D) and VEN or FIS treatment in spleen (Fig. 7F).

[0124] It was next tested whether senolytic treatments mitigated age-associated expression of Il23r (Fig. 8). Within aged female kidney, liver, adipose, and spleen and male kidney and adipose, AP treatment significantly reduced Il23r expression (Fig. 8). In comparison of the senolytic effect on Il23r expression in tissue, it was found that VEN significantly mitigated the age-dependent increases m ll2 r in female kidney, liver, spleen, and adipose. In males, VEN reversed the age-dependent expression c> ll23r in kidney and adipose. In examination of the flavonol senolytics, FIS and LUT significantly reduced Il23r expression in old female kidney and spleen compared to controls (Fig. 8A and 8E). LUT also reduced Il23r expression in old female adipose (Fig. 81). Antithetical to the peripheral tissues, in female cortex, both AP and LUT significantly increased Il23r compared to old controls, returning toward the higher level of expression in young mice (Fig. 8K). Altogether, the senolytic-driven changes in plasma polypeptide and gene expression suggest that CCL5 and IL23R are important aging biomarkers that co-express with senescence factors and are candidate progeronic mediators of organ crosstalk.

[0125] Taken together, these results demonstrate that the circulating polypeptide signature of a mammal (e.g., a human) can be used as an indicator of the systemic senescent cell burden of that mammal. For example, 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.

[0126] METHODS

[0127] Animals

[0128] 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. Blood samples were centrifuged (1300 x g for 15 min, 4°C), and the plasma fraction was separated and stored at - 80°C. Following blood collection, 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.

[0129] Senotherapeutic treatment of mice

[0130] For 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. For senolytic compounds, 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. treatments, with drug treatment groups also receiving vehicle by i.p. or by gavage. Young and old pl6-InkAttac control mice received the same vehicle dosing strategy (Fig. 1). NAV (ABT-263, #11500) and LUT (3 ',4', 5,7- Tetrahydroxyflavone, #10004161) were acquired from Cayman Chemical. VEN (ABT-199, #A8194) was acquired from APExBio Technology. FIS (3,7,3’,4’-Tetrahydroxyflavone, #T0121) was acquired from TCI America. AP20187 was acquired from WuXi AppTec. All other reagents, unless otherwise denoted, were acquired from Sigma-Aldrich / Millipore. For the gene expression and plasma profiling focused study, the treatment strategy was 5 consecutive daily administrations for one week, two weeks off, and then another 5 -day treatment. Treatments were discontinued one week prior to necropsy.

[0131] For pharmacokinetic study of drug accumulation / elimination in tissue, 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.

[0132] Real-time polymerase chain reaction (RT-PCR)

[0133] Tissue 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. For all tissues, 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!6mk4aprimer denotes coverage of the Cdkn2a variant 1 and pi6totaldetects both Cdkn2a variant 1 and variant 2. 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'AACTvalue for each tissue sample. In studies involving RT-PCR analyses in young versus old tissues, we report CT values in addition to relative expression results. In system- wide comparisons across multiple drug treatments, we found it very informative to examine total abundance of the transcripts according to tissue of origin and age. This qualitative comparison is feasible since cDNA was synthesized from the same amount of input RNA per tissue sample (2 pg) (Fig. 9).

[0134] Plasma analysis

[0135] 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.

[0136] Mouse embryonic fibroblast cell culture and senescence confirmation

[0137] A fibroblast culture model was used to validate hallmark markers of senescence (41Baker 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). P4 cells were plated in 6-well plates or T75 flasks. At 40% confhiency, 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 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. For SA-P-gal staining, cells in 6-well plates were fixed with 4% PF A for 10 minutes, rinsed with PBS, and stained with solution containing 40 mM citric acid / Na2HPO4 buffer at pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCh, 2 mM MgCh, and 1 mg / mL (25 mM) X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside, VWR) in water. Cells were stained for 27.5 hours at 37 °C with low shaking. Cells were rinsed with PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI) at 1 pg / mL for nuclear colocalization. Images were acquired with 405 nm fluorescence and bright-field on an Eclipse Ti inverted microscope (Nikon Instruments) to determine densitometry of the SA-P-gal- converted chromogenic compound.

[0138] Pharmacokinetic analyses by high-performance liquid chromatography (HPLC) and mass spectrometry (MS)

[0139] 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.

[0140] Statistics

[0141] For both plasma polypeptide and tissue gene expression analyses, a two-step approach was implemented. First, identify factors altered by age using two-tailed statistical models, and second, identify factors altered by each senolytic intervention in aged samples using one-tailed statistical models. One-tailed tests are substantiated by the logic that we are testing the hypothesis that senotherapeutic treatments will reverse the aging trajectory in a specific direction. In comparison of both age-, sex-, and treatment-dependent factors, conservative parameters were implemented, including non-parametric and multiple comparison testing. GraphPad Prism 9 and R 4.1.2 were used for statistical analyses.

[0142] 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. Factors that demonstrated evidence of a p < 0.10 age-specific difference in at least one sex (13 polypeptides identified in females, 23 polypeptides identified in males) were applied to statistical testing that interrogated senolytic intervention responsivity in aged mice only. Among these age-modified polypeptides, Kruskal-Wallis rank sum tests were used to identify polypeptides with evidence of being altered by any of the five drugs as a group difference (OLD AP, OLD VEN, OLD NAV, OLD FIS, OLD LUT), with sexes stratified (p < 0.10; 6 polypeptides in females, 11 polypeptides in males). 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.

[0143] 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. Dunnett test correction was applied to account for the multiple testing by the five treatment groups. Sample sizes were 5-8 mice per tissue and treatment group. Correlations between RT-PCR and plasma polypeptide data were interrogated using Spearman correlations and significance was determined with the criterion p < 0.05.

[0144] MEF experiments. For gene expression from cultured MEFs or from their conditioned media, comparisons were made between sham control and irradiated samples using Mann Whitney test with significance at p < 0.05, adjusted for multiple comparisons with the Holm- Sidak method, n = 6 samples per group.

[0145] Example 2: Senotherapeutic control of aging biomarkers in circulation and tissues of old mice

[0146] The results in this Example re-present and expand on at least some of the results provided in other Examples.

[0147] RESULTS

[0148] Circulating plasma proteins increased and decreased in abundance with aging

[0149] To identify circulating biomarkers that were altered by aging between young (3-4 months) and old (16-20 months) mice, Olink proximity extension assays were used. The protein panel included SASP, inflammatory, metabolic, developmental, cell scaffolding, and signaling factors (Fig. 18). The utility of this approach was to probe both senescence-related and generalized factors of systemic health in aging. Relative to young females, old females’ plasma showed increased levels of four proteins (CCL5, CCN1, IL23R, GCG) and reduced levels of three proteins (CA13, CNTN4, MIA) (Fig. 18A). Relative to young males, old males’ plasma showed increased levels of four proteins (CCL3, CCN1, IL17A, IL23R) and reduced levels of 12 proteins (CCN4, CDH6, CNTN4, CLMP, CPE, FSTL3, LPL, MIA, NOTCH3, TGFBR3, TNFRSF12A, VSIG2) (Fig. 18B). Across both sexes, IL23R, CCL5, and CCN 1 were higher and CNTN4 and MIA were lower in aged plasma. It was next sought to measure the transcriptional expression of these and other aging biomarkers in tissues. Genes that encode age-associated plasma proteins increased with senescence-related transcripts in aged mouse tissues

[0150] To test whether the proteins that increased in aged plasma originated from senescent cells, 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. Compared to young mice, higher expression of p!6mk4asN&s detected in old mouse kidney (Fig. 19A), liver (Fig. 19B), spleen (Fig. 19C), and brain cerebral cortex (Fig. 19D) of both sexes. pl6mk4aexpression was also higher in perigonadal adipose of old male mice (Fig. 19E). Expression of the broader Cdkn2a locus detecting both / ? / 9‘"' and pl6,nk4aclosely tracked with the expression of p!6ink4aper organ (Figs. 19A-19H). In examination of relative cycle threshold values, the aged spleen harbored the greatest abundance of pl6 transcript across young and old tissues (Fig. 25). However, ordinal prediction models demonstrate the largest age-associated change in p!6mk4aand Cdkn2a relative expression in kidney, followed by liver (Figs. 19G and 19H). 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). Moreover, 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). Expression of SASP chemokines Ccl3 (Fig. 19K) and Ccl5 (Fig. 19L), Il lb (Fig. 19M), and 116 (Fig. 19N) were systemically increased across multiple aged tissues, including kidney, liver, spleen, cortex, and adipose, with Ccl5 more consistently increased relative to other analyzed chemokines.

[0151] To determine whether plasma protein levels were associated with tissue-specific transcript abundance in young and old mice, Spearman’s correlation was applied (Fig. 26). Multiple age-altered plasma proteins that positively correlated with their corresponding transcripts were shown. In females, U23r expression in several tissues positively correlated with encoded protein abundance in plasma (kidney r = 0.78, p = 0.004; liver r = 0.87, p < 0.001; adipose r = 0.70, p = 0.014; lung = 0.74, p = 0.008). Male liver Ccl5 positively correlated with CCL5 plasma protein (r = 0.56, p = 0.047). The associations between age- altered plasma proteins and expression of canonical senescence markers were also assessed. The plasma IL23R protein correlated with Cdkn2a and pldnk4atranscript abundance across tissues (Fig. 26), with the greatest positive association observed between plasma IL23R and kidney Cdkn2a expression in female mice (r = 0.83, p = 0.002; Fig. 26A). The kidney was characterized by consistent age-elevated senescence gene signatures (Fig. 19), in which pl6mk4a, 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. 26).

[0152] Since Cdkn2a / pl6 and Il23r (established and emerging aging biomarkers, respectively) both increased in aged kidney, fluorescent in situ hybridization was performed to localize and validate their expression in aged kidney (Fig. 27). pidnk4aexpression was abundant throughout the nephrons and its interstitium. Moreover, 1123r was highly concentrated in cells found in the renal sinus cavity and perivascular tissue, in which pl6mk4awas co-localized. Furthermore, it was observed that Il23r co-localized with Cd3e+ cells in immune nodes throughout the aged kidney, present in glomeruli, and pervasive in the mesenchymal tissue, whereas the young kidney lacked the presence of Il23r (Figs. 27B-27J).

[0153] Senescent cells expressed 1123r and Ccl5

[0154] To determine if IL23R or CCL5 originated from senescent cells, an established cell culture model of irradiation (IR)-induced senescence was implemented in mouse embryonic fibroblasts (MEFs) (Fig. 28). Senescence-associated P-galactosidase (SA-P-gal) staining, nuclear morphology assessments, RT-PCR gene expression profiling, and multiplexed immunoassay analysis of the SASP in conditioned media showed that IR-exposed MEFs were senescent. 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). For gene expression profiling, the established senescence and SASP genes, as well as genes encoding proteins that displayed age-associated changes in plasma, were analyzed (Fig. 28E). Relative to sham, pl6mk4a, Cdkn2a, p21, Gdfl5, 116, and Mmp3 expressions increased in IR MEFs. Coinciding with this senescence signature, 1123r expression was 2.4 ± 0.3-fold greater, and 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. The abundance of canonical SASP proteins and emerging biomarkers secreted from the IR senescence and control cells were measured in the conditioned media using multiplexed Luminex and Olink immunoassays (Figs. 28F-28G). Across the two assays, CCL2, CCL3, CCL5, CCL11, CCL20, CXCL1, CXCL9, GDF15, IL23R, MMP2, MMP12, OPN, and TNFRSF1A proteins significantly increased in IR MEF conditioned media. Collectively, Cc75 / CCL5 and / / 23 / 7IL23R expression were induced in and secreted from the senescent cells, suggesting that the elevated CCL5 and IL23R in aged blood circulation could originate from senescent cells.

[0155] Age -associated changes in plasma proteins were suppressed by senotherapeutic interventions

[0156] It was next tested whether putative age-associated plasma proteins are modulated by drugs that target senescent cells in vivo. In old pl6-InkAttac mice, a head-to-head comparison of AP20187 (AP)-mediated transgenic versus senolytic interventions through a short-term, intermittent delivery strategy was performed (Fig. 20A). All four compounds (VEN, NAV, FIS, and LUT) demonstrated selective ability to induce apoptosis via cleaved caspase-3 activity in etoposide-induced senescent lung fibroblast in vitro (Fig. 29). It was tested whether the senotherapeutic agents reversed the direction of age-related changes of plasma proteins in vivo. Drug responses were tested for proteins demonstrating an age-dependent change using non-parametric, one-tailed statistical tests with correction for multiple comparisons (Figs. 20B-20M). In old female mice, AP, VEN, and LUT reduced plasma abundance of IL23R (Fig. 20B). In old male mice, VEN reduced plasma abundance of IL23R (Fig. 20C). AP and NAV reduced plasma abundance of CCL5 in old females (Fig. 20D). VEN, FIS, and LUT reduced plasma abundance of GCG in old females (Fig. 20F). In old males, VEN, NAV, FIS, and LUT reduced plasma abundance of IL17A (Fig. 201). 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). Thus, 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. Among these data, 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. In male plasma, FIS further increased the age-dependent change in CDH6, CPE, FSTL3, MIA, QDPR, and CCN4. In female plasma, VEN further increased the age-dependent change in CDH6 and CNTN4.

[0157] Drugs targeting senescent cells concentrated in secretory organs

[0158] Pharmacokinetic analysis was performed in plasma and tissues using mass spectrometry at 20 minutes, 2 hours, 6 hours, and 3 days following four consecutive days of treatment. 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). In mice treated with VEN, maximum concentration in plasma (2.9-6.0 pM), kidney (2.8-4.1 pM), and liver (18.8-29.5 pM) was detected 2 hours after the final dose, and most of the drug was eliminated by 72 hours. These measurements were derived from 18-month-old mice that were drug treated for only four days. Older or diseased mice treated longer-term were expected to display distinct drug kinetics. Nevertheless, these results demonstrated that AP and VEN, drugs targeting senescent cells, concentrated in and persisted in kidney and liver, which harbored a significant burden of senescent cell markers (Fig. 19) and thus, may be more likely to induce on-target effects in these tissues.

[0159] Il23r and Ccl5 are senescence-associated tissue biomarkers that increased with age and were reduced by senotherapeutics

[0160] It was examined whether the senotherapeutic compounds reduced the abundance of transcripts encoding age- and senolytic-altered plasma proteins in register with transcripts encoding canonical senescence factors. To test whether the senolytic treatments reduced age- increased transcript levels relative to old vehicle-treated mice, a hypothesis directed one- tailed, non-parametric pairwise comparisons with multiple comparison corrections for females and males per tissue was conducted. The results showed that AP treatment reduced Cdkn2a expression in the aged female kidney (Fig. 21A). VEN, NAV, and LUT also reduced Cdkn2a expression in female kidney (Fig. 21A), and FIS reduced Cdkn2a in male kidney (Fig. 2 IB). In female spleen, NAV reduced Cdkn2a (Fig. 2 IE), and VEN and NAV reduced p!6nk4a(Fig. 21G). AP, FIS, and LUT each reduced Cdkn2a in cortex of male mice (Fig. 21J), and FIS reduced p!6mk4a(Fig. 21L). Senolytic-mediated changes were in liver, lung, or perigonadal adipose Cdkn2a or p!6mk4aexpression (Fig. 30). The trend toward age-dependent increase in p21 expression in the spleen and adipose were reduced by NAV and / or LUT (Fig. 31). The lower age-associated p21 expression in male cortex was reverted toward youthful levels by VEN or FIS (Fig. 31).

[0161] Additional tissue-specific senotherapeutic modulation of senescence gene expression can be found in Figures 34-39. VEN exhibited the greatest efficacy in mitigating senescence- related gene expression in the acute treatment paradigm, with partial phenocopy of AP- mediated senescent cell clearance in kidney. To further discern senolytic-dependent shifts in age-related factors, principal component analysis was performed on the combined kidney gene expression and plasma protein variables from young, old, and treated old mice. It was observed that the aged mice treated with AP or senotherapeutic drugs tended to cluster in between aged or young mice treated with VEH (Figure 40).

[0162] It was next determined if 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). In aged female spleen, 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).

[0163] It was also tested whether senotherapeutics mitigated age-associated Il23r transcriptional changes (Fig. 23). In aged female kidney and liver, 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). In males, 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). Antithetical to the peripheral tissues, AP and LUT increased 1123r expression in old female cortex, and VEN increased 1123r expression in old male cortex, toward a higher level of cortical expression in young mice (Figs. 23K and 23L). Altogether, the age- and senotherapeutic-mediated changes in plasma protein and gene expression showed that IL23R and CCL5 could be aging and senescence biomarkers and candidate mediators of interorgan signal transduction.

[0164] IL23R protein increased in aged human plasma

[0165] The expression level of IL23R was examined using enzyme-linked immunosorbent assay (ELISA) in human plasma from a cross-sectional sample of 80 study participants. IL23R increased with age in the combined sample (r = 0.337, p = 0.002) and separately in females (r = 0.321, p = 0.043) and males (r = 0.323, p = 0.045) (Fig. 24). Next, the IL23R protein abundance was estimated in young and aged mouse, and human samples. In mouse samples, Olink assays were performed that included plasma standards spiked with IL23R protein. It was found that average IL23R plasma concentration for 22-24 month female mice was 78 ± 5 pg / mL (n = 17), whereas the young plasma samples (n = 7) were below the lower limit of detection of the standard curve. Using ELISAs with IL23R protein standards, it was determined that average IL23R in human plasma was 37 ± 3 pg / mL, in aged females was 66- 82, and in males aged 67-82 years old was 46 ± 4 pg / mL. Therefore, IL23R increased with age in both mice and humans.

[0166] METHODS

[0167] Animals

[0168] 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. Blood samples were centrifuged (1300 x g for 15 minutes, 4°C), and the plasma fraction was separated and stored at -80°C. Following blood collection, 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.

[0169] Senotherapeutic treatment of mice

[0170] For 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. For senolytic compounds, 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. treatments, with AP mice receiving gavage vehicle and senolytic drug groups also receiving vehicle by i.p. Young and old p!6-InkAttac control mice received the same vehicle dosing strategy (Fig. 20A). NAV (ABT-263, #11500) and LUT (3',4',5,7-Tetrahydroxyflavone, #10004161) were acquired from Cayman Chemical. VEN (ABT- 199, #A8194) was acquired from APExBio Technology. FIS (3,7,3’,4’-Tetrahydroxyflavone, #T0121 ) was acquired from TCI America. AP20187 was acquired from WuXi AppTec. All other reagents, unless otherwise denoted, were acquired from Sigma- Aldrich / Millipore.

[0171] For the gene expression and plasma profiling focused study, 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). 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.

[0172] Real-time polymerase chain reaction (RT-PCR)

[0173] Tissue 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. For all tissues, 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). The p!6mk4aprimer denoted coverage of the Cdkn2a variant 1 and ‘Cdkn2a" primer detected both Cdkn2a variant 1 and variant 2. 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’AACTvalue 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).

[0174] In situ hybridization

[0175] 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 ' ldnk4aprobe (#447491, Mm-Cdkn2a-tv2) was used in channel 1 and paired with TSA Vivid 520 fluorophore (#323271). U23r (#403751-C2, Mm-I123r-C2) was used in channel 2 and paired with TSA Vivid 570 fluorophore (#323272). Cd3e (#314721-C3, Mm-Cd3e-C3) was used in channel 3 and paired with TSA Vivid 650 (#323273). Negative control probe DapB (#320871) was used on parallel sections as control. 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.

[0176] Mouse plasma protein analysis

[0177] The Olink Target 96 Mouse Exploratory panel was used to analyze 92 protein biomarkers from mouse plasma by proximity extension assays (PEA). 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.

[0178] Human samples and plasma protein analysis

[0179] 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 / m2or greater than 40.0 kg / m2and men with BMI less than 18.5 kg / m2or greater than 38.0 kg / m2were 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. 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).

[0180] Mouse embryonic fibroblast cell culture and senescence confirmation

[0181] A 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). 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. For SA-P-gal staining, cells in 6-well plates were fixed with 4% PFA for 10 minutes, rinsed with PBS, and stained with solution containing 40 mM citric acid / Na2HPO4 buffer at pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCL, 2 mM MgCh, and 1 mg / mL (25 mM) X-gal (5-bromo-4-chloro-3-indolyl-P- D-galactopyranoside, VWR) in water. Cells were stained for 27.5 hours at 37°C with low shaking. Cells were rinsed with PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI) at 1 pg / mL for nuclear co-localization. Images were acquired with 405 nm fluorescence and bright-field on an Eclipse Ti inverted microscope (Nikon Instruments) for visualization of SA-P-gal-converted chromogenic compound. iLastik pixel classification workflow was used to create a binary mask of brightfield images (Berg et al, Nat Meth, 16: 1226-1232 (2019)). Unstained regions and SA-P-Gal stained regions were manually added for training, with one image per well for both sham and IR included in the training set. 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.

[0182] For cell preparation, 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.

[0183] Conditioned media from sham and IR cells was used for multiplexed sandwich ELISAs using the Luminex platform (Bio-techne). Analyte-specific antibodies were precoated onto magnetic beads embedded with fluorophores. Microparticles, standards, and media samples were pipetted into wells and analytes of interest bound by immobilized antibodies. After washing off unbound material, biotinylated antibody cocktail was added to each well. Following another wash step, Streptavidin-PE conjugate was added. After a final wash, microparticles were resuspended in buffer and read using the Intelliflex xMAP to magnetically capture the beads and read the fluorophore signals unique to each analyte. Conditioned media, with samples run in duplicate, was also tested with the Olink PEA assay Target 96 Mouse Exploratory panel. For PCR and conditioned media analyte measurements, six sham and six irradiated experiment samples were used. Senolytic drug testing in senescent lung fibroblasts

[0184] 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. After 72 hours, the cells were fixed with 4% formalin, permeabilized with 0.25% triton X-100, blocked with 1% BSA, and immunostained with an antibody for cleaved caspase-3 (Cell Signaling, #9661, RRID:AB_2341188). Alexa Flour-488 secondary anti-rabbit (Thermo Fisher, #A-11008, RRID:AB_143165) was incubated along with DAPI. Images were collected using a Cytation5 automated microscope (Biotek) using a 4X objective and cleaved caspase-3 intensity was quantified using onboard software (Gen5). Alternatively, 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.

[0185] Pharmacokinetic analyses

[0186] Quantitation of AP and VEN in mouse plasma, kidney, liver, brain, and muscle was performed on a Sciex 6500 MS / MS interfaced with a Sciex ExionLC 2.0 liquid chromatography system. Plasma samples were extracted, and proteins were precipitated by mixing with three times v:v acetonitrile containing carbamazepine as an internal standard. Samples were allowed to sit on ice for approximately thirty minutes with occasional agitation prior to filtration through a 0.2 pm filter plate. Standards were prepared by spiking AP and VEN standards into blank plasma and processed using the same procedure as the samples. Kidney, liver, brain, and muscle, liver samples were 3-fold diluted (w:v) with PBS and dissociated with a probe tip sonicator. The homogenate was processed similarly to the plasma samples. VEN was detected in positive ion mode following the parent ion 868.3 and fragment ion 636.2 and AP used parent ion 742 and fragment ion 209.2.

[0187] Statistics

[0188] A stepwise statistical approach for assessing age- and treatment-dependent changes in plasma protein and tissue gene expression was used. GraphPad Prism 9 and R 4.1.2 were used for statistical analyses. Correlations between RT-PCR and plasma protein data were interrogated using Spearman correlations and significance was determined with the criterion p < 0.05. Sample sizes were 5-8 mice per group.

[0189] For gene expression from cultured MEFs or protein measurement from their conditioned media, comparisons were made between sham control and irradiated samples using Mann Whitney test with significance at p < 0.05, adjusted for multiple comparisons with the Holm-Sidak method, n = 6 samples per group.

[0190] 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.

[0191] Statistical methods for protein and gene expression analysis

[0192] A three-step statistical approach was implemented to identify factors altered by age and then determine drug treatments that were efficacious in reducing the age-accumulated proteins and transcript signatures (Figure 32). The analyses were run separately for male and female mice (a priori decision). Data were not assumed to have outcomes normally distributed and each dataset was analyzed using non-parametric tests. First, age-dependent effects were explored between vehicle-treated young and old mice and only those proteins that differed between the two groups were examined in the next step. After establishing the factors in plasma and tissue that changed in aged animals, senolytic drug-treated groups were compared to the old vehicle-treated mice. Only proteins which suggested an overall group difference among all the older mice groups were examined in the next step. Finally, onesided tests were used to identify treated groups which brought values closer to those of the young mice. One-tailed tests were substantiated by the logic and hypothesis that senotherapeutic treatments would reverse the aging trajectory. Step 1. Age-dependent effects in plasma. To determine which of the 67 plasma proteins showed evidence of age dependent difference, a two-sided Kruskal-Wallis rank sum test was used to compare YNG VEH vs. OLD VEH mice at the p<0.05 significance level, stratified by sex. At this point, a protein was considered age altered if it met the significance criterion. Beta value estimates and confidence intervals are displayed in Figure 18. Median values for each protein in which either sex showed a significant difference between young and old.

[0193] Step 2. Group-level effects in old plasma. We separately investigated the group differences in senolytic-treated mice compared to OLD VEH controls. Using the list of age- altered proteins from step 1, two-sided Kruskal- Wallis rank sum tests were used to determine group differences for any of the proteins, separately for each sex. This indicated if there was evidence that at least one group is different from the five others (OLD VEH, OLD AP, OLD VEN, OLD NAV, OLD FIS, OLD LUT). Only those proteins in which there is an overall group level p value < 0.10 were selected for testing reversal by senotherapeutic treatment resulting in 13 proteins among females and 23 among males (28 unique). Sample sizes were 5-8 mice per group.

[0194] 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. Among the six female proteins and 11 male proteins with some evidence of a group difference determined above, 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. Using ordinal logistic regression, 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. It was also determined if any of the treatments exacerbated the age-dependent effects, with the alternative hypothesis that the mean value of the treated group was further away from the young vehicle values. This test was again performed by one-sided pairwise comparison with ordinal logistic regression, with the Dunnett test multiple comparisons correction. Age-dependent effects in tissue gene expression. For gene expression profiling a similar approach was used. First, a tissue-specific, sex-stratified comparison of young vehicle and old sample data was conducted to initially assess evidence of age differences using ordinal logistic regression models that included the terms sex, tissue type, and group (young versus old) fit for each gene including all two-way and three-way interactions. 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 including all two-way and three-way interactions. 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. Dunnett test correction was applied to account for the multiple testing by the treatment groups. Sample sizes were 5-8 mice per tissue and treatment group. Correlations between RT-PCR and plasma protein data were interrogated using Spearman correlations and significance was determined with the criterion p < 0.05. Due to use of rank-based non-parametric tests throughout, there was less concern about the influence of outliers. However, data were examined graphically prior to any analysis to assess whether there were sample-specific results that appeared abnormal.

[0195] Principal component analysis

[0196] Principal component analyses for combined RT-PCR and plasma protein data were generated using Graphpad Prism 10, with variables selected from young vehicles, old vehicles, and one old senolytic-treated group per analysis (Figure 40). Density based spatial clustering of applications with noise (R package “dbscan”, eps = 1.5, minPts = 3, borderPoints = true) was performed on loadings vectors of principal components 1 and 2 in order to derive similarly distributed gene and protein variables per plot. Cluster 0 denotes a noise variable that did not have any proximal neighbors to cluster with.

[0197] Example 3: Assessing the Efficacy of an Anti -Senescence Treatment

[0198] 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.

[0199] 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.

[0200] When 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.

[0201] When the circulating polypeptide signature of the second blood sample 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 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 not be an effective treatment for that human. In this case, the human can discontinue that particular anti- senescence treatment. Optionally, that human also can initiate an increased dosing regimen of that particular anti-senescence treatment or can initiate a different anti-senescence treatment.

[0202] OTHER EMBODIMENTS

[0203] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying an effective treatment within a mammal, wherein said mammal received an anti-senescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises:(a) determining that said mammal comprises 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, and(b) classifying said anti-senescence treatment as being an effective anti-senescence treatment for said mammal.

2. The method of claim 1, wherein said mammal is a human.

3. The method of any one of claims 1-2, wherein said determining step (a) comprises determining that said mammal comprises 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.

4. The method of any one of claims 1-3, wherein said determining step (a) comprises determining that said mammal comprises a decreased level of circulating GCG polypeptides.

5. The method of any one of claims 1-4, wherein said determining step (a) comprises determining that said mammal comprises a decreased level of circulating CCL5 polypeptides.

6. The method of any one of claims 1-5, wherein said determining step (a) comprises determining that said mammal comprises a decreased level of circulating IL17A polypeptides.

7. The method of any one of claims 1-6, wherein said determining step (a) comprises determining that said mammal comprises an increased level of circulating CA 13 polypeptides.

8. The method of any one of claims 1-7, wherein said determining step (a) comprises determining that said mammal comprises 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.

9. The method of any one of claims 1-8, wherein said determining step (a) comprises comparing a level of said circulating IL23R polypeptides within a first sample obtained from said mammal when or at a time prior to when said mammal received said anti-senescence treatment to a level of said circulating IL23R polypeptides within a second sample obtained from said mammal at a time after the time when said mammal received said anti-senescence treatment, to determine that said mammal comprises said decreased level of said circulating IL23R polypeptides.

10. The method of claim 9, wherein said determining step (a) comprises comparing a level of said circulating GCG polypeptides within said first sample to a level of said circulating GCG polypeptides within said second sample to determine that said mammal comprises said decreased level of said circulating GCG polypeptides.

11. The method of any one of claims 9-10, wherein said determining step (a) comprises comparing a level of said circulating CCL5 polypeptides within said first sample to a level of said circulating CCL5 polypeptides within said second sample to determine that said mammal comprises said decreased level of said circulating CCL5 polypeptides.

12. The method of any one of claims 9-11, wherein said determining step (a) comprises comparing a level of said circulating IL17A polypeptides within said first sample to a level of said circulating IL17A polypeptides within said second sample to determine that said mammal comprises said decreased level of said circulating IL17A polypeptides.

13. The method of any one of claims 9-12, wherein said determining step (a) comprises comparing a level of said circulating CAI 3 polypeptides within said first sample to a level of said circulating CA I 3 polypeptides within said second sample to determine that said mammal comprises said increased level of said circulating CA I 3 polypeptides.

14. The method of any one of claims 9-13, wherein said first sample and said second sample are blood samples.

15. The method of claim 14, wherein said blood samples are plasma samples.

16. The method of any one of claims 9-15, wherein said first sample was obtained from said mammal when said mammal received said anti-senescence treatment.

17. The method of any one of claims 1-16, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

18. The method of any one of claims 1-17, wherein said method further comprises administering said effective anti-senescence treatment to said mammal after said determining step.

19. A method for identifying an ineffective treatment within a mammal, wherein said mammal received an anti-senescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises:(a) determining that said mammal 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 said anti-senescence treatment as being an ineffective anti-senescence treatment for said mammal.

20. The method of claim 19, wherein said mammal is a human.

21. The method of any one of claims 19-20, wherein said determining step (a) comprises comparing a level of said circulating IL23R polypeptides, a level of said circulating GCG polypeptides, a level of said circulating CCL5 polypeptides, a level of said circulating 1L17A polypeptides, and a level of said circulating CAI 3 polypeptide within a first sample obtained from said mammal when or at a time prior to when said mammal received said antisenescence treatment to a level of said circulating IL23R polypeptides, a level of said circulating GCG polypeptides, a level of said circulating CCL5 polypeptides, a level of said circulating IL17A polypeptides, a level of said circulating CNTN4 polypeptides, and a level of said circulating CA 13 polypeptide within a second sample obtained from said mammal at a time after the time when said mammal received said anti- senescence treatment, to determine that said mammal comprises said decreased level of said circulating IL23R polypeptides, said decreased level of said circulating GCG polypeptides, said decreased level of said circulating CCL5 polypeptides, said decreased level of said circulating IL17A polypeptides, and said increased level of said circulating CA13 polypeptides.

22. The method of claim 21, wherein said first sample and said second sample are blood samples.

23. The method of claim 22, wherein said blood samples are plasma samples.

24. The method of any one of claims 21-23, wherein said first sample was obtained from said mammal when said mammal received said anti-senescence treatment.

25. The method of any one of claims 19-24, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

26. The method of any one of claims 19-25, wherein said method further comprises administering a revised anti-senescence treatment that is different from said anti-senescence treatment to said mammal after said determining step.

27. The method of claim 26, wherein said revised anti-senescence treatment comprises administering a higher amount of said one or more senotherapeutic agents of said antisenescence treatment to said mammal.

28. The method of claim 26, wherein said revised anti-senescence treatment comprises administering one or more senotherapeutic agents that are different from said one or more senotherapeutic agents of said anti-senescence treatment.

29. A method for treating a mammal, wherein said mammal received a prior antisenescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises:(a) determining that said mammal comprises 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, and(b) administering said prior anti-senescence treatment or a slightly revised version of said prior anti- senescence treatment to said mammal.

30. The method of claim 29, wherein said mammal is a human.

31. The method of any one of claims 29-30, wherein said determining step (a) comprises determining that said mammal comprises 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.

32. The method of any one of claims 29-31, wherein said determining step (a) comprises determining that said mammal comprises a decreased level of circulating GCG polypeptides.

33. The method of any one of claims 29-32, wherein said determining step (a) comprises determining that said mammal comprises a decreased level of circulating CCL5 polypeptides.

34. The method of any one of claims 29-33, wherein said determining step (a) comprises determining that said mammal comprises a decreased level of circulating 1L17A polypeptides.

35. The method of any one of claims 29-34, wherein said determining step (a) comprises determining that said mammal comprises an increased level of circulating CA I3 polypeptides.

36. The method of any one of claims 29-35, wherein said determining step (a) comprises determining that said mammal comprises 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.

37. The method of any one of claims 29-36, wherein said determining step (a) comprises comparing a level of said circulating IL23R polypeptides within a first sample obtained from said mammal when or at a time prior to when said mammal received said anti-senescence treatment to a level of said circulating IL23R polypeptides within a second sample obtained from said mammal at a time after the time when said mammal received said anti-senescence treatment, to determine that said mammal comprises said decreased level of said circulating IL23R polypeptides.

38. The method of claim 37, wherein said determining step (a) comprises comparing a level of said circulating GCG polypeptides within said first sample to a level of said circulating GCG polypeptides within said second sample to determine that said mammal comprises said decreased level of said circulating GCG polypeptides.

39. The method of any one of claims 37-38, wherein said determining step (a) comprises comparing a level of said circulating CCL5 polypeptides within said first sample to a level of said circulating CCL5 polypeptides within said second sample to determine that said mammal comprises said decreased level of said circulating CCL5 polypeptides.

40. The method of any one of claims 37-39, wherein said determining step (a) comprises comparing a level of said circulating LL17A polypeptides within said first sample to a level of said circulating IL17A polypeptides within said second sample to determine that said mammal comprises said decreased level of said circulating IL17A polypeptides.

41. The method of any one of claims 37-40, wherein said determining step (a) comprises comparing a level of said circulating CA I 3 polypeptides within said first sample to a level of said circulating CA I 3 polypeptides within said second sample to determine that said mammal comprises said increased level of said circulating CA I 3 polypeptides.

42. The method of any one of claims 37-41, wherein said first sample and said second sample are blood samples.

43. The method of claim 42, wherein said blood samples are plasma samples.

44. The method of any one of claims 37-43, wherein said first sample was obtained from said mammal when said mammal received said anti-senescence treatment.

45. The method of any one of claims 29-44, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

46. The method of any one of claims 29-45, wherein said method further comprises administering said prior anti-senescence treatment to said mammal after said determining step.

47. A method for treating a mammal, wherein said mammal received a prior antisenescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises administering a subsequent administration of said prior antisenescence treatment or a slightly revised version of said prior anti-senescence treatment to said mammal, wherein said mammal was identified as comprising 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 said prior anti-senescence treatment.

48. The method of claim 47, wherein said mammal is a human.

49. The method of any one of claims 47-48, wherein said mammal was identified as comprising 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.

50. The method of any one of claims 47-49, wherein said mammal was identified as comprising a decreased level of circulating GCG polypeptides.

51. The method of any one of claims 47-50, wherein said mammal was identified as comprising a decreased level of circulating CCL5 polypeptides.

52. The method of any one of claims 47-51, wherein said mammal was identified as comprising a decreased level of circulating IL17A polypeptides.

53. The method of any one of claims 47-52, wherein said mammal was identified as comprising an increased level of circulating CA I 3 polypeptides.

54. The method of any one of claims 47-53, wherein said mammal was identified as comprising 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.

55. The method of any one of claims 47-54, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

56. A method for treating a mammal, wherein said mammal received a prior antisenescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises:(a) determining that said mammal 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 anti-senescence treatment different from said prior antisenescence treatment to said mammal.

57. The method of claim 56, wherein said mammal is a human.

58. The method of any one of claims 56-57, wherein said determining step (a) comprises comparing a level of said circulating IL23R polypeptides, a level of said circulating GCG polypeptides, a level of said circulating CCL5 polypeptides, a level of said circulating IL17A polypeptides, and a level of said circulating CA I 3 polypeptide within a first sample obtained from said mammal when or at a time prior to when said mammal received said antisenescence treatment to a level of said circulating IL23R polypeptides, a level of saidcirculating GCG polypeptides, a level of said circulating CCL5 polypeptides, a level of said circulating IL17A polypeptides, a level of said circulating CNTN4 polypeptides, and a level of said circulating CAI 3 polypeptide within a second sample obtained from said mammal at a time after the time when said mammal received said anti- senescence treatment, to determine that said mammal comprises said decreased level of said circulating IL23R polypeptides, said decreased level of said circulating GCG polypeptides, said decreased level of said circulating CCL5 polypeptides, said decreased level of said circulating IL17A polypeptides, and said increased level of said circulating CA13 polypeptides.

59. The method of claim 58, wherein said first sample and said second sample are blood samples.

60. The method of claim 59, wherein said blood samples are plasma samples.

61. The method of any one of claims 56-60, wherein said first sample was obtained from said mammal when said mammal received said anti-senescence treatment.

62. The method of any one of claims 56-61, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

63. The method of any one of claims 56-62, wherein said revised anti-senescence treatment comprises administering a higher amount of said one or more senotherapeutic agents of said anti-senescence treatment to said mammal.

64. The method of any one of claims 56-62, wherein said revised anti-senescence treatment comprises administering one or more senotherapeutic agents that are different from said one or more senotherapeutic agents of said anti-senescence treatment.

65. A method for treating a mammal, wherein said mammal received a prior antisenescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises administering a revised anti-senescence treatment different from said prior anti-senescence treatment to said mammal, wherein said 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 circulatingCAI 3 polypeptides following said prior anti- senescence treatment.

66. The method of claim 65, wherein said mammal is a human.

67. The method of any one of claims 65-66, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

68. The method of any one of claims 65-67, wherein said revised anti-senescence treatment comprises administering a higher amount of said one or more senotherapeutic agents of said anti-senescence treatment to said mammal.

69. The method of any one of claims 65-67, wherein said revised anti-senescence treatment comprises administering one or more senotherapeutic agents that are different from said one or more senotherapeutic agents of said anti-senescence treatment.

70. A method for continuing a prior anti-senescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises administering an antisenescence treatment that is the same as said prior anti-senescence treatment, or a slightly revised version of said prior anti-senescence treatment, to a mammal identified as comprising 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 CCL5polypeptides, a decreased level of circulating IL17A polypeptides, and an increased level of circulating CA I 3 polypeptides following said prior anti- senescence treatment.

71. The method of claim 70, wherein said mammal is a human.

72. The method of any one of claims 70-71, wherein said mammal was identified as comprising one or more of a decreased level of circulating GCG polypeptides, a decreased level of circulating CCL5 polypeptides, a decreased level of circulating 1L17A polypeptides, and an increased level of circulating CAI 3 polypeptides.

73. The method of any one of claims 70-72, wherein said mammal was identified as comprising a decreased level of circulating GCG polypeptides.

74. The method of any one of claims 70-73, wherein said mammal was identified as comprising a decreased level of circulating CCL5 polypeptides.

75. The method of any one of claims 70-74, wherein said mammal was identified as comprising a decreased level of circulating IL17A polypeptides.

76. The method of any one of claims 70-75, wherein said mammal was identified as comprising an increased level of circulating CA13 polypeptides.

77. The method of any one of claims 70-76, wherein said mammal was identified as comprising 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 C A I 3 polypeptides.

78. The method of any one of claims 70-77, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

79. A method for changing an ineffective use of a prior anti-senescence treatment comprising administration of one or more senotherapeutic agents, wherein said method comprises administering an anti-senescence treatment that is different from said prior antisenescence 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 said prior anti-senescence treatment.

80. The method of claim 79, wherein said mammal is a human.

81. The method of any one of claims 79-80, wherein said one or more senotherapeutic agents are selected from the group consisting of dasatinib, quercetin, navitoclax, A1331852, Al 155463, fisetin, luteolin, geldanamycin, tanespimycin, alvespimycin, piperlongumine, panobinostat, FOX04-related peptides, nutlin-3a, ruxolitinib, metformin, and rapamycin.

82. The method of any one of claims 79-81, wherein said revised anti-senescence treatment comprises administering a higher amount of said one or more senotherapeutic agents of said anti-senescence treatment to said mammal.

83. The method of any one of claims 79-82, wherein said revised anti-senescence treatment comprises administering one or more senotherapeutic agents that are different from said one or more senotherapeutic agents of said anti-senescence treatment.

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