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senolytics might become an entirely new path for alleviating currently untreatable chronic diseases and enhancing human health span.
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After we reported the first senolytic drugs, D + Q [32], a number of additional senolytics were found by us and others. Many more clinical trials for different indications and with different senolytic drugs and combinations need to be completed to confirm the findings from the three clinical trials of senolytics reported in peer-reviewed journals so far.
Here, in an open-label Phase 1 pilot study, we show for the first time that senolytic drugs decrease senescent cell abundance in humans. A 3-day oral course of D + Q in subjects with diabetic kidney disease (DKD) reduced adipose tissue senescent cell burden 11 days later, as indicated by decreases in cells with markers of senescence: p16INK4A-and p21CIP1-expressing cells, cells with senescence-associated β-galactosidase (SAβgal) activity, and adipocyte progenitors with limited replicative potential.
Much remains to be done to optimize senolytic regimens and to identify the possibly broad range of effects and side effects of these agents in humans. If senolytic agents can be shown to be effective for several individual age-related conditions, they may prove to have a role beyond alleviating single diseases: they may be effective in reducing the multimorbidity common in elderly patients. In the first demonstration that healthspan can be improved by removing senescent cells from naturally-aged mice, we found that clearing senescent cells with D + Q improves cardiac function and vascular reactivity in old mice, alleviates frailty and increases intervertebral disc proteoglycans in progeroid mice, and enhances treadmill endurance in single-leg radiation-injured mice in early 2015 [32].
In mouse models of age- and senescence-related chronic diseases, D + Q alleviated metabolic dysfunction in high fat diet-fed as well as genetically obese mice(in part by decreasing insulin resistance), DKD manifested by podocyte dysfunction and proteinuria, high fat diet-induced renal fibrosis, renal cortical hypo‑oxygenation, and increased creatinine, hepatic steatosis, neuropsychiatric dysfunction in high fat-fed and genetically obese mice, high fat diet-induced cardiac dysfunction and vascular hyporeactivity, Alzheimer's-like dementia in mice with brain tau or β-amyloid protein aggregates, failure of the arteriovenous fistulae used for vascular access during hemodialysis, bleomycin-induced pulmonary fibrosis (a mouse model of IPF), and hyperoxia-induced pulmonary dysfunction, among others [7,9,20,23,24,[32], [33], [34], [35], [36], [37]]. Thus, senolytic agents might have the potential to delay, prevent, or treat age-related diseases as a group, instead of one-at-a-time. To advance toward this goal, it is critical to focus on developing senolytic agents that are safe and effective when administered systemically (as opposed to locally by injection) in humans.
The intended target of senolytics is senescent cells. We present evidence showing that the drugs, D + Q, which are senolytic in mice and cell or tissue culture models, decrease senescent cells in humans. No individual markers of senescent cells appear to be fully sensitive and specific. Furthermore, individual factors associated with the SASP can be produced by non-senescent cells, such as immune cells. We therefore assayed several key cellular senescence markers and circulating SASP factors in tandem as well as markers of macrophages, which can exhibit features similar to those of senescent cells [59]. Macrophages are attracted, activated into a senescent-like state, and anchored by senescent cells in adipose tissue [23].
Key markers of senescent cell burden were decreased in adipose tissue and skin biopsied from subjects 11 days after completing the 3-day course of D + Q, as were key circulating SASP factors, compared to before administration of these senolytic drugs.
Although we are optimistic about the prospects for introducing senolytics and other agents that modulate fundamental ageing processes into clinical practice in the future, particularly in the near future for serious diseases for which there are currently no effective interventions, we must conclude with a note of caution. The field of senolytics is new. The first clinical trial of senolytic agents was only reported in January 2019. The findings reported here are preliminary results from an ongoing clinical trial of senolytics for treating dysfunction in patients with diabetic chronic kidney disease. Fewer than 150 subjects have been treated with these drugs in the context of clinical trials that we are aware of so far. In addition to side effects related to individual senolytic drugs known from other contexts in which those drugs have been used, there could turn out to be serious side-effects of senolytics as a class, which are not yet known. We caution against the use of senolytic agents outside the context of clinical trials until more is known about their effects and side effects.
https://www.sciencedirect.com/science/article/pii/S2352396419305912
By definition, the target of senolytics is senescent cells, not a molecule or a single biochemical pathway. The first senolytic drugs, Dasatinib (D) and Quercetin (Q), were discovered using a mechanism-based approach instead of the random high-throughput screening usually used for drug discovery [32]. Through a bioinformatics strategy that leveraged proteomic and transcriptomic data, we discovered networks of senescent cell anti-apoptotic pathways (SCAPs) that enable senescent cells to survive despite their own SASP. We verified this role of these networks in senescent cells by using RNA interference to show that targeting SCAP nodes, such as BCL-xL in the case of human umbilical vein endothelial cells, kills senescent cells.
Because senescent cells can take weeks to months to develop and do not divide, and because even eliminating only 30% of senescent cells can be sufficient to alleviate dysfunction in preclinical studies [3,5,7,9,23,24,28,[32], [33], [34], [35], [36], [37]], D + Q is as effective in mice if administered intermittently, for example every 2 weeks to a month, as continuously, even though D and Q have elimination half-lives of only 4 and 11 h, respectively
Implications of all the available evidence
Interventions targeting fundamental ageing processes such as cellular senescence could delay, prevent, or alleviate multiple age-related diseases and disorders as a group, instead of one-at-a-time, as per the Geroscience Hypothesis. Increasingly in mice, this hypothesis appears to be true. Combined with the first clinical trial of senolytic agents showing that D + Q improves physical function in patients with IPF published earlier this year in this journal, our current article showing that D + Q actually decreases senescent cell burden in humans is consistent with the possibility that the Geroscience Hypothesis may also hold true for humans. If clinical trials over the next few years support and extend our findings to show that these agents can alleviate additional age- and cellular senescence-related disorders and diseases (beyond IPF) and reduce senescent cell burden (beyond adipose tissue and skin and as reflected by decreased SASP factors in blood), senolytics might become an entirely new path for alleviating currently untreatable chronic diseases and enhancing human healthspan.
Cellular senescence is a cell fate that entails essentially irreversible replicative arrest and that was first reported in serially sub-cultured human cells in 1961 [1]. Insults such as serial passaging, DNA damage, exposure to the damage-associated molecular pattern molecules that accumulate in injured or chronically-diseased tissues, and metabolic insults can cause cells to become senescent [2]. Senescent cells and pre-senescent cells with limited replicative potential accumulate with ageing in multiple tissues in which they are linked to functional declines, including in adipose tissue, where abundance of adipocyte progenitors with decreased proliferative capacity increases with ageing
Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease
Abstract
Background
Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in multiple chronic diseases. Senolytics, including the combination of Dasatinib and Quercetin (D + Q), selectively eliminate senescent cells by transiently disabling pro-survival networks that defend them against their own apoptotic environment. In the first clinical trial of senolytics, D + Q improved physical function in patients with idiopathic pulmonary fibrosis (IPF), a fatal senescence-associated disease, but to date, no peer-reviewed study has directly demonstrated that senolytics decrease senescent cells in humans.
Methods
In an open label Phase 1 pilot study, we administered 3 days of oral D 100 mg and Q 1000 mg to subjects with diabetic kidney disease (N = 9; 68·7 ± 3·1 years old; 2 female; BMI:33·9 ± 2·3 kg/m2; eGFR:27·0 ± 2·1 mL/min/1·73m2). Adipose tissue, skin biopsies, and blood were collected before and 11 days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed.
Findings
D + Q reduced adipose tissue senescent cell burden within 11 days, with decreases in p16INK4A-and p21CIP1-expressing cells, cells with senescence-associated β-galactosidase activity, and adipocyte progenitors with limited replicative potential. Adipose tissue macrophages, which are attracted, anchored, and activated by senescent cells, and crown-like structures were decreased. Skin epidermal p16INK4A+ and p21CIP1+ cells were reduced, as were circulating SASP factors, including IL-1α, IL-6, and MMPs-9 and -12.
Interpretation
"Hit-and-run" treatment with senolytics, which in the case of D + Q have elimination half-lives <11 h, significantly decreases senescent cell burden in humans.
Fund
NIH and Foundations.
After we reported the first senolytic drugs, D + Q [32], a number of additional senolytics were found by us and others [37,[64], [65], [66], [67], [68], [69], [70]]. For example, based on our report that BCL-xL is an endothelial cell SCAP pathway target in early 2015 [32], we subsequently found that the BCL-2 family inhibitor, Navitoclax (N; also known as ABT-263), and the more specific BCL-xL inhibitors, A1331852 and A1155463, are, like Q, senolytic in the case of senescent human endothelial cells, but not senescent human adipocyte progenitors [64,71]. Thus, the range of senescent cells targeted by BCL-xL inhibitors on their own is restricted. Our rationale for using D + Q as opposed to other drugs in the trial reported here follows. D + Q is more specifically senolytic than other drugs that act as "panolytics", including some agents that bind MDM2, preventing p53 destabilization (e.g., Nutlin3a) and some BCL-2 family inhibitors (e.g., N). Some such agents, for example nutlins, can cause severe (class 3/4) off-target reductions in non-senescent cell types, including platelet deficiency causing bleeding, neutropenia causing infections, and deficiencies of other cell types [72,73]. This impact on multiple cell types (over and above senescent cells) of drugs that act principally on MDM2 can make it difficult to attribute clinical effects of such drugs to selective targeting of senescent cells (as opposed to non-senescent cells). While D can also sometimes cause transient and reversible platelet deficiency, this is uncommon. Even with hematological malignancies that themselves can cause platelet depletion, significant (class 3/4) bleeding occurs in <1% of patients on continuous D treatment. This usually only occurs after months of uninterrupted D administration, unlike the intermittent D regimen that we found here to be senolytic (Web Reference 1). Additionally, some senolytics (e.g., Q) and panolytic drugs (e.g., N) do not target certain types of senescent cells, such as senescent human adipocyte progenitors [71], which are a major senescent cell type in adipose tissue, and which were studied in the clinical trial reported here, another reason for combining D with Q in our study. A benefit of D + Q is that this senolytic combination more comprehensively disables SCAP networks than N, increasing the range of senescent cell types selectively eliminated by D + Q [32]. We therefore studied systemic administration of D + Q as opposed to panolytic drugs (e.g., Nutlin3a), since the latter may have to be injected locally instead of systemically to avoid adverse effects caused by their off-target depletion of non-senescent cell types.
A combination of the BCL-2 inhibitors ABT-263 and A-1210477 inhibited cell proliferation in the HeLa, C33A, SiHa and CaSki human cervical cancer cell lines. Drug sensitivity was initially tested using 2-dimensional (2D) cell culture models. As ABT-263 binds to both BCL-2 and BCL-XL at high affinity, it was unclear whether the synergism of the drug combination was driven either by singly inhibiting BCL-2 or BCL-XL, or inhibition of both. Therefore, we used the BCL-2 selective inhibitor ABT-199 and the BCL-XL selective inhibitor A1331852 to resolved the individual antitumor activities of ABT-263 into BCL-2 and BCL-XL dependent mechanisms. A-1210477 was substituted with the orally bioavailable S63845. The SiHa, C33A and CaSki cell lines were resistant to single agent treatment of all three drugs, suggesting that none of these anti-apoptotic proteins singly mediate survival of the cells. HeLa cells were resistant to single agent treatment of ABT-199 and A1331852 but were sensitive to S63845 indicating that they depend on MCL-1 for survival. Co-inhibition of BCL-XL and MCL-1 with A1331852 and S63845 significantly inhibited cell proliferation of all four cell lines. Similar data were obtained with 3-dimensional spheroid cell culture models generated from two cervical cancer cell lines in vitro. Treatment with a combination of A1331852 and S63845 resulted in inhibition of growth and invasion of the 3D spheroids. Co-inhibition of BCL-2 and MCL-1 with ABT-199 and S63845, also inhibited cell proliferation of all cancer cell lines, except SiHa. However, the effect of the combination was not as pronounced as combination of A1331852 and S63845. Collectively, our data demonstrate that the combination of MCL-1-selective inhibitors with either selective inhibitors of either BCL-XL or BCL-2 may be potentially useful as treatment strategies for the management of cervical cancer.
https://www.biorxiv.org/content/10.1101/824649v1.full
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