Strategies for Late Phase Preclinical and Early Clinical Trials of Senolytics

Abstract

Cellular senescence and the hallmarks of aging contribute to age-related disease and dysfunction. The Unitary Theory of Fundamental Aging Mechanisms highlights the interdependence among the hallmarks of aging and suggests that by intervening in one fundamental aging process, most or all of the other processes could be impacted. Accumulation of senescent cells is associated with frailty, cardiovascular disease, obesity, diabetes, cognitive decline, and other age- and/or chronic disease-related disorders, suggesting that senescent cells are a target for intervention. Early preclinical data using senolytics, agents that target senescent cells, show promising results in several aging and disease models. The first in-human trials using the senolytic combination of Dasatinib and Quercetin indicated reduced senescent cell burden in adipose tissue of diabetic kidney disease patients and improved physical function in patients with idiopathic pulmonary fibrosis. Clinical trials with other senolytics, including the flavonoid Fisetin and BCL-xL inhibitors, are underway. These results from preclinical and early clinical trials illustrate the potential of senolytics to alleviate age-related dysfunction and diseases. However, multiple clinical trials across different aging and disease models are desperately needed. Parallel trials across institutions through the Translational Geroscience Network are facilitating testing to determine whether senolytics can be translated into clinical application.

1. Introduction

1.1. Impact of aging/disease: Need for senolytics

As the aging population steadily increases, age-related diseases and disorders are becoming a major subset of health problems [1]. Since 2002, the leading causes of death in individuals aged 65 or older has been age-related diseases, with cardiovascular disease, cancer, and chronic lower respiratory disease accounting for over half of these deaths [2]. Frailty, neurodegenerative diseases, cardiac dysfunction, respiratory diseases, cancers, and other chronic diseases are directly impacted by senescent cell accumulation [3]. Additionally, senescent cell burden negatively impacts healthspan and lifespan [4]. As more people suffer from these chronic conditions, the burden on the healthcare system grows. Current treatment options for many of these conditions are focused on treating them one-disease-at-a-time. This is as opposed to intervening against multiple disorders together, since multimorbidity often occurs in older people, by targeting root causal fundamental aging processes shared across these conditions. Targeting cellular senescence using senolytics, agents that selectively clear senescent cells, could be a potential therapeutic option to delay, prevent, or treat multiple age-related disorders.

2. Geroscience Hypothesis

2.1. Hallmarks of Aging

Fundamental aging mechanisms can be grouped into hallmarks or pillars of aging. One such grouping includes nine hallmarks: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication [5, 6]. Each of these pillars can contribute to age-related disease and dysfunction (Figure 1).

Figure 1:

The hallmarks of aging may underpin a variety of age-related diseases and disorders, as well as chronic disease in younger individuals.

2.2. Unitary Theory of Fundamental Aging Processes

The nine hallmarks of aging may not be independent processes, but rather interdependent [7]. Our Unitary Theory of Fundamental Aging Processes posits that by targeting one fundamental aging process, it may be feasible to impact several or all others [7][8]. Cellular senescence is bi-directionally linked to other hallmarks of aging, including epigenetic alterations [5], mitochondrial dysfunction [9][10][11][12], dysfunctional nutrient processing [13–15], stem cell exhaustion [16][17][18], altered cellular communication [19][19][5], genomic instability [20][21], telomere attrition [22][23][24][25], and loss of proteostasis [26][27]. Because of its known and well documented interactions with other aging hallmarks, cellular senescence appears to be an appropriate target for intervention.

2.3. Cellular senescence as a target

Cellular senescence is a cell fate that entails cell cycle arrest first observed in human fibroblasts by Hayflick and Moorehead in 1961 [28]. Senescent cells resist apoptosis but can be removed by the immune system [29][30]. Our ‘Threshold Theory of Senescent Cell Burden’ posits that there is a point beyond which senescent cell accumulation exceeds capacity of the immune system to clear senescent cells, leading to rising senescent cell burden and associated acceleration of age- and disease-related dysfunction [31][7][13].

Cellular senescence presents a unique target for intervention at two points: 1) clearance of senescent cells and 2) inhibition of production of senescence-associated secretory phenotype (SASP) factors. The accumulation of senescent cells can cause immune system dysfunction (inducing further senescent cell accumulation), inflammation, tissue damage, and cell impairment due to the SASP [32]. The SASP, secreted by some, but not all, senescent cells, can lead to stem and progenitor cell damage, as well as tissue and systemic dysfunction [33][34][7]. The SASP includes a variety of cytokines, chemokines, proteases, activins and inhibins, growth factors, bioactive lipids, micro-RNAs, cell-free mitochondrial DNA, other non-coding nucleotides, and microsomes and exosomes [32][35][7][36]. Senescent cells and their SASP are found at etiological sites in a host of age-related diseases and disorders including frailty, cardiovascular disease, osteoporosis, cancers, diabetes/obesity, and other chronic diseases, even in children and younger adults [37][38][39][40]. Senotherapeutics include agents that target both of these processes: senolytics selectively target senescent cells and senomorphics (e.g., metformin or rapamycin), act as SASP inhibitors [41].

Senomorphics can suppress effects of senescence in one of two ways, (i) controlling the regulatory network of the SASP, and (ii) by targeting a specific SASP component. Although senomorphics may be beneficial to individuals who take them regularly, it is unclear if intermittent dosing would provide the same benefits [42]. More research is needed to determine the efficacy of senomorphics.

3. Discovery of senolytic drugs

Prompted by the finding that senescent cells are linked to declines in healthspan [4], which suggested senescent cells a potential target for intervention, our team began the search for potential senolytic interventions. We turned to a hypothesis-driven, mechanism-based drug discovery approach for identifying possible senolytics. Using the principles that senescent cells resist their own removal through Senescent Cell Anti-Apoptotic Pathway (SCAP) networks, we targeted several different SASP pathways by RNA interference to identify key nodes in the SCAP network [43]. Once identified, we used bioinformatics approaches to identify compounds that target these nodes and could possibly be effective in enabling apoptosis of those senescent cells that have a pro-apoptotic SASP [44][43]. Eventually, 46 compounds were so identified as being potentially senolytic. We focused on advancing those compounds that are natural products or drugs already used in humans to expedite translation to clinical application.

The first generation of senolytics were mostly agents or combinations of agents with multiple SCAP targets, rather than being agents with a single molecular target that follow a traditional one target-one drug-one disease model [7]. Initially Dasatinib (D), an anti-cancer agent, was identified as senolytic against human senescent adipocyte progenitors, and Quercetin (Q), a naturally occurring flavonoid, senolytic against umbilical vein endothelial cells, with the combination, D+Q, being senolytic against a broader range of senescent cell types than either alone [45]. Fisetin, a naturally occurring flavanoid related to Q, was found to also be senolytic [46]. Later, we and another group identified Navitoclax, which has several BCL-2-related targets, as being a potential senolytic [7, 27, 45]. However, Navitoclax induces apoptosis in HUVECs but not human preadipocytes, remains to date an investigational agent that is not yet on the formulary, and can have severe off-target effects on non-senescent cells, including severe neutropenia and platelet deficiency (although there has been progress in devising a solution for the latter) [46][45][47][7]. After the first senolytics were identified using the mechanism-based approach, a second approach for identifying senolytics, using high-throughput drug library screens, is currently being developed and appears to be successful [7][48]. The first FDA-registered clinical trial of the combination of D+Q was a cancer trial [42]. This 2006 trial included patients who had B lymphoma as well as other certain types of leukemia. While senolytics specifically clear senescent cells, if the Unitary Theory of Fundamental Aging Processes is correct, senolytics could act to alleviate adverse effects of other fundamental aging mechanisms.

4. Preclinical trials

4.1. Senolytics alleviate multiple disorders in mouse models

Senolytics have been tested in a variety of different preclinical models [7]. In naturally-aged mice, treatment with D+Q improved cardiac function; in radiation-exposed mice, D+Q improved exercise capacity; in progeroid Ercc1(−/Δ) (accelerated aging) mice, D+Q delayed age-related dysfunction [45][39]. Idiopathic pulmonary fibrosis (IPF) is characterized by senescent cell accumulation in the lungs, and in bleomycin-injured mice, D+Q effectively killed senescent fibroblasts, leading to improvement in physical and pulmonary function [49]. In obese mice, D+Q alleviated metabolic and adipose tissue dysfunction [15]. D+Q also helped decrease senescent cell burden in atherosclerosis mouse models [39]. Furthermore, in osteoporosis mouse models, treatment with D+Q increased bone mass and strength [40].

Fisetin has also had favorable preclinical results. Fisetin induced apoptosis in senescent human umbilical vein endothelial cells (HUVECs) [49]. In wild-type mice, Fisetin decreased age-related pathology and increased lifespan [50]. There is also growing preclinical data about the effectiveness of Fisetin in alleviating neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, ALS, and Huntington’s disease [51].

4.2. Translation to humans

Because of the promising preclinical data showing the efficacy of senolytics on a variety of different disease models, senolytics were considered for clinical trials. Due to the novel nature of senolytic agents and the unknown risk/benefit ratio, senolytics were first considered for clinical trials in patients with serious conditions including diabetes, IPF, and osteoporosis [7]. Fisetin and Quercetin, naturally occurring flavanoids, have favorable safety profiles and therefore have been considered for a variety of clinical trials, including with healthy older adults. Dasatinib has been used in clinical practice since 2006 and has a well-established safety profile. With approval from the Food and Drug Administration (FDA), the first clinical trials using Fisetin and D+Q have begun and many more are in the planning stages.

5. Current and Planned Clinical Trials

5.1. Active trials, completed trials, and early results

The first in-human trial of senolytics showed improved physical function in idiopathic pulmonary fibrosis (IPF) patients after treatment with D+Q [52]. Another early clinical trial showed treatment with D+Q reduces senescent cell burden in adipose tissue of diabetic kidney disease patients [53]. Recently, an open label pilot phase 1 trial of D+Q for Alzheimer’s disease, SToMP-AD [54], has been completed with results that are about to be submitted for publication and the Phase 2 component of this trial is about to begin. Currently active trials with senolytics include, but are not limited to, those listed in Table 1.

Table 1.

Active Translational Geroscience Network (TGN) Trials with Senolytics.

Dasatinib + Quercetin (D+Q) Population/Disease	Lead Site	ClinicalTrials.gov #
Senescent Cell Burden in Hematopoietic Stem Cell Transplant Survivors	• Mayo Clinic	NCT02652052
Senolytic Therapy to Modulate Progression of Alzheimer’s Disease	• The University of Texas Health Science Center at San Antonio	NCT04063124
Senescence, Frailty, and Mesenchymal Stem Cell Functionality in Chronic Kidney Disease: Effect of Senolytic Agents	• Mayo Clinic	NCT02848131
Fisetin (F)
Use of Senolytic and Anti-Fibrotic Agents to Improve the Beneficial Effect of Bone Marrow Stem Cells for Osteoarthritis	• The Steadman Clinic	NCT04815902
Senolytic Drugs Attenuate Osteoarthritis-Related Articular Cartilage Degeneration: A Clinical Trial	• The Steadman Clinic	NCT04210986
COVID-FISETIN: Pilot in SaRS-CoV-2 of Fisetin to Alleviate Dysfunction and Inflammation	• Mayo Clinic	NCT04476953
Alleviation by Fisetin of Frailty, Inflammation, and Related Measures in Older Adults (AFFIRM-LITE)	• Mayo Clinic	NCT03675724
Alleviation by Fisetin of Frailty, Inflammation, and Related Measures in Older Women (AFFIRM)	• Mayo Clinic	NCT03430037
Inflammation and Stem Cells in Diabetic and Chronic Kidney Disease	• Mayo Clinic	NCT03325322
Both Arms
Targeting Cellular Senescence with Senolytics to Improve Skeletal Health in Older Humans	• Mayo Clinic	NCT04313634

5.2. Planned trials

The first clinical trials provided safety and early efficacy data [52, 53]. Since the initial data was published, many trials using senolytics have been planned (Table 2). Most recently, our team found application of senolytics (both Fisetin and D+Q) effective on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mouse models [55]. With the COVID-19 pandemic still very much present, several trials for COVID-19 application are in the planning stages, with one ongoing for hospitalized patients (COVID-FISETIN: Pilot in SARS-CoV-2 of Fisetin to Alleviate Dysfunction and Inflammation, NCT04476953).

Table 2.

Planned Translational Geroscience Network (TGN) Trials with Senolytics.

Dasatinib + Quercetin (D+Q) Population/Disease	Lead Site	ClinicalTrials.gov #
ALSENLITE: An Open-Label Pilot Study of Senolytics for Alzheimer’s Disease	• Mayo Clinic	NCT04785300
Senolytic Therapy to Modulate the Progression of Alzheimer’s Disease (SToMP-AD) Study	• Wake Forest Health Sciences	NCT04685590
Fisetin (F)
COVFIS-HOME: COVID-19 Pilot Study of Fisetin to Alleviate Dysfunction and Decrease Complications	• Mayo Clinic	NCT04771611
COVID-FIS: Pilot in COVID-19 (SARS-CoV-2) of Fisetin in Older Adults in Nursing Homes	• Mayo Clinic	NCT04537299
Both Arms
An Open-Label Intervention Trial to Reduce Senescence and Improve Frailty in Adult Survivors of Childhood Cancer	• St. Jude Children’s Research Hospital	NCT04733534

6. Translational Geroscience Network

Due to the need for effective interventions and the slow nature of preclinical and clinical trials, a Translational Geroscience Network (TGN) was formed to conduct clinical trials of interventions targeting fundamental aging mechanisms in parallel (Figure 2). The TGN comprises 8 institutions: Mayo Clinic, Harvard, John Hopkins, Wake Forest, Universities of Minnesota, Michigan, and Connecticut, and University of Texas Health Sciences Center at San Antonio, as well as partners, including St. Jude Children’s Cancer Hospital and the Steadman Clinic. There are several subcommittees in the TGN including a Data Management and Statistical Analysis Subcommittee, the Diversity, Equity, and Inclusion Subcommittee, and Regulatory, Website, Biobanking, and Facility for Geroscience Analysis (FGA) Subcommittees.

Figure 2:

The Translational Geroscience Network (TGN) has subcommittees to achieve the four aims of the network.

The FGA is a laboratory core that develops and conducts innovative new assays for aging and disease markers. These tests are currently being used to assess target engagement and efficacy of senolytics in preclinical and early phase clinical trials. The FGA is exploring new markers of fundamental aging mechanisms and related diseases. With unified infrastructure and resources across multiple institutions, the TGN may be able to quickly navigate obstacles of initiating and completing trials of interventions targeting basic aging mechanisms, including senolytics.

7. Conclusions and Future Directions

The hallmarks of aging may be a mechanistic contributor to age-related phenotypes and multiple diseases and disorders. If the Unitary Theory of Fundamental Aging Mechanisms is true, intervening against one pillar will affect most, if not all, of the other pillars of aging. Cellular senescence may be a good interventional target and it is hoped that senotherapeutics, including senolytics and senomorphics, will prove to be efficacious. In future trials, the impact of combining multiple aging therapeutics to treat different aging processes needs to be determined. These combinations may have reduced efficacy or result in additive or synergistic effects compared to individual interventions for targeting multiple age-related or chronic conditions and diseases. As the number of people suffering from age-related disease and disorders continues to increase, it is becoming even more important to establish collaborations and infrastructure like the TGN to facilitate and accelerate translation of agents that target fundamental aging mechanisms into clinical practice.

Highlights.

• Accumulation of senescent cells is associated with age-related diseases and disorders

• Senolytics, including senolytics and senomorphics, are potential interventions for delaying, preventing, or treating age-related dysfunction

• Senolytics show efficacy in pre-clinical and early clinical trials

• Running trials in parallel across several institutions as modeled by the Translational Geroscience Network (TGN) could fast-track clinical application of senolytics

Abbreviations

D

Dasatinib

D+Q

Dasatinib and Quercetin

FDA

Food and Drug Administration

FGA

Facility for Geroscience Analysis

HUVECs

Human Umbilical Vein Endothelial Cells

IPF

Idiopathic Pulmonary Fibrosis

Q

Quercetin

SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus-2

SASP

Senescence-Associated Secretory Phenotype

SCAP

Senescent Cell Anti-Apoptotic Pathway

TGN

Translational Geroscience Network