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Preface

This risk-benefit analysis is part of Forever Healthy's "Rejuvenation Now" initiative that seeks to continuously identify potential rejuvenation therapies and systematically evaluate their risks, benefits, and associated therapeutic protocols to create transparency.

Section 1: Overview 

Motivation

Cellular senescence, a state of essentially irreversible replicative arrest, is one of the hallmarks of aging. Senolytics are drugs that act by selectively facilitating apoptosis of senescent cells by transiently disabling one or more of the senescent cell anti-apoptotic pathways (SCAPs) that enable senescent cells to survive.

Dasatinib & Quercetin (D+Q) were the first senolytic drugs to be discovered and as they have been shown to affect different SCAPs in vitro, targeting different types of senescent cells, they are often employed in combination.

It is supposed that intermittent dosing of D+Q in combination leads to the elimination of senescent cells in humans and by doing so, has the potential to delay, prevent or alleviate multiple age-related diseases and increase the healthy lifespan. 

Key Questions 

This analysis seeks to answer the following questions:

• Which benefits result from D+Q senolytic therapy? 

• Which risks are involved in D+Q senolytic therapy (general and method-specific)?

• What are the potential risk mitigation strategies?

• Which method or combination of methods is the most effective for D+Q senolytic therapy?

• Which of the available methods are safe for use? 

• What is the best therapeutic protocol?

• What is the best treatment monitoring strategy available at the moment?

Impatient readers may choose to skip directly to Section 5 for the presentation of the results. 

Section 2: Methods

Analytic model

This RBA has been prepared based on the principles outlined in A Comprehensive Approach to Benefit-Risk Assessment in Drug Development (Sarac et al., 2012). 

Literature search

A literature search was conducted on PubMed and the Cochrane Library using the search terms shown in Table 1 and includes results available as of April 17, 2020. Titles and abstracts of the resulting studies were screened and relevant articles downloaded in full text. The references of the full-text articles were manually searched in order to identify additional studies that may have been missed by the search terms.

Inclusion criteria: All studies (clinical, preclinical, in vitro) that tested D or Q or the combination as senolytics were included. In order to assess the adverse effects of each compound, we also included studies in humans that were performed for the usual indications of the drugs.

Exclusion criteria: We excluded studies that used combined chemotherapy regimens from our analysis as well as preclinical studies in our assessment of adverse effects. 

Table 1: Literature Search 

Recommended Reading

General Introduction

The following sites offer information on Dasatinib & Quercetin senolytic therapy at a consumer level and are useful as an introduction to the topic:

• The Scripps Research Institute – Dasatinib and Quercetin - lifespan.io

• First-in-human trial of senolytic drugs encouraging Small pilot study points to feasibility of larger trials in age-related diseases - sciencedaily.com

• Senolytics - thelri.org 

• Young anti-aging field takes big step with Mayo Clinic senolytics showcase - Endpts.com
  
  
  

Scientific Overview 

The following scientific reviews provide a more detailed overview of the topic of senolytic therapy:

• Discovery, development, and future application of senolytics: theories and predictions

• Cellular Senescence: A Translational Perspective

Abbreviation list

Section 3: Existing evidence

Summary of results

We screened 3,343 papers and included 156 in our analysis. We identified 118 relevant human studies that used D or Q, 111 of which were related to side-effects or safety. In total, there have only been 3 trials that used D+Q as senolytics in human subjects. Two of the clinical trials were of relatively high quality but were both small, phase I, open-label studies (n= 9,14) on subjects with pre-existing diseases (lung and chronic kidney) (Hickson et al., 2019; Justice et al., 2019). A corrigendum with a reanalysis of data from one of the trials was also included (Hickson et al., 2020). The third trial is a randomized control trial (RCT) of low quality but did have 4 test groups (D+Q, D+placebo, Q+placebo, placebo+placebo) and enrolled healthy participants (Tkemaoadze & Apkjazava, 2019).

A fourth study in which senescent cell markers from skin biopsies were measured retrospectively (dasatinib only) was also chosen for inclusion. Additionally, there are 4 trials listed on clinicaltrials.gov that are expected to publish results over the next 3 years. 

We identified 31 preclinical trials related to the use of D+Q as senolytics, alone or in combination. 12 of the studies investigated the senolytic effects of Q alone. We included another 7 preclinical studies that provided possible mechanisms for side effects encountered in clinical trials. 

Clinical trials

Table 2: Clinical trials

Preclinical trials

Table 3: Preclinical trials

Section 4: Risk-Benefit Analysis

Decision Model

Risk and benefit criteria

The decision profile is made of up risk and benefit criteria extracted from the outcomes of the above-mentioned papers. The benefit criteria are organized by category and include the type, magnitude, and duration of the benefit as well as its perceived importance to the patient. The risk criteria are organized by category, type, severity, frequency, detectability, and mitigation. All are assigned numerical values: 

1 = low

2 = moderate

3 = high

The numerical values for both risk and benefit criteria are then summarized serving as the justification for the weighting in the following column.

Weight

The criteria are weighted on a value scale to enable comparison (based on the relative importance of a difference). Risk and benefit criteria are assigned to either low (1-1.66), medium (1.67-2.33), or high (2.34-3) weighted categories.

Weighting is independent of data sets and the final weights are based on consensus with justification based on the preceding columns of the table.

Score

Each category is assessed according to the performance of D+Q senolytic therapy against the comparator (physiological aging) whereby a numerical value is assigned for each criterion -1 (inferior), 0 (equivalent or non-inferior), and +1 (superior) to the comparator.

Uncertainty

Uncertainty is determined according to the amount and quality of the evidence, whether it came from human or animal studies and whether methodological flaws, conflicting studies, or conflicts of interest (funding) by the authors are present. Evidence that is based on human RCTs or systematic reviews is initially assigned an uncertainty score of 1, evidence from open-label trials is assigned a score of 2, and evidence that is based on observational studies, and preclinical trials is assigned a score of 3. The uncertainty score is then adjusted by upgrading or downgrading using the above-mentioned criteria. 

Weighted score

The weights and scores are multiplied to produce weighted scores that enable direct comparison (-3 → +3) and then adjusted using the uncertainty score. Weighted scores may be upgraded where the uncertainty of the evidence is low or downgraded where the uncertainty of the evidence is high. 

Benefit assessment 

Our analysis identified a total of only 8 benefits that have been documented in human studies and another 46 benefits from preclinical trials (Table 4). Of the 8 benefits in humans, 5 were actually various measurements of markers of senescence or the SASP, hypothesized to translate to clinically beneficial effects. Only 3 benefits had any direct clinical relevance and they were of low magnitude. Based on the current state of evidence, the beneficial effects of D+Q seem to be extremely limited in humans.

However, the benefits identified in the preclinical studies are significant and encompass many organ systems. Of note, several of the benefits only occurred in diseased populations (ie. diabetic mice) and did not extend to control populations that received treatment with D+Q.

Table 4: Benefit assessment

Benefits from clinical trials

Markers of Senescence

An open-label phase 1 clinical trial (n=9) of a 3-day oral course of D+Q (100 mg + 1000 mg) in patients with chronic kidney disease (aged 50-80) was the first to measure a decrease in the number of several key markers of senescence (Hickson et al., 2019).

The number of p16INK4a+ cells was reduced by 35% in adipose tissue biopsies and 20% in the epidermal layer (although the result did not reach statistical significance). Epidermal p16INK4a cells have been associated with cardiovascular disease (CVD) risk and "aging" (Waaijer et al., 2012). 

The number of p21CIP1+ cells was also decreased (Hickson et al., 2019). The raw values were reduced by 17% in adipose tissue biopsies and 31% in the epidermis. That the reductions occurred in both adipose tissue and skin suggests that D+Q treatment works systemically to decrease senescent cell burden.

D+Q also reduced the number of SABgal+ cells by 62% and decreased the number of macrophages per adipocyte by 28% (Hickson et al., 2019). Senescent cells have been shown to attract, activate, and anchor macrophages in adipose tissue (Hickson et al., 2019).

Senescent cells and macrophages contribute to the formation of the "crown-like structures" (CLS) characteristically found in adipose tissue in diabetes and obesity. The study reported 86% fewer CLS per adipocyte following treatment with D+Q (Hickson et al., 2019). 

Senescent and pre-senescent cells have no or limited replicative potential, resulting in increased population doubling times as they accumulate. The trial also found there was an increase in the number of primary adipocyte progenitors which is consistent with the effects of removing senescent cells (Hickson et al., 2019).

While as of yet, there is no ideal marker for senescent cells, the changes in the several markers mentioned above indicate that treatment with D+Q is likely effective as a senolytic in humans. It appears that senolytics work by facilitating apoptosis of senescent cells due to their SASP, not by targeting all cells expressing pINK4a (Hickson et al., 2019).

The changes in multiple tissues (skin, adipose tissue, plasma) suggest that oral administration of D+Q decreases overall senescent cell burden rather than targeting cells within a single organ or structure (Hickson et al., 2019).

Decreases in circulating SASP factors/gene expression

An open-label trial (n=9) found that there was a decrease in circulating SASP factors (plasma IL-1a, IL-2, IL- 6, IL-9 and MMP 2, MMP 9, and MMP 12) following 3 days of senolytic treatment (Hickson et al., 2019). FGF-2, GM-CSF, and IL-1RA also tended to be lower but did not reach significance. A recently published reanalysis of the data found that the composite score of the SASP was significantly reduced despite only MMP-12 being decreased significantly in isolation (Hickson et al., 2020).

A second open-label trial (n=14) in patients with idiopathic pulmonary fibrosis (IPF) found that select SASP proteins including IL-6, MMP-7 and TIMP2 showed a trend towards reduction (8 participants had reductions in circulating amounts) following treatment with D+Q 3 days per week for 3 weeks (Justice et al., 2019).

An analysis of SASP gene signatures in skin biopsies from a trial (n=12) that used D (100 mg) for 169 days to treat systemic sclerosis-associated interstitial lung disease (Martyanov et al., 2019) found that in the subset of patients that responded to D treatment (n=3) SASP levels were both higher at baseline and, significantly lower post-treatment compared with non-improvers. 80.3% (53/66) of the SASP gene signatures showed a decrease in expression post-treatment which was correlated with clinical improvements (vs. 53% (35/66) in non-improvers).

Cardiovascular system

One RCT (n=64) in healthy volunteers (over the age of 36 years) reported a significant reduction in post-exercise systolic blood pressure at 10 and 20 minutes in the group that received treatment with D+Q for 5 days (Tkemaoadze & Apkjazava, 2019). The initial blood pressure in all groups was approximately 115 mmHg and decreased to 108 mmHg in the D+Q group at 10 minutes after the completion of the "stair-ascending test" while the BP of the control group decreased to 112 mmHg. The difference was still significant at 20 minutes post-exercise but reached the same value in all groups by 30 minutes.

Increased physical function in IPF

An open-label trial reported improvements in physical function that included improved 6-min walk distance, 4-m gait speed, and 5-repeated chair-stand times (Justice et al., 2019). These improvements were consistent with preclinical findings of improvements in treadmill endurance and frailty following senescent cell removal in various murine models.

"Lightness" in joints

One RCT (n=64) in healthy volunteers reported that nearly all participants in the D+Q group experienced a feeling of "lightness" in the joints the day after treatment (Tkemaoadze & Apkjazava, 2019).

Benefits in preclinical trials

Health/Lifespan

A trial that used intermittent treatment with D+Q (5 mg/kg + 50 mg/kg) weekly in an accelerated aging mouse model found that healthspan was significantly extended (Zhu et al., 2015). They reported a significant reduction in a composite score of age-related symptoms that included kyphosis, dystonia, tremors, loss of grip strength, coat condition, ataxia, urinary incontinence, impaired gait, hind limb paralysis, and poor body condition. The extension of healthspan was due to both the delay in onset of symptoms and the attenuation of their severity (Zhu et al., 2015).

A second study reported that bi-weekly administration of D+Q (5 mg/kg + 50 mg/kg) starting at 24-27 months of age (equivalent to age 75-90 years in humans) resulted in a 36% higher median post-treatment lifespan and lower mortality hazard (64.9% compared to the control group) (Xu et al., 2018).

Central nervous system

Three preclinical trials in mice reported beneficial effects in the CNS due to the elimination of senescent cells (Ogrodnik et al., 2019; Zhang et al., 2019; Musi et al., 2018). The first trial demonstrated that obesity results in the accumulation of senescent glial cells in the region of the lateral ventricle and that senescent glial cells exhibit excessive fat deposits. Clearance of senescent cells using D+Q (5 mg/kg+ 50 mg/kg) for 5 days every two weeks over 8 weeks restored neurogenesis and alleviated anxiety-related behavior (Ogrodnik et al., 2019).

A second trial (Zhang et al., 2019) found that exposure to amyloid-beta (Aβ) plaques triggered senescence in oligodendrocyte progenitor cells (OPCs) and that short-term treatment with D+Q (12 mg/kg + 50 mg/kg) daily for 9 days reduced SA-BGal activity and levels of Olig2 and p21. When Alzheimer's disease (AD) mice received D+Q over a longer period of 11 weeks, there was a decrease in Aβ load, and neuroinflammation (as evidenced by decreases in IL-1, 6, TNFa) as well as improvements in cognition.

Using AD transgenic mouse models, a third trial (Musi et al., 2018) found that neurofibrillary tangles (NFT), but not Aβ plaques, display a senescence‐like phenotype and that intermittent treatment with D+Q (5 mg/kg+ 50 mg/kg) in 6 sessions over 12 weeks reduced the number of NFT-containing cortical neurons by 35%. The gene expression of the NFT-associated senescence gene array was also reduced. The reduction in NFT-containing neurons corresponded with a decreased ventricular volume pathology of 28% and a reduction in cortical brain atrophy. Aberrant cerebral blood flow was improved to the point that it no longer differed significantly from controls and the D+Q treated mice displayed higher levels of neurogenesis markers (Musi et al., 2018).

Cardiovascular system

Four preclinical studies reported benefits to the cardiovascular system following treatment with D+Q (Roos et al., 2016; Zhu et al., 2015; Kim et al., 2020; Lewis-McDougall et al., 2019).

The first trial, assessed the effect of D+Q ( 5 mg/kg + 10 mg/kg) once per month for 3 months in aged and atherosclerotic mice (Roos et al., 2016). The authors reported a significant reduction in senescent cell markers in the medial layer of the aorta but not in intimal atherosclerotic plaques although intimal plaque calcification was decreased.

A single dose of D+Q (5 mg/kg + 50 mg/kg) has been shown to improve left ventricular ejection fraction in mice by approximately 10% (from 68% baseline up to 78% following treatment) due to improvements in end-systolic cardiac dimensions (Zhu et al., 2015).

D+Q treatment also improved vasomotor function in two trials (Zhu et al., 2015; Roos et al., 2016) as measured by a greater response to stimulation with acetylcholine and nitroprusside (Zhu et al., 2015). The data suggest that senolytic treatment improves nitric oxide signaling in aged mice, however, the molecular mechanisms are unclear. 

In vitro, Q has been shown to alleviate oxidative-stress induced vascular smooth muscle cell senescence through activation of AMPK (Kim et al., 2020). 

Elimination of senescent cardiac progenitor cells (CPCs) using D+Q has been shown in vitro to abrogate the SASP and in vivo, to activate resident CPCs (Lewis-McDougall et al., 2019). 

Improved cardiac diastolic function following D+Q treatment was reported by a study in obese mice (Palmer et al., 2019). 

Genes

Incubation with Q (3-12 µM for 24 hours) has been shown to increase the expression of SIRT1 and thioredoxin in a dose-dependent manner in human kidney cells (Abharzanjani et al., 2017).

Immune system

One trial reported a decrease in the inflammatory aspects of IPF in bronchoalveolar lavage (BAL) fluid following treatment with D+Q. White blood cell counts were significantly increased in vehicle-treated bleomycin-exposed mice, and treatment with D+Q attenuated this increase. Although cytokine levels within the BAL fluid were highly variable, the increases in MCP-1 and IL-6 were diminished following treatment with D+Q (Schafer et al., 2017).

In vitro studies of Q also reported a decrease in the level of reactive oxygen species (ROS) (Geng et al., 2019; Sohn et al., 2018). One trial reported decreased ROS levels and restoration of the heterochromatin architecture in a model of Werner's syndrome in human mesenchymal stem cells (Geng et al., 2019). A second study demonstrated that treatment with Q (5 uM) significantly decreased the relative ROS level when cells were exposed to H202 (Sohn et al., 2018). 

An in vitro study reported that cancer cells became more sensitive to radiation therapy following treatment with D+Q (Wang et al., 2020).

Markers of senescence

As in the human trials, a large number of "benefits" are related to reductions in markers of senescence or increases in cell proliferation capacity. Senescent cells often express p16INK4a, a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible (Coppé et al., 2011). Several in vivo (Nath et al., 2018; Schafer et al., 2017; Kim et al., 2019; Zhu et al., 2015) and in vitro (Parikh et al., 2018; Schafer et al., 2017; Suvakov et al., 2019; Geng et al., 2019; Kim et al., 2020; Yang et al., 2014 ) studies have demonstrated decreased p16Ink4a expression following treatment with or exposure to D+Q. This decrease has been measured in fetal airway cells, veins, lung fibroblasts, mesenchymal stem cells, renal tubular cells, liver, and muscle. 

Several in vivo (Ogrodnik et al., 2019; Xu et al., 2018; Zhu et al., 2015) and in vitro (Chondrogianni et al., 2010; Parikh et al., 2018; Abharzanjani et al., 2017; Geng et al., 2019; Kim et al., 2020; Sohn et al., 2018) studies also reported a decrease in the number of SABgal+ cells, another important marker of senescence. In a mouse model, the decrease in SABGal+ cells in perigonadal adipose tissue was approximately 7% following D+Q treatment (Ogrodnik et al., 2019) while another study reported a decrease of approximately 9.5% in human explanted adipose tissue (Xu et al., 2018). A third study also reported a decrease in SABgal+ cells in the inguinal fat of irradiated mice following a single dose of D+Q (Zhu et al., 2015).

In vitro, treatment of HLF-1 cells with Q resulted in only 13.5% of cells staining positive for SABgal after 55 days (compared to treatment with DMSO or CAP that showed >75% SABgal+ staining) (Chondrogianni et al., 2010). Dose-dependent decreases in SABgal+ cells following treatment with D and/or Q have been seen under various senescence-inducing conditions including hyperglycemia, hyperoxia and chemotherapy (Abharzanjani et al., 2017; Geng et al., 2019; Yang et al., 2014; Parikh et al., 2018). 

Several studies also reported a decrease in p21+ cells following treatment with D+Q (Zhang et al., 2019; Hohmann et al., 2018; Parikh et al., 2018; Geng et al., 2019; Kim et al., 2020; Yang et al., 2014). The mean intensity of p21+ cells decreased from 2800 down to 800 following short term (9 days) of D+Q treatment in AB plaques in a mouse model of Alzheimer's disease (Zhang et al., 2019). Treatment with Q (30 mg/kg intraperitoneally, over a period of 1 or 3 weeks also reduced p21 expression in bleomycin-induced lung injury in aged mice at 14 days (Hohmann et al., 2018).

In vitro studies also showed a decrease in levels of p21 following treatment with Q alone (Geng et al., 2019; Kim et al., 2020) and demonstrated an inhibitory effect on vascular smooth muscle cell (VSMC) senescence via activation of AMPK (Kim et al., 2020). Immunofluorescence analysis of D+Q incubated fetal airway smooth muscle cells showed decreased nuclear co-localization of p21 and p-γH2A.X from 65% down to 45% (Parikh et al., 2018).

Q has also been shown to reduce the expression of p19-ARF in the lungs (Hohmann et al., 2018) and kidneys (Kim et al., 2019) of aged and high-fat diet-fed mice, respectively. 

Three studies on Q also reported a significant decrease in p53 expression following exposure to Q, in oxidative (H202) or high-fat diet-induced metabolic stress (Kim et al., 2019; Kim et al., 2020) and in adriamycin and replicative senescence (Yang et al., 2014 ). However, one study reported an increase in p53 expression following D+Q treatment (Cavalcante et al., 2019).

In vitro, Q has also been shown to reduce markers of DNA damage including yH2AX and 53BP1 (Geng et al., 2019). The relative expression of cells double-positive for both markers decreased from 1 to 0.6 following exposure to Q (Geng et al., 2019). In mice, D+Q treatment has been shown to reduce yH2AX in liver biopsies 17% down to 11% (Ogrodnik et al., 2017). 

Telomeres

An in vitro study demonstrated that telomere length was increased by 70% and cell proliferation was increased by >50% in a Werner Syndrome (WS) model of human mesenchymal stem cells when exposed to Q at a concentration of 100 nmol/L (Geng et al., 2019). 

Telomere-associated foci (TAFs) are sites of DNA damage within telomeres and are believed to be a more specific marker of senescence than SABgal (Xu et al., 2018). Several studies have reported a decrease in TAF cells in various tissues including the brain, aorta, and liver in mice and human explanted tissue (Ogrodnik et al., 2019; Roos et al., 2016; Xu et al., 2018; Ogrodnik et al., 2017). Levels of TAF+ cells were decreased from 34% down to 18% in perigonadal adipose tissue of obese mice (Ogrodnik et al., 2019), from 42% to 22% in the medial layer of the aorta in aged atherosclerotic mice (Roos et al., 2016), and from 16% to 5% in the liver of aged mice (Ogrodnik et al., 2017). Explanted human omental tissue from obese individuals exposed to 1 uM + 20 uM D+Q for 48 hours also showed a reduced number of TAF+ cells compared to controls (Xu et al., 2018).

SASP

Several studies found a decrease in a variety of SASP components in mice (Zhang et al., 2019; Hohmann et al., 2018; Schafer et al., 2017; Palmer et al., 2019), in ex vivo human tissue (Xu et al., 2018; Suvakov et al., 2019; Geng et al., 2019 ) and in vitro. 

Summary of measured SASP components