







Fisetin Senolytic Therapy

Risk-Benefit Analysis





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Version 1.2

September 18, 2019













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Table of content









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.





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Senolytics are agents that selectively induce apoptosis of senescent cells. Fisetin is a flavonoid polyphenol found in many types of fruits and vegetables (Arai et al., 2000) that is believed to act as a senolytic in addition to its numerous other known benefits. Although natural senolytics are less potent, compared to the targeted senolytics, they have lower toxicity and are thus, likely to be more readily translatable to clinical medicine. This RBA focuses on the risks and benefits of using fisetin as a senolytic rather than its more common use as a supplement.





Key Questions





This RBA seeks to answer the following questions:

Which health and/or longevity benefits result from the use of fisetin as a senolytic?

Which risks are involved in the use of fisetin as a senolytic (general and method-specific)?

What are the potential risk mitigation strategies?





What is the best therapeutic protocol available at the moment?

Impatient readers may choose to skip directly to Section 6 for the conclusion and tips on practical application.









Analytic model





prepared based on the principles outlined in A Comprehensive Approach to Benefit-Risk Assessment in Drug Development





Literature search





A literature search was conducted on Pubmed and the Cochrane Library using the search terms . 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 trials that may have been missed by the search terms.

Because of the small number of papers available on the topic, we also searched for studies in which fisetin was found to influence any of the 6 known Senescent Cell Anti-Apoptotic Pathways (SCAPs).





Table 1: Literature Search

Number of publications fisetin AND senolytic 5 fisetin AND aging 26 fisetin AND BCL 18 fisetin AND PI3 AND AKT 3 fisetin AND apoptosis 147 fisetin AND MDM2 1 fisetin AND p53 23 fisetin AND p21 7 fisetin AND serpine 5 fisetin AND HIF 4 fisetin AND toxicity 99 fisetin AND pharmacokinetic* 41 fisetin AND benefit* 23 fisetin AND risk* 23 Other sources (names cited in the text) A manual search of the reference lists of the selected papers























Abbreviation list





MEF murine embryonic fibroblasts HUVECs human umbilical vein endothelial cells SCAPs senescent cell antiapoptotic pathways SASP senescence-associated secretory phenotype BW bodyweight IMR90 human embryonic lung fibroblasts SA-β-gal senescence-associated beta-galactosidase p16Ink4a a tumor suppressor protein p21Cip1 cell-cycle regulatory protein that Interacts with cyclin-CDK2 and -CDK4, inhibiting cell cycle progression at G1 MCP-1 monocyte chemotactic protein-1





Summary of ongoing clinical trials





There are currently 3, phase 2 clinical trials underway (clinicaltrials.gov) and the first data is expected to be reported in about a year. The data from the phase 1 trials has not been published. All trials are being conducted by the same investigators at the Mayo clinic using the same treatment protocol.

















(animals & in vitro)





Only 2 papers directly related to the use of fisetin as a senolytic were identified, neither of which were conducted in humans (Yousefzadeh et la., 2018; Zhu et al., 2017). The other 5 studies included in the table relate to pharmacokinetics, risk, and lifespan extension.





Table 2: Preclinical trials





: 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. risk criteria are organized by category, type, severity, frequency, detectability, and mitigation. All are assigned numerical values:

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





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 fisetin 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. Human evidence is initially assigned a score of "1", evidence from rodent studies, "2", and in vitro or lower animal studies, "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





Benefit assessment





Table 4: Benefit assessment









Senescent cells

Accumulation of senescent cells is one of the major phenomena underlying aging and chronic disease. Senescent cells are resistant to apoptosis by virtue of 6 known senescent cell antiapoptotic pathways (SCAPs). SCAPs allow the survival of senescent cells despite the secretion of the proapoptotic SASP. The SCAPs required for senescent cell resistance to apoptosis vary according to cell type.

Agents targeting a single SCAP may not eliminate all types of senescent cells and so far, all senolytics that have been tested have exhibited a certain degree of cell-type specificity. Fisetin has been found to interfere with 4 of the 6 known SCAPs in several in vitro studies (Pal et al., 2013; Zhu et al., 2017; Zhang et al., 2016; Li et al., 2011; Triantafyllou et al., 2008; Min et al., 2017; Sabarwal et al., 2017) leading to interest in its use as a senolytic agent.

Fisetin was shown to inhibit BCL-2 (an inhibitor of apoptosis) (Verma et al., 2017; Shih et al., 2017; Tsai et al., 2019) by binding to a hydrophobic groove on the protein (Verma et al., 2017). It has also been shown to induce apoptosis through ROS production (Sabarwal et al., 2017) and increased activity of caspase-3, -8 and -9 (Tsai et al., 2019). Fisetin also increased apoptosis via p53 mediated up-regulation of DR5 expression at the transcriptional level (Min et al., 2017).

In studies directly examining its effects on senescence, fisetin has been shown to significantly reduce markers of senescence such as p16Ink4a expression, SA-β-gal activity/expression, and SASP markers in fat, spleen, liver, kidney and CD3+ T cells in mice as well as ex vivo, in human adipose tissue (Yousefzadeh et al., 2018). It has also been shown to reduce the fraction of senescent cells (T-cells, stem cells, natural killer cells, and endothelial cells but not macrophages or dendritic cells) in white adipose tissue of naturally aged mice (Yousefzadeh et al., 2018).

In vitro, fisetin was found to dose-dependently increase apoptosis, reduce cell viability, and reduce senescent cell number in HUVECs but not IMR90 (fibroblasts) or primary human preadipocytes indicating a degree of cell-type specificity.





Metabolism

Positive effects on pancreatic and liver homeostasis were also seen in significantly lower levels of ALT and amylase and decreased oxidative stress in the liver (Yousefzadeh et al., 2018).





Lifespan

Fisetin administration beginning at 85 weeks of age (approximately equivalent to human age 75) was shown to increase lifespan in mice by about 10% (Yousefzadeh et al., 2018). In vitro, fisetin 10 µm did not change the lifespan of flies but 100 µmfisetin increased lifespan by up to 23% (Wood et al., 2004).





Risk assessment





Table 5: Risk assessment

Category Species Risk Frequency Detectability Mitigation Sum Weight Score Weighted Score 1 Wound healing mice ↑ time to wound closure 2 2 1 1 (intermittent dosing during periods of good health) 6 1.5 -1 2 rodents: Demaria et al., 2014 -1.5 2 Wound healing mice ↑ fibrosis after wound healing 2 2 1 1 (intermittent dosing during periods of good health) 6 1.5 -1 2 rodents: Demaria et al., 2014 -1.5 3 Liver toxicity mice ↑ ALT/AST 1 1 1 1 (intermittent dosing or use a form with increased bioavailability) 4 1 -1 2 rodents: Sun et al., 2018 -1





Individual risks





Very little is known about the potential side effects of senolytic drugs as a class. A study of genetic clearance of senolytic cells has shown a delay in wound healing and increased fibrosis after the wound is healed (Demaria et al., 2014). A second study shows that senescent cells function to limit fibrosis during liver regeneration and that impairment of this function leads to increased fibrosis (Krizhanovsky et al., 2008). A third, purely hypothetical risk is cell lysis syndrome due to the sudden death of many cells. This is, however, highly unlikely because even in aged tissue, the proportion of senescent cells is about 15% (Herbig et al., 2006) and senolysis has been shown to lead to a reduction of about 30-40% of senolytic cells (Zhu et al., 2015) . These risks are relatively easy to mitigate through intermittent dosing that is limited to periods of good health.

Only one animal study on fisetin has reported any form of toxicity from fisetin use and the authors concluded that the elevations in ALT/AST levels (indications of liver toxicity) were in large part due to the vehicle used to administer the fisetin (DMSO). However, the fisetin + vehicle group showed significantly higher elevations than the vehicle alone group indicating that high doses of fisetin may additionally burden the liver because of its poor bioavailability (Sun et al., 2018). At a lower dose (112 mg/kg), fisetin didn't significantly increase apoptosis or lead to liver toxicity (Sun et al., 2018). Intermittent dosing and use of a form of fisetin with increased bioavailability are likely to mitigate the risk of liver toxicity.





Summary of the evidence on pharmacokinetics, form, dose, and duration





Pharmacokinetics

To the best of our knowledge, there haven't been any studies published on the pharmacokinetics of fisetin in humans.

Fisetin is a potent bioactive compound with limited bioavailability because of its low aqueous solubility and poor absorption from the gut. The absolute bioavailability of fisetin was calculated as 7.8% and 31.7% after oral administration of 100 and 200mg/kg fisetin, respectively (Jo et al., 2017). Fisetin concentrations achieved in a mouse study without toxicity (2.7- 349.4 µM) (Touil et al., 2011) are higher than those found to be senolytic in vitro (Zhu et al., 2017). The biological activity of fisetin depends on the presence of hydroxyl groups at 3, 7, 3′, 4′ positions and the oxo group at position 4 with a double bond between C2 and C3 (Kashjap et al., 2018). The hydroxyl group at C-7 and the double bond between C2 and C3 are essential for its antioxidant activity (Sengupta et al., 2004).

Several studies have reported that the solubility and bioavailability of fisetin can be improved by cocrystallization with caffeine, isonicotinamide, and nicotinamide, complexation with cyclodextrins and encapsulation with nanoparticles (Kashjap et al., 2018). Fisetin micelles demonstrate a more sustained and prolonged in vitro release behavior, as well as enhanced cytotoxicity, cellular uptake, and fisetin-induced apoptosis (Chen et al., 2015).

The metabolism of fisetin was first determined in rats following intravenous and oral administration (Shia et al., 2009). Flavonoids are known to be extensively metabolized following oral consumption resulting in glucuronidated, sulfated and methylated metabolites. Following intravenous injection in rats (10 mg/kg), blood levels of fisetin rapidly declined with the appearance of sulfate and glucuronide-containing conjugates. Following oral administration (50 mg/kg), fisetin was only transiently present in rat serum and was replaced by conjugated sulfates and glucuronides. The serum concentration of fisetin sulfates/glucuronides was maintained at 10 uM for >24 h (Maher, 2017). The half-life of fisetin in female mice (3.12 h) following i.v. administration was longer than in male mice (Kashjap et al., 2018).

Significant accumulation of fisetin and its metabolites in multiple tissues was also seen. The main metabolites were glucuronidated fisetin, geraldol (3,4′,7-trihydroxy-3′-methoxyflavone) and glucuronidated geraldol. Geraldol was as active as fisetin in several biological assays.

In macaques fed a single oral dose of 25 mg/kg bw, it was found that sulfated and/or glucuronidated forms of fisetin reach concentrations of 30 µM in the cerebrospinal fluid with a plasma half-life of 8 h (Maher, 2017).

Due to the commercial unavailability of most conjugates, only a few studies have been performed on the biological effects of fisetin metabolites. Results varied showing, on one hand, significantly lower activity against erythrocyte hemolysis for sulfates/glucuronides (Shia et al., 2009) but somewhat stronger cytotoxic activity on lung carcinoma cells as compared to intact fisetin. Geraldol could also inhibit endothelial cell migration and proliferation. Combined with fisetin's rapid half-life of 9 minutes this suggests that fisetin metabolites play an important role in it's in vivo activities (Touil et al., 2011).





Form

Fisetin has been administered by i.v. and orally in animals. Current clinical trials use oral supplements and many investigations are being carried on the theme of increasing its bioavailability. Suppositories have also been suggested as a means of increasing bioavailability and are the method of choice of some well-known health care professionals (Joseph Mercola). However, there are no studies that have measured the amount of fisetin absorbed rectally in humans.

Dose

The clinical trials currently in progress use a dose of 20 mg/kg bw, administered on two consecutive days (Table 2).

The pharmacologic activity of fisetin has been shown to vary according to the concentration achieved within the cells. Similar to quercetin, fisetin, showed protective effects against H2O2-induced cytotoxicity, DNA strand breaks, and apoptosis at concentrations as low as 10–25 µmol/L. On the other hand, these flavonoids induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation, and caspase activation at concentrations between 50 and 250 µmol/L. Data suggests that cytoprotective concentrations of some flavonoids are lower by a factor of 5–10 than their DNA-damaging and proapoptotic concentrations (Waetjen et al., 2005).

Duration

Although yet to be confirmed for fisetin, intermittent administration of some senolytic compounds has been shown to be adequate ( Zhu et al., 2015 ). Since a brief disruption of pro-survival pathways is enough to kill senescent cells, senolytics don't have to be present continuously to be effective ( Zhu et al., 2015 ). The frequency of senolytic treatment will depend on rates of senescent cell reaccumulation, which probably varies according to conditions ( Kirkland et al., 2017 ). Advantages of intermittent administration include less chance of developing adverse and off-target effects and the ability to choose periods of good health for administration.





Toxicity

There is no evidence at the present time for either short- or long-term toxicity from fisetin supplementation. Mice that were orally administered fisetin at 2000 mg/kg bodyweight, examined for 48 h and then sacrificed showed no indication of toxicity ( Maher, 2017 ). The effects of long-term feeding of fisetin (∼25 mg/kg bodyweight over nine months) have also been evaluated in mice. No significant difference in body weight was seen in animals supplemented with fisetin. Additionally, multiple tissues (lungs, spleen, liver, kidneys, heart, stomach, intestine, testes and ovary) were examined using standard toxicological criteria and no toxicity was found. Furthermore, fisetin has no inhibitory effects on the activities of cytochrome P450s 3A4, 2C9 and 2D6 at concentrations up to 10 µM ( Maher, 2017 ).

The LD50 of fisetin in rats by i.v. is 180 mg/kg ( www.caymanchem.com ).





Drug Interactions

There is a lack of information on drug interactions with fisetin. As fisetin has been shown to lower blood glucose in diabetic rats (given at 10 mg/kg bodyweight for 30 days) (Prasath et al., 2014) it is conceivable that it could potentiate the effects of glucose-lowering drugs such as metformin. This risk is likely lessened by the intermittent dosing schedule.























To view the tornado diagram as a pdf please click on the thumbnail below:





For those who would prefer to view the document in excel, we have included the original .xls file.

Fisetin RBA v1.xlsx





Section 6:





Main benefits





Fisetin has been shown to decrease senescent cell biomarkers as well as the numbers of senescent cells in a variety of tissues, including ex vivo, human adipose tissue as well as in vivo, in mice.



Main risks





Reduction of senescent cells has been associated with delayed wound healing and an increased level of fibrosis after healing (Demaria et al., 2014). Fisetin was associated with liver toxicity in mice at doses of 223 mg/kg administered over 3 weeks (Sun et al., 2018). There is also the possibility of drug interactions with glucose-lowering drugs such as metformin.





Risk Mitigation Strategies





Wait to commence therapy until clinical trial data has been published that describes the possible benefits and adverse effects

Consult your physician before beginning therapy

Frequency of therapy is to be determined by the results of the senescent cell biomarker lab panel

Measure baseline senescent cell load biomarkers including SA-β-gal activity, p16Ink4a expression, p21Cip1 expression, and SASP factors: IL-6, IL-8, MCP-1 when possible

Repeat screening at regular intervals to determine the effectiveness of the therapy, and the appropriateness of the dose

Measure blood values, liver & kidney function and electrolyte values at regular intervals

Use caution when combining fisetin with glucose-lowering drugs (monitor blood sugar levels)

Avoid or use with extreme caution if nonalcoholic fatty liver disease has been diagnosed or is suspected

Cease therapy if any identifiable adverse effects occur





Section 7: Practical Application





Form & Dose





Follow the risk mitigation strategies outlined in Section 6

At the current time, the only form and dose that has been tested in phase 1 clinical trials is the so-called "Mayo Protocol"

The Mayo Protocol consists of taking 20 mg/kg body weight of oral fisetin on two consecutive days and repeating the same dose, one month later















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