COVFIS-HOME: A Phase 2 Placebo-Controlled Pilot Study in COVID-19 of Fisetin to Alleviate Dysfunction and Decrease Complications in At-Risk Outpatients
Phase: PHASE2
Conditions: Covid19, Coronavirus Infection
Sponsor: James L. Kirkland, MD, PhD

 
 
COVFIS-HOME: A PHASE 2 PLACEBO-CONTROLLED PILOT 
STUDY IN COVID-19 OF FISETIN TO ALLEVIATE 
DYSFUNCTION AND DECREASE COMPLICATIONS IN AT-RISK 
OUTPATIENTS 
 
 
 
NCT04771611 
 
4/18/2022 

COVFIS-HOME: COVID-19 PILOT STUDY OF FISETIN TO ALLEVIATE DYSFUNCTION AND DECREASE COMPLICATIONS 
Version 4.0, 18NOV2021 
 
 
 
COVFIS-HOME: A PHASE 2 PLACEBO-CONTROLLED PILOT 
STUDY IN COVID-19 OF FISETIN TO ALLEVIATE DYSFUNCTION 
AND DECREASE COMPLICATIONS IN AT-RISK OUTPATIENTS 
 
 
Co-Principal Investigator 
and Regulatory Sponsor: 
 
 
 
 
 
 
Co-Principal Investigator: 
 
 
 
 
James L. Kirkland, M.D., Ph.D., 
Kogod Center on Aging, 
Gerontology and Geriatric Medicine, 
Mayo Clinic, 
200 First Street, SW, 
Rochester, MN 55905 
 
 
Avni Y. Joshi, M.D., M.S., 
Allergy and Immunology 
Department of Medicine and Pediatrics &Adolescent 
Medicine, 
Mayo Clinic, 
200 First Street, SW, 
Rochester, MN 55905 
 
 
Study Product: 
Fisetin 
Protocol Number (IRBe): 
 
IND Number: 
149813 
 
 
Initial Version (Original): January 21, 2021; Version 1.0 
Revised Version: 
 
May 11, 2021; Version 2.0 
Revised Version: 
 
June 15, 2021; Version 3.0 
Revised Version:  
 
November 18, 2021; Version 4.0 
Revised Version:  
 
April 18th, 2022; Version 5.0 

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COVFIS-HOME: A PHASE 2 PLACEBO-CONTROLLED PILOT 
STUDY IN COVID-19 OF FISETIN TO ALLEVIATE DYSFUNCTION 
AND DECREASE COMPLICATIONS IN AT-RISK OUTPATIENTS 
 
 
 
James L. Kirkland, M.D., Ph.D., 
 
 
 
 
Department of Medicine 
 
(Signature) 
(Date) 
 
 
 
 
 
Avni Y. Joshi, M.D., M.S., 
 
 
 
 
Department of Medicine and  
    (Signature) 
(Date) 
Pediatrics &Adolescent Medicine 
 
 
 

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Table of Contents 
INVESTIGATOR’S SIGNATURE ........................................................... 2 
SYNOPSIS .......................................................................................... 5 
TIME PERIOD AND NUMBER OF PATIENTS: ........................................ 9 
1 
INTRODUCTION ......................................................................... 10 
1.1 
Background ................................................................................... 10 
1.2 
Background on Fisetin .................................................................. 16 
1.3 
Fisetin Dosage .............................................................................. 29 
2 
STUDY OBJECTIVES .................................................................... 31 
3 
STUDY DESIGN ........................................................................... 31 
3.1 
Study Schedule ............................................................................. 31 
3.2 
Enrollment .................................................................................... 31 
3.3 
Screening Evaluations and Randomization .................................... 31 
3.4 
Active Treatment Period ............................................................... 32 
Table 1 Timetable of Visits and Requirements ................................ 33 
4 
STUDY POPULATION .................................................................. 34 
4.1 
Source and Number of Patients ..................................................... 34 
4.2 
Patient Selection Criteria .............................................................. 34 
4.2.1 Inclusion Criteria ......................................................................... 34 
4.2.2 Exclusion Criteria ......................................................................... 34 
4.3 
Prohibited, Allowable and Concurrent Medications ....................... 35 
5 
STUDY METHODOLOGY ............................................................... 36 
5.1 
Efficacy Outcomes ......................................................................... 36 
5.2 
Safety Monitoring ......................................................................... 36 
5.2.1 Adverse Event Reporting ............................................................... 36 
5.2.2 Other Safety Monitoring ................................................................ 38 
5.3 
Withdrawal of Patients from the Study ......................................... 39 
5.4 
Study Completion .......................................................................... 39 
6 
STUDY MEDICATION .................................................................. 39 
6.1 
Medication Dispensing .................................................................. 39 
6.2 
Dosage Regimen ........................................................................... 40 
7 
DATA COLLECTION ..................................................................... 40 
8 
DATA ANALYSIS AND STATISTICAL CONSIDERATIONS .............. 40 
8.1 
Statistical Power and Sample Size Considerations ........................ 40 
8.2 
Analysis population ....................................................................... 40 
8.2.1 Intent to Treat (ITT) population ..................................................... 40 
8.2.2 Safety population ........................................................................ 40 
8.3 
Data analysis ................................................................................ 41 

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8.3.1 Analysis of efficacy outcomes ........................................................ 41 
8.3.2 Analysis of safety outcomes .......................................................... 41 
8.3.3 Interim analysis .......................................................................... 41 
9 
STUDY COORDINATION .............................................................. 41 
REFERENCES .................................................................................... 42 
LIST OF APPENDICES....................................................................... 48 
Appendix A Detailed Study Safety Parameters ............................... 49 
Appendix B Administrative Procedures for the Reporting of Adverse 
Events ............................................................................................. 51 
Appendix C Excluded Medications and Dosing Modifications……..……55 
 

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PROTOCOL SYNOPSIS 
TITLE: COVFIS-HOME: A PHASE 2 PLACEBO-CONTROLLED PILOT STUDY IN 
COVID-19 OF FISETIN TO ALLEVIATE DYSFUNCTION AND DECREASE 
COMPLICATIONS IN AT-RISK OUTPATIENTS 
 
INDICATION: Reduction of complications in patients with COVID-19 infection. 
 
OBJECTIVES: The primary objective of this study is to determine whether short-term treatment 
with Fisetin reduces the rate of death and long-term complications related to COVID-19. The 
secondary objective is to determine the safety of treatment with Fisetin in this patient population. 
The exploratory objective is to evaluate links between aging biomarkers and treatment effects. 
 
PATIENT POPULATION: Males and females, at least 18 years of age, who have been diagnosed 
with a COVID-19 infection and have at least one high-risk criterion. 
 
Inclusion Criteria: 
 
• Males and females, at least 18 years of age, capable and willing to provide informed consent; 
• Patient must have received a diagnosis of COVID-19 infection within the last 10 days; 
• Outpatient setting (not currently hospitalized); 
• Patient must possess at least one of the following high-risk criteria: 60 years or more of age, 
obesity (BMI ≥ 30 kg/m2), diabetes mellitus, uncontrolled hypertension (systolic blood pressure 
≥150 mm Hg), known respiratory disease (including asthma, chronic obstructive pulmonary 
disease, or present or past smoking), known heart failure, known coronary disease, fever of 
≥38.4°C within the last 48 hours, dyspnea at the time of presentation, the combination of high 
neutrophil count and low lymphocyte count; 
• Female patient is either not of childbearing potential, defined as postmenopausal for at least 1 
year or surgically sterile, or is of childbearing potential and practicing at least one method of 
contraception and preferably two complementary forms of contraception including a barrier 
method (e.g. male or female condoms, spermicides, sponges, foams, jellies, diaphragm, 
intrauterine device (IUD)) throughout the study and for 30 days after study completion; 
• Patient or their caregiver must be able and willing to comply with the requirements of this study 
protocol. 
 
Exclusion Criteria: 
 
• Patient currently hospitalized or under immediate consideration for hospitalization; 
• Patient currently in shock or with hemodynamic instability; 
• Patient with severe hepatic disease (as per clinical judgement) or liver enzymes >2x the upper 
limit of normal; 
• Female patient who is pregnant, breast-feeding, or is considering becoming pregnant during the 
study or for 1 day after the last dose of study medication; 
• Patient currently taking Sirolimus, Tacrolimus, or other mTOR inhibitors for other indications 
(mainly chronic indications represented by organ transplantation or autoimmune diseases); 
• On Warfarin therapy; Subjects taking any of the medications listed in Appendix C may 
participate if they are otherwise eligible AND the medication can be safely held during the 
following times: 

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o Immediately before the 1st IP administration (Day 0) until at least 10 hours after 
the 2nd IP administration (Day 1) 
o Immediately before the 3rd IP administration (Day 8) until at least 10 hours after 
the 4th IP administration (Day 9) 
• Patient with a history of an allergic reaction or significant sensitivity to Fisetin; 
• Patient undergoing chemotherapy for cancer; 
• Patient is considered by the investigator, for any reason, to be an unsuitable study candidate. 
• History of diverticulitis or diverticulosis with GI bleeding, as per clinical judgement. 
 
STUDY DESIGN: This will be a randomized, double-blind, placebo-controlled study. Following 
signature of the informed consent form, approximately 300 subjects meeting all inclusion and no 
exclusion criteria will be consented with a goal of randomizing 150 subjects to receive either Fisetin 
or placebo (1:1 allocation ratio) on Days 0 and 1 and 8 and 9 (4 doses over 10 days). Follow-up 
phone contacts will occur at 15 and 30 days following randomization for evaluation of the occurrence 
of any trial endpoints or other adverse events, with unblended follow-up contacts at Days 60. 
 
OUTCOMES: The primary endpoint will be the rate of complications related to COVID-19 (CoV) 
disease using a 7 point score adapted from the WHO Ordinal Scale for Clinical Improvement of CoV 
(Appendix A) in the 30 days following randomization. The secondary endpoint is to determine safety 
and tolerability of treatment with Fisetin in this patient population. Exploratory objectives are to 
evaluate links between soluble and genetic markers of senescent cell abundance, SASP factors, 
inflammation, viral load, and treatment effects and if Fisetin treatment during acute CoV infection 
decreases Long-hauler syndrome at Days 60. 
 
STATISTICAL RATIONALE AND ANALYSIS: All main analyses in COVFIS-HOME will be 
conducted on an intention-to-treat basis. Assuming 75 subjects/ group with a significance level of 
0.05, the study will have 80% power to detect an odds ratio of 2.24 comparing the placebo- to Fisetin-
treated group, using the 7-point severity score at Day 30 as the endpoint in an ordinal logistic 
regression model. 
 
A fully independent 5-member Data and Safety Monitoring Board (DSMB) will be established and 
will review unblinded safety data as detailed in the DSMB charter. An interim analysis is planned 
after approximately 50% of randomized patients have completed 30 days of follow-up. 
 
The DSMB charter will pre-specify the methods of interim efficacy analyses and the rules for early 
study termination, approved by all board members. The stopping rules for efficacy will be based on 
the O’Brien-Fleming α-spending function or on a similar conservative approach so that the impact 
on the final alpha level will be negligible. Futility will be assessed by computing conditional power. 
The DSMB will have the option of recommending early study termination because of overwhelming 
efficacy, early termination for futility, or continuation of the trial as planned. 
 
The primary analysis of efficacy will be based on the intent-to-treat principle. The primary endpoint 
will be compared between the two treatment groups using a chi-square test. Secondary endpoints will 
be analyzed similarly. 
 
ANTICIPATED TOTAL NUMBER OF RANDOMIZED PATIENTS: Approximately 150 
patients in total will be randomized in this study. 
 
STUDY LOCATION: Mayo Clinic Minnesota. 

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GLOSSARY 
 
AE 
 
Adverse Event/Adverse Experience 
AFFIRM 
Alleviation by Fisetin of Frailty, Inflammation, and Related Measures Trial 
ALT 
 
Alanine Aminotransferase 
ARDS  
Acute Respiratory Distress Syndrome 
AST 
 
Aspartate Aminotransferase 
BAP  
Biospecimens Accessioning and Processing Laboratory 
CFR 
 
Code of Federal Regulations 
CK 
 
Creatine Kinase 
CoV 
 
Coronavirus-19 
CRF 
 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CVA  
Cerebrovascular Accident 
D 
 
Dasatinib 
DAMP  
Damage-Associated Molecular Profile 
DSMB  
Data and Safety Monitoring Board 
ECMO  
Extra-Corporeal Membrane Oxygenation 
eGFR  
Estimated Glomerular Filtration Rate 
ESR 
 
Erythrocyte Sedimentation Rate 
FDA  
Food and Drug Administration 
γ-H2.AX 
γ-Histone 2.A DNA Damage Foci 
GCP  
Good Clinical Practice 
GCSF  
Granulocyte Colony Stimulating Factor 
GMP  
Good Manufacturing Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
HUVEC 
Human Umbilical Vein Endothelial Cell 
IB 
 
Investigator’s Brochure 
IL  
 
(e.g., IL-6) 
Interleukin (e.g., Interleukin-6) 
IND 
 
Investigational New Drug Application 
IP 
 
Investigational Product 
IPF 
 
Idiopathic Pulmonary Fibrosis 
IRB 
 
Institutional Review Board 
LAR  
Legally Authorized Representative 
LPS 
 
Lipopolysaccharide 
MCP-1 
Monocyte Chemoattractant Protein-1 
MHV  
Mouse Hepatitis Virus, a Mouse β-Coronavirus 
MIP1-α 
Macrophage Inflammatory Protein 1-α 
miRNA 
Micro-RNA 
MMP  
Matrix Metallopeptidase 
MSC  
Mesenchymal Stem Cell 
NME  
Normal Microbial Experience 
PAMP  
Pathogen-Associated Molecular Profile 
PHI 
 
Protected Health Information 
PI 
 
Principal Investigator 
Q 
 
Quercetin 

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SA-βGal 
Senescence-Associated β-Galactosidase 
SAE 
 
Serious Adverse Event/Serious Adverse Experience 
SASP  
Senescence-Associated Secretory Phenotype 
SCAP  
Senescent Cell Anti-Apoptotic Pathway 
sHLH  
Secondary Hemophagocytic Lymphohistiocytosis 
SOP 
 
Standard Operating Procedure 
TAF 
 
Telomere-Associated DNA Damage Foci 
TNF-α  
Tumor Necrosis Factor-α 
U of M 
University of Minnesota 
UPIRSTO 
Unanticipated Problems Involving Risk to Subjects or Others 
WCBP  
Women of Childbearing Potential

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TIME PERIOD AND NUMBER OF PATIENTS: 
 
A. Anticipated Starting Date of Study: 1Q21 
B. Anticipated Completion Date: 4Q22 
C. Anticipated Number of Patients per Site: Centralized recruitment and follow-up 
D. Anticipated Number of Sites: Mayo Clinic Minnesota. Centralized recruitment and follow-up. 
 
Generic 
Name
Strength and 
 Dosage Form 
 
Therapeutic 
 Classification
 
Fisetin 
100 mg capsules, 20 mg/kg body 
weight/ day, as intact capsules or 
opened with powder mixed into room 
temperature or chilled drinks or food 
(e.g., pudding, apple sauce, rice 
pudding) 
Senolytic 
Placebo to match 100 mg capsules 
NA 

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1 
INTRODUCTION 
 
1.1 
Background 
 
Coronavirus-19 (CoV): Coronaviruses constitute the subfamily Orthocoronavirinae, in the family 
Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-
sense single-stranded RNA genome and a nucleocapsid of helical symmetry. They are the largest 
among known RNA viruses. As of May 5, 2020, there have been at least 25,486 confirmed deaths 
and more than 3,646,206 confirmed cases in the β-coronavirus-19 (CoV) pandemic. CoV can cause 
physical dysfunction (frailty) and death from hyper-inflammation, cytokine storm, and associated 
acute respiratory distress syndrome (ARDS) and myocarditis with troponin leak, particularly in older 
or chronically-ill individuals8. Mouse hepatitis virus (MHV) is a β-coronavirus that is related to 
CoV, which causes an epidemic murine illness with high mortality, especially among colonies of 
laboratory mice9. We previously found that cellular senescence leads to physical dysfunction 
(frailty) and hyper-inflammation (cytokine storm), factors that contribute to morbidity and mortality 
from CoV. We are currently conducting a clinical trial of Fisetin in elderly subjects for these 
indications, AFFIRM (Alleviation by Fisetin of Frailty, Inflammation, and Related Measures in 
Older Adults; ClinicalTrials.gov identifier NCT03430037; IND 134052). 
 
Longhauler Syndrome: Initial reports from WHO-China in February described clinical recovery 
from time of onset of COVID symptoms to be between 2-6 weeks, the latter being patients with 
more severe cases (https://www.who.int/docs/default-source/coronaviruse/situation-
reports/20200306-sitrep-46-covid-19.pdf?sfvrsn=96b04adf_4). As more information becomes 
available, patients are coming forward with experiences of longer recovery times and lingering 
symptoms, Long Covid, Long-haul Covid, or Long-haulers10-12. Though largely undefined, “Long-
haulers” denotes patients who experience persistent symptoms after having acute COVID-19. While 
the full impact of long COVID is widely unknown, some sources are reporting upwards of 10% of 
patients experiencing symptoms lasting greater than 3 weeks12. An earlier study done in Italy 
showed that 87% of hospitalized patients reported at least one ongoing symptom 60 days after onset 
of first COVID-19 symptoms, with 55% of patients reporting 3 or more ongoing symptoms13. These 
long lasting symptoms can occur in adults across all age groups, regardless of pre-existing 
conditions14. The long term sequelae of the virus are largely unknown, but early reports of 
significant lung damage15,16 and persistent myocardial inflammation17 are concerning, making the 
discovery of long term impact at the mechanistic level all the more urgent. 
 
Cellular Senescence: Cellular senescence contributes to age-related dysfunction and multiple 
diseases throughout the lifespan. Senescent cell burden is very low in young individuals but 
increases with aging in several tissues. These include adipose tissue, skeletal muscle, heart, kidney, 
brain, bone, and skin5,18-24. Senescent cells accumulate at sites of pathology in multiple chronic 
diseases, including, among others, the lungs in chronic obstructive pulmonary disease, asthma, 
idiopathic pulmonary fibrosis (IPF), and smoking, adipose and other tissues in diabetes/ obesity, the 
brain in Alzheimer’s and other dementias, and blood vessels and the heart in cardiovascular disease. 
 
Senescent cells were discovered in 196125 when serially-subcultured human embryonic fibroblasts 
were shown to lose replicative capacity, yet remain viable. This prompted work to test if aging leads 
to accumulation in vivo of pre-senescent cells, which have limited remaining replicative potential, 
and senescent cells, which cannot replicate but are metabolically active. In 1979, this was reported 
to be true for fibroblasts in skin biopsied from older than younger subjects6 and by us in 1990, for 
primary adipocyte progenitors (preadipocytes) cloned from adipose tissue of rats across the age 

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spectrum that had been raised under controlled conditions7. Senescent cells appear at pathogenic 
sites of many major diseases, including chronic lung disease, diabetes/ obesity, cardiovascular 
diseases, Alzheimer’s disease, cancers, osteoporosis, renal disease, and cirrhosis in preclinical 
species and humans5,21,23, conditions associated with physical dysfunction (frailty), inflammation, 
and CoV morbidity and mortality. 
 
Senescence is essentially a cell fate, like differentiation, proliferation, apoptosis, or necrosis. 
External and internal signals can contribute to driving a cell into senescence. These are generally 
cell or tissue damage-related, including DNA alterations (dysfunctional telomeres, strand breaks, 
etc.), metabolic dysfunction (reactive oxygen species [ROS], high glucose, bioactive lipids, 
mitochondrial dysfunction), protein alterations (aggregates, misfolding, failed autophagy), 
inflammatory signals, mechanical/shear stress, pathogen-associated molecular pattern factors 
(PAMPs), including viruses, bacterial/fungal proteins, lipopolysaccharide (LPS), etc., damage-
associated molecular pattern factors (DAMPs; tissue damage signals [extracellular nucleotides, 
etc.]), cellular damage signals (DNA damage, telomeric dysfunction, protein aggregates, 
mitochondrial dysfunction), oncogenes, and mitogens (insulin-like growth factor-1, etc.). Once 
initiated, senescence takes 10 days to 6 weeks to become established through transcription factor 
cascades (that may, but do not always, include p16INK4a/ retinoblastoma protein and/or p53/p21CIP1), 
causing extensive changes in gene expression, histone modifications, altered organelle function 
(e.g., mitochondria, endoplasmic reticulum, nucleolus, nuclear envelope), elevated protein 
production due to increased mTOR and decreased autophagy, and profound morphological and 
metabolic shifts21,26. After drug-induced removal, senescent cells take from 10 days to 6 weeks to re-
occur if cellular senescence inducers remain present, at least in vitro. 
 
There are no fully sensitive or specific markers for identifying senescent cells. These cells are 
generally large, may have high p16INK4a and/or p21CIP1 (but not always), and exhibit DNA damage 
foci, particularly in telomeres (telomere-associated DNA damage foci [TAFs]), senescence-
associated distention of pericentromeric satellite DNA, and increased senescence-associated β-
galactosidase (SA-βGal)21. Like cancer cells, senescent cells frequently are metabolically-shifted 
from fatty acid utilization toward glycolysis, resulting in ROS generation, lipid accumulation, 
lipotoxicity, and dysdifferentiation into adipocyte-like but insulin-resistant “MAD” (mesenchymal 
adipocyte-like default) cells27. Senescent cells are resistant to apoptosis28. 
 
Senescent Cell Characteristics: Cellular senescence leads to inflammation, fibrosis, DNA damage, 
mitochondrial dysfunction, ROS generation, NAD+ depletion, protein aggregation, failed autophagy, 
lipotoxicity, and stem and progenitor cell dysfunction. In turn, cellular senescence can be caused by 
other fundamental aging processes, including DNA damage, low NAD+, mitochondrial dysfunction, 
ROS, protein aggregates, and lipotoxicity. Thus, from our and others’ data, fundamental aging 
processes appear to be interlinked, findings that led us to formulate a “Unitary Theory of 
Fundamental Aging Processes”, which posits that interventions, such as senolytics, targeting any one 
of these processes, such as cellular senescence, will affect many of the rest of the aging processes 
that predispose to aging phenotypes, frailty, hyper-inflammation, chronic diseases, and morbidity 
and mortality from acute insults, including viral infection. Effects of the study agent in our clinical 
trial will be ascertained not only on senescent cell abundance and effects, but also on these other 
fundamental aging processes. 
 
Senescence-Associated Secretory Phenotype (SASP): Accumulation of senescent cells can cause 
local and systemic inflammation, tissue destruction, immune system inhibition, and stem and 
progenitor cell dysfunction due to their SASP3,4,21,29,30. Generally 30-70% of senescent cells develop 

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a SASP that entails release of pro-inflammatory, pro-apoptotic cytokines (TNFα, IL-1α, IL-6, IL-7, 
IL-8, IFN, and many more), chemokines (e.g., MCP-1, MIP1-α, RANTES, RARRES2) that attract, 
activate, and anchor immune cells (including macrophages, dendritic cells, T-lymphocyte subsets, 
and neutrophils), tissue-destroying proteases (MMP-3, -9, -12, etc.), pro-coagulant factors (PAI-1, 
etc.), factors that impede stem cell/ progenitor function (Activin A, TGF-β-related proteins), ferritin, 
growth factors (e.g., GCSF), bioactive lipids (bradykines, saturated ceramides, prostanoids), micro-
RNA’s (miRNA’s), tissue-damaging non-coding nucleotides (including release of mitochondrial 
DNA, which attracts, activates, and anchors dendritic cells31), microvesicles including exosomes, 
and other factors that cause tissue necrosis, systemic inflammation, stem cell/ progenitor 
dysfunction, and spread of senescence to non-senescent cells. These SASP factors include those that 
result in frailty, hyper-inflammation/ cytokine storm/ ARDS/ myocarditis with troponin leak in CoV 
patients8. 
 
Likely due to their SASP, only a small number of senescent cells can cause considerable 
dysfunction. In pre-clinical experiments, transplanting senescent cells around the knee joints of 
young mice was sufficient to induce an osteoarthritis-like phenotype, while transplanting non-
senescent cells did not32. Transplanting 106 radiation- or chemotherapy-induced senescent 
autologous ear fibroblasts or syngeneic adipocyte progenitors intraperitoneally into lean, adult mice, 
so that fewer than 1/10,000 of all cells in the transplanted mice were senescent, induced impaired 
physical function (frailty) and premature death due to early onset of the same age-related diseases 
that cause death in naturally-aged mice32. This also occurred after human senescent (vs. non-
senescent) cells were transplanted into immune-deficient (SCID) mice. When labelled senescent 
cells are transplanted intraperitoneally, they remained within the peritoneum, yet the transplant 
recipients developed non-labeled senescent cells in their limbs, demonstrating that senescence can 
spread from cell to cell, even at a distance32. 
 
We established that adipose tissue from obese individuals have an increased burden of senescent 
adipocyte progenitors33. Furthermore, these senescent cells have a potent pro-inflammatory SASP. 
This leads to the hypothesis that an older or obese individual or any other individual with an 
increased burden of senescent cells could be predisposed to frailty, hyper-inflammation/ cytokine 
storm/ ARDS/ myocarditis with troponin leak after CoV infection. 
 
Clearance of Senescent cells by the Immune System: Senescent cells are usually cleared by the 
immune system34. They can attract, activate, and anchor immune cells, including macrophages, 
dendritic cells, T-lymphocytes, and neutrophils through such SASP chemokines as MCP-1, MIP1-α, 
RANTES, and RARRES2, cytokines such as IL-6, and TNFα, extracellular mitochondrial DNA, 
possibly miRNAs, and other factors31,32,34,35. However, above a threshold burden, senescent cells 
interfere with the immune system and its ability to remove them. For example, IL-6, a SASP 
component, interferes with macrophage migration, MMPs can cleave FAS ligand and other immune 
cell surface proteins, senescent cells cause fibrosis (impeding immune cell infiltration and trapping 
immune cells within inflammatory foci), and senescent cells can express “don’t eat me” signals34. 
Consistent with this, senescent cells can drive cancer initiation and progression, which senolytics 
are highly effective at preventing, with substantial delays in death from multiple forms of cancer in 
mice5,32,36. Fisetin, which selectively clears senescent cells, delays death of older mice, 50% of which 
die from cancer, by over 17%36. 
 
Threshold Theory of Senescent Cell Burden: Based on the above, we formulated a “Threshold 
Theory of Senescent Cell Burden”, which holds that once senescent cell abundance is sufficient to 
cause spread of senescence that exceeds capacity of the immune system to keep up with clearing 

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these cells, an accelerated aging-like state ensues32,34. Consistent with this: 1) the number of 
senescent cells that needs to be transplanted to cause frailty, limit healthspan, and cause premature 
death, including from cancer, is higher in young than old mice and in young lean than young obese 
mice. Old mice or young, obese mice have more pre-existing senescent cells than young, lean 
mice32. 2) Senescent cell abundance remains low in skin until early old age in humans, followed by 
an upward inflection in senescent cell burden in subjects who are in their late 60’s through the mid-
70’s37. This precedes the age-related increase in multi-morbidity38. 3) The lag between induction of 
senescence by chemotherapy and development of age-related morbidities is longer in childhood 
cancer survivors than adults who received higher chemotherapy doses in preparation for bone 
marrow transplantation39,40. These findings support the hypothesis that there is a threshold above 
which senescent cell burden due to spread of cellular senescence becomes self-amplifying. This 
causes increased risk for senescence- and age-related phenotypes, physical dysfunction/ frailty, 
hyper-inflammation, and diseases, perhaps contributing to age-related multi-morbidity38. Potentially, 
attraction, activation, and anchoring of immune cells is accelerated after senescent cells exceed this 
threshold, predisposing to excessive inflammation in frailty and during viral infection, plus 
continuing and exacerbating production of the SASP factors that underlie frailty, hyper-
inflammation/ cytokine storm, ARDS, and cardiomyopathy, as occurs in fatal CoV cases, as well as 
subsequent fibrosis, e.g., of the lungs. 
 
Pathogen-Associated Molecular Profile Factors (PAMPs): Of note, the SASP is modifiable: 
drugs, hormones, and especially the PAMPs associated with infections such as coronaviruses, can 
modify the SASP. We found that PAMPs can exacerbate the SASP in previously relatively quiescent 
human senescent cells by multiple fold (much greater than the effect of cytokines on the SASP), 
with greatly increased secretion of factors underlying cytokine storm (IL-6, IL-8, MCP-1) and 
increased attraction and activation of innate immune cells, potentially driving the frailty, hyper-
inflammation, cytokine storm, and ARDS caused by coronavirus (Fig. 1). 
 
 
 
Fig. 1. Senescent cells challenged with PAMPs highly express SASP factor mRNAs. Human 
subcutaneous adipocyte progenitors from different subjects were irradiated or not to generate senescent or 

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proliferating cells, respectively (n=3). Then cells were treated with different concentrations of the prototype 
PAMP, LPS, for 3 hours before mRNA isolation. One-way ANOVA and Dunnett multiple comparison post-
tests were performed against the control (0 ng) for each condition (proliferating or senescent). * = p<0.05, ** 
= p<0.01. Human non-senescent and radiation-induced senescent preadipocytes were treated with human 
recombinant IL-6, TNF-α, and IFN-γ for 3h. 
 
Similar results were obtained in vivo when progeroid mice, which have senescent cells to the same 
extent and in the same tissues as aged mice, were challenged with the PAMP, LPS (Fig. 2). In two 
tissues examined (kidney and liver), levels of senescence markers (p16Ink4, p21Cip1) and the SASP 
(Il1 , Il6, TNF
 and Mcp1) were greater in the progeroid mice compared to age-matched 
controls, illustrating an amplification of the response to PAMPs in organisms with increased 
senescent cell burden. In another study focused on neuroprotection in the face of LPS challenge, rats 
were administered Fisetin 20 i.p. mg/kg/day x 14 days, while being challenged with LPS41. In 
Fisetin-treated animals, inflammation and neurodegeneration were suppressed, while memory 
improved. 
 
Fig. 2. Pre-existing senescence causes an exaggerated response to pathogens in vivo. Eleven-week-old 
wild-type (blue) and progeroid Ercc1-/  (red) mice were injected with 500 ng/kg of LPS or vehicle (PBS) i.p. 
(N=4/ group). Twenty-four hours post-injection, mice were euthanized and tissues were collected. Total RNA 
was isolated from tissues and senescence and SASP marker expression were quantified in the (A) kidney and 
(B) liver by real-time qPCR using the ∆∆Ct method. Gapdh was used as a control for gene expression 
analysis. (C) Circulating levels of IL-6 and MCP-1 were quantified in serum by ELISA. All values represent 

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the mean ± SD. Statistical significance was determined by two-way ANOVA. * p<0.05, ** p<0.01, *** 
p<0.001, ****p<0.0001. (unpublished data, M. Yousefzadeh, B. Hughes, P. Robbins, L. Niedernhofer). 
 
Amplifier/ Rheostat Hypothesis of Cellular Senescence: The SASP depends on the type of cell 
that became senescent, how senescence was induced, and its milieu. The SASP changes over time in 
response to internal (e.g., transposons) and external cues (e.g., PAMPs, DAMPs, glucocorticoids, 
SASP inhibitors such as rapamycin). PAMPs are particularly effective at exacerbating the SASP 
(Figs. 1&2), perhaps accounting for severe morbidity in previously highly-functioning elderly 
individuals with an increased (but quiescent) senescent cell burden that is activated by infections 
such as CoV or septicemia. Based on these considerations, we advanced an “Amplifier/ Rheostat 
Hypothesis” of cellular senescence: that the increased senescent cell burden in elderly and 
chronically ill patients contributes to their excessive, frailty/morbidity/mortality-inducing hyper-
inflammatory (cytokine storm) response to infections that have much less cytokine-inducing effects 
in younger, previously healthy people (Fig. 3). 
 
 
 
Figure 3: Amplifier/Rheostat hypothesis 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 3. SASP Amplifier/Rheostat Hypothesis. Among the 
10 leading causes of mortality are influenza, pneumonia, 
genitourinary infections, and septicemia. The elderly and 
patients with cellular senescence-associated diseases 
(e.g., diabetes, dementias, smoking, asthma) are more 
susceptible to adverse outcomes in response to infections 
(e.g., ARDS, cytokine storm, myocardial damage with 
troponin leak, delirium, hypotension, thrombosis, delayed 
recovery) and develop a more exaggerated inflammatory 
state than young patients without pre-existing chronic 
diseases. These adverse outcomes of infections in the 
elderly or patients with senescence-associated chronic 
diseases have generally been attributed to immune 
system dysfunction or to chronic inflammation. Our 
“Amplifier/ Rheostat” Hypothesis is that PAMPs (e.g., LPS, 
viral antigens) cause a shift in the SASP of pre-existing 
senescent cells (SC) into a highly inflammatory, pro-
apoptotic, pro-fibrotic SASP. This may not only exacerbate 
acute systemic inflammatory responses and cytokine 
release by innate immune system cells (macrophages, 
dendritic cells, T lymphocyte subsets, neutrophils), but 
could also amplify spread of senescence to other cells. 
The resulting additional senescent cells may act to 
exacerbate and prolong inflammation, attenuate or delay 
recovery, lead to persistent frailty, cause long term tissue 
fibrosis if the afflicted individual survives long enough, 
and contribute to multi-organ failure (L. Langhi, T. 
Tchkonia, J.L. Kirkland, October, 2018; unpublished). 

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ß-Coronavirus Causes Infected Non-Senescent Cells to become Senescent: We found that mice 
infected with a mouse ß-coronavirus closely related to human COVID-19 develop increased 
senescent cell burden (Fig. 4). Similarly, human cells exposed to COVID-19 Spike-1 antigen or 
infected with an attenuated SARS-COVID-19 virus become senescent, as manifest by increased 
cellular SA-βGal (Fig. 5). 
 
 
Fig. 4: Specific pathogen-free (SPF) mice exposed to a normal microbial experience (NME) that includes 
acute mouse ß-coronavirus infection develop an increased abundance of senescent cell. Young (3-month-old) 
and old (20-24-month-old) male and female WT mice were exposed to NME bedding produced from pet 
store mice for 7 days. Abundance of p16Ink4a and p21Cip1 senescent cells was assayed in the liver, lung, and 
kidney of SPF or NME young and old mice. The ß-coronavirus-infected old mice had a particularly marked 
increase in senescent cell burden, with trends evident even in younger mice. Two-way ANOVA. *p<0.05, 
**p<0.01, ***p<0.001. 
 
Fig. 5: Both COVID-19 Spike-1 antigen and attenuated SARS-CoV cause non-senescent human kidney 
endothelial cells to become senescent within 10 days as manifested by increased cellular SA-βGal. Two-way 
ANOVA. *p<0.05, ***p<0.001. 
 
Senolytics: In 2004 Sharpless et al.42 showed that senescent cell accumulation is delayed in Ames 
dwarf mice (with pituitary hormone deficiencies) and calorically-restricted mice, models with 
increased healthspan and lifespan. This was critical in prompting us to test the hypothesis that 
targeting senescent cells may alleviate multiple age-related disorders. Our efforts to discover 
senolytics, drugs that selectively eliminate senescent cells, began in 2004/5, with initial attempts to 

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create fusion proteins comprising a senescent cell surface-binding domain coupled to a toxin, high 
throughput compound library screens for candidates that eliminate senescent but not non-senescent 
cells, and other approaches. These traditional approaches were not initially successful, so we turned 
to a hypothesis-driven discovery paradigm to discover the first senolytic drugs21,43,44. We 
hypothesized that since senescent cells kill cells around them and damage tissues due to their 
inflammatory, apoptotic SASP, yet these senescent cells do not die, they must have pro-survival 
networks that defend them against their own SASP: senescent cell anti-apoptotic pathways 
(SCAPs)5,43-45. 
 
From proteomic/transcriptomic databases and using bio-informatics methods, we discovered the 
SCAP network43. Through RNA interference analyses, we demonstrated that transiently disabling 
this SCAP network leads to apoptosis of senescent cells, while normal cells without a SASP are not 
affected, the “Achilles’ heel” of senescent cells. We next used bioinformatics approaches to identify 
drugs and natural products that act on critical SCAP nodes. Among these are Fisetin, Quercetin (Q), 
Dasatinib (D), and now over 20 others. We found these agents selectively eliminate senescent cells 
in mice and humans. They alleviate multiple conditions in mice, including diabetes and its 
complications, cardiovascular disorders (including heart failure and vascular dysfunction), 
dementias, pulmonary dysfunction (including bleomycin-induced lung disease), diabetes and its 
complications, frailty, kidney and liver damage or dysfunction, osteoporosis, osteoarthritis, and 
delay cancer and extend healthspan and lifespan5,21-24. 
 
We intentionally selected drugs for further development from among those identified as being 
senolytic. We focused on drugs that: 1) were already FDA-approved or were natural products with a 
history of safe human use, 2) could be administered orally, and 3) have a short elimination half-life 
(T½). Fisetin met these criteria. It is a natural product with a favorable safety profile, is effective 
orally, and has an elimination T½ ~3 hours, meaning it is cleared within a half day. 
 
The target of senolytics is senescent cells, not a single receptor, enzyme, or biochemical pathway. 
By targeting pro-survival networks instead of single molecules, specificity for senescent cells can be 
increased, side-effect profiles flattened, and off-target effects on non-senescent cells reduced. Drugs 
that have single or limited targets, such as Navitoclax (ABT-263), which we discovered is senolytic 
at the same time as another group46,47, or Nutlin3a, have off-target apoptotic effects on multiple non-
senescent cell types, making them “panolytic”. Navitoclax, a BCL-2/ BCL-w/ BCL-xL inhibitor, 
eliminates only a restricted range of senescent cells, e.g., BCL-2 inhibitors are not senolytic against 
human adipocyte progenitors, one of the most abundant senescent cell types in aged humans or 
people with diabetes and obesity. Our strategies for developing senolytics have more in common 
with approaches for developing antibiotics than the conventional one-target/one-drug/one-disease 
drug development approach. 
 
Description of the population to be studied. Adult women and men with PCR positive SARS-
CoV-2 infection, will be screened for inclusion in this study. Subjects who meet screening criteria 
will be randomized to receive Fisetin 20 mg/kg/day orally (capsules or powder dissolved in water or 
added to apple sauce, pudding, or yogurt) or by nasogastric (NG-tube) or transabdominal duodenal 
tube (D-tube; powder dissolved in water within 30 minutes, to achieve transient, high tissue Fisetin 
levels in a “hit-and-run” approach or placebo for 2 days (Days 0-1 and 8-9) and will be followed 
until study Day 30 or death. Subjects will continue to be followed for 2 months. The primary 
outcome is need for hospitalization and death with COVID-19. Secondary outcomes will be 
assessment for long hauler syndrome. Buccal swabs, urine and stool tests will be conducted before 
and after initiating treatment with Fisetin or placebo serially. 

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1.1 
Investigational Product (IP) 
An IP is defined as a pharmaceutical form of an active substance or placebo being tested or used as a 
reference in a clinical study, including products already with a marketing authorization but used or 
assembled (formulated or packaged) differently than the authorized form, or used for an 
unauthorized indication, or when used to gain further information about the authorized form. The IP 
will be stored in a secure area according to local regulations. Investigators will have responsibility 
for ensuring that IP is only dispensed to study subjects. The IP (Fisetin and placebo) will be 
dispensed only by authorized personnel according to regulations that apply at Mayo Clinic 
Rochester. In this protocol, the IP are Fisetin and placebo. 
 
Fisetin: Fisetin 
 is a member of the flavonoid family, a family of 
naturally-occurring polyphenolic compounds. As a natural product, it is widely sold, including by 
Amazon, from which it is available in containers of 30 capsules of 100 mg Fisetin/ capsule at <$20 
per container. The structure of Fisetin is similar to that of Q, which is one of the first two senolytic 
drugs discovered43 (Fig. 6). Fisetin, a high Trolox-equivalent antioxidant, is present in many fruits 
and vegetables such as apples, persimmon, grapes, onions, and cucumbers, with the highest 
concentration found in strawberries (160 µg/g)48,49 (Fig. 7). The average dietary intake of naturally-
occurring Fisetin was approximately 0.8 mg/day in an earlier Japanese study50,51. However, the 
average total intake of polyphenols, comprised mainly of flavonoids, is around 1 gram per day, 
although it may be quite variable52-54. Fisetin has little or no odor or taste. 
 
Due to its being a hydrophobic compound, Fisetin easily penetrates cell membranes, entering into 
cells to exert antioxidative and other effects55,56. Other promising biological activities include anti-
hyperlipidemic56-58, anti-inflammatory57, neurotrophic, apoptotic59, and antiproliferative effects36. 
 
There are reports of genotoxicity due to some flavonols, but Fisetin may exert little or no genotoxic 
effects in vivo60. Indeed, Fisetin has been noted to be anticarcinogenic48,61,62. We found that chronic 
Fisetin administration delays cancer in mice and increases remaining lifespan in old mice by over 
17%63. Since up to 50% of most mouse strains die from cancers, this suggests that the beneficial 
effects of Fisetin on such pro-carcinogenic factors as senescent cell burden and tissue chronic 
inflammation appear to more than counteract any theoretical effects of flavonol-associated 
genotoxicity. In the same mouse study, Fisetin was administered to a murine model of a human 
progeroid syndrome63. These mice are profoundly sensitive to genotoxic agents64, yet their health 
and lifespan were improved by long-term, high dose Fisetin63. 
 
Fig. 6. Structure of the senolytic flavonol, Q. 
Quercetin, one of the first two senolytic drugs 
identified (along with Dasatinib [D]), and 
which is present in apple peels, has a bitter taste 
and is less bioavailable and has a longer 
elimination T½ than Fisetin. 
Fig. 7. Structure of Fisetin. Fisetin differs from Q 
by an hydroxyl group. It has little or no odor or 
taste. Its terminal T½ in mice is ~3.1 hrs1. This short 
half-life makes Fisetin ideal as a senolytic drug to 
ablate pro-inflammatory, pro-apoptotic, non-
dividing senescent cells in a “hit-and-run” fashion5. 

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Fisetin is available as a dietary supplement. Other flavonols such as Q have been safely used in 
clinical trials in amounts up to 500 mg twice daily for up to 8 weeks. To date, no reports of 
significant toxicity for Fisetin in vivo have been made. Since Fisetin along with other flavones and 
flavonoids, has been shown to inhibit cytochrome P450 2C9, CYP3A4, and others (Section 3.9.4), 
precaution will be taken to avoid significant drug-drug interactions, e.g., with warfarin65. 
 
We demonstrated that Fisetin is a senolytic agent and alleviates multiple senescence-associated 
disorders in mice, decreases physical dysfunction if administered to old mice, attenuates age-related 
tissue damage and pathology, and extends remaining lifespan by 17%44,63. We have not found any 
adverse effects in mice (at up to 25 times the dose used in the mouse effectiveness studies [below] or 
the dose for humans proposed here), old Rhesus monkeys (at up to 5 times the dose for humans 
proposed here), or humans so far. We obtained a full IND for Fisetin from the FDA and have a GMP 
drug supply handled by Mayo Research Pharmacy. 
 
Fisetin 100 mg capsules or Fisetin (with cellulose) powder (in water, other chilled or room-
temperature drinks, apple sauce, pudding, yogurt, or enteral hydration/ nutrition solutions) will be 
administered orally for 2 consecutive days on study Days 0 and 1. To reduce senescent cell 
abundance by a “hit-and-run” approach, the full daily doses of Fisetin will be administered within 30 
minutes. The investigational supplies will be obtained from Vital Nutrients, a contracted GMP 
manufacturer. Fisetin capsules supplied by Vital Nutrients are opaque blue in color. Fisetin powder 
is a green yellow color. We established stability parameters for Fisetin in collaboration with Mayo 
Clinic’s Department of Laboratory Medicine and Pathology. We acquired an IND for Fisetin after 
establishing botanical sourcing, GMP isolation by Vital Nutrients, assurance of lack of 
contaminating pesticides, heavy metals, and solvents, and stability, degradation, and accelerated 
toxicity testing. 
1.2 
Preclinical Data 
In our article about the first senolytics discovered, we demonstrated D+Q reduces senescent cell 
burden in multiple tissues and improves function in naturally-aged animals43. Since then, D+Q, 
Fisetin (which is closely related to Q), Navitoclax, and other senolytics have been shown to alleviate 
multiple diseases and disorders in animal models (Table 1). Among effects are: restoration of 
mesenchymal stem cell (MSC) function; improved cardiac ejection fraction, fractional shortening, 
and regeneration in old mice; enhanced vascular reactivity in old mice, decreased vascular 
calcification and restored vascular reactivity in hypercholesterolemic, high fat-fed ApoE-/- mice; 
decreased loss of intervertebral disc glycosaminoglycans and spondylosis in progeroid Ercc1-/Δ 
mice; decreased gait disturbance in mice following radiation damage to a leg and hematological 
dysfunction caused by whole body radiation; decreased age-related changes of skin; improved 
pulmonary function and reduced pulmonary fibrosis in mice with bleomycin-induced lung damage, a 
model of IPF, plus decreases in: liver fibrosis in Mdr-/- cirrhotic mice; insulin resistance, hepatic 
steatosis, and renal dysfunction in high fat-fed mice; neuropsychiatric dysfunction and impaired 
neurogenesis in high fat-fed mice; neuro-inflammation, impaired neurogenesis, microvascular 
impairment, and cognitive dysfunction in mouse models of dementia; age-related osteoporosis; 
uterine fibrosis; physical frailty; and muscle weakness in naturally-aged, progeroid, radiated, and 
senescent cell-transplanted young mice as well as delayed cancer and increased healthspan and 
lifespan in old mice5,20-23,32,35,43,47,66-73. 
 
 
 
 

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Most studies of senolytics for various disease 
states, including in humans, have been with D+Q 
since D and Q were the first senolytics. However, 
while D+Q is relatively safe, it is likely not as 
safe as Fisetin for very ill patients. Unlike Fisetin, 
D cannot be administered safely to patients with a 
prolonged QTc (>450 msec). Many elderly and 
chronically-ill patients have a prolonged QTc, 
meaning they would be excluded from studies 
involving treatment with D. Additionally, there 
are multiple other drug interactions with D that 
would result in fewer patients being able to be 
treated than with Fisetin. Navitoclax has a poorer safety profile than Fisetin or D+Q: unlike D, 
which was approved by the FDA in 2006, Navitoclax has failed to gain FDA approval so far as a 
generally-available prescription drug, largely because of extensive and unpredictable induction of 
apoptosis in non-senescent cell types. For example, Navitoclax can cause life-threatening 
neutropenia or hemorrhage due to platelet depletion. Fisetin so far has had an exceptional safety 
profile in our clinical trials for elderly patients with multi-morbidity. Therefore, we decided to 
analyze Fisetin here rather than D+Q or Navitoclax. 
 
“Hit and run”: D+Q or Fisetin are effective if administered once every few days or weeks since 
they do not need to be continuously present to occupy a receptor or interfere with an enzyme, 
reducing off-target effects. Brief disruption of pro-survival pathways is sufficient to kill senescent 
cells in mouse and human cell cultures, in vivo in mice and monkeys, and in human adipose explants 
freshly-isolated from obese subjects21,43,63,68. In mice, monthly D+Q is as effective as daily 
administration to alleviate age-related osteoporosis95, reducing potential side-effects. These points, 
together with satisfying a modified set of Koch’s postulates (Table 2), show that senolytics alleviate 
Condition 
Reference
s 
Diabetes/ Obesity 
35,71,74-77 
Cardiac Dysfunction 
66,67,78 
Vascular Hyporeactivity/ Calcification 
67 
AV Fistulae 
79 
Frailty 
32,43,63,68 
Age-Related Muscle Loss (Sarcopenia) 
80 
Chemotherapy Complications 
32,40,47,68,81 
Radiation Complications 
82 
Cancers 
32 
Bone Marrow Transplant Complications 
40 
Organ Transplantation Complications 
31,83 
Myeloma/ MGUS 
84 
Age-Related Cognitive Dysfunction 
85 
Alzheimer’s Disease 
86,87 
Parkinson’s Disease 
88 
Amyotrophic Lateral Sclerosis 
89 
Ataxia 
43 
Obesity-Related Neuropsychiatric 
Dysfunction 
90 
Renal Dysfunction 
91,92 
Urinary Incontinence 
43 
Osteoporosis 
93-95 
Osteoarthritis 
96 
Age-Related Intervertebral Disk Disease 
43,97 
Idiopathic Pulmonary Fibrosis 
68,98 
Hyperoxic Lung Damage 
99 
Chronic Obstructive Pulmonary Disease 
100 
Tobacco 
101 
Hepatic Steatosis 
70 
Cirrhosis 
69 
Primary Biliary Cirrhosis 
102 
Progerias 
43,63 
Pre-eclampsia 
103 
Macular Degeneration 
104,105 
Glaucoma 
106-108 
Cataracts 
109 
Prostatic Hypertrophy 
110-112 
Psoriasis 
113 
Healthspan 
32,43,63 
Lifespan 
32,43,63 
Table 1. Conditions with emerging evidence for a causal 
contribution of cellular senescence or that are alleviated by 
senolytic drugs. Cellular senescence contributes to age-
related dysfunction and diseases throughout the lifespan. 
Senescence can contribute to local and systemic 
inflammation, dysfunction, and spread of senescence 
through release of cytokines, chemokines, proteases, 
ferritin, stem cell/progenitor toxins, reactive metabolites, 
miRNA’s, mitochondrial DNA, other nucleotides, and 
extracellular vesicles, the SASP2-4. Pre-senescent cells, 
which have limited remaining replicative potential, and 
senescent cells, which cannot replicate, accumulate to a 
greater extent in vivo in skin biopsies from older than 
younger subjects6. Senescent and pre-senescent adipocyte 
progenitors cloned from adipose tissue increase with aging 
in rats raised under controlled conditions7. Senolytics have 
been shown to alleviate many of these conditions by us 
(bold) and others. 

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dysfunction by removing senescent cells, not other off-target mechanisms. Unlike microbes or 
cancer cells, since senescent cells do not divide, they are unlikely to acquire replication-dependent 
drug resistance. Fisetin is well-suited for a “hit-and-run” administration protocol since it has an 
elimination T½ of ~3 hours, we demonstrated that it takes only 30 minutes of exposure of human 
tissues for senolytics to initiate the apoptosis process32, and senescent cells take over 1 week to re-
accumulate. Of note, no steady state levels for Fisetin need to be achieved and the goal is to 
eliminate senescent cells in a “hit-and-run” fashion, not to achieve a steady state blood level. The 
critical outcome here is target engagement, i.e., do we kill senescent cells?, not do we achieve a 
particular drug or tissue level? Therefore, as in our other trials, traditional blood pharmacokinetic 
parameters will not be assayed. Rather, the critical pharmacodynamic measure, senescent cell 
abundance, will be assayed. This approach has been tacitly agreed to by the FDA regarding our 
other clinical trials of senolytics, including with Fisetin. 
 
Table 2. A Modified Set of Koch’s Postulates 
To establish causation: 
• 
Are senescent cells present in animals or humans with the disorder? 
• 
Do individuals without senescent cells have the disorder? 
• 
Is the disorder reproduced by inducing local accumulation of senescent cells (e.g., by transplanting 
senescent cells, focal irradiation, or tissue-specific genetic approaches)? 
• 
Does removing these transplanted or induced senescent cells prevent or alleviate the disorder? 
• 
Does targeting naturally-occurring senescent cells alleviate the disorder? 
• 
Does administering the potentially senolytic candidate have few or no effects related to the disorder 
being tested in individuals with few or no senescent cells (e.g., young mice)? 
• 
Does the potentially senolytic candidate alleviate the condition if given intermittently, at intervals 
longer than the drug’s half-life, since senescent cells can take 10 days to 6 weeks to re-accumulate (at 
least in culture)? In the case of D+Q, senolytic drugs are as effective if administered monthly as 
continuously, at least in the case of age-related osteoporosis95. 
• 
Does the candidate alleviate multiple age-related conditions? (If a candidate is truly senolytic and the 
Geroscience Hypothesis is true, then it should alleviate multiple age- and chronic disease-related 
disorders). 
 
The above criteria have been met for senolytic drugs for frailty, diabetes, and age-related 
osteoporosis and many have been met for osteoarthritis and neurodegenerative diseases in mice. 
 
Fisetin selectively causes apoptosis of human cultured senescent cells: Fisetin reduces viability 
of 30-70% of senescent human umbilical endothelial cells (HUVECs) in vitro44 (Fig. 8). Note that 
30-70% elimination of senescent cells was achieved in our INK-ATTAC mice by activating the 
suicide gene expressed only in p16Ink4a+ cells (many or most of which are senescent) in these 
mice114. This extent of elimination was sufficient to alleviate multiple age-related conditions in 
chronologically-aged and progeroid mice20,67,68,114. 

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Fisetin selectively eliminates senescent cells from mice: Fisetin reduces age-related senescent cell 
accumulation in mice (Figs. 9 & 10). 
 
 
 
 
 
 
 
 
 
Fig. 8. Fisetin reduces human 
senescent cell viability through 
apoptosis. Senescent and proliferating 
non-senescent cultured HUVEC’s were 
treated with Fisetin 20 µM or vehicle, 
respectively, for <12 hours and the 
percentage of apoptotic cells was 
measured by TUNEL assay. 
Fig. 9. Fisetin reduces senescent cell abundance in 
mouse adipose tissue. Inguinal adipose tissue from a 
27 month old mouse that had been treated with Fisetin 
as daily pulse doses (tissue above) for 2 consecutive 
days once a month for 3 months (from age 24 
months) was assayed for SA-βGal activity 4 days 
after the last dose of Fisetin. Adipose tissue from a 
control 27 month-old littermate treated with vehicle is 
shown below. Fisetin also attenuated age-related 
lipodystrophy, as is evident by the larger adipose 
depot in the treated mouse. SA-βGal makes adipose 
tissue appear blue. Representative of 3 pairs of Fisetin 
and vehicle-treated littermates. A 27 month-old 
mouse is equivalent to an 80-90 year old human. 
Fig. 10. Fisetin reduces SA-βGal+ cells 
in adipose tissue from old mice. 
Twenty-four month-old mice were 
treated with vehicle (N=3) or Fisetin 
100 mg/kg (N=2) as daily pulse doses 
for five consecutive days by oral 
gavage. Mice were sacrificed 3 days 
after the last dose. Numbers of SA-
βGal+ cells were assayed in inguinal 
adipose tissue. 

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Fisetin selectively eliminates senescent cells from monkeys: Fig. 11. 
 
Fisetin alleviates cellular senescence-related phenotypes and extends healthspan and lifespan 
in mice: Ercc1−/∆ mice, which model human XFE progeria, have features resembling accelerated 
aging, with a lifespan of 6 months115. They develop progressive age-related chronic degenerative 
diseases116 and accumulate senescent cells in the same tissues as aged wild-type mice, albeit more 
rapidly117. To track senescent cell abundance in vivo, Ercc1−/∆ mice were crossed with p-16-luc 
transgenic mice. p-16-luc mice express luciferase under the control of the p16Ink4a promotor (p16Ink4a 
is increased in many or most senescent cells). After administering luciferin i.v., p16Ink4+ cells 
expressing the transgene emit light and can be seen and quantified in vivo using a light-detection 
box. The light signal (radiance) indicates the intensity and location of p16Ink4+ cells. Note that oral 
exposure to Fisetin at this dose, which is higher than that proposed for our human studies, does not 
cause toxicity even in these frail mice. There were no adverse effects on mobility, feeding, 
appearance, or weight. 
 
Fisetin decreased senescent cell burden in Ercc1-/∆; p16-luc mice as determined by decreased 
radiance (Fig. 12). Ercc1-/∆; p16-luc mice on C57Bl/6 Albino:FVB background at 3-4 weeks of age 
were fed ad libitum with a standard Teklad 2020 chow diet (“Lifespan” diet; N=15) or the standard 
Teklad 2020 chow diet containing Fisetin 500 ppm (N=2). Fisetin 60 mg/kg/day was consumed by 
each mouse based on Fisetin 500 ppm (500 mg/kg) being in the diet and 3-5 g chow eaten/ day. 
Senescent cell burden was measured at baseline and biweekly in control (lifespan) and Fisetin-
treated mice. Imaging of luciferase activity indicates a significant decrease in senescence signal. 
 
Fig. 11. SA-βGal+ cell number in subcutaneous 
adipose tissue biopsies of Rhesus macaques at 
baseline and after treatment with pulse doses 
of Fisetin. There is a greater decrease of 
senescent cells in the adipose tissue of Rhesus 
macaques at day 38 than at day 8 after the first 
dose of Fisetin 100 mg/kg. Animals were 
treated with Fisetin as daily pulse doses for 3 
consecutive days. Biopsies were done at 
baseline, 8 days, and 38 days after the first dose 
of Fisetin. 

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Fig. 12. Fisetin reduces age-related senescent cell accumulation in p16Luc/-;Ercc1-/ mice. Intermittent 
treatment of progeroid mice with Fisetin reduces senescent cell burden. (A) Representative image of age-
matched, 12 week-old male p16+/Luc;Ercc1−/∆ mice fed a diet containing Fisetin 500 ppm (500 mg/kg), or 
drug-free control diet. (B) Luciferase signal was measured biweekly in p16+/Luc;Ercc1−/∆ mice fed either 
control chow or chow containing Fisetin 500 ppm, N = 4–10 mice/ group and time point. The Fisetin was 
administrated intermittently for two weeks at a time (yellow bars). Otherwise the mice were fed a control diet. 
(C) The same data as seen in (B), but plotted as the percent change in luciferase signal as the animals aged. 
Values represented as the mean ± SEM. Two-tailed unpaired Student's t-test. ***p < .001, ****p < .0001. 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Fisetin increased skin hydration and elasticity and increased healthspan in Ercc1-/ mice (Figs. 13-
15). 
 
 
 
 
 
 
Fisetin increases remaining lifespan in old mice, consistent with a delay in cancers, which account 
for 50% of deaths in mice, and other causes of death (Fig. 16). 
 
Fig. 13. Skin hydration decreases with 
aging. Fisetin alleviated age-related loss of 
skin hydration. Hairless Shk-/-;K14-Cre+/-
;Ercc1-/fl mice lack the DNA repair protein 
ERCC1 in the epidermis, so that they are 
photosensitive. Mice were UV-irradiated 3X/ 
week at a dose that provides the equivalent 
of 30 years of photo-aging in humans. Mice 
(N=3) were treated topically with Fisetin 5 
mg/ml in a gel (100 µL), 3X/ week. No Tx = 
UV + Vehicle only. Fisetin caused a 
significant increase in skin hydration. 
Fig. 14. Fisetin alleviated the age-
related decline in skin elasticity. Mice 
(N=3) were treated topically with 
Fisetin 5 mg/ml in a gel (100 µL), 3X/ 
week. No Tx = UV + Vehicle only. 
Fisetin caused a significant increase in 
skin elasticity. 
Fig. 15. Fisetin extends healthspan measured by 
a composite frailty score in the Ercc1−/∆ mouse 
model of accelerated aging. At 3-4 weeks of age, 
Ercc1−/∆ mice on a C57Bl/6:FVB background were 
fed ad libitum with a standard Teklad 2020 chow 
diet (N=5) or standard Teklad 2020 chow diet 
containing Fisetin 500 ppm (N=5). Fisetin 60 
mg/kg/day is consumed by each mouse based on 
Fisetin 500 ppm (500 mg/kg) in the diet and 3-5 g 
of chow eaten/ day. The composite frailty score for 
mice includes assays of mobility, cognition, 
appearance, continence, tremor, gait disorder, and 
other variables. This score was measured in both 
groups at baseline and biweekly by an investigator 
blinded as to the treatment group. 

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Fig. 16. Fisetin increases survival in chronologically-aged wild-type mice. Late-life intervention with 
Fisetin in aged wild-type mice extends health span and lifespan. (A) At 85-weeks of age (>20 months; 
equivalent to age >70 years in humans), male and female mice were administered a diet containing Fisetin 
500 ppm (500 mg/kg) or fed a control diet with no drug. Lifespan was measured. N = 8–9 mice/ group. Log 
rank (Mantel-Cox) test. (B) Median lifespan of the same cohort of mice. Each dot represents an individual 
animal. Black bars indicate the mean ± S.E.M. Two-tailed unpaired Student's t-test. (C) Clinical chemistry of 
blood from the above mice to measure markers of liver (alanine aminotransferase [ALT]) and pancreatic 
(amylase [AMY]) dysfunction. N = 3–6 mice/ group. Two-tailed unpaired Student's t-test. (D) Composite 
lesion scores for aged-related pathologies in multiple tissues determined by histopathologic analysis 
according to the criteria of the Geropathology Grading Platform118. N = 3–8 mice/ group. Two-tailed unpaired 
Student's t-test. (E) Representative images of the kidney of a mouse fed control chow or Fisetin chow. In the 
control mouse, arrows (from left to right) indicate increased cellularity at a segment of the glomerular capsule 
border, moderate levels of lymphoid aggregates, and tubular cell vacuolization. In the Fisetin-treated mouse, 
the arrow indicates only mild segmental cellularity at the glomerular capsule border and a few scattered 
lymphoid cells near the glomerulus (200× magnification). *p < .05. 
 
Idea generation: The generation of this study began with our Amplifier/ Rheostat Hypothesis (Fig. 
3), which we validated in September, 2018 by testing if PAMPs exacerbate the SASP (Fig. 1). 
Together with the finding that coronavirus causes deaths from cytokine storm/ ARDS, we 
formulated the hypothesis that the senolytic drug, Fisetin (Fig. 7), could alleviate complications of 
coronavirus due to an excessive inflammatory reaction in February, 2020. This led us to search for a 
mouse model of coronavirus. Our collaborators at the U of M had an infected colony and had done 
an experiment in December, 2019 showing that young mice survive, but old mice die beginning 7 
days after exposure to the mouse β-coronavirus, Mouse Hepatits Virus (MHV; Fig. 4) and develop 
high circulating IL-6, unlike infected young mice (Fig. 5). We administered Fisetin vs. vehicle to old 
mice and sent them to the MHV-infected colony at U of M (Fig. 17). We and our collaborators 
discovered that Fisetin substantially increased survival of β-coronavirus-infected old mice (Figs. 18, 
20, &22). 
 
Aged, infected prevention results: We conducted an experiment in which 10 male and 10 female 
mice >24 months old, equivalent to humans aged >80 years, were administered Fisetin 20 mg/kg as 
daily pulse doses by oral gavage for 2 days and 10 control >24 month old male and 10 female mice 
were administered vehicle at Mayo Clinic. After 2 more days, the mice were moved from Mayo 

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Clinic to the mouse β-coronavirus-contaminated facility at U of M. Since they fight, the males were 
placed in bedding in which infected male mice had been housed. The females were placed directly in 
contact with contaminated female mice, and so had a much higher exposure than the males. 
Similarly to the male experiment, in a second experiment, 10 female mice pretreated with Fisetin 
and 10 female mice with vehicle at Mayo Clinic were placed in bedding in which infected female 
mice had been housed. Those mice pretreated with Fisetin or vehicle by gavage before being 
transferred from Mayo Clinic to the infected colony at U of M were administered Fisetin 500 ppm or 
vehicle, respectively, in food while they were in the contaminated facility (Fig. 17). 
 
Fig. 17. Mouse coronavirus Fisetin experiment design. 
 
All of the 20 vehicle-treated mice in the first experiment died within 12 days of pathogen exposure. 
However, 5 of the 20 Fisetin-treated bedding-exposed male (p<0.0001) and infected mouse-exposed 
female mice (p=0.2) were still alive after 15 days of continuous infection exposure plus a further 12 
days after being switched to clean bedding at Day 16 (Fig. 18). In the second experiment (Fig. 20), 
all of the 10 vehicle-treated bedding-exposed female mice died within 10 days of infection exposure. 
However, 7 of the 10 Fisetin-treated female mice were still alive (p<0.0002) after 13 days of 
continuous infection exposure. Thus, Fisetin prevents mortality in old mice caused by viral exposure 
that includes the mouse β-coronavirus. All mice tested had blood test-proven mouse β-coronavirus 
infection. Tests for multiple other pathogens were negative. 
 
 
Fig. 18. Survival of male mice exposed to bedding contaminated with mouse β-coronavirus is increased 
substantially by Fisetin. (A) Old male mice (age>24 months; N=10) were administered Fisetin 20 
mg/kg/day as daily pulse doses, and 10 control old male mice vehicle, by oral gavage beginning 4 days before 
being transported from Mayo Clinic to a contaminated facility at U of M, where they were exposed to 
bedding from β-coronavirus-infected mice. Males cannot be exposed directly to infected mice because they 
fight. At the U of M facility, the Fisetin-treated mice were maintained on a Fisetin 500 ppm diet and the 
vehicle-treated mice were fed regular chow. Male mice treated with Fisetin had a significant improvement in 

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survival vs. vehicle-treated old male mice in this prevention study (p < 0.0001; log-rank Mantel Cox test). (B) 
Old female mice (age >24 months; N=10) were also administered Fisetin at Mayo Clinic beginning 4 days 
before being transported to U of M and exposed directly to infected female mice (likely resulting in a much 
higher viral load than in the males described above that were exposed only to infected bedding). The females 
exposed directly to infected female mice tended to have better survival vs. vehicle-treated old female mice 
(N=10; p=0.2). Of note, by day 11, when the vehicle-treated mice had died, the only pathogen that the female 
co-housed mice had antibodies against was the mouse β-coronavirus, MHV (Fig. 19). 
 
 
 
 
Fig. 20. Fisetin increases survival of female mice exposed to bedding contaminated with mouse β-
coronavirus. As for males in the above experiment (Fig. 20), old female mice (age >24 months; N=10) were 
administered Fisetin 20 mg/kg/day as daily pulse doses or vehicle (N=10) by oral gavage beginning 4 days 
before being transported from Mayo to the contaminated facility at U of M, where they were continuously 
exposed to bedding from β-coronavirus-infected mice. At the U of M facility, Fisetin-treated mice were fed 
Fisetin 500 ppm chow and vehicle-treated mice were fed regular chow. Like males, female mice treated with 
Fisetin had significant improvement in survival vs. vehicle-treated mice in this prevention study (p < 0.0002; 
log-rank Mantel Cox test). 
 
Aged, infected treatment results: In this experiment, we tested if Fisetin intervention in old mice 
after they have been infected and as they become symptomatic slows death is underway. Old mice 
(24 months) were transferred from Mayo Clinic to the contaminated facility at U of M and, 3 days 
after introduction to that facility from Mayo Clinic (study Day 0), were treated for 3 consecutive 
days with Fisetin 20 mg/kg/day as daily pulse doses vs. vehicle by gavage on study Days 3 to 5, 10 
to 12, and 17 (Figs. 21&22). After each series of high daily Fisetin doses by gavage, they were 
maintained on Fisetin 500 ppm in food or regular chow, respectively, on study Days 6 to 9 and 13 to 
16. 
Fig. 19. Fisetin increases the antibody response of 
old female mice co-housed with mouse β-
coronavirus-infected mice. Old female mice (24 
months) had less antibody response against 
coronavirus than young mice by 11 days after both 
groups were co-housed with mice infected with 
mouse β-coronavirus. However, at Day 16, the 24-
month old female mice treated with Fisetin (as in Fig. 
17) had an antibody response similar in magnitude to 
the young mice. All old female mice treated with 
vehicle had died by Day 16. Thus, Fisetin treatment 
does not prevent antibody response of old mice to 
coronavirus infection and allows time for the older 
mice to develop an antibody response similar to that 
of young mice. 

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Fig. 21. Experiment to determine if senolytics administered after exposure to β-coronavirus slows 
mortality in old mice. 
 
Fig. 22. Fisetin treatment slows death of old mice infected with β-coronavirus. (A) Male 24 month old 
mice were transported from Mayo Clinic to the mouse β-coronavirus contaminated colony at U of M, 
depicted here as Day 0. All mice were exposed to bedding used by infected male mice. Beginning 3 days after 
transfer from Mayo Clinic, they were treated for 3 days with Fisetin 20 mg/kg/day as daily pulse doses vs. 
vehicle by oral gavage (N=10 mice/ group) on Days 4 to 6. On Days 7 to 9, the Fisetin-treated mice were fed 
with chow containing Fisetin 500 ppm and the vehicle-treated mice were fed chow only. On Days 10 to 12 
and 13 to 16, animals were treated with Fisetin 20 mg/kg as daily pulse doses vs. vehicle by oral gavage on 
Days 10 to 12 and 13 (all vehicle-treated mice were dead at Day 14). On Day 17 animals were treated with 
Fisetin 20 mg/kg as daily pulse doses. (B) Female 24 month old mice were treated in the same way. Fisetin 
significantly increased survival of β-coronavirus-infected mice (males: p < 0.0001; females: p < 0.001; log-
rank Mantel Cox test). 
 
Fisetin treatment of old, β-coronavirus-infected mice slowed death in an experiment close to the 
clinical study design planned here (Fig. 22). Of note, in separate, parallel studies in which animals 
were euthanized after cohousing or exposure to dirty bedding, transmission of MHV was 100% and 
antibodies against the virus were detected by day 11 (Fig. 19). No antibodies to other common 
mouse pathogens were detectible. 
1.3 
Clinical Data to Date 
Senescent cell clearance from humans: In the first clinical study published about senolytics, a 

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pilot, open-label study in 14 patients with IPF, we found that 9 pulse-doses of oral D+Q over 3 
weeks led to improved 6-minute walk distance, walking speed, ability to rise from a chair, and short 
physical performance battery by 5 days after the final dose119. These results led to initiation of a 
Phase IIb randomized, placebo-controlled, double-blind trial that is currently underway. 
 
Interim results of a Phase 1, open-label, clinical trial of D+Q for subjects with diabetic kidney 
disease (DKD) underway at Mayo (ClinicalTrials.gov Identifier: NCT02848131) showed a 3 day 
oral course of D+Q administered as pulse doses in 9 subjects with DKD reduced adipose tissue 
senescent cell burden assayed by p16INK4a+ and SA-βGal+ cells by 11 days after the last dose, 
compared to biopsies before D+Q administration76. A composite score of 10 circulating SASP 
factors was significantly decreased 11 days after completing the 3 day D+Q intervention. 
Furthermore, activated CD68+ macrophage tissue infiltration was significantly decreased. This trial 
is continuing (goal=30) to test effects of senolytics on adipose tissue and skin senescent cell 
abundance and urine SASP factors, metabolic and renal function, inflammation, quality of life, 
safety (drug toxicity), and tolerability. No serious drug side effects have emerged so far. Evidence 
continues to show clearance of senescent cells. Each subject will be followed for 4 months after the 
single course of D+Q to provide data for a larger Phase IIb randomized, placebo-controlled, double-
blind trial of senolytics for DKD. 
 
The senolytic drug to be used in our study here is Fisetin. We found that frailty in elderly women is 
associated with senescent cell burden and the associated inflammation in adipose tissue biopsies98. 
Furthermore, senolytics alleviated frailty in the subjects in our IPF trial120. Therefore, a Phase IIb 
double-blind, placebo-controlled clinical trial of Fisetin to reduce senescent cell burden and alleviate 
frailty and inflammation, AFFIRM, has commenced. Oral Fisetin 20 mg/kg/day for 2 days vs. 
placebo is being administered as daily pulsed doses for frailty (gait speed <0.8 m/sec) and 
inflammation in elderly women with multi-morbidity. This trial at Mayo Clinic is FDA-approved 
(ClinicalTrials.gov Identifier: NCT03430037) and has enrolled over 20 subjects who have received 
Fisetin or placebo. The trial is not yet unblinded. No participants have had severe or serious adverse 
events so far at the same doses of Fisetin as proposed here for the same indications, clearing 
senescent cells and alleviating physical dysfunction and inflammation (in CoV patients). 
 
1.4 
Dose Rationale 
Others have treated mice with Fisetin 50 mg/kg/day for weeks without evidence of toxicity121. We 
treated 4 male and 4 female 8 month old C57/Bl6 mice with Fisetin 500 mg/kg/day (25-fold higher 
than the dose to be used in this clinical trial) by oral gavage for 2 consecutive days compared to 4 
male and 4 female mice treated with vehicle. We found no evidence of substantial toxicity, as 
assessed by monitoring activity, food intake, and respiratory quotient in metabolic cages 
(Comprehensive Laboratory Animal Monitoring System, Columbus Instruments) for 48 hours after 
the last Fisetin dose. We administered 100 mg/kg/day for 2 consecutive days by mouth as daily 
pulse doses, 5 times the dose proposed in in this clinical trial, to 2 older Rhesus monkeys (ages 19 
and 28 years, which is old for this species) at the National Primate Center. In over 1 month of close 
monitoring, neither monkey had adverse effects such as reduced appetite, altered activity, vomiting, 
or diarrhea. Acknowledging the higher rates of drug metabolism in mice compared to humans and 
differences in physiology between non-human primates and humans, in an effort to avoid side 
effects, a daily dose of ~20 mg/kg was selected here. We verified that this dose is sufficient to 
reduce senescent cell burden in mice. Our studies in very old coronavirus-infected mice 
demonstrated that this dose of Fisetin dramatically enhances survival (Figs. 18, 20, and 22). 

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1.5 
Risks and Benefits 
There are appear few if any risks of Fisetin at the doses to be used that we have evidence for, 
provided the subjects meet the exclusion criteria in the protocol regarding medications that Fisetin 
could interfere with. We have not seen serious or severe adverse events so far in elderly frail women 
enrolled in our AFFIRM studies from Fisetin 20 mg/ kg/ day for 2 consecutive days in the over 20 
subjects randomized to Fisetin or placebo. We did not see adverse effects such as altered food 
intake, weight, or behavior in old Rhesus macaques given 5 times the dose proposed here or in mice 
given 25 times the dose. 
 
Based on our studies in mice, there may be decreased morbidity and mortality due to β-coronavirus 
infection after Fisetin administration. All subjects in this study will be elderly (and/or chronically-
ill) and hospitalized with mild or moderate CoV infection that could progress to severe or critical 
disease. The potential benefit to individual subjects in this trial and other people might be large. 
 
In this study, we will examine the effect of oral/ enteral Fisetin as a senolytic agent in humans, 
which may have immense potential for and reducing frailty/ physical dysfunction and inflammation 
and alleviating morbidity and mortality from CoV or other infections in older subjects and those 
with other conditions and diseases associated with accumulation of senescent cells. As an additional, 
advantageous, off-target effect, removal of senescent cells may allow for functional improvement of 
neighboring or distant tissues and cells, including MSC’s, accelerating recovery. We found that 
senolytics restore function of MSC’s20,73. Our collaborator, G. Ellison, working with investigators in 
China, noted that transplanting normal MSC’s can alleviate symptoms and signs in severely ill CoV-
infected patients122. Thus, an additional benefit of senolytics for CoV could be alleviation of stem 
cell and progenitor dysfunction. 
 
Based upon review of the published literature and our pre-clinical data, we do not anticipate the 
occurrence of serious adverse events with the prescribed regimen in this study. We have identified a 
variety of drug-drug interactions (e.g., current use of warfarin) for which we established exclusion 
criteria or modification plans to minimize associated risks. We could find no reports of Fisetin 
toxicity in the literature. Some flavonols have been associated with DNA damage in vitro, but 
Fisetin exerts little or no genotoxic effects60. To the contrary, Fisetin has been noted to be 
anticarcinogenic48,61,62. Indeed, we found that chronic Fisetin administration delays cancer in mice 
and increases remaining lifespan in old mice as well as DNA damage/ genotoxicity highly-prone 
Ercc1-/Δ mice by over 17%63. Since up to 50% of most mouse strains die from cancers, this shows 
that the beneficial effects of Fisetin on such pro-carcinogenic factors as senescent cell burden and 
tissue chronic inflammation appear to more than counteract any theoretical effects of flavonol-
associated genotoxicity. Fisetin actually reduces cancer risk, as opposed to increasing it, as would 
be expected if Fisetin-induced DNA damage were a risk for the subjects in the clinical study 
proposed here. 
 
Overall, we do not anticipate substantial risk from the natural product, Fisetin, which is present in 
many foods, including cucumbers, strawberries, and other fruits and vegetables, albeit at levels 
lower than those that will be used here. Thus, the risks of using this drug are likely minimal 
compared to the anticipated benefits for patients with CoV infection severe enough to require 
hospitalization and the knowledge that may be gained from these clinical investigations. 
 
2 
STUDY OBJECTIVES 
 
The primary objective of this study is to determine whether short-term treatment with Fisetin reduces 

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the rate of complications related to COVID-19 (CoV) disease using a 7 point score adapted from the 
WHO Ordinal Scale for Clinical Improvement of CoV (Appendix A). The secondary objective is to 
determine safety and tolerability of treatment with Fisetin in this patient population. Exploratory 
objectives are to evaluate links between soluble and genetic markers of senescent cell abundance, 
SASP factors, inflammation, viral load, and treatment effects. 
 
3 
STUDY DESIGN 
 
This will be an enterprise wide, randomized, double-blind, placebo-controlled study. Following 
signature of the informed consent form, approximately 150 subjects meeting all inclusion and no 
exclusion criteria will be randomized to receive either Fisetin or placebo (1:1 allocation ratio) for 
days 0 and 1 AND days 8 and 9. Follow-up phone assessments will occur at 15, 30, and 60 days 
following randomization for evaluation of the occurrence of any trial endpoints or other adverse 
events. Exploratory objectives are to evaluate links between soluble and genetic markers of senescent 
cell abundance, SASP factors, inflammation, viral load, and treatment effects and if Fisetin treatment 
during acute CoV infection decreases Long-hauler syndrome at Days 60 . 
 
3.1 Study Schedule 
 
The schedule of visits for this study is outlined in Table 1. However, a patient may be evaluated at 
any time for safety concerns. 
 
3.2 Enrollment 
 
Informed consent will be obtained from patients who volunteer to participate in the study prior to 
the conduct of any study-specific procedures. The patient will be considered “enrolled” into the 
study at the time an informed consent is provided. 
 
Biomarker samples collection including senescent biomarker analysis will be optional for 
participants. The informed consent form will reflect this option and patients will specifically be 
asked to consent to this biomarker evaluation. 
 
 
3.3 Screening Evaluations and Randomization 
 
Screening evaluations will include a review of the patient’s medical history and assessment of 
concomitant medications to determine if the patient qualifies for the study. Patients who meet all 
inclusion criteria and no exclusion criteria will be randomized to receive the study drug or placebo. 
All patients must be randomized within 10 days of the diagnosis of COVID-19 infection. 
 
Women of childbearing potential must have a negative urine pregnancy test result after the time of 
randomization, but before IP administration in order to qualify. Qualifying patients will be 
randomized to receive placebo or fisetin administered in a blinded manner. Blinded randomization 
will be performed through an automated Interactive Web Response System (IWRS). 75 patients 
will be randomized to active treatment and 75 patients will be randomized to placebo for a total of 
150 randomized patients. 
 
 

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3.4 Active Treatment Period 
 
All patients will receive study medication dosing (either Fisetin 100 mg or matching placebo) (PO) 
ON day 0 and 1 And Day 8 and 9. If a dose is missed, it should not be replaced. Throughout the 
study, patients will undergo phone visits (at 15, 30, and 60 days) to assess for potential study 
endpoints and other adverse events (AEs). Patients will be dispensed the entire supply of study 
medication on Day 0 to use for the duration of the study.  
 
At each phone contact, patients will be: 
1) questioned in a non-specific manner for the occurrence of AEs and any change in 
concomitant medications 
2) 7 point score adapted from the WHO Ordinal Scale for Clinical Improvement of CoV. 
 
Fecal, urine and buccal swab samples for biomarker are optional for the participants. The informed 
consent form will reflect this option and patients will specifically be asked to consent to this 
biomarker evaluation.  
 
Please refer to Table 1 and Appendix A for Timetable of Visits and Requirements. 

 
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Table 1 Timetable of Visits and Requirements 
 
Visits 
1 
Screening/Ra
ndomization 
Phone call 
2a 
Randomization/ 
Baseline Phone 
call 
 
3b  
Phone   
contact 
 
4b  
Phone 
contact 
5b  
Phone contact 
6b  
Phone  
contact 
Days 
0 
1 
8 
(-/+ 2 daysd) 
9 
 
15 
(-/+ 2 days) 
30 
(-2/+7 days) 
60 
(-/+ 14 days) 
Informed consent 
X 
 
 
 
 
 
 
 
Medical/Surgical history 
X 
 
 
 
 
 
 
 
Review Concomitant Medications 
X 
X 
 
X 
 
X 
X 
X 
Phone contact 
X 
X 
 
X 
 
X 
X 
X 
Review Inclusion/Exclusion criteria 
X 
X 
 
 
 
 
 
 
Urine pregnancy test (only women of 
childbearing potential) 
 
X 
 
X 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
SF-36 Health Survey 
 
X 
 
X 
 
X 
X 
X 
Buccal, fecal and urine sample for biomarkers 
(optional) 
 
X 
 
 
 
X 
X 
X 
Record potential study endpoints (ordinal scale) 
 
X 
 
X 
 
X 
X 
X 
Review Adverse Events 
 
X 
 
X 
 
X 
X 
X 
Study medication dispensing 
 
X 
 
 
 
 
 
 
Study medication dosing 
 
X 
X 
X 
X 
 
 
 
a) 
Screening Visit 1 and Visit 2 may be performed on the same day; under these circumstances, requirements at both Screening Visit 1and Visit 2 will be performed once. 
b) 
Phone contact  
c) 
Saliva sample for pharmacogenomics evaluation will be collected using saliva collection kit. 
d) 
Phone contact can occur + or – 2 days; however study drug administration should still occur on days 8 and 9. 
 
 
 
 
 
 

 
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4 
STUDY POPULATION 
 
4.1 Source and Number of Patients 
 
A total of 150 patients will be randomized. 
 
4.2 Patient Selection Criteria 
 
4.2.1 Inclusion Criteria: 
 
All of these criteria must be met: 
• Males and females, at least 18 years of age, capable and willing to provide informed 
consent; 
• Patient must have received a diagnosis of COVID-19 infection within the last 10 days; 
• Outpatient setting (not currently hospitalized or under immediate consideration for 
hospitalization); 
• Patient must possess at least one of the following high-risk criteria: 70 years or more of 
age, obesity (BMI ≥30 kg/m2), diabetes mellitus, immune deficient state (primary or 
secondary immunodeficiency), history of hypertension (systolic blood pressure ≥150 mm 
Hg), known respiratory disease (including asthma or chronic obstructive pulmonary 
disease, current or previous smoking), known heart failure, known coronary disease, 
fever of ≥38.4°C within the last 72 hours, dyspnea at the time of presentation, bicytopenia, 
pancytopenia, or the combination of high neutrophil count and low lymphocyte count; 
• Female patient is either not of childbearing potential, defined as postmenopausal for at least 
1 year or surgically sterile, or is of childbearing potential and practicing at least one method 
of contraception and preferably two complementary forms of contraception including a 
barrier method (e.g. male or female condoms, spermicides, sponges, foams, jellies, 
diaphragm, intrauterine device (IUD)) throughout the study and for 30 days after study 
completion; 
• Patient must be able and willing to comply with the requirements of this study protocol. 
 
4.2.2 Exclusion Criteria: 
 
None of these exclusion criteria should be met: 
• Patient currently hospitalized or under immediate consideration for hospitalization; 
• Patient currently in shock or with hemodynamic instability; 
• Patient with severe hepatic disease (as per clinical judgement) or liver enzymes >2x the upper 
limit of normal; 
• Female patient who is pregnant, breast-feeding, or is considering becoming pregnant during 
the study or for 1 day after the last dose of study medication; 
• Patient currently taking Sirolimus, Tacrolimus, or other mTOR inhibitors for other indications 
(mainly chronic indications represented by organ transplantation or autoimmune diseases); 

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• On Warfarin therapy; Subjects taking any of the medications listed in Appendix C may 
participate if they are otherwise eligible AND the medication can be safely held during the 
following times: 
o Immediately before the 1st IP administration (Day 0) until at least 10 hours after the 
2nd IP administration (Day 1) 
o Immediately before the 3rd IP administration (Day 8) until at least 10 hours after the 
4th IP administration (Day 9) 
• Patient with a history of an allergic reaction or significant sensitivity to Fisetin; 
• Patient undergoing chemotherapy for cancer; 
• Patient is considered by the investigator, for any reason, to be an unsuitable study candidate. 
• History of diverticulitis or diverticulosis with GI bleeding, as per clinical judegement. 
 
4.3 Prohibited, Allowable, and Concurrent Medications 
 
The use of concomitant medications at the time of randomization will be recorded in the eCRF 
(with the exceptions of concomitant medication taken “when necessary” (PRN)). 
 
5 
STUDY METHODOLOGY 
 
5.1 Efficacy Outcomes 
 
The primary endpoint will be the composite of death or the need for hospitalization due to COVID-
19 infection in the first 30 days after randomization. The secondary endpoints will consist of the 
components of the composite primary endpoint; and the need for mechanical ventilation in the first 
30 days after randomization. Exploratory endpoints will include associations between senescent 
biomarkers and long hauler syndrome effects. 
 
Biomarkers endpoints: 
Samples received from participants will be kept frozen until evaluation of biomarkers. 
Samples will be stored and processed at Mayo Clinic Rochester. 
 
5.2 Safety Monitoring 
 
Drug safety will be assessed by an evaluation of types, frequencies, severities and duration of 
any reported AEs. Patients will be monitored for signs and symptoms of drug toxicity. 
 
 
For all toxicities that require the study therapy to be temporarily or permanently discontinued, 
relevant clinical and laboratory tests will be repeated as clinically needed until there is final 
resolution, stabilization of the toxicity, or another cause for the abnormality is determined. 
 
5.2.1 Adverse Event Reporting 
 
Information regarding AEs will be collected from the first dose of study medication (at Visit 2) 
through and including the last visit. Any AEs prior to randomization will be recorded in the medical 
history and kept in the patients’ chart. 

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In addition to standard reporting of Serious Adverse Events (SAEs) as defined and outlined in 
Section 5.2.1.1 below, information regarding SAEs that occur within 30 days following the last 
study visit, and reported to the investigational site, will be collected. Each patient will be observed 
and queried in a non-specific fashion at each visit during the study for any new or continuing 
symptoms since the previous visit. 
 
All SAEs will be recorded on the appropriate eCRF section. The only other AEs to be recorded 
on the eCRF are those that are either related to the gastrointestinal system, that are judged related 
to the study medication by the investigator or laboratory abnormalities judged clinically significant 
by the investigator. Information collected will include the onset, duration, severity, relationship to 
study drug, and the management as outlined in Appendix C. 
 
5.2.1.1 Serious Adverse Events 
 
Serious Adverse Events (SAE) are those that meet any of the following International Council for 
Harmonisation (ICH) criteria: 
 
• Is fatal or immediately life-threatening; 
• Results in persistent or significant disability/incapacity; 
• Requires or prolongs inpatient hospitalization; 
• Is a congenital anomaly/birth defect in the offspring of the patient; 
• Is a cancer; 
• Is an overdose (intentional or accidental); 
• Is judged to be medically important. 
 
Medically important events may not be immediately life-threatening or result in death or 
hospitalization but may jeopardize the patient or may require intervention to prevent one of the 
outcomes listed in the definition above. Serious Adverse Events are to be reported if they are 
known to occur within 30 days following the last study visit. 
 
Medical and scientific judgment should be exercised in deciding whether other AEs meet these 
criteria and are immediately reportable to the sponsor or designee. 
 
In the event of a serious or life-threatening adverse event, or in the event of death, immediately 
report the event on the appropriate SAE form in the eCRF. 
 
 
If any SAE occurs, the study treatment may be interrupted or discontinued at the Investigator’s 
discretion. If an acute medical emergency occurs, the investigator should make every effort to 
reach the contact person listed above, before breaking the randomization code, via the automated 
Interactive Web Response System (IWRS). However, the investigator may break the 
randomization code (via IWRS) at any time if this is required for proper treatment of the patient 
and even if the contact person could not be reached. 
 
Reports to the Health Authorities must be made within 7 calendar days, (for death and life-
threatening events) and within 15 calendar days (for other serious events) after being informed of 

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an SAE by the investigator. 
 
5.2.1.2 Lack of Efficacy of study medication 
 
Any signs or symptoms defined in the protocol as lack of efficacy or collected as efficacy 
parameters (endpoints) will be captured as AE in the database. 
 
5.2.1.3 Adverse Event Follow-Up 
 
Record all reportable serious and non-serious AEs on the appropriate eCRF including the onset, 
duration, severity, relationship to the study drug, and ultimate management. All AEs reported 
during the treatment phase should be recorded and followed until the AE has subsided, or stabilized 
or until the end of the study, whichever occurs first. The study will be stopped at any time if new 
knowledge is gained and the risk-benefit ratio is no longer favorable for the participating patients 
 
5.2.2 Other Safety Monitoring 
 
5.2.2.1 Laboratory Evaluations 
 
Optional biomarker with buccal, urine and stool samples will be collected for the biomarker 
analysis. At day 0/1, day 15, day 30 and day 60. The samples will be kept in a laboratory at 
Rochester campus. These samples may be used to assess (but not limited to) markers of 
inflammation and senescence. 
 
5.3 Withdrawal of Patients from the Study 
 
Patients have the right to withdraw from the study at any time during the course of the study. 
However, every effort should be made, within the bounds of safety, patient choice and the 
provisions of informed consent, to have each patient complete the study up to and including the 
last protocol-specified study visit. If the study medication jeopardizes the patient’s health or if the 
patient wishes to discontinue for any reason, study medication can be discontinued but the patient 
should be encouraged to remain in the study for the follow-up visit up to the End of Study visit. If 
the patient does not wish to pursue the protocol follow-up visit, the option for a last contact at the 
end of the study to obtain their vital status should be proposed. Patients who are not compliant 
during the active treatment period should be counseled on the importance of complying with study 
requirements and be allowed to remain in the study. No patient who has withdrawn their consent 
from the study during the active treatment period should be replaced. 
 
In the case of an adverse event or safety concern, the study medication may be withheld 
temporarily, or the dose reduced, as per investigator judgment.  
 
Patients withdrawn at any time from the study during the active treatment period should complete 
all last protocol-specified visit requirements (End of Study Visit, the reason for withdrawal from 
the study, the date of the last visit and the date of the last dose of double-blind medication will be 
clearly documented in the eCRF). 
 
 

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5.4 Study Completion 
 
The study will end when the last randomized patient will have completed his (or her) 60 day 
follow-up phone contact. Completion of the study by a patient should be clearly indicated in the 
eCRF, along with the date of the last visit and the date of the last dose of double- blind medication. 
 
Patients will be remunerated for participation in the study; they will receive $25 after completing 
Day 15, Day 30 and Day 60. 
 
6 
STUDY MEDICATION 
 
Mayo Research pharmacy will prepare and dispense study medication (fisetin and placebo) 
with appropriate labeling to include a statement that these products are for investigational use 
only. 
 
6.1 Medication Dispensing 
 
Study medication may be dispensed by the designated pharmacist or a qualified investigative site 
representative, according to a detailed set of dispensing instructions. Study medication may also 
be shipped directly to the patient’s home depending on evolving circumstances related to COVID-
19. 
 
6.2 Dosage Regimen 
 
At randomization, patients will be dispensed Fisetin capsules or placebo to match Fisetin capsules. 
Patients will be instructed to take 20mg/kg on days 0 and 1 and another 20mg/kg on days 8 and 9 
according to the detailed set of dispensing instructions outlined on the label. 
 
7 
DATA COLLECTION 
 
Electronic Case Report Forms (eCRF) for all patients will be supplied. These are to be completed 
as instructed. Original source documents and other study documentation will be maintained at the 
study site. Risk-based monitoring will be performed by the study team. 
 
8 
DATA ANALYSIS AND STATISTICAL CONSIDERATIONS 
 
8.1 Statistical Power and Sample Size Considerations 
 
All main analyses in COVFIS-HOME will be conducted on an intention-to-treat basis. Assuming 75 
subjects/ group with a significance level of 0.05, the study will have 80% power to detect an odds ratio 
of 2.24 comparing the placebo- to Fisetin-treated group, using the 7-point severity score at Day 30 as the 
endpoint in an ordinal logistic regression model. 
Analysis population 
 
 

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8.1.2  Intent to Treat (ITT) population 
 
All patients randomized will be included in the ITT population. Patients will be assigned to 
treatment groups as randomized for analysis purposes. 
 
8.1.3 Safety population 
 
All patients who received at least one dose of study medication will be included in the safety 
analysis population. Patients will be assigned according to the true treatment received for 
analysis purposes. 
8.2 Data analysis 
 
8.2.1 Analysis of efficacy outcomes 
 
The primary analysis of efficacy will be based on the intent-to-treat principle. The primary 
endpoint will be compared between the two treatment groups using a chi-square test. Logistic 
regression models might also be used to compare the primary endpoint between the two treatment 
groups while accounting for important baseline characteristics. Secondary endpoints will be 
analyzed similarly. Exploratory analysis (biomarkers) will include logistic regression models with 
the primary/secondary endpoints as dependent variables and with treatment group, biomarker and 
treatment group x biomarker interaction as independent variables. 
All statistical tests will be two-sided and conducted at the 0.05 significance level, with the 
exception of the primary analysis that will be conducted at a slightly lower level to account for the 
interim analysis. Statistical analyses will be done using SAS version 9.4 or higher. 
 
8.2.2 Analysis of safety outcomes 
 
Safety of Fisetin will be evaluated by presenting descriptive statistics for various safety endpoints 
broken down by group. This will be done for the population of patients who received at least one 
dose of study medication (safety population). 
 
8.2.3 Interim analysis 
 
A fully independent 5-member Data and Safety Monitoring Board (DSMB) will be established 
and will review unblinded safety data as detailed in the DSMB charter. An interim analysis is 
planned after approximately 50% of randomized patients have completed 30 days of follow-up. 
The DSMB charter will pre-specify the methods of interim efficacy analyses and the rules for early 
study termination, approved by all board members. The stopping rules for efficacy will be based 
on the O’Brien-Fleming alpha-spending function or on a similar conservative approach so that the 
impact on the final alpha level will be negligible. Futility will be assessed by computing 
conditional power. The DSMB will have the option of recommending early study termination 
because of overwhelming efficacy, early termination for futility, or continuation of the trial as 
planned. 
The final analysis of the primary endpoint will be conducted at a significance level slightly below 

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the 0.05 level to account for the interim analysis. However, since this will have a negligible impact 
on power, the sample size calculation was calculated using a significance level of 0.05. 
 
9 
STUDY COORDINATION 
 
Mayo Clinic Rochester will be responsible for processing and quality control of the data. The 
handling of data, including data quality control, will comply with all applicable regulatory 
guidelines, and the study Data Management Plan. 

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LIST OF APPENDICES 
 
Appendix 
Title 
A 
Detailed Study Safety Parameters 
B 
Administrative Procedures for the Reporting of Adverse Events 
C 
Excluded Medications and Dosing Modifications 

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Appendix A 
Detailed Study Safety Parameters 
 
1. Demographics and Medical/Surgical History 
 
The following elements of demographics, medical/surgical history will be recorded at the 
Screening Visit: 
• age 
• ethnic origin 
• sex 
• smoking history 
• history of diabetes 
• history of hypertension 
• history of dyslipidemia 
• prior cardiac events/heart failure 
• prior obstructive pulmonary disease 
• prior other respiratory disease 
 
2. Physical Appearance 
1. weight 
2. height 
3. BMI ≥ 30 kg/m2 
 
4. Detailed Visit Summary 
Screening Visit (Days -2 to 0) 
Patients will be pre-screened and inclusion and exclusion criteria will be reviewed prior to being recruited for the 
study.   
• 
Screening Labs (within 6 months prior to randomization): CBC with diff and comprehensive metabolic 
panel with assessment of liver and kidney function tests 
• 
Pregnancy test (women of childbearing potential) 
 
Subjects who qualify should be immediately randomized. Randomization and dosing should occur on the same 
day if possible. 
 
Baseline (Day 0 & Day 1) – IP Administration 
 
• 
Mail in kit for buccal swab, urine and stool sample prior to first dose of IP administration, if applicable 
• 
SF 36 Health Survey 
• 
Determination of Ordinal Scale Score. 
• 
The 1st IP administration will occur on Day 0. 
• 
The 2nd IP administration will occur on Day 1 (12 -36 hours after the 1st IP administration).  
 
• 
Day 8 & Day 9 – IP Administration Phone call visit with the study team as a reminder for IP 
administration. This call can occur within a 2 day window (+ or -) of day 8; study drug administration will 
still occur on day 8 and day 9. 
• 
Determination of Ordinal Scale score. 
• 
Review of adverse events. 
• 
SF-36 Health Survey 

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• 
The 3rd IP administration will occur on Day 8.  
• 
The 4th IP administration will occur on Day 9 (12- 36 hours after the 3rd IP administration). 
 
Day 15 – (±2 days) 
• 
Determination of Ordinal Scale score. 
• 
Review of adverse events. 
• 
SF-36 Health Survey 
• 
Mail in kit for buccal swab, urine and stool sample, if feasible 
 
Day 30 (-2 to +7 days) 
• 
Determination of Ordinal Scale score. 
• 
Review of adverse events. 
• 
SF-36 Health Survey 
• 
Mail in kit for buccal swab, urine and stool sample, if feasible 
 
Day 60 (±14 days) 
• 
Determination of Ordinal Scale score. 
• 
Review of adverse events. 
• 
SF-36 Health Survey 
• 
Mail in kit for buccal swab, urine and stool sample, if feasible 
 
 
Appendix B 
Administrative Procedures for the Reporting of Adverse Events 
 
The following administrative procedures for reporting AEs are to be followed during the 
conduct of this clinical trial. 
 
1 
ADVERSE EVENTS DURING THE TRIAL 
 
Each patient will be observed and queried in a non-specific manner by the investigator or 
study coordinator at each visit for any new or continuing AE since the previous visit. Any 
AEs prior to the first dose of study medication will be recorded in the medical history and 
kept in the patients’ chart. All SAEs will be recorded in the appropriate eCRF section. In 
addition to SAEs, the only AEs to be recorded in the eCRF are those that are either related to 
the gastrointestinal system, that are judged related to the study medication by the investigator 
or that are laboratory abnormalities judged clinically significant by the investigator. 
Information collected will include the onset, duration, severity, relationship to study drug, 
and the management. SAEs are to be reported if they are known to occur within 30 days 
following the last study visit. 
 
The investigator will review the clinical laboratory test results in a timely fashion when 
received from the laboratory. Those results qualifying as AEs as defined in this appendix will 
be recorded on the AE eCRF section and will be handled according to these AE reporting 
procedures. 
 
The investigator will review concomitant medications being taken by the patient. 
 
Definitions 

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1.1 Pre-existing condition 
 
A pre-existing condition is one that is present prior to randomization. A worsening of a pre- 
existing condition after taking the first dose of investigational product should be reported as 
an AE. 
 
1.2 Adverse Event (AE) 
 
An AE is defined as any unfavorable and unintended sign (including a clinically meaningful 
abnormal laboratory finding), symptom, or disease temporally associated with the use of an 
investigational product, whether or not related to the investigational product. 
 
A medical procedure is not considered and should not be reported as an AE. However, the 
medical condition which led to the procedure should be considered as an AE and be reported 
as such. 
 
1.3 Related Adverse Event 
 
A related AE is one where, according to the Investigator, there is a reasonable possibility that 
the event may have been caused by the study drug. 
 
1.4 Serious Adverse Event (SAE) 
 
Serious Adverse Events (SAE) are those that meet any of the following International Council 
for Harmonisation (ICH) criteria: 
 
• Is fatal or immediately life-threatening (NOTE: the term "Life-Threatening" 
refers to an event in which the patient was at immediate risk of death at the 
time of the event; it does not refer to an event which could hypothetically have 
caused death had it been more severe); 
• Results in persistent or significant disability/incapacity; 
• Requires or prolongs patient hospitalization; 
• Is a congenital anomaly/birth defect in the offspring of the patient; 
• Is a cancer; 
• Is an overdose (intentional or accidental); 
• Is judged to be medically important. 
 
Medically important events may not be immediately life-threatening, result in death or 
hospitalization, but may jeopardize the patient or may require intervention to prevent one of 
the outcomes listed in the definition above. Serious adverse events are to be reported if they 
are known to occur within 30 days following the last study visit. 
 
Medical and scientific judgment should be exercised in deciding whether other AEs meet 
these criteria and are immediately reportable to the sponsor or its designee. 
 

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Hospitalization is defined as a patient admission to a hospital for medical treatment or 
observation; a visit to the emergency room for an outpatient consultation is not considered a 
hospitalization. Moreover, the following hospitalizations are not considered SAEs: 
 
• Hospitalizations for diagnostic or elective surgical procedures for a pre-
existing condition. 
• Hospitalization for therapy of the target disease(s) of the study if the 
protocol explicitly anticipated and defined the symptoms or episodes. 
• Hospitalization for study efficacy measurement, as defined in the protocol. 
 
1.5 Life-Threatening Adverse Event 
 
A life-threatening AE is an AE that, in the opinion of the investigator, places the patient at 
immediate risk of death from the reaction as it occurred. 
 
1.6 Unexpected Adverse Event 
 
An unexpected AE is any AE that is not consistent with findings previously observed. 
 
1.7 Clinical Laboratory Adverse Event 
 
A clinical laboratory abnormality is regarded as an AE if it has been confirmed by at least 1 
repeat test and suggests a disease and/or organ toxicity severe enough to require active 
management. 
 
1.8 Treatment-Emergent Signs and Symptoms (TESS) 
 
A TESS event is any AE that was not present prior to randomization or that worsens in 
character, intensity or frequency while the patient is in an active treatment period. 
 
1.9 Post-treatment Adverse Event 
 
A post-treatment AE is any AE that occurs after treatment is discontinued. 
 
2 
HANDLING OF ADVERSE EVENTS 
 
2.1 Treatment-Emergent Signs and Symptoms 
 
Any condition/diagnosis that meets the definition of a TESS event is captured in the 
appropriate eCRF AE form and kept in the patient’s chart. 
 
2.2 Serious Adverse Events 
 
All SAEs are to be immediately reported. Serious Adverse Events, within 24 hours of the 
Investigator’s first knowledge of the event. 
 

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2.3 Intensity 
 
The following criteria are used to assess the intensity of each AE: 
 
• Mild: The patient is aware of the sign or symptom, but finds it easily tolerated. 
• Moderate: The patient has enough discomfort to cause interference with or 
change in usual activities. 
• Severe: The patient is incapacitated and unable to work or participate in 
many or all usual activities. 
 
2.4 Relationship to Study Drug – Physician’s Assessment 
 
There are 3 categories for the physician’s assessment of the causal relationship between study 
drug and an AE as follows: not related, possibly and probably. 
 
2.5 Clinical Outcome 
 
The following categories are used to assess the clinical outcome of each AE: 
 
• Death related to the adverse event 
• Recovered – The patient has fully recovered from the AE without 
observable residual effects. 
• Recovered with sequelae – The patient has recovered from the 
AE with observable residual effects. 
• Not Recovered – The patient is still being treated for the residual effects of 
the original AE. This does not include treatment for pre-existing conditions 
including the indication for the study drug. 
• Recovering 
• Unknown 
 
3 
CAPTURING AVDERSE EVENTS 
 
3.1 Pre-Existing Condition 
 
A pre-existing condition should be captured in the medical history and kept in the patient’s 
chart. If the frequency, intensity, or character of the condition worsens during study 
treatment, and is either related to the gastrointestinal system, is judged related to the study 
medication by the investigator or is a laboratory abnormality judged clinically significant by 
the investigator, it must be documented in the appropriate eCRF AE form. 
 
3.2 Lack of Efficacy of Study Medication 
 
Signs or symptoms defined in the protocol as lack of efficacy or collected as efficacy 
parameters (endpoints) will be captured as AEs. 
 
3.3 Clinical Laboratory Adverse Event 

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A clinical laboratory abnormality should be reported as an AE only if it is considered to be 
clinically significant by the investigator and confirmed by repeat testing. 
 
3.4 Hospitalization or Surgery/Procedure 
 
Any AE reported as study endpoint should not be reported as an SAE. Any 
condition/diagnosis responsible for surgery/procedure should be reported as an AE if it meets 
the criteria for an AE. A medical procedure is not considered and should not be reported as 
an AE. The surgery/procedure itself will be reported as a Clinical Outcome of the underlying 
event. Events that prolong any hospitalization are reported as SAEs. 
 
3.5 Death 
 
The cause of death should be reported as an AE. 
 
 
3.6 Other Adverse Events 
 
Study endpoints are to be reported in the eCRF within 24 hours of awareness of the event. 
 
In addition to SAEs and study endpoints, the only AEs to be recorded on the eCRF are those 
that are either related to the gastrointestinal system, that are judged related to the study 
medication by the investigator or that are laboratory abnormalities judged clinically 
significant by the investigator. Information collected will include the onset, duration, 
severity, relationship to study drug, and the management. 
 
3.7 Follow-Up Period 
 
For SAEs, the patient must remain under observation until the SAE has subsided or stabilized 
and all serious findings have returned to normal or stabilized. Any follow-up information to 
an initial SAE report must be updated in the eCRF. Serious Adverse Events are to be reported 
if they are known to occur within 30 days following the last study visit. 
 
 
 
 
 
 
 
 

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Appendix C. Excluded Medications and Dosing Modifications 
Subjects taking any of the medications listed in Appendix C may participate if they are otherwise 
eligible AND the medication can be safely held during the following times: 
• 
Immediately before the 1st IP administration (Day 0) until at least 10 hours after the 2nd IP 
administration (Day 1) 
• 
Immediately before the 3rd IP administration (Day 8) until at least 10 hours after the 4th IP 
administration (Day 9) 
 
Cardiac:  
• 
Digoxin 
• 
Flecainide 
• 
Amiodarone  
Psychiatric: 
• 
Duloxetine (Cymbalta)  
• 
Lithium  
• 
Clozapine (Clozaril)  
• 
Thioridazine (Mellaril or Melleril) 
Neurologic: 
• 
Carbamazepine (Tegretol)  
• 
Phenobarbital (Luminal and Solfoton)  
• 
Phenytoin (Dilantin, Phenytoin Sodium, Phenytek)  
• 
Fosphenytoin (Cerebyx) 
• 
Riluzole (Rilutek; Used to treat amyotrophic lateral sclerosis ALS) 
Antimicrobial/fungal: 
• 
Aminoglycosides (Eg. amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, 
tobramycin)  
• 
Azole antifungals (fluconazole, miconazole, voriconazole) – (if absolutely necessary, levels must be 
subtherapeutic or therapeutic) 
• 
Macrolides (clarithromycin) 
• 
Antivirals (nelfinavir, indinavir, saquinavir)  
Oral hypoglycemic/Anti-Diabetes drugs: 
• 
Glimepiride (Amaryl)  
• 
Glyburide (DiaBeta, Glynase PresTab, Micronase)  
Anticoagulants/ Antiplatelet: 
• 
Warfarin  
• 
Full dose aspirin  
Others: 
• 
Cyclosporine  
• 
Methotrexate  
• 
Nitroglycerin  
• 
St. John’s Wort 
• 
Theophylline 
• 
Tyrosine Kinase Inhibitors (list included further down in this appendix) 
• 
Tizanidine (Zanaflex)  
• 
Tacrine 

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• 
Diclofenac 
 

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Continuation of Excluded Medications 
Sensitive Substrates CYP2C9, CYP2C19, CYP1A2, Other (OATP1B1) 
 
 
Sensitive substrate 
 
Drugs with a narrow therapeutic 
range  
CYP2C9 
 
Glyburide 
Losartan  
 
Fosphenytoin  
Phenytoin  
Warfarin  
Glimepiride 
Diclofenac 
Bosentan  
CYP2C19 
 
Phenytoin  
CYP 1A2 
 
Clozapine  
Olanzapine 
 
Theophylline  
Tizanidine 
Warfarin 
Ramelteon 
Tacrine 
Duloxetine 
Mexiletine 
Riluzole 
Other 
(OATP1B1) 
Corticosteroids 
Thyroid hormones 
Eluxadoline 
Eltrombopag 
Pioglitazone (Actos) - monitor blood glucose levels closely or 
½ dose QD  
Enzalutamide 
Colchicine (½ dose QD until 10 hours after the last (2nd) IP 
administration 
Venlafaxine (skip dose on study drug Day 2; then resume as 
usual) 
Atorvastatin (QOD or ½ dose QD until 10 hours after the last 
(2nd) IP administration; unless willing to modify as directed.) 
Rosuvastatin (QOD or ½ dose QD until 10 hours after the last 
(2nd) IP administration unless willing to modify as directed.) 
Desipramine (QOD or hold until 10 hours after the last (2nd) IP 
administration) 
Atomoxetine (hold) 
Repaglidine- monitor blood glucose level closely (CYP2C8) 
 
 
Tyrosine Kinase Inhibitor List (CYP2C8)  
afatinib 
axitinib 
Bosulif 
bosutinib 
cabozantinib 
Caprelsa 
Cometriq 
crizotinib 
dasatinib 
erlotinib 
Gilotrif 
Gleevec 
ibrutinib 
Iclusig 
imatinib 
Imbruvica 
Inlyta 
lapatinib 
Nexavar 
nilotinib 
pazopanib 
ponatinib 
regorafenib 
sorafenib 
Sprycel 
Stivarga 
sunitinib 
Sutent 
Tarceva 
Tasigna 
Tivopath 
tivozanib 
Tykerb 
vandetanib 
Votrient 
Xalkori 
Zaltrap 
ziv-
aflibercept