Phase II Trial of Effects of the Nutritional Supplement Sulforaphane on Doxorubicin-Associated Cardiac Dysfunction (CRI18-026)
Phase: PHASE1
Conditions: Anthracycline Related Cardiotoxicity in Breast Cancer
Sponsor: Texas Tech University Health Sciences Center

 1.9.2019 
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Phase II trial of effects of the Nutritional Supplement Sulforaphane on 
Doxorubicin-Associated Cardiac Dysfunction  
 
 
 
 
 

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Specific Aim 
Determine whether nutritional supplement sulforaphane (SFN) is safe to administer to 
breast cancer patients undergoing doxorubicin (DOX) chemotherapy.  We have identified 
biomarkers for presymptomatic detection of DOX cardiotoxicity in breast cancer patients and reported 
that SFN alleviates DOX-induced cardiac toxicity while maintaining its anti-tumor activity in an animal 
model of breast cancer.  SFN is in various stages of preclinical and clinical trials against different types 
of cancer but its safety and effects on identified markers have never been tested in patients treated with 
DOX.  Sulforaphane is a generally recognized as a safe (GRAS) compound and this compound is 
currently in 61 different clinical trials including, Cystic Fibrosis, COPD, Melanoma, Breast and prostate 
cancer 
etc. 
(https://clinicaltrials.gov/ct2/results?term=sulforaphane&pg=1). 
Avmacol, 
(Nutramax 
Laboratories Consumer Care, Inc. Edgewood, MD 21040) an over-the-counter dietary supplement 
containing broccoli seed and sprout extracts that is rich with sulforaphane, will be used in this study. This 
is an FDA regulated trial using an investigational drug under IND 141682. Dose considerations were 
made on the basis of the ongoing clinical trial “Effects of Avmacol® in the Oral Mucosa of Patients 
Following 
Curative 
Treatment 
for 
Tobacco-related 
Head 
and 
Neck 
Cancer” 
(https://clinicaltrials.gov/ct2/show/NCT03268993).  Furthermore, Avmacol has never been tested as an 
adjuvant to try and protect the heart from DOXs harmful effects. Therefore, we are proposing to do an 
early-clinical trial to assess SFN safety in DOX-treated patients and possibly prevent DOX-cardiotoxicity 
in breast cancer patients. 
Our hypothesis is that SFN protects the heart from DOX-mediated cardiac injury without affecting its 
antitumor efficacy. Here, we will test SFN in combination with DOX-based chemotherapy for breast 
cancer; we will assess its safety, cardio-protective properties, and anti-cancer efficacy. We will also 
examine the effect of SFN on the expression of several biomarkers that indicate when a patient cannot 
tolerate DOX.1 To test our hypothesis, we propose the following Specific Aims. 
Aim 1: Demonstrate that co-administration of SFN protects the heart and is not associated 
with toxicity or reduced tumor response in breast cancer patients undergoing DOX 
chemotherapy.  
We propose a pilot clinical trial to assess the safety of SFN as a co-treatment with DOX chemotherapy. 
Strategy: Up to seventy breast cancer patients prescribed DOX chemotherapy will be consented then 
randomly assigned to receive either SFN or placebo during DOX chemotherapy. This sample size 
includes an additional 10 participants to allow for potential attrition. There will be a 50-50 chance of 
receiving SFN.  The patients will be recruited from the Breast Cancer Clinic (Dr. Awasthi) in the Southwest 
Cancer Center at TTUHSC/UMC Lubbock. Cardiac function with echocardiography will be assessed and 
compared between arms for evidence of substantive decrease in DOX cardiotoxicity with SFN compared 
to placebo. Tumor size will also be compared (based on RECIST criteria) between treatment arms. We 
will monitor treatment-emergent symptoms, hematological parameters of cancer therapy toxicity, and 
renal and hepatic function. Blood samples will be collected to assay markers of cardiotoxicity, including 
B-type natriuretic peptide and troponin.  
Aim 2: Determine if SFN alters the levels of known biomarkers of DOX cardiotoxicity and 
affects the expression of SIRT1- and Nrf2-target genes.  
We will measure changes in transcript and protein biomarkers of pre-symptomatic DOX-cardiotoxicity in 
response to SFN, and will determine if SFN promotes SIRT1- and Nrf2-dependent gene expression. 

 1.9.2019 
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Because Nrf2 upregulates certain detoxifying enzymes, we will measure the plasma levels of 
cardiotoxic metabolites, doxorubicinol (DOXol). Strategy: Blood will be collected before the start of DOX 
chemotherapy and after each treatment cycle to isolate peripheral blood mononuclear cells (PBMCs) and 
prepare plasma. In lieu of tissue biopsies, PBMCs will be used to examine SIRT1 and Nrf2 activity and 
target gene expression. Multiplex arrays will be used to measure plasma cytokine levels, and plasma 
DOX metabolites will be assessed with ultrahigh performance liquid chromatography-tandem mass 
spectrometry. These experiments will provide initial evidence to indicate that SFN activates SIRT1 and 
Nrf2 pathways in non-cancer tissue of breast cancer patients. 
Impact: This will be the first clinical study to assess the safety of SFN as a co-treatment with DOX to 
mitigate cardiotoxicity in breast cancer patients. Positive findings will be used to justify a larger 
randomized controlled trial. Subjects will be randomized at a 1:1 ratio. 
 
Background 
 DOX toxicity and its impact on human health.  Cardiotoxic side-effects are among the main 
complications of doxorubicin chemotherapy and this limits its use in high-dose cancer treatment, and 
strongly impacts the quality of life and survival of cancer patients.2-12  The occurrence of cardiac events 
in cancer patients associated with DOX-therapy is estimated between 10% and 25%.13,14  The prevalence 
of left ventricular contractile dysfunction in patients with a cumulative DOX dose of approximately 430–
600 mg/m2 is about 50–60%, in which a significant incidence of cardiomyopathic episodes is 
observed.13,14  DOX-induced cardiotoxicity can develop immediately, within months, or even years after 
completion of therapy.14  Dexrazoxane is the only USFDA approved agent for reducing DOX-
cardiomyopathy in women with metastatic breast cancer, but it has its own side effects.15,16 Clearly, 
devising a strategy to prevent DOX-induced cardiotoxicity would have tremendous benefit in terms of 
limiting medical complications and improving lives of cancer patients. 
DOX cardiotoxicity and anti-cancer activity.  The mechanism of anti-cancer action of DOX seems 
mostly distinct from the mechanisms responsible for its cardiotoxicity.  DOX anti-cancer activity is based 
on producing DNA damage and inhibiting cell replication.17-22 These effects are almost nonexistent in 
non-replicating cardiomyocytes.  Instead, the primary cause of cardiotoxicity is DOX-induced oxidative 
stress, resulting in part from its inhibition of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression 
(also supported by our new findings).23  DOX exposure causes initial mitochondrial injury, which 
compromises cellular bioenergetic capacity, and initiates a delayed progression of cardiac damage.24,25  
Since the mechanisms of DOX-mediated cardiotoxicity and anti-cancer activity are different, it is likely 
that cardiac and cancer cells employ different protective mechanisms.  Therefore, elucidations of such 
mechanisms will not only help in devising novel strategies to protect the heart from the harmful effects of 
DOX, but also help to combat cancer more effectively.  
Sulforaphane (SFN) is protective to normal cells, but toxic to cancer cells.  SFN, a safe 
phytochemical, protects and enhances Nrf2 signaling and consequent cytoprotective gene activation to 
counter DOX cardiotoxicity in animal models.23,26-33  SFN also exhibits anti-cancer activities related to 
enhancing the transcription of tumor suppressor genes, possibly by influencing DNA methylation, 
activation of caspase-3, induction of cell cycle arrest, reactivation of estrogen receptor alpha (ERα), and 
by inhibition of DNA methyltransferase (DNMT) and telomerase reverse transcriptase (TERT).34-42  As 
well, SFN may possibly influence DOX metabolism in cancer cells to potentiate its anti-tumor activity.  
While SFN is in various stages of testing in clinical trials of different types of cancer 

 1.9.2019 
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(https://clinicaltrials.gov/ct2/results?term=sulforaphane&pg=1), 26,42,43 it has not yet been exploited as an 
adjuvant with doxorubicin in breast cancer treatment regimens. 
Overall Rationale  
Metastasis and DOX-induced cardiotoxicity are the most common causes of mortality for DOX-treated 
cancer patients.44 SFN is a nontoxic compound isolated from cruciferous vegetables that exhibits 
anticancer, antidiabetic, and antimicrobial activity in many animal models of disease.26,42,43,45 Our 
preliminary data suggest that SFN not only protects the heart from DOX toxicity but enhances the 
effectiveness of DOX against breast tumors in vivo.  
 SFN is in various stages of preclinical and clinical trials against different types of cancer, including 
prostate cancer.46 However, its safety has never been tested in patients treated with DOX, and it has 
never been tested for a cardioprotective effect in combination 
with DOX. Therefore, we propose a pilot clinical trial to 
assess the safety and efficacy of SFN in DOX-treated breast 
cancer patients. In this trial, we will assess cardiac function 
as an indicator of whether SFN prevents DOX-induced 
cardiotoxicity.  
 
 
 
Preliminary Studies 
SFN prevents DOX-induced cardiomyopathy in tumor-
bearing rats (Accepted manuscript in PLOS one). Because 
DOX is used to treat breast cancer in humans, we examined 
the cardioprotective action of SFN during DOX chemotherapy 
in a rat model of breast cancer. MAT B III breast cancer cells 
were injected into a mammary gland of female Sprague 
Dawley rats (6-8 weeks old), and the animals were treated 
when tumors reached 5-6 mm3 in diameter.47 SFN (4 mg/kg 
in 0.1 ml saline) or vehicle was administered orally 5 
days/week for 5 weeks. After 1 week of SFN (or vehicle) 
treatment, the rats were injected intraperitoneally with DOX 
(5 mg/kg) or vehicle once per week for the remaining 3 weeks 
(total of 20 mg/kg, similar to a dose of 120 mg/m2 for 
humans). Two days after completing treatment, cardiac 
function was assayed, and tissues were collected for further 
analysis. DOX treatment dramatically reduced cardiac 
contractility, as measured by fractional shortening (FS%), left 
ventricular ejection fraction (LVEF%), cardiac output (CO), 
and stroke volume (SV). The cardiotoxic effects of DOX were 
significantly attenuated with SFN co-treatment (Fig. 1). 
Figure 1. SFN+DOX co-treatment of tumor-bearing 
rats preserves cardiac function. MAT B III breast 
cancer cells were injected into a mammary gland of 
adult rats (n = 8). SFN (4 mg/kg in 0.1 ml saline) or 
vehicle was administered orally 5 days/week for 5 
weeks. After 1 week, rats were injected i.p. with DOX 
(5 mg/kg or vehicle once per week for the remaining 3 
weeks. Cardiac function was assessed with high-
resolution echocardiography (Vevo 700) before tumor 
inoculation and treatment and after the final treatment. 
DOX
SFN+DOX
0
25
50
75
100
Ejection Fraction
 Before tumor inoculation & treatment
 After  treatment
*p<0.05 vs   Before tumor inoculation & treatment
**p<0.05 vs DOX after treatment
DOX
SFN+DOX
0
10
20
30
40
50
60
**
*
Fractional Shortening
**
*
DOX
SFN+DOX
30
40
50
60
Cardiac output
**
*
DOX
SFN+DOX
0
30
60
90
120
150
Stroke Volume
**
*

 1.9.2019 
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SFN enhances the ability of DOX to 
regress breast tumors. To determine 
if SFN affected the anticancer 
activity of DOX, we treated tumor-
bearing rats with vehicle, SFN, DOX, 
or SFN+DOX (as just described) and 
monitored tumor growth. SFN alone 
had no significant effects on tumor 
growth at the doses intended in the 
study (albeit it does have mild 
antitumor activity at higher doses), 
and DOX alone reduced tumor 
growth 
(Fig. 
2A). 
However, 
SFN+DOX eradicated the tumors 
from all rats by day 35 after tumor 
implantation (Fig. 2A). Thus, SFN 
co-treatment does not impair the 
ability of DOX to kill cancer cells, and 
SFN can be used safely with DOX to 
attenuate cardiotoxicity. We also found that the DOX dose can be lowered by 50% when combined with 
SFN to eradicate tumors (Fig. 2B). Thus, SFN+DOX co-treatment reduces DOX cardiotoxicity and 
decreases tumor volume in rats. 
 
SFN improves mitochondrial function during DOX treatment. In cardiac cells, DOX is converted to a more 
reactive 
semiquinone 
by 
mitochondrial complex I of the 
electron 
transport 
chain 
(ETC), increasing oxidative 
stress.48-51 We reasoned that 
these effects on the ETC 
decrease the level of ATP in 
cardiomyocytes. 
However, 
SFN is known to increase 
respiration 
linked 
to 
ATP 
synthesis.52-55 To determine if 
SFN stabilizes ATP levels in 
the cardiomyocytes of DOX-
treated rats, we examined 
ATP levels in treated and 
control animals. We found that 
SFN maintains ATP levels 
during DOX treatment (Fig. 
3A).  
 
Figure 2.  SFN co-treatment lowers the effective DOX dosage in a rat breast 
cancer model. Effect of metronomic SFN (4 mg/kg, 5 days/week), DOX (A, 5 mg/kg 
weekly or B, 2.5 mg/kg weekly, total 10 or 20 mg/kg), or SFN+DOX on MAT B III-
induced mammary tumors. Data represent the mean tumor volume ± SD. Red arrows 
indicate DOX injections. (*p = 0.002 [SFN+DOX vs. DOX], **p = 0.003 [Vehicle vs. DOX]; 
n = 8). 
  
  
Figure 3.  SFN increases ATP content in the rat heart during DOX treatment. A) 
ATP content was evaluated in fresh heart biopsies. B) SFN improves ETC function 
during DOX treatment. According to oxygen flux measures, co-treatment of rats with 
SFN+DOX improved complex I, II+III, and IV respiration compared to rats treated only 
with DOX. Each bar represents the mean ± SD (n = 8); statistical significance 
determined with a t-test.  
0
7
14
21
28
35
0
5
10
15
20
25
30
35
40
0
7
14
21
28
35
0
5
10
15
20
25
30
35
40
B
**
**
*
*
Tumor Volume (mm3)
Days
 Vehicle
 DOX (5mg/kg; total 20mg/kg)
 SFN+DOX
 SFN
A
*
Tumor Volume (mm3)
Days
 Vehicle
 DOX (2.5mg/kg; total 10mg/kg)
 SFN+DOX
 SFN
*

 1.9.2019 
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Pharmacological activation of Nrf2-target gene expression by SFN should remove excess ROS produced 
during DOX therapy by elevating phase 2 detoxification and antioxidant enzyme activities and preserving 
ETC complex function. To evaluate this, ETC activity was measured in left ventricular biopsies of SFN- 
and/or DOX-treated rats with high-resolution respirometry using the OROBOROS Oxygraph-2k 
(Oroboros Instruments).56 SFN clearly protected the ETC from damage by DOX (Fig. 3B), as indicated 
by an increase in oxygen flux. We confirmed that these protective effects were mediated by Nrf2-
dependent signaling by comparing the enzymatic 
activities of SOD and catalase, two Nrf2 target gene 
products (data not shown).  
 SFN upregulates key Nrf2-target antioxidant genes in the 
PBMCs of DOX-treated tumor-bearing rats. SFN 
preserves nuclear Nrf2 activity in DOX-treated rat 
cardiomyocytes but not in DOX-treated breast tumor 
tissue (data not shown), and this promotes the 
upregulation 
of 
antioxidative 
and 
antielectrophile 
defenses in heart cells. Notably, we demonstrated that the 
gene-expression 
profiles 
of 
cardiomyocytes 
and 
peripheral blood mononuclear cells (PBMCs) are very 
similar but differ from the expression profile of breast 
tumors,22 and this supports our approach to use PBMCs 
as a surrogate tissue for assessing biomarkers of DOX 
cardiotoxicity (Fig. 4).  
Specifically, 
we 
compared 
the 
transcript 
levels 
of 
key 
Nrf2-target 
mitochondrial 
antioxidant/antielectrophile genes between tumor cells, cardiomyocytes, and PBMCs of DOX- and 
SFN+DOX-treated tumor-bearing rats. In the heart and PBMCs, there were significant increases in the 
expression of key antioxidant and antielectrophilic genes (Fig. 4). However, transcript levels of these 
Nrf2-dependent enzymes were unchanged in the SFN-treated tumor tissue. These data suggest that 
protective Nrf2 signaling is activated in the cardiomyocytes and PBMCs of DOX-treated animals, not in 
tumor cells.  
We conclude that SFN does not induce protective mechanisms in tumor cells, and we predict that it can 
be used safely as an adjuvant with DOX.   
Above animal studies, by our University of Arkansas for Medical Sciences (UAMS) group has provided 
strong evidence that DOX-toxicity correlates with impairment of antioxidant defenses caused by a defect 
in functions of Nrf2.23,57  Nrf2 is a regulatory protein that coordinates the body’s defense mechanisms 
against oxidative stress and its resultant damage.  SFN, an Nrf2-related activator of cytoprotective genes, 
provides protection in several disease states26,30,31,33 and is in various stages of clinical trials.26,43,58-64  
SFN is a metabolic product of glucoraphanin, a glucosinolate found in broccoli and cauliflower, is 
commercially available as a dietary supplement, and has an excellent safety profile.  We recently reported 
that elevated Nrf2 activity drives cellular defenses to provide protection against DOX-induced 
cardiomyopathy.57  In addition, our pilot studies in cardiac cells reveal that SFN modifies PGAM5, initiating 
nuclear translocation of Nrf2 to activate protective gene expression and confer resistance to DOX.  Also, 
we discovered that SFN therapy protected rats from DOX-induced cardiomyopathy. In an additional study 
in rats, SFN enhanced the chemotherapeutic effects of DOX on implanted breast cancer cells. 
 
Figure 
4. 
Differential 
expression of antioxidant 
and 
antielectrophilic 
genes in SFN+DOX and 
DOX-treated 
tumor-
bearing rats. Transcript 
levels were measured with 
qRT-PCR on 6 animals per 
group and were normalized 
to Rsp3 for each animal. 
The bars represent ratios (± 
SD of the ratio) of mean 
transcript 
levels 
of 
statistically 
significant 
genes. p < .05; values were 
calculated with two-factor 
ANOVA on the basis of two 
independent experiments. 
0
1
2
3
4
5
6
*
*
*
*
*
*
PBMC
0
1
2
3
4
5
6
*
*
*
*
*
Heart
Aldh2
Akr1
Cat
Sod1
Sod2
Gpx1
Gpx4
0
1
2
3
4
5
6
Transcript level (ratio SFN+DOX treated/DOX treated)
Tumor

 1.9.2019 
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These preliminary studies are exciting and could have immense translational impact in terms of use of 
SFN to lessen DOX-toxicity; however, it has never been tested in patients receiving DOX, and it has not 
yet been exploited as an adjuvant in cancer treatment regimens. Therefore, we propose to use SFN in 
breast cancer individuals undergoing DOX-therapy to see if it will alleviate DOX-associated oxidative 
damage and improve cardiac function. Our goal is to aid in the development of a safe strategy to reduce 
cardiac side effects of DOX. 
Study Design and Procedures 
Study Design.  We intend to include up to 70 DOX-naïve women diagnosed with breast cancer 
undergoing neoadjuvant chemotherapy with no prior cardiac disease and who will receive DOX without 
Her-2 receptor antagonists (to eliminate possibility of secondary side effects) as part of their clinical care.  
These potential subjects will be recruited in a randomized, controlled, double blinded pilot study 
comparing SFN to placebo.  The study will be conducted at the Texas Tech University Health Sciences 
Center, and University Medical Center.  The trial will be approved by our local IRB and registered at 
clinicaltrials.gov. UMC cancer center pharmacist Ajoke A. Tijani, RPh. (Tijani@umchealthsystem.com) 
will receive placebo and test compound from supplier and will dispense them to test subjects.  
Randomization will be achieved with assignment of a subject ID to the study subject. The list of subject 
IDs will be subsequently assigned either study drug or placebo using a randomizer software such as 
(www.randomizer.org). The pharmacist dispensing the medication will be aware of the study IDs in 
relation to the patient identity, in order to be able to dispense the test drug or the placebo per their 
assigned status as test or control subjects.  
Study Drug and Placebo. Processed SFN-rich extract will be purchased in form of caplets from 
Nutramax Laboratories, Inc. 2208 Lakeside Blvd Edgewood, MD 21040. Caplets containing SFN-rich 
broccoli sprout extracts or microcrystalline cellulose (placebo) also from Nutramax Labs will be dispensed 
to participants in sealed bottles with instructions to keep them in a household freezer.  Size of the caplet 
will be about the size of a 1000 mg Vit C pill (about 2 cm in length). The participants will be dosed, based 
on weight, in a double-blind fashion with identical appearing placebo or SFN caplets in a daily dose for 
12 weeks of: two caplets for individuals <100 lb., four caplets for individuals 100–200 lb. and eight caplets 
for individuals >200 lb.  Avmacol or placebo will be prescribed by Dr. Awasthi and will be dispensed by 
local pharmacy or study coordinators at TTUHSC/UMC Lubbock. We will be doing pill counts to make 
sure that volunteers have used as directed. We will measure the Sulforaphane level in plasma by 
well-established method. 26 
The drug and the placebo will be stored at the South West Cancer Center pharmacy based on 
manufacturer based guidelines and dispensed to each participant at their baseline visit then at DOX 
infusion visits 1, 2, and 3. Patients will be expected to maintain at least an 80% adherence to the 
medication regimen, in the absence of prohibitive toxicity. Adherence will be monitored through 
therapeutic drug level (Plasma sulforaphane levels of 120 ng/ml) monitoring as mentioned above 
and pill counts.  For subjects who do not meet 80% compliance, they will be instructed on the 
importance of taking the pills as directed, but if their next visit demonstrates less than 80% 
compliance, they will be withdrawn from the study.  As long as participants complete their 4th cycle 
of DOX chemotherapy treatment we will still use their study data during analysis.  If side effects are 
noted, patient will be asked to notify the study team of the same and will be evaluated within a 
suitable time frame based on severity of side effects. 

 1.9.2019 
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SFN is a safe natural isolate. Above doses  (30mg/caplet) are considered adequate to maintain intended 
therapeutic drug levels, while maintaining a simple study design and without significant concerns for drug 
toxicity It has been used in several clinical trials from doses ranging from 2-200 µmol/day for 2-28 weeks 
in 
25-80 
years 
old 
subjects 
without 
significant 
side-effects 
or 
toxicity 
(https://clinicaltrials.gov/ct2/results?term=sulforaphane&Search=Search), yet never in combination with 
DOX. 
Statistical Considerations 
Data for the primary endpoint (DOX cardiotoxicity) and other binary outcomes will be summarized by 
treatment arm as number and proportion per arm. The change in DOX-cardiotoxicity rate with SFN 
compared to placebo will be assessed with a 1-sided Z-test (see Sample-size justification). Tumor size, 
plasma cardiac biomarkers, cardiac ejection fraction, DOX metabolite levels will be summarized by 
treatment arm and time point as means and standard deviations and graphed as box plots and profile 
plots. We will adhere to ANOVA distributional assumptions so that appropriate data transformations can 
be applied. Data for each continuous variable will be analyzed for differences in group means at each 
time point with ANOVA or Mann-Whitney-Wilcoxon non-parametric tests at each time point. These tests 
will employ a more-stringent alpha = 0.02 significance level to adjust for the multiple comparisons without 
overinflating type II error. For subjects withdrawn from the study or do not complete the study for any 
reason, as long as they completed their 4th cycle of DOX chemotherapy treatment we will still use 
their study data during analysis. 
Sample-size justification. The number of subjects (35 per treatment arm; 70 total; we expect some 
attrition, which this sample size addresses) is based on the primary endpoint of DOX-induced cardiac 
dysfunction, the rate of which was recently observed at our previous institution (UAMS) to be ~25%1. We 
expect that SFN will decrease this to 5%. The cardiotoxicity rates for each arm will be compared via 1-
sided pooled-variance Z-test at an alpha = 0.10. The statistical power of this test, conducted as described, 
needs to exceed 80% in order for our study to be generally recognized as having adequate statistical 
power. Our sample size provides the 1-sided pooled-variance Z-test with 83.2% power at 10% alpha to 
detect the expected 20-point decrease in DOX-toxicity rates from 25% in the placebo arm to 5% in the 
SFN arm. 
The formal consent of each subject, using the IRB-approved consent form, will be obtained before that 
subject is submitted to any study procedure.  The study schedule is detailed in Table 1. 
 
 
 
 
 
 
 
 
 
 

 1.9.2019 
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Table 1:  Study Schedule 
Procedures 
 
Screening Baseline  
First 
DOX 
infusion 
Second 
DOX 
infusion 
Third 
Dox 
infusion 
Fourth 
Dox 
Infusion 
1 week 
S/P 4th  
Dox 
infusion 
1 year 
follow-
up 
 
 
Day 0        
(within 3 
weeks +/- 3 
days from 
screening) 
1st week 
+/- 3 
days 
Week 
3rd-4th 
+/- 3 
days 
Week 
5th-6th 
+/- 3 
days 
Week 
7th-8th 
+/- 3 
days 
Week 
8th-9th 
+/- 3 
days 
Week 
60-62 
+/- 3 
weeks. 
ICF 
X 
 
 
 
 
 
 
 
HX review 
X 
 
 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
 
X 
ECHO 
X 
 
 
 
 
 
X 
X 
PET CT 
X 
 
 
 
 
 
X 
 
Urine 
pregnancy test 
X 
 
 
 
 
 
 
 
I&E review 
 
X 
 
 
 
 
 
 
Randomization 
to SFN or 
Placebo 
 
X 
 
 
 
 
 
 
SF 36 
questionnaire 
 
X 
 
 
 
 
X 
 
Blood work 
(CBCd, CMP, 
Research) 
If still on DOX, 
draw prior to 
DOX 
Chemotherapy 
infusions 
 
X  
(prior to 
first dose of 
study 
medication) 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
Weight 
 
X 
X 
X 
X 
X 
X 
X 
Dispense 
Study 
medication. 
(# of tablets 
based on 
weight criteria 
see below) 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
Pill count 
 
 
X 
X 
X 
X 
 
 
Assess 
Adverse 
events 
 
 
X 
X 
X 
X 
X 
X 
 
DOX Chemotherapy goal is every 2 week infusions Week 1, Week 3, Week 5, and Week 7. This is 
Dose- Dense Chemotherapy only (for a uniform patient population on uniform chemotherapy dosing).  
WT = <100 pounds 2 tablets per day 
WT 100 to 200 pounds 4 tablets per day 
WT greater than 200 pounds 8 tablets per day 

 1.9.2019 
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Goal for medication compliance is =/over 80%  
 
The study drug (SFN or placebo) will be administered for a total of 12 weeks. Patients will be monitored 
every 2 weeks at their DOX chemotherapy infusion visits for toxicity checks for the initial 8 weeks and 
“as needed” thereafter. Please refer to “safety monitoring” topic for further details. 
 
Study subjects will be instructed to avoid taking all over the counter supplements during the 12 weeks of 
the study when taking the trial drug/placebo. 
Questionnaires 
The subject will complete a general health questionnaire (SF 36) at the baseline visit and a week after 
completing the 4th infusion. Non-English speakers will be provided a translator (either in person or on 
phone/video) to aid with filling out the questionnaire. 
Blood samples.  Approximately 20 ml of non-fasting blood will be drawn from the participants by 
someone trained in phlebotomy: about 10 ml of this sample will be used for routine laboratory monitoring 
(complete blood count with differential and comprehensive metabolic panel). Remaining sample will be 
used for doxorubicin associated cardiotoxicity assessment as detailed below. Blood will be collected at: 
 
Before starting SFN (or placebo) at baseline 
 
Before the first DOX chemotherapy infusion 
 
Before the second DOX chemotherapy infusion 
 
Before the third DOX chemotherapy infusion 
 
Before the fourth DOX chemotherapy infusion 
 
About one week after the fourth DOX chemotherapy infusion 
 
About one year after completion of DOX chemotherapy 
Peripheral blood cells (PBCs) are isolated from EDTA anti-coagulated blood using standard Ficoll-Paque 
Plus gradient centrifugation (density 1.073 g/mL) according to the instructions of the manufacturer (GE 
Healthcare, USA).  Total RNA will be extracted from PBC using RNeasy columns (Qiagen; Valencia, CA) 
and samples with RNA integrity number (RIN) score>7 will be used for gene expression analysis by 
qPCR.  Differentially expressed genes (DEG) of patients who developed DOX-associated cardiotoxicity 
after the completion of chemotherapy were compared with DEG of patients who did not. We have 
identified 67 unique up or down regulated genes.  The most enriched “biological functions” of this dataset 
were cell-to-cell signaling, cellular movement, free radical scavenging, inflammatory diseases, skeletal 
and muscular disorders, immunological diseases and cellular development.  We will perform qRT-PCR 
to determine the effect of SFN on identified genes in patients with abnormal LVEF in comparison with the 
patients with normal LVEF. 
2D ECHO - We will evaluate cardiac function in the oncology clinic at UMC Lubbock with 2D echo. 2D 
echo is undertaken when cardiac structural details are desired alongside an estimation of cardiac ejection 
fraction. 2D echo will be done with either a GE Logic E9 or a Philips Epic machine by experienced 
technicians. One blinded cardiologist, certified in reading ECHO, will read the report. The same machine 
and protocol will be used for all patients.  
ECHO will be done per standard of care: 
 
before starting SFN (or placebo) at baseline 
 
one week after the forth DOX chemotherapy infusion 

 1.9.2019 
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 
one year after completion of DOX chemotherapy 
FDG – PET CT  
We will evaluate tumor response objectively by baseline and post treatment (one week +/- 3 days from 
4th DOX infusion) completion FDG – PET CT. Testing will be done in house by a radiologist with expertise 
in FDG-PET. He will be blinded to the treatment regimen as well.  
Evaluation of tumor response will be based on standard PERCIST (version 1.0) criteria. 
https://pubs.rsna.org/doi/10.1148/radiol.2016142043 
The FDG – PET CT, questionnaires, specific blood draws, and the nutritional supplements or placebos 
are study related.  All other tests and activities are part of routine breast cancer treatment care. 
Sulforaphane and Placebo Information 
SFN in the precursor glucosinolate form is widely available as a nutritional supplement. However, 
effective conversion to the isothiocyanate form depends on the intestinal flora and may vary among 
individuals. Therefore, we will administer Avmacol®, an innovative supplement that contains 
glucoraphanin and active myrosinase enzyme to maximize and stabilize the amount of SFN provided to 
each patient. Data on humans provided by the manufacturer has shown that Avmacol® creates an 
average of 44± 4 micromoles of SFN per recommended serving (2 caplets). 65-71 Avmacol® or 
microcrystalline cellulose (placebo) will be purchased in form of caplets from Nutramax Laboratories, Inc. 
(Edgewood, MD). Placebo and test compound will be dispensed to participants in sealed bottles with 
instructions to keep them in a household freezer. The participants will be dosed in a double-blind fashion 
with identical appearing placebo or SFN caplets in a dose of two caplets for individuals with weight of 
<100 lb, four caplets for individuals with a weight of 100–200 lb, and eight caplets for individuals weighing 
>200 lb daily for 12 weeks.  UMC Cancer Center (Lubbock) pharmacist Ajoke A. Tijani, RPh. will receive 
and store placebo and test compound from the supplier.   
Study Population 
We intend to include seventy DOX-naïve breast cancer patients undergoing neoadjuvant chemotherapy 
with no prior cardiac disease and who will receive doxorubicin without Her-2 receptor antagonists as part 
of their clinical care will be recruited in a randomized controlled, double blinded pilot study comparing 
SFN to placebo. Study participants will be randomized 1:1 to receive placebo or test compound.  
Inclusion Criteria 
 
Age 18 to 89 years 
 
No prior diagnosis of coronary artery, carotid artery or peripheral artery disease 
 
Not pregnant or breastfeeding (urine pregnancy test will be done if female of childbearing 
potential) 
 
Breast cancer requiring treatment with DOX-containing regimen above 
 
Women in child bearing age group (18-50 years) will agree to use birth control for duration of 
study  
 
Study subjects must be willing and able to swallow caplets, up to 8 daily. 
 

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Exclusion Criteria 
 
Pregnant (by urine pregnancy test) 
 
Baseline ejection fraction of less than 50%, evidence of left ventricular hypertrophy or baseline 
EKG reported as abnormal per cardiologist.                        
 
Inability to provide informed consent. 
 
Prior history of chest radiation therapy 
 
Diabetes or Hypertension or prior Myocardial infarction 
 
Trastuzumab patients  
 
Taking other  supplements 
 
Inability to follow up for safety monitoring 
 
Prisoners  
 
Previous or current use of cocaine or any illicit drug 
 
Unable or unwilling to provide blood samples 
 
Taking medications known to have cardiac effects, such as but not limited to, beta blockers, anti-
arrhythmic agents, non dihydropyridine calcium channel blockers, ace inhibitors, NSAIDS, diuretic 
agents. 
 
Unable to follow the protocol 
 
Inability to receive anthracycline due to any reason (underlying baseline cardiac dysfunction due 
to other reasons, with an EF under 50%) 
 
Patients already taking SFN OTC  
Potential Benefits 
While no direct benefit to the study participants is intended, the primary effects of sulforaphane in 
conjunction with doxorubicin may provide additional benefits to that particular patient group. However, 
the magnitude of such benefit, if any, is hypothetical and hence the need for above testing for further 
confirmation. 
This study will shed light on the role of anti-oxidant therapy (Sulforaphane) in improving cardiac function 
in the patients undergoing DOX-therapy. It may provide a safe, readily available resource to potentially 
reduce the decline in cardiac function with DOX chemotherapy. 
 
Potential Risks 
 
Sulforaphane:  This product has a very safe effect profile and is available over the counter as a 
nutritional supplement.  Previous clinical trials do not report any serious side effect this 
supplement except some minor incidents of vomiting, abdominal pain, constipation, increased 
appetite etc. (https://clinicaltrials.gov/ct2/show/results/NCT01474993?sect=X430156#othr). 
Since it has not been tested in breast cancer patients before, patients will be monitored for any 
previously unreported side effects. The complete blood count (CBC), and CMP tests will be 
used as a broad screening test to determine an individual's general health status.   

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 
Microcrystalline cellulose (placebo): This product is chemically an inert substance, if taken in 
large quantities it can cause diarrhea in some individuals. 
 
Blood Draw: Venipuncture can cause some pain, discomfort, redness, infection, bleeding, bruising 
and swelling at the draw site, lightheadedness, or fainting 
As with most studies, there is also a potential risk of breach of confidentiality. 
Safety Monitoring 
A physician investigator will monitor study subjects prior to each treatment dose (about every 2 weeks) 
for assessment of tolerance, possible side effects and review of laboratory data (complete blood count 
with differential and comprehensive metabolic panel). Patients will also be provided with a hotline number 
where a physician will be accessible 24 hours a day and 7 days a week for reporting possible serious 
adverse effects. Such calls will then be triaged by the physician (symptomatic treatment versus ER visit) 
based on type and severity of symptoms reported. Such events will be noted in the patient’s file and will 
be reported in the trial results. 
It will be at the discretion of the physician investigator to delay or discontinue treatment depending on the 
severity of the noted adverse effects (delayed for up to grade 3 CTCAE and discontinued for grade 4 
CTCAE events).  
All adverse events including side effects attributed to treatment, treatment delays or discontinuation will 
be duly recorded in the study records for further reference. 
Adverse events will be reported to the IRB as described in the TTUHSC Human Research Protection 
Manual. 
Data Handling and Recordkeeping 
The Principal Investigator will carefully monitor study procedures to protect the safety of research 
subjects, the quality of the data and the integrity of the study.  All study subject material will be assigned 
a unique identifying code or number (study ID).  The key to the code will be kept in a locked file in the 
Principal Investigator’s office, if hardcopy, or on a password-protected TTUHSC server (iRIS), secured 
behind the TTUHSC firewall, both located behind locked doors in a restricted access area of the TTUHSC 
campus.  Only authorized individuals will have access to the code and information that identifies the 
subject in this study. Data will be kept for 3 years as per TTUHSC IRB regulations. 
Basic health screening data will be collected using standardized paper forms that will be identified with 
the participant’s study ID.  Paper data will be entered into the computer independently by research staff 
trained to perform data entry.  The computer data will be stored on a secure drive behind the TTUHSC 
firewall and downloaded by the Data Manager, who will then transfer the data to SPSS and check for 
missing data.  The computer(s) used to collect data will be kept in a locked office or locked cabinet when 
not in use. 
Blood samples, identified only with subject ID, will be obtained and analyzed in the investigator’s lab.  
Results of the assays along with week, date, and time of sample collection will be entered into the study 
dataset.  The Data manager (Sharda Singh) will employ a verification program to determine and correct 
any discrepancies based on source data.  The study monitor will also help with data verification.  
Echocardiograms will be identified using participant study ID only and will be stored on the archiving 
system of the cardiac noninvasive lab at TTUHSC.  All analysis will be performed in the lab and the 

 1.9.2019 
Page 14 of 20 
 
results will be entered in a paper form with the patient ID. 
Electronic and hardcopy study data will be maintained as explained above until manuscript publication, 
after which electronic files will be erased from the TTUHSC server and hard copy documentation will be 
shredded according to TTUHSC Policy.  Blood samples not completely consumed by analyses will be 
mixed with sodium hypochlorite solution and disposed of down a sink drain according to TTUHSC policy. 
Ethical Considerations 
This study will be conducted in accordance with all applicable government regulations and Texas Tech 
University Health Sciences Center research policies and procedures.  This protocol and any amendments 
will be submitted and approved by the TTUHSC Institutional Review Board (IRB) to conduct the study.  
The informed consent of each subject, using the IRB-approved consent form, will be obtained before that 
subject is submitted to any study procedure.  The consent form will be translated into Spanish to 
accommodate Spanish reading only participants; and a Spanish translator will be used when necessary 
to facilitate communications. All subjects for this study will be provided a consent form describing this 
study and providing sufficient information in language suitable for subjects to make an informed decision 
about their participation in this study.  The person obtaining consent will thoroughly explain each element 
of the document and outline the risks and benefits, alternate treatment(s), and requirements of the 
study.  The consent process will take place in a quiet and private location, and subjects may take as 
much time as needed to make a decision about their participation.  Participation privacy will be maintained 
and questions regarding participation will be answered.  No coercion or undue influence will be used in 
the consent process.  A copy of the signed consent will be given to the participant, scanned into his or 
her medical record and the iRIS system; and the informed consent process will be documented in each 
subject’s medical record. 
 
Monitoring Plan:  
To ensure compliance of the study protocol, GCP guidelines, and TTUHSC Human Research Protection 
Program research policies and procedures during the conduct of the study, and quality data a monitor in 
the Clinical Research Institute will conduct the monitoring of the study. The first monitoring visit will be 
conducted within two weeks after the first subject has been enrolled into the study. The succeeding 
monitoring visits will be scheduled periodically, but no less than every 2 months when there is an active 
study participant, at a mutually agreed timeframe by the PI and study monitor. All data collected will be 
100% source document verified. The study monitor may inspect and audit all study documents, i.e. data 
collection forms, drug accountability, and medical records, within the applicable confidentiality 
regulations. 
Dissemination of Data 
Results of this study may be used for presentations, posters, or publications. The publications will not 
contain any identifiable information that could be linked to a participant. 
Funding:  This IRB protocol approval is needed to submit a grant application to NIH, DoD and intramural 
funding. Approved study will start upon receipt of the funds. The use of Clinical Research Institute 
resources will be acknowledged in any publications. 

 1.9.2019 
Page 15 of 20 
 
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