Protective Effects of the Nutritional Supplement Sulforaphane on Doxorubicin-Associated Cardiac Dysfunction

Determine whether nutritional supplement sulforaphane (SFN) is safe to administer to breast cancer patients undergoing doxorubicin (DOX) chemotherapy. The investigators 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 Extra Strength, (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 Extra Strength® in the Oral Mucosa of Patients Following Curative Treatment for Tobacco-related Head and Neck Cancer" (https://clinicaltrials.gov/ct2/show/NCT03268993). Furthermore, Avmacol Extra Strength has never been tested as an adjuvant to try and protect the heart from DOXs harmful effects. . Therefore, the investigators 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, the investigators will test SFN in combination with DOX-based chemotherapy for breast cancer; the investigators will assess its safety, cardio-protective properties, and anti-cancer efficacy. The investigators 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, the investigators 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.

The investigators 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. Jones and Philipovskiy) 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. The investigators 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.

The investigators 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. Because Nrf2 upregulates certain detoxifying enzymes, the investigators 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.

Study Design and Procedures Study Design. The investigators 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. ([email protected]) 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., three caplets for individuals 100-200 lb. and four caplets for individuals >200 lb. Avmacol Extra Strength or placebo will be prescribed by Dr. Jones and Philipovskiy and will be dispensed by local pharmacy or study coordinators at TTUHSC/UMC Lubbock. The investigators will be doing pill counts to make sure that volunteers have used as directed. The investigators 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 the investigators 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.

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. The investigators 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 the investigators will still use their study data during analysis.

Sample-size justification. The number of subjects (35 per treatment arm; 70 total; the investigators 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. The investigators 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.