Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Animal Science
Non Technical Summary
The overall goal of W-4122 researchers is to examine the effects that bioactive components of the diet such as phytochemicals, foodborne toxicants, microbial metabolites, and specific macro- and micronutrients exert on human health and in the safety of the food supply. W-4122 participants collectively utilize mechanistic, preclinical, and clinical research methods to provide a comprehensive translational approach towards understanding the role of natural chemicals in human health and food safety. This project (Whelan-TN) is an extension and addition to the overall goal of this multistate initiative. Whelan (TN) explores the impact of phytochemicals and macronutrients on human health, their safety, and the potential underlying mechanisms responsible for their effects utilizing mechanistic, preclinical, and clinical research methods. The phytochemicals are in the form of a commercial product called Zyflamend™ (Whelan 2014). Zyflamend (New Chapter, Brattleboro, VT) is well defined and highly characterized dietary product derived from the extracts of ten different herbs (holy basil, turmeric, ginger, green tea, rosemary, Hu Zhang, barberry, oregano, baikal skullcap, and Chinese goldthread). Zyflamend was originally designed by combining herbs reported to have antioxidant and anti-inflammatory properties. For example, Zyflamend reduces the production of proinflammatory eicosanoids (Yang 2007; 2008), inhibits the activities of both isoforms of cyclooxygenase (COX-1 and COX- 2), as well as 12- and 5-lipoxygenases (Yang 2007; 2008), and inhibits the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (Burke 2015; Kunnumakkara 2012; Sandur 2007) and the subsequent production of cytokine-inducible nitric oxide (iNOS) (Ekmekcioglu 2011). Furthermore, Zyflamend has been reported to reduce C-reactive protein (CRP) levels in humans (Capodice 2009). CRP is an acute-phase reaction protein produced in response to inflammation (Thiele 2015).The macronutrient Dr. Whelan works with is in the form of fish oil called Wholemega™ (New Chapter, Brattleboro, VT). Omega-3 fats from fish oils are also anti-inflammatory, but their mechanisms of action may involve pathways different from Zyflamend, thus their combination may act synergistically to have an enhanced health benefit, particularly when it involves inflammation. Wholemega™ (New Chapter, Brattleboro, VT) is composed of extra-virgin wild Alaskan salmon oil, extracted using a mechanical separation process similar to that of extra virgin olive oil; combined with supercritical extracts of rosemary and oregano to provide antioxidant stability. Both products (Zyflamend and Wholemega) are "foods" and not distilled down to individual bioactives - it is food that people eat, not isolated chemicals. Thus, the scope of this research adds an additional dimension to the W-4122 research team and is not just duplicative, but additive.Consistent with the overall goal of W-4122, Whelan (TN) has determined and verified a mathematical model for the extrapolation of human equivalent doses of bioactive nutrients preclinically and clinically, such that, when experiments are performed in preclinical models, there is a greater likelihood that the results can directly apply to people (these results have been verified). He has established that these combinations are safe following clinical trials with patients, in part, by evaluating metabolomics and looking for adverse symptoms (Tague 2016). He has published multiple articles describing the epigenetic, molecular and signaling mechanisms behind the health benefits of Zyflamend and fish oils and that these effects translate from cell culture to preclinical and clinical models (as reviewed in Whelan 2017). He has described these mechanisms using human equivalent doses and not pharmacological doses, critical to the translational piece of W-4122 (as reviewed in Whelan 2017). He has published on the synergistic action of the components in food such that when the bioactive is provided in a food matrix, it is thousands of times more effective than when used in isolation (eg. purified compound) (Zhao 2014). In fact, he has also determined when two isolated phytochemicals derived from the same food are put together (one biologically active and the other inactive when they are used in isolation), the inactive phytochemical becomes the most biologically active of the two when combined, mimicking what happens in the food matrix. Similarly, one can observe divergent effects with unintended consequences when bioactives are used in isolation versus as part of a food. For example, foods that contain vitamin E reduce the risk of skin cancer, while vitamin E supplements increase the risk. Foods that contain beta-carotene decrease the risk of lung cancer, while supplements of beta-carotene increase the risk. It is not the single bioactive in a food that has the most health promoting effect, but the combination of all the constituents. Zyflamend and Wholemega are complete extracts of the original matrix composed of many bioactives in a food.Lower extremity peripheral arterial disease (PAD) is estimated to affect ~8-12 million people in the US, and increases with age, cigarette smoking, diabetes, hypertension and obesity. Each year individuals with PAD undergo surgery to improve blood flow to areas where there is blockage in a blood vessel. One particular technique involves the insertion of a stent. A stent is a wire mesh that is inserted in the blood vessel that was blocked to reintroduce (increase) blood flow. However, a problem with treatment is that over time it leads to localized inflammation resulting in a reblockage of the blood vessel (this is called restenosis). This process involves the recruitment of immune cells to the area and they release a variety of chemicals that entice the muscle cells of the blood vessels to multiply and invade the area adjacent to the blood (the 'intima"). This thickening of the intima is a biomarker of this inflammatory process and is a main contributor of restenosis. When this happens, the individual has to go back into surgery to correct the situation.In a preclinical model mimicking PAD, preliminary experiments involving Whelan and colleagues (Mountain 2018) show that the combination of Zyflamend and Wholemega at human equivalent doses significantly inhibits intimal thickening and significantly reduces proinflammatory compounds (cytokines) produced by activated immune cells. Because of these stunning results, the next step is to investigate the translational feasibility in the human condition (clinically), viz, individuals with PAD. If we can demonstrate that a combination of bioactives from foods can increase the longevity of these stents in the peripheral blood vessels (outside the heart) and the effect is related to their anti-inflammatory properties, this would increase the confidence that the same benefits could apply to stents that were used to unblock coronary arteries in the heart. This would not only have a huge health-promoting benefit, but also a tremendous financial impact for both the patient and insurance companies; thus, the target of this proposed research.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Identify cellular mechanisms and host molecular targets of beneficial or adverse dietary components that influence human health.
Project Methods
This is a double-blind, randomized control trial. Sixty individuals (≥ 50 yo) receiving percutaneous lower extremity revascularization treatment will be recruited from the Division of Vascular Surgery at the University of Tennessee Medical Center, Knoxville, TN. They will be evenly divided into a control group receiving a placebo (n=30) and an experimental group receiving the combination of the bioactive natural products, Zyflamend™ and Wholemega™ (n=30). The experimental group will be referred to as the "Zyflamend Heart" group. The group size was determined based on statistical power calculations. The treatment assignments will be generated with the use of a pseudo-random-number generator with randomly permutated blocks that will be used to ensure balance between the numbers of subjects assigned to each of the two treatments. The Placebo control group will be given 6 capsules/day of pure extra virgin olive oil (1.0 gram/capsule) at two daily intervals, 4 capsules with breakfast and 2 capsules with dinner. The Zyflamend Heart group will take 2 capsules each of Zyflamend and Wholemega in the morning and 1 capsule each of Zyflamend and Wholemega in the evening (total 3 capsules each or 6 total capsules).Preliminary studies with preclinical rodent models clearly demonstrate that the combination of Zyflamend™ and Wholemega™ fish oil significantly attenuates intimal hyperplasia at human equivalent doses, and these affects are associated with a reduction in proinflammatory cytokines. Zyflamend™ (New Chapter, Brattleboro, VT) is well defined and highly characterized dietary supplement derived from the extracts of ten different herbs (holy basil, turmeric, ginger, green tea, rosemary, Hu Zhang, barberry, oregano, baikal skullcap, and Chinese goldthread). A detailed description and characterization of the preparation of Zyflamend and quality assurance of the mixture has been described previously. Wholemega™ (New Chapter, Brattleboro, VT) is composed of extra-virgin wild Alaskan salmon oil, extracted using a mechanical separation process similar to that of extra virgin olive oil; combined with supercritical extracts of rosemary and oregano to provide antioxidant stability.The subject's eligibility will be based on inclusion/exclusion criteria (age, appropriate diagnosis, scheduled for revascularization surgery, dietary intakes, etc). They will be slated to have a stent placed in the femoral or popliteal artery for isolated infrainguinal femoropopliteal lesion (any TASC lesion) without significant aortoiliac arterial disease and must be available for 12 months of surveillance and follow up.At the initial visit, the physician will confirm PAD diagnosis, perform physical exam and preoperative arterial duplex. They will discuss the study with the patient, and if willing, they will be referred to the study team to be contacted to establish data on all inclusion/exclusion criteria. At a follow up visit, the patients will complete the following: eligibility questionnaire, informed consent, patient contact information and questionnaires, blood draw for biomarkers and metabolomics, randomization, dispense study drug and instructions. Day of Surgery Postoperative Baseline lower extremity arterial duplex or with ankle brachial index assessment (Instent Arterial Assessment). Standard Duplex measurement for femoral or popliteal instent arterial assessment including flow velocity (cm/seconds) and intraluminal arterial wall thickness: native artery proximal, mid and distal to stent. Follow up in-person visits will be at 3 mo, 6 mo and 1 yr with routine anthropometric measurements and a blood sample to measure inflammation-related cytokines, apoptotic markers and small molecules routinely analyzed in metabolomic measurements, along with a standard noninvasive vascular lab study arterial duplex with ankle-brachial index (Instent Arterial Assessment), and confirmation of antiplatelet (aspirin and/or Plavix) and statin medication therapies.Non-in-person contact will occur every two weeks to discuss any potential side effects from the study pills, and to evaluate any changes to subjects' medical conditions relevant to the exclusion criteria. The participants will fill out a weekly diary regarding clinical indicators of response.When blood samples are collected, blood will be drawn by the clinical research nurse and processed (plasma/serum) by the vascular lab. Samples will be processed accordingly, plasma/serum collected, aliquoted, and frozen at -80C. All inflammatory marker analyses will be performed by ELISA or a functional laboratory equivalent.Primary endpoints include safety assessment of the supplements and restenosis defined by a peak systolic velocity greater above 300cm/sec in the treated artery, velocity ratio greater than 2.4 or ankle brachial index decrease greater than 0.15. This will be evaluated with noninvasive arterial duplex and arterial brachial index. Secondary Endpoints include clinical response defined as wound healing, resolution of rest pain and ambulatory distance and biomarkers of inflammation. Other study variables include: demographics, health history, medication usage after enrollment, select dietary and behavioral factors, and any side-effects.Results from the above assays will be examined to determine if there is an increase in the risk of graft occlusion secondary to neointimal hyperplasia. Both univariate (Kaplan-Meier method) and multivariate analyses (Cox proportional hazards model) will determine the association among preoperative variables, graft patency, and changes in inflammatory markers.Upon completion, we will compare the treatment and placebo groups with respect to restenosis rates, clinic response and baseline covariates for any meaningful differences. The difference in these percentages will be tested with Fishers exact test. The relative risk of restenosis will be calculated. Other secondary endpoints will be compared using the Wilcoxon rank sum test. The proportion of participants experiencing various side-effects will be compared using Fishers exact test. All analyses will use an intent-to-treat approach. Stratified analyses using Cochran-Mantel-Haenszel statistics will be performed to evaluate the association between study endpoints and treatment group for males and females, respectively. Multiplex analysis of cytokine messages using the low-density arrays will generate an extensive number of data points. More sophisticated analyses employing repeated-measures methodology will be utilized to analyze laboratory data.Adverse events tabulated by severity and by relationship to treatment. If missing data becomes an issue, then we will conduct sensitivity analyses to encompass different scenarios of assumptions and discuss consistency or discrepancy among them.The first 3 months of the current study involve creating the manual of operations, forms, data entry screens, and other study materials. Also, during this time, staff are trained, and pre-test procedures and refinements are carried out. The last subjects to enter the clinical trial will do so at the 15-month point of the study so that the last subjects will complete the study around the 25-month point. Analysis and data entry and editing will occur throughout the study and will be completed within 3 months after the final study subject's completion of the study.