Steven K. Clinton, M.D., Ph.D. The Ohio State University

1. Clinical Trials: What you need to know to study diet and nutritional variables (emphasis on fruits and vegetables and their components) Steven K. Clinton, M.D., Ph.D. The Ohio State University

2. Introduction

3. What is a Clinical Trial:Essential Characteristics “a prospectivestudy involving human subjects designed to answer specific questions about the effects or impact of particular biomedical or behavioral interventions; these may include drugs, treatments, devices, or behavioral or nutritional strategies” - NIH / NCI Definition

4. Biblical Description of a Clinical Trial Daniel 1:12-16 Fragment of Daniel’s Protocol

5. The “First” Clinical Trial in Nutrition “Please test your servants for ten days: Give us nothing but vegetables to eat and water to drink. Then compare our appearance with that of the young men who eat the royal food, and treat your servants in accordance with what you see. So he consented them and tested them for ten days. At the end of the ten days they looked healthier and better nourished than any of the young men who ate the royal food. So the guard took away their choice food and the wine they were to drink and gave them vegetables instead.” Daniel 1:12-16

6. The “First” Clinical Trial in Nutrition It’s often difficult to translate clinical trial results into practice, but in Daniel’s case, we remember it today: “Eat your vegetables!” 6

7. Definitions • Chemoprevention • Pharmaceuticals (chemically defined pure agents) • Natural • Synthetic • Nutrients • Pharmacologic dosages (acting like a drug) • Nutritionally relevant dosages (acting like a nutrient) • Dietary prevention • Dietary pattern (orchestration of your entire diet) • Foods (complex mixtures of bioactives) • Traditional foods • Functional foods • Strategically altered and composed to target a disease process

8. Levels of the Evidence Randomized Controlled Double Blind Studies * Randomized Controlled Studies * Cohort Studies Case Control Studies Case Series Case Reports Ideas, Opinions * Buttressed by basic science

9. Evaluating the Evidence CHAPTER 3 PART 1 • BACKGROUND Judging the evidence The task of expert committees responsible for reports such as this is to collect, discuss, and judge scientific evidence, as a basis for recommendations made in the public interest. The purpose of this third introductory chapter is to summarize the process the Panel has used … WCRF/AICR Report - Released Nov. 1-2, 2007

10. Translatingthe Evidence PART 3 • RECOMMENDATIONS CHAPTER 8 Policies and actions The prevention of cancer worldwide is an urgent and feasible task of great importance. It requires concerted and integrated international, national and local action... Figure 8.1 The nine actors: impact of concerted action

11. Controversyand Wisdom wis·dom  (wzdm)n. The sum of learning through the ages; knowledge: "In those homely sayings was couched the collective wisdom of generations" (Maya Angelou).

13. Cohort Studies Case-Control Cross-Sectional Human Research / Clinical Trials Uncontrolled Trials Clinical Trials Controlled Trials Human Research Observational Studies

14. Team Building

15. Principle investigator and team Clinical collaborators Laboratory collaborators (agent development, analytics, …) Biological sample procurement Biostatistics Single institution / multi-institution investigators Institutional support / shared resources / NIH or USDA General Clinical Research Center (GCRC) Clinical and Tanslational Science Award (CTSA) Comprehensive Cancer Center (NIH CCC) Funding agencies Team Building: Everyone Deserves Credit!

16. Design and writing of a clinical trial Help write a grant to fund the trial Necessary for IRB process Collect, store, monitor & analyze data quality Interim analysis in large studies Data andSafety Monitoring Board (DSMB) Help write / review abstracts, reports, or publications Biostatisticians should be involved in your study from the beginning.

17. Remember this Model:A,B,C and Ds of a Dietary or Nutritional Clinical Trail • Agents • Diet pattern, specific food item • Nutrient or phytochemical (pure) • Extract • Concentrate • Neutricuetical (manipulated, processed) • Biomarkers and Outcomes • Cohort • Dollars

18. The AgentWorking with whole foods is more complex.

19. Agent: Key Issues • Standardization of study intervention • EZ for “drug” or “nutrient” or “phytochemical” • Form, safety, dose response, placebo possible. • Complex for “food product” • Variation due to source, cultivar, season, growing conditions, soil, processing, cooking, stability over time. • Placebo and double blinding may be problematic • Dietary Patterns • Heterogeneity among participants, compliance • Control group is problematic.

20. Agent: Key Issues • Known composition of a food is critical • Homogeneous and equal for all participants • Over the time of the study (batch to batch) • During storage • Reproducible by others (report methods) • Quality control • Composition and ingredients • Microbial and toxic contaminants

21. Example:Berries Acai Berry, Baneberry, Barbados Cherry, Barberry, Bearberry, Bilberry, Bittersweet, Blackberry, Blueberry, Black Mulberry, Black Raspberry, Boysenberry, Buffalo Berry, Bunchberry, Chokeberry, Chokecherry, Cloudberry, Cowberry, Cranberry, Currant, Dewberry, Elderberry, Farkleberry, Goji Berry, Gooseberry, Grape, Holly Berry, Huckleberry, Indian Plum, Ivy Berry, Juneberry, Juniper Berry, Lingonberry, Logan Berry, Mistletoe Berry, Nannyberry, Oregon Grape, Persimmon, Pokeberry, Privet Berry, Raspberry, Red Mulberry, Salmonberry, Strawberry, Sugarberry, Tayberry, Thimbleberry, White Mulberry, Wineberry, Wintergreen, Yew Berry, Youngberry

22. Phylogeny of Black Raspberries Species: Rubusoccidentalis Native to eastern North America Different in composition and taste compared to the red raspberry and blackberry. High in anthocyanins and ellagic acid Low yield per acre  expensive to grow.

24. Challenges for Berry Studies • Standardization of intervention agent • Variable chemical composition • Genetics • Environment (temperature, water, pests….) • Harvest (maturity, sensecence,…..) • Storage and shipping • Processing • What is the critical bioactive(s) to measure • Complex mechanism(s) of action and uncertaintly about desired composition of food product.

26. Components of Black Raspberries by Season (mg/100 g)

27. Components of Black Raspberries (mg/100g) a Concentration of components is expressed as mg/100g : selenium is expressed as ug/100g b Cultivar used for inhibition of oral tumors in HCP c Cultivar used for inhibition of esophageal tumors in rats (complete chemoprevention assay) d Cultivar used for inhibition of esophageal tumors (post-initiation assay)

28. What are the bioactive compounds in black raspberries? Steven Schwartz, Ph.D. and Ken Riedl, Ph.D. Dept. Food Science and Technology OSUCCC Nutrient and Phytochemical Analytic Shared Resource

29. Anthocyanins in Black Raspberry Fractions Cyanidin 3-O-glucoside Cyanidin 3-O-sambubioside Cyanidin 3-O-rutinoside Cyanidin 3-O-(2G-xylosylrutinoside)

31. Many anthocyanin chemical structures Anthocyanidin + Sugar = Anthocyanin (non-acylated) Anthocyanidin Anthocyanidin + Sugar + Acid = Anthocyanin (acylated) From Monica Giusti, Ph.D.

32. Berry Ellagitannins and Ellagic Acid From : Ken Riedl, Ph.D. Ellagitannins – high MW polyphenols with hexahydroxy-diphenic acid or ellagic acid attached to glucose core. Hydrolysis of Ester Bonds Liberates HHDA which Lactonizes to EA

33. The analytical chemist is critical to your success. Careful documentation of chemical composition is essential.

34. Application of Food Science and Technology is Critical • Enhanced delivery of bioactives • Target tissue • Systemic distribution • Improved compliance • Quality control.

35. Several Berry Clinical Trials

36. Human Clinical Trials:Strawberries and Esophageal Cancer Esophageal Squamous Cell Dysplasia in China • Tong Chen, M.D., Ph.D.

37. Esophageal Cancer • 6th most common malignant neoplasm worldwide • Squamous cell carcinoma- arises in the proximal and mid-esophagus from dysplastic lesions in the squamous epithelium • Adenocarcinoma- arises distally from from Barrett’s esophagus (metaplastic precancerous lesion) • The incidence of esophageal SCC shows marked variation in its geographic distribution. • Males have a 3- to 4- fold greater risk for developing esophageal SCC than females. • In the United States, African Americans have more than a 5-fold higher incidence of esophageal SCC than Caucasians.

38. Etiology of Esophageal SCC • Tobacco • Alcohol • Ingestion of salt-pickled, salt-cured and moldy food • Nutritional deficiency (Zn, etc) • Consumption of temperature hot beverage • Human papilloma virus infection

39. Esophageal SCC in China Peking Union Medical College Hospital

40. Peking Union Medical College Hospital

43. Randomized Phase II Trial of Lyophilized Strawberries inPatients with Dysplastic Precancerous Lesions of theEsophagus Chen et al. Cancer Prev Res; 5(1); 41–50. 2011

44. Nutricuticals:Combination Agents

45. Preclinical Study

46. Preclinical Results

47. Tomato-soy food products for cancer prevention studies Bohn et al. Phase I Trial (Nutrition and Cancer, 2013)

48. Department of Horticulture Selection of Tomato Cultivar Phytochemical content Growth characteristics Disease resistance Processing issues OSU Farms Plant and harvest 1 can = 150 ml juice (6 oz) 22.5 mg lycopene and 33 mg isoflavones Department of Food Science and Technology Process into paste Phytochemical analysis Reconstitution and Formulation Solubility Taste Texture Taste Panel Soy protein and extracts Phytochemical analysis

49. Bioavailability of phytochemical constituents from a novel soy fortified lycopene rich tomato juice developed for targeted cancer prevention trials. Bohn T, Blackwood M, Francis D, Tian Q, Schwartz SJ, Clinton SK. Nutr Cancer. 2013;65(6):919-29.

50. Agent Bioavailability Studies