br Results br Discussion Our study confirmed the safety

Results
Discussion Our study confirmed the safety and tolerability of highly pure VEDT (99%) at doses up to five times greater than those given to patients in previous studies, which used a mixture of tocotrienols (Rahmat et al., 1993; Qureshi et al., 1995, 2002; Zaiden et al., 2010). The relatively short time to Tmax and half-life of pure VEDT are similar to results reported with lower doses of mixed tocotrienols (Qureshi et al., 1995, 2002; Zaiden et al., 2010). The fact that levels in the plasma did not correlate well with administered dose suggests that there are factors affecting the metabolism and circulation of this nutrient that remain unknown. Nevertheless, the concentrations obtained in plasma in this study are comparable to those obtained in mice treated with VEDT in which tumor growth was delayed and apoptosis was induced (Husain et al., 2009). The finding that pancreatic tumors from several patients treated with VEDT displayed much higher percentages of apoptotic tumor Digoxigenin-11-dUTP than those from untreated patients suggests that plasma levels obtained may be sufficient to have the desired biological effect. The challenge now is to understand why some patients treated at a higher dose failed to show a higher percentage of apoptotic tumor cells. This could be due to tumor genetic heterogeneity, altered susceptibility to the biological effect of VEDT, or intrinsic variability among individuals in the metabolism and clearance of the drug at high doses. Higher concentrations could also modulate other cellular targets, which counteract the VEDT pro-apoptotic effects. Regarding dose, our selection was designed to ensure the greatest margin of safety and to focus on identifying the biologic effective dose as defined by biological activity in tumor samples. Our starting dose (100mg twice a day) was based on the rationale described in the Food and Drug Administration (FDA) guidance “General Guide for Starting Dose Selection for a Cytotoxic Agent in Cancer Patients” (available at www.fda.gov/cder/cancer/docs/doseflow.pdf) as well as on the no-observed-adverse-effect level (NOAEL) determined in a published repeat-dose toxicity study in rats (Nakamura et al., 2001). In this 13-week toxicity study, the NOAELs for VEDT were concluded to be 0.19% in the diet (120mg/kg body weight/day for male rats and 130mg/kg body weight/day for female rats). The human equivalent doses based on equivalent relative body surface areas of 1:6:2 will yield 19.4mg/kg/day dosed daily for up to 13weeks. Therefore, for a 65-kg subject the NOAEL dose of VEDT will be 1261mg/day. In mice, VEDT dosed at 100mg/kg/day once daily (via oral gavage) resulted in significant inhibition of tumor growth and induction of apoptosis in the neoplastic cells. Using the recommended body surface area (BSA) normalization method for allometric dose translations when starting new clinical studies (Reagan-Shaw et al., 2008), the formula below was used: Human Equivalent Dose (mg/kg)=Animal dose (mg/kg)×Animal Km/Human Km (where Km values based on body surface area for a mouse is 3 and an adult human is 37 (60kg)). Converting a dose of VEDT (100mg/kg) given to a mouse into the human equivalent dose based on body surface area is: HED=100 (mg/kg)×3 (mouse Km)/37 (Human Km); The HED=8.11mg/kg. A 70kg subject would therefore require 567mg/day. Dose escalation to a maximum of 3200mg/day will be 5.6 times the predicted bioactive dose from the preclinical studies. The ability of VEDT to induce apoptosis in pre-malignant lesions (dysplastic tissue) but not in adjacent normal tissue has particular promise for pancreatic cancer prevention. Pancreatic carcinogenesis is a multistep process that involves the accumulation of a set of genetic changes that convert normal cells first to premalignant cells and then to malignant cells (Iacobuzio-Donahue, 2012; Iacobuzio-Donahue et al., 2012). In a recent study, an average of 63 mutations per cancer was altered in 24 human PDAs (Jones et al., 2008). The most widely accepted explanation for the accumulation of multiple mutations in the same cell is the model of clonal evolution, when cells with one crucial mutation expand clonally (Aparicio and Caldas, 2013). By increasing in number through mitosis and suppression of apoptosis, cells with the first mutation become more likely to develop a second mutation, which predisposes to a third mutation and so on. The continuing clonal expansion, selection, and heterogeneity allow accumulation of multiple mutations in the same cell and the ultimate generation of malignant clones (Iacobuzio-Donahue, 2012). Therefore, reducing or eliminating the number of premalignant cells could be an effective strategy to reduce pancreatic cancer risk (Wu and Lippman, 2011). One intriguing finding demonstrated consistently by us and others is the selective killing effect of tocotrienols against cancer cells over normal cells (Hodul et al., 2013; Husain et al., 2011; Srivastava and Gupta, 2006; Yap et al., 2008, 2010). Tocotrienol was found to induce apoptosis in prostate and breast cancer cells but not in nonmalignant breast and prostate epithelial cells (Srivastava and Gupta, 2006; Yap et al., 2008, 2010). Because no effective chemoprevention therapy for the medical treatment of pre-malignant pancreatic lesions is available, patients face the choice of potentially life-changing pancreatectomy versus observation with the risk of transformation to invasive adenocarcinoma and metastasis. In addition, no effective surveillance strategies are available to identify transformation before metastasis. The ability of VEDT to induce apoptosis in the neoplastic cells of intraductal papillary mucinous neoplasm and pancreatic intraepithelial neoplasia lesions with minimal toxicity establishes this micronutrient as a candidate chemoprevention agent for the medical treatment of patients with these lesions who opt to not have surgery. The tolerability of VEDT with longer-term administration of high doses will be the subject of future investigation.