**7. Other considerations**

for already proven indications as well as for subsequently approved disease indications, including for example, PCa. AMR-101, also marketed as Vascepa, received USFDA approval in July, 2012 [205]. Similar to Lovasa, this approval was based on effective treatment of hypertriglyceridemia but with the added ability to impact people with high triglycerides; the latter indication has been filed and is under review pending outcomes in clinical trials [206]. Epanova, another promising n-PUFA formula, is undergoing clinical trials for similar indica‐ tions as previously mentioned for Lovasa, and USFDA approval is forthcoming [207]. Other dietary supplements, containing various amounts and ratios of n-3 PUFA which are sold over the counter in the United States and Canada would likely be based on values issued by the Institute of Medicine and National Academies as described below. Such unregulated formu‐ lary preparations do not undergo clinical trials. Studies relating to n-3 PUFA pharmaceutical preparations have focused on treating cardiovascular disease with no clinical consideration for PCa. Albeit, men diagnosed with PCa have accepted the use of such nutraceuticals and thus there is a role in ensuring that this approach constitutes safe and effective therapies. Although studies around the prescription formulas have demonstrated a very good safety and tolerability profile, the focus has been in treating cardiovascular disease and thus there has

Another proposed explanation for inconsistent findings in the role of n-3 PUFA in prostate cancer relates to absolute intake versus ratio of n-3 to n-6 PUFA. The latter may be more relevant for prostate cancer risk considering that the recommended dietary ratio of n-6/n-3 PUFA for health benefits are variable and range from 1:1-4:1, yet dietary intake in such studies, relative to the Western diet could contain 10 or more times the amount of n-6 compared to n-3 PUFA [208]. Also consider that in gathering and thoroughly reviewing all information toward this chapter, we were unable to find an *official* recommended daily allowance for n-3 PUFA for adults or children. In Canada and the United States, the general public and health professionals (including healthcare policy makers and public health officials) use a set of five nutrient based reference values issued by the Institute of Medicine and National Academies for a wide variety of food and dietary applications [209]. These dietary reference intakes (DRI), are inclusive of components including: estimated average requirements (EAR), recommended daily allowance (RDA), adequate intake (AI), tolerable upper intake level (UL) and an acceptable macronutrient distribution range (AMDR). Considered to be the official standard for federal agencies, these values are used to issue dietary guidance or policy directives for the health and well-being of individuals. In the absence of a DRI specific to EPA and DHA, the National Academies have recommended an AMDR of 10% for another n-3 PUFA, namely alpha-linolenic acid (ALA) to be consumed as EPA and/or DHA equating to a much lower value (about 100 mg/day) than currently recommended by many groups worldwide. For example, a number of nutritionist and physicians recommend as much as 450 milligrams of n-3 PUFA in either DHA or EPA form per day to promote a healthy diet [79, 210], a fact alone which warrants further studies to determine DRI, specifically UL. More importantly, if the effects of n-3 PUFA represent different disease specifications, then it is crucial to establish DRIs for DHA and EPA individ‐ ually, and in combination. Considering the contradictory evidence that excessive DHA may be a possible cause for some types of aggressive prostate cancer, the expansion of this field of research may want to revisit the incorporation of such basic factors as the ratio of EPA to DHA

been no concern over potential adverse effects for other diseases.

232 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

#### **7.1. Markers of tumor growth**

Epidemiologic studies have shown that lower serum insulin-like growth factor (IGF)-1 levels and increased IGF binding protein-3 (IGFBP-3) levels are associated with decreased PCa risk [211]. Unlike humans, rodents do not have a PSA counterpart for monitoring prostate cancer initiation and progression, however prostatic secretory protein (PSP)-94 has been established as a serum tumor marker [212]. However, measurement of mouse serum for IGF-I, IGFBP-3 and PSP-94 can be assayed using enzyme-linked immunosorbent assay (ELISA) using mousespecific antibodies and recombinant mouse standards (where applicable) and by methods previously described [213]. Therefore, such biomarkers could be used as indicators in both human and mouse studies in conjunction with other parameters and may be correlated with fatty acid analysis.

#### **7.2. Fatty acid analysis: Serum blood levels of EPA and DHA**

Selection of fatty acid concentration and methods of introducing fatty acids to cell cultures in such experiments are documented [214, 215]. Like most *in vitro* studies, data obtained may provide indicators of how to progress to the next levels of research. More relevant to human disease outcome, the fatty acid composition of serum (or plasma) phospholipid is an estab‐ lished valid biochemical marker for assessing the physiological status of various fatty acids including predictive correlations with the dietary intakes omega-3 PUFA, including EPA and DHA. [216, 217]. Serum fatty acid analysis by gas chromatography seems to be the standard for such experiments [218, 219] for both humans and animals.
