ClinicalTrials.gov NCT01070355.

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The majority of evidence linking anti-colorectal cancer (CRC) activity with omega-3 polyunsaturated fatty acids (O3FAs) has focussed on decreased CRC risk (prevention). More recently, preclinical data and human observational studies have begun to make the case for adjuvant treatment of advanced CRC. Herein, we review latest data regarding the effect of O3FAs on post-diagnosis CRC outcomes, including mechanistic preclinical data, evidence that O3FAs have beneficial effects on efficacy and tolerability of CRC chemotherapy, and human epidemiological data linking dietary O3FA intake with CRC outcomes. We also highlight ongoing randomised controlled trials of O3FAs with CRC endpoints and discuss critical gaps in the evidence base, which include limited understanding of the effects of O3FAs on the tumour microenvironment, the host immune response to CRC, and the intestinal microbiome.

Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89 Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2-targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89 Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues.

BACKGROUND AND PURPOSEThe omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis. EXPERIMENTAL APPROACHA BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasoundguided intrasplenic injection of 1 ¥ 10 6 MC-26 cells (n = 16 each group). KEY RESULTSTreatment with 5% (w w -1 ) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE2 levels (with concomitant increased production of PGE3). Liver tumours from 5% EPA-FFA-treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50-200 mM) in vitro was rescued by exogenous PGE2 (10 mM) and PGE1-alcohol (1 mM). CONCLUSIONS AND IMPLICATIONSEPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a 'PGE2 to PGE3 switch' in liver tumours. Inhibition of PGE2-EP4 receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA. AbbreviationsAA, arachidonic acid; BrdU, 5-bromo-2

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone ''trigger'' by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N -{4-[bis-(2-chloroethyl)-amino]-phenyl}-B,B,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V max and K m values of 11.86 F 3.09 Mmol/min/mg and 2.70 F 1.14 Mmol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time-and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase.In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 of 32 A c c e p t e d M a n u s c r i p t Abbreviations:1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDC), 4-(Dimethylamino) pyridine (DMAP), 4-[2-(N,NDimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE), alphalinolenic acid (LNA) arachidonic acid (AA), colorectal cancer (CRC), coefficient of variation (CV), deuterated alpha-linolenic acid , docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), EPA for Metastasis Trial EMT fatty acid FA free fatty acid FFA gas chromatography tandem mass spectrometry GC MS liquid chromatography electrospray ionization triple quadrupole tandem mass spectrometry (LC-ESI-MS/MS) limit of detection LOD limit of quantification LOQ linoleic acid (LA), oleic acid (OA), palmitic acid (PA), stearic acid (SA).A liquid chromatography-tandem mass spectrometry method to measure fatty acids in biological samples. A c c e p t e d M a n u s c r i p t Milene HIGHLIGHTS We have developed a LC-ESI-MS/MS method to measure fatty acid content of biological samples, particularly relevant to laboratory and clinical studies of PUFAs. We compared it directly with an established GC-MS protocol. The method is reproducible and suited for clinical application. The methodology allows measurements of total and unesterified fatty acids  Distribution of free versus bound FAs vary and may impact on biological activity. INTRODUCTIONLong-chain (>14 carbon atoms) polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5 -3) and arachidonic acid (AA, C20:4 -6) play crucial roles in normal human physiology and the We also confirmed applicability of the LC-ESI-MS/MS method to animal and human samples in the context of EPA supplementation experiments. Finally, we highlight the use of the LC-ESI-MS/MS method to compare relative changes in free (unesterified) and total (free + esterified) cellular PUFA content after exogenous EPA exposure. METHODS Reagents.EPA free fatty acid (FFA) was provided by SLA Pharma AG (Watford, UK). Use of EPA-FFA in in vitro experiments has been described previously [17]. Acetonitrile, methanol, 2-propanol, water and chloroform usedPage 5 of 32A c c e p t e d M a n u s c r i p t for fatty acid extraction were purchased from Sigma Aldrich (St. Louis, MO, USA) and were UHPLC-MS grade.Deionised purified water was generated using an Elga Maxima and Elga Purelab Option purifying system (18.2 MΩ-cm) (Veolia Water Technologies UK, High Wycombeaminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE) and fatty acid standards; eicosapentaenoic acid (EPA ...

Measurement of red blood cell eicosapentaenoic acid (EPA) levels in a randomised trial of EPA in patients with colorectal cancer liver metastases, Prostaglandins Leukotrienes and Essential Fatty Acids, http://dx.doi.org/10.1016/j.plefa.2016.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractWe investigated red blood cell (RBC) PUFA profiles, and the predictive value of RBC EPA content for tumour EPA exposure and clinical outcomes, in the EMT study, a randomised trial of EPA in patients awaiting colorectal cancer (CRC) liver metastasis surgery (A.J. Cockbain et al, 2014). There was a significant increase in RBC EPA in the EPA group (n=43; median intervention 30 days; mean absolute 1.26 [±0.14]% increase; P<0.001), but not in the placebo arm (n=45). EPA incorporation varied widely in EPA users and was not explained by treatment duration or compliance.