Maleimide-Thiol Linkages Alter the Biodistribution of SN38 Therapeutic Microbubbles Compared to Biotin-Avidin While Preserving Parity in Tumoral Drug Delivery.

Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome-MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide-thiol linkage, that is currently used in antibody-drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety.

The effect of aspirin and eicosapentaenoic acid on urinary biomarkers of prostaglandin E2 synthesis and platelet activation in participants of the seAFOod polyp prevention trial.

Urinary prostaglandin (PG) E metabolite (PGE-M) and 11-dehydro (d)-thromboxane (TX) B2 are biomarkers of cyclooxygenase-dependent prostanoid synthesis. We investigated (1) the effect of aspirin 300 mg daily and eicosapentaenoic acid (EPA) 2000 mg daily, alone and in combination, on urinary biomarker levels and, (2) whether urinary biomarker levels predicted colorectal polyp risk, during participation in the seAFOod polyp prevention trial. Urinary PGE-M and 11-d-TXB2 were measured by liquid chromatography-tandem mass spectrometry. The relationship between urinary biomarker levels and colorectal polyp outcomes was investigated using negative binomial (polyp number) and logistic (% with one or more polyps) regression models. Despite wide temporal variability in PGE-M and 11-d-TXB2 levels within individuals, both aspirin and, to a lesser extent, EPA decreased levels of both biomarkers (74% [P ≤ .001] and 8% [P ≤ .05] reduction in median 11-d-TXB2 values, respectively). In the placebo group, a high (quartile [Q] 2-4) baseline 11-d-TXB2 level predicted increased polyp number (incidence rate ratio [IRR] [95% CI] 2.26 [1.11,4.58]) and risk (odds ratio [95% CI] 3.56 [1.09,11.63]). A low (Q1) on-treatment 11-d-TXB2 level predicted reduced colorectal polyp number compared to placebo (IRR 0.34 [0.12,0.93] for combination aspirin and EPA treatment) compared to high on-treatment 11-d-TXB2 values (0.61 [0.34,1.11]). Aspirin and EPA both inhibit PGE-M and 11-d-TXB2 synthesis in keeping with shared in vivo cyclooxygenase inhibition. Colorectal polyp risk and treatment response prediction by 11-d-TXB2 is consistent with a role for platelet activation during early colorectal carcinogenesis. The use of urinary 11-d-TXB2 measurement for a precision approach to colorectal cancer risk prediction and chemoprevention requires prospective evaluation.

Polymorphisms in Cyclooxygenase, Lipoxygenase, and TP53 Genes Predict Colorectal Polyp Risk Reduction by Aspirin in the seAFOod Polyp Prevention Trial.

Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin E2. We investigated whether 35 SNPs in oxylipin metabolism genes such as cyclooxygenase (PTGS) and lipoxygenase (ALOX), as well as 7 SNPs already associated with colorectal cancer risk reduction by aspirin (e.g., TP53; rs104522), modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomized 2 × 2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. 542 (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with nonaspirin users was restricted to rs4837960 (PTGS1) common homozygotes [IRR, 0.69; 95% confidence interval (CI), 0.53-0.90); q = 0.06], rs2745557 (PTGS2) compound heterozygote-rare homozygotes [IRR, 0.60 (0.41-0.88); q = 0.06], rs7090328 (ALOX5) rare homozygotes [IRR 0.27 (0.11-0.64); q = 0.05], rs2073438 (ALOX12) common homozygotes [IRR, 0.57 (0.41-0.80); q = 0.05], and rs104522 (TP53) rare homozygotes [IRR, 0.37 (0.17-0.79); q = 0.06]. No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalized, predictive models of colorectal cancer chemoprevention by aspirin. PREVENTION RELEVANCE: Single-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most.

Cyclooxygenase activity mediates colorectal cancer cell resistance to the omega-3 polyunsaturated fatty acid eicosapentaenoic acid.

PURPOSE: The naturally-occurring omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and inexpensive, making it an attractive anti-cancer intervention. However, EPA has only modest anti-colorectal cancer (CRC) activity, when used alone. Both cyclooxygenase (COX) isoforms metabolise EPA and are over-expressed in CRC cells. We investigated whether COX inhibition increases the sensitivity of CRC cells to growth inhibition by EPA. METHODS: A panel of 18 human and mouse CRC cell lines was used to characterize the differential sensitivity of CRC cells to the growth inhibitory effects of EPA. The effect of CRISPR-Cas9 genetic deletion and pharmacological inhibition of COX-1 and COX-2 on the anti-cancer activity of EPA was determined using in vitro and in vivo models. RESULTS: Genetic ablation of both COX isoforms increased sensitivity of CT26 mouse CRC cells to growth inhibition by EPA in vitro and in vivo. The non-selective COX inhibitor aspirin and the selective COX-2 inhibitor celecoxib increased sensitivity of several human and mouse CRC cell lines to EPA in vitro. However, in a MC38 mouse CRC cell tumour model, with dosing that mirrored low-dose aspirin use in humans, thereby producing significant platelet COX-1 inhibition, there was ineffective intra-tumoral COX-2 inhibition by aspirin and no effect on EPA sensitivity of MC38 cell tumours. CONCLUSION: Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs represents a therapeutic opportunity to augment the modest anti-CRC activity of EPA. However, intra-tumoral COX inhibition is likely to be critical for this drug-nutrient interaction and careful tissue pharmacodynamic profiling is required in subsequent pre-clinical and human studies.

A liquid chromatography-tandem mass spectrometry method to measure fatty acids in biological samples.

As pre-clinical and clinical research interest in ω-3 polyunsaturated fatty acids (PUFA) increases, so does the need for a fast, accurate and reproducible analytical method to measure fatty acids (FA) in biological samples in order to validate potential prognostic and predictive biomarkers, as well as establishing compliance in ω-3 PUFA intervention trials. We developed a LC-ESI-MS/MS method suitable for high throughput development to measure FAs and validated it in the context of treatment with the ω-3 PUFA eicosapentaenoic acid (EPA). Uniquely we directly compared the LC-ESI-MS/MS method to a GC-MS protocol. We demonstrated the LC-ESI-MS/MS method is accurate and reproducible, with coefficients of variation consistently below 15% for each PUFA analysed. The relative FA content values correlated well with those obtained by GC-MS (r2=0.94, p<0.001 for EPA) in vitro. The data obtained following analysis of FA content of liver tissues from mice fed an eicosapentaenoic acid enriched diet showed similar results to that of published studies in which GC-MS was used. The LC-ESI-MS/MS method allows concomitant analysis of unesterified (free, unbound) and esterified (bound) FAs in biological samples, allowing investigation of different PUFA pools in cells and tissues.

Anticolorectal cancer activity of the omega-3 polyunsaturated fatty acid eicosapentaenoic acid.

BACKGROUND: Oral administration of the omega-3 fatty acid eicosapentaenoic acid (EPA), as the free fatty acid (FFA), leads to EPA incorporation into, and reduced growth of, experimental colorectal cancer liver metastases (CRCLM). DESIGN: We performed a Phase II double-blind, randomised, placebo-controlled trial of EPA-FFA 2 g daily in patients undergoing liver resection surgery for CRCLM. The patients took EPA-FFA (n=43) or placebo (n=45) prior to surgery. The primary end-point was the CRCLM Ki67 proliferation index (PI). Secondary end-points included safety and tolerability of EPA-FFA, tumour fatty acid content and CD31-positive vascularity. We also analysed overall survival (OS) and disease-free survival (DFS). RESULTS: The median (range) duration of EPA-FFA treatment was 30 (12-65) days. Treatment groups were well matched with no significant difference in disease burden at surgery or preoperative chemotherapy. EPA-FFA treatment was well tolerated with no excess of postoperative complications. Tumour tissue from EPA-FFA-treated patients demonstrated a 40% increase in EPA content (p=0.0008), no difference in Ki67 PI, but reduced vascularity in 'EPA-naïve' individuals (p=0.075). EPA-FFA also demonstrated antiangiogenic activity in vitro. In the first 18 months after CRCLM resection, EPA-FFA-treated individuals obtained OS benefit compared with placebo, although early CRC recurrence rates were similar. CONCLUSIONS: EPA-FFA therapy is safe and well tolerated in patients with advanced CRC undergoing liver surgery. EPA-FFA may have antiangiogenic properties. Remarkably, limited preoperative treatment may provide postoperative OS benefit. Phase III clinical evaluation of prolonged EPA-FFA treatment in CRCLM patients is warranted. TRIAL IDENTIFIER: ClinicalTrials.gov NCT01070355.

Mucosal biomarkers of colorectal cancer risk do not increase at 6 months following sleeve gastrectomy, unlike gastric bypass.

OBJECTIVE: The hypothesis that sleeve gastrectomy (SG) is not associated with an increase in mucosal colorectal cancer (CRC) biomarkers, unlike Roux-en-Y gastric bypass (RYGB), was tested. DESIGN AND METHODS: Rectal mucosa, blood, and urine were obtained from morbidly obese patients (n = 23) before and after (median 28 months) SG, as well as from nonobese controls (n = 20). Rectal epithelial cell mitosis and apoptosis, crypt size/fission, and pro-inflammatory gene expression were measured, as well as systemic inflammatory biomarkers, including C-reactive protein (CRP). RESULTS: The mean pre-operative body mass index in SG patients was 65.7 kg/m2 (24.7 kg/m2 in controls). Mean excess weight loss post-SG was 38.2%. There was a significant increase in mitosis frequency, crypt size, and crypt fission (all P < 0.01) in SG patients versus controls, as well as evidence of a chronic inflammatory state (raised CRP and mononuclear cell p65 NFκB binding), but there was no significant change in these biomarkers after SG, except CRP reduction. Macrophage migration inhibitory factor mRNA levels were increased by 39% post-SG (P = 0.038). CONCLUSIONS: Mucosal biomarkers of CRC risk do not increase at 6 months following SG, unlike RYGB. Biomarkers of rectal crypt proliferation and systemic inflammation are increased in morbidly obese patients compared with controls.