Targeted microbubbles carrying lipid-oil-nanodroplets for ultrasound-triggered delivery of the hydrophobic drug, combretastatin A4.

The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC-MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery.

Ultrasound-triggered therapeutic microbubbles enhance the efficacy of cytotoxic drugs by increasing circulation and tumor drug accumulation and limiting bioavailability and toxicity in normal tissues.

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, 89Zr 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. 89Zr 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.

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk.

Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (- 1.9 (- 2.7; - 1.1) mmHg in men and - 2.5 (- 3.3; - 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale.

Evaluation of (-)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake.

Data from dietary intervention studies suggest that intake of (-)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (-)-epicatechin metabolites (SREM), particularly (-)-epicatechin-3'-glucuronide, (-)-epicatechin-3'-sulfate and 3'-O-methyl-(-)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (-)-epicatechin intake. SREMB in urine proved to be a specific indicator of (-)-epicatechin intake, showing also a strong correlation with the amount of (-)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (-)-epicatechin as SREMB in 24 h urine was 10% (IQR 7-13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (-)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (-)-epicatechin.

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

A study of inter-individual variability in the Phase II metabolism of xenobiotics in human skin.

Understanding skin metabolism is key to improve in vitro to in vivo extrapolations used to inform risk assessments of topically applied products. However, published literature is scarce and usually covers a limited and non-representative number of donors. We developed a protocol to handle and store ex vivo skin samples post-surgery and prepare skin S9 fractions to measure the metabolic activity of Phase II enzymes. Preincubation of an excess of cofactors at 37 °C for fifteen minutes in the S9 before introduction of the testing probe, greatly increased the stability of the enzymes. Using this standardised assay, the rates of sulphation (SULT) and glucuronidation (UGT) of 7-hydroxycoumarin, methylation (COMT) of dopamine and N-acetylation (NAT) of procainamide were measured in the ng/mg protein/h (converted to ng/cm2/h) range in eighty-seven individuals. Glutathione conjugation (GST) of 1-chloro-2,4-dinitrobenzene was assessed in a smaller pool of fifty donors; the metabolic rate was much faster and measured over six minutes using a different methodology to express rates in µg/mg protein/min (converted to µg/cm2/min). A comprehensive statistical analysis of these results was carried out, separating donors by age, gender and metabolic rate measured.