Intravenous lipid emulsions: engineering and biological characterisation of new generation emulsions

目的：本项目旨在提供基本理解，以支持临床静脉注射用新一代脂肪乳剂的设计和早期开发，这些脂肪乳剂基于工业申办者感兴趣的组分。它将提供新的和基本的理解乳剂制剂从新的组成部分，并连接这与生物学特性的制剂相关的临床应用。背景资料：静脉内乳剂是甘油三酯的水包油乳剂，临床上用作药物给药的载体或患者（特别是早产儿）的胃肠外营养。在过去十年中，基于对脂肪乳剂代谢的了解增加，静脉乳剂的典型组成已发生变化，以减少不良反应（例如20% vs 10%组成，因为后者具有较高水平的游离脂质，导致脂蛋白脂肪酶过饱和）。该领域目前的概念是替代通常使用的甘油三酯并探索新的组分以减少不良反应（例如减少炎性类二十烷酸或避免亚油酸过载）和/或提供积极的治疗效果（例如ω-3酸对胎儿和婴儿视网膜和认知发育的影响）。然而，许多最近的出版物使用经典的配方方案，特别添加新的组分，没有证据表明在基础水平上研究这些新的添加物对乳液配方和性能的影响。此外，需要研究这些新添加物对脂解、细胞摄取和途径以及免疫学的影响，以评估新制剂的潜在临床用途。拟议项目的目标将分三个阶段实现。第一阶段。处方和理化特性：将应用实验设计和实验室分析来评估几种不同脂质混合的效果（包括巴克斯特感兴趣的化合物）对（i）界面行为，（ii）脂相粘度及其对粉碎过程效率的影响，（iii）乳液液滴的电荷和尺寸分布，（iv）乳液稳定性，粘度和乳液“分解”。物理化学表征将包括：粒度、zeta电位、形态、流变学、界面张力和界面流变学。第二阶段。体外生物学表征：已确定许多因素（包括脂质组成）会影响脂肪乳剂的清除和代谢。因此，2期研究重点关注“1期”脂肪乳剂的脂解和细胞代谢的体外研究。将测量游离脂质水平。将在掺入3 H-胆固醇油酰醚和/或荧光探针后研究细胞缔合和内化。在不同条件（例如外源性apoE）下，将标记乳剂应用于培养细胞（J774巨噬细胞、HUVEC内皮细胞和原代肝细胞）。将评估细胞沉积和脂质利用。第三阶段潜在免疫调节作用的分析：新型静脉乳剂的潜在不良免疫调节特性存在安全性问题，特别是因为这些药物经常用于免疫功能低下的患者。将通过对单核细胞、T细胞、树突状细胞（DC）和中性粒细胞的影响评估“第1阶段”乳剂的免疫调节作用，如下所示。将用乳剂样品刺激外周血单核细胞，并将使用细胞内细胞因子染色和多色FACS分析来测量细胞因子产生的水平。将评估用乳液样品处理后的DC功能特性（内吞作用和T细胞活化）。将研究乳剂对中性粒细胞呼吸爆发、吞噬能力和细胞因子谱的影响。

Aim: This project aims to provide fundamental understanding to underpin the design and early development of new generation lipid emulsions for intravenous application in clinic, which are based on components of interest to the industrial sponsor. It will provide new and fundamental understanding of emulsion formulations from new components and connect this with the biological properties of the formulations relevant for clinical application. Background: Intravenous emulsions are oil-in-water emulsions of triglycerides and are clinically applied as vehicles for administration of drugs or as the parenteral nutrition in patients, and particularly, premature neonates. In the last decade, and based on increased understanding of lipid emulsion metabolism, the typical compositions of intravenous emulsions have been changed to reduce adverse effects (eg 20% vs 10% composition as the latter has higher level of free lipids causing oversaturation of the lipoprotein lipase). The current notion in the field is to replace typically used triglycerides and to explore new components to reduce adverse effects (eg reduce inflammatory eicosanoids or avoid linoleic acid overload) and/or to provide a positive therapeutic effect (eg omega-3 acids effects on retinal and cognitive development of foetus and infants). However, a number of recent publications use classical formulation protocols with an ad hoc addition of new components, without evidence of study at the fundamental level what effects these new additions have on the emulsion formulation and properties. Moreover, studies on the effect of these new additions on the lipolysis, cellular uptake and pathways, and immunology will be required to assess the potential clinical usefulness of the new formulations. The aim of the proposed project will be achieved in three phases. Phase 1. Formulation and physicochemical characterisation: Experimental design and laboratory analyses will be applied to assess the effect that mixing of several different lipids (including compounds of interest to Baxter) has on (i) interfacial behaviour, (ii) lipid phase viscosity and its impact on the efficiency of the comminution process, (iii) charge and size distribution of the emulsion droplets (iv) emulsion stability, viscosity and emulsion 'break-down'. The physicochemical characterisation will include: particle size, zeta potential, morphology, rheology, interfacial tension and interfacial rheology. Phase 2. In vitro biological characterisation: A number of factors, including lipids composition, have been identified to impact the clearance and metabolism of lipid emulsions. Phase 2 hence focuses on in vitro studies of lipolysis and cellular metabolism of lipid emulsions from 'Phase 1'. The level of free lipids will be measured. Cellular association and internalization will be studied following incorporation of 3H-cholesteryl oleoyl ether, and/or a fluorescent probe. Labelled emulsions will be applied to the cells in culture (J774 macrophage, HUVEC endothelial and primary hepatocytes) under different conditions (eg exogenous apoE). Cell deposition and utilization of lipids will be assessed. Phase 3. Analyses of potential immuno-modulatory effects: The potential adverse immune-modulatory properties of new intravenous emulsions present a safety concern, particularly as these are often used in immune-compromised patients. Immune-modulatory effects for emulsions from 'Phase 1' will be assessed through their impact on monocytes, T cells, dendritic cells (DC) and neutrophils, as follows. Peripheral blood monocytes will be stimulated with emulsion samples and intracellular cytokine staining and multi-colour FACS analysis will be used to measure the level of cytokine production. DCs functional properties (endocytosis and T cell activation) after treatment with emulsion samples will be assessed. The impact of emulsions on neutrophils' respiratory burst, phagocytic ability and cytokine profile will be studied.