Application of microarrays to develop an in vitro in vivo correlation screening toolbox

该项目旨在进行一种体外测试，可以预测通过胃肠道转运体网络吸收的重新配方药物的吸收速度和程度，从而有效地测量药物的渗透性和吸收。调节药物吸收的两个关键参数是剂量对溶解度(主要是难于溶解的药物)和细胞膜通透性(主要是通过转运体网络吸收的药物)，这两个参数分别决定了吸收的速度和程度。在体外试验和体内数据之间，被动渗透胃肠道的药物分子显示出高度的相关性。然而，通过载体介导的转运系统(促进扩散、主动转运)吸收的药物在体外表现出虚假的体内相关性(IVIVC)，从而导致对大量动物实验的高度依赖。该项目将使用重新配制为口服液制剂时生物利用度数据非常少或很差的候选药物(血管紧张素转换酶抑制剂)。Pharmaspec专门从事针对老龄化人口和儿童的现有药物的药物重新配方，并热衷于合作研究体外、强大和高效的药物渗透性评估方法。这将使Pharmaspec能够建立和提供准确的剂量方案数据，以期提高所提供的医疗保健的质量。作为起点，Pharmaspec已经起草了一份药物分子清单，这些药物分子经常作为重新配方的口服液药物开给老年人，但缺乏关于重新配方后生物学性能的足够数据。腺癌细胞系(Caco 2)是监管机构和行业批准的渗透性研究的金标准。然而，使用这种细胞系的研究低估了药物的吸收，因为它测量的是药物吸收的量，而没有考虑涉及的转运蛋白机制。使用微阵列研究控制药物和离子运输的基因表达谱(包括时间过程分析)将是对Caco 2细胞系的一大改进，因为它们将能够使用演示跨胃肠道运输的体外模型来评估转录变化。这项拟议的研究源于阿斯顿大学进行的一项初步研究，该研究涉及研究通过载体介导的转运吸收的模型药物(吲哚美辛)在体外吸收过程中发生的转录变化(Khan等人，2009年)。研究表明，药物分子的高渗透性是由于溶质载体转运体和ATP结合盒转运体系统之间的协同作用。以往的研究表明，吲哚美辛的通透性独立于其他转运体系统，并受ATP结合盒转运体系统的影响。目前的方案将利用基因芯片研究渗透过程中转运蛋白网络系统内基因表达的变化，并建立并验证渗透研究实验装置。这项研究的目的是调查在体外和体内研究中表达的程度(定量分析)与实际吸收的药物量之间的相关性。通过调查药物运输过程中发生的潜在基因组变化来开发IVIVC还将导致建立一种适用于指导和优化药物产品开发的质量控制工具，并作为生物利用度研究的替代工具。Khan等人，(2009)渗透性研究中配方对基因组签名影响的调查。药学与药理学杂志，61，1，69-70

This project aims to work towards an in vitro test that can predict the rate and extent of absorption of reformulated drugs that are taken up via the transporter network through the gastrointestinal tract, allowing, drug permeability and absorption to be measured effectively. The two key parameters that regulate drug absorption are dose to solubility (mainly for poorly water soluble drugs) and cell membrane permeability (mainly for drugs that are absorbed with the aid of a transporter network) which in turn dictate the rate and extent of absorption respectively. Drug molecules that permeate passively across the gastrointestinal tract exhibit a high degree of correlation between in vitro tests and in vivo data. However drugs that are absorbed via the carrier mediated transport systems (facilitated diffusion, active transport) show spurious in vitro in vivo correlation (IVIVC) thereby resulting in high reliance on a large number of animal experiments. The project will utilise drug candidates (angiotensin converting enzyme inhibitors) for which there is very little or poor bioavailability data when reformulated as oral liquid preparations. Pharmaspec specialise in drug reformulation of existing medicines specifically for the ageing population and children and are keen to collaborate in studying in vitro, robust and efficient methods of assessing drug permeability. This will enable the Pharmaspec to establish and provide accurate dose regimen data with a view to improving the quality of healthcare provided. As a starting point, Pharmaspec have drawn up a list of drug molecules that are frequently prescribed to the elderly population as reformulated oral liquid medicines but that lack sufficient data on biological performance upon reformulation. The adenocarcinoma cell line (Caco2) is an approved gold standard by both the regulatory authorities and industry for permeability studies. However, studies using this cell line underestimate drug absorption as it measures the amount of drug absorbed without any consideration of transporter mechanisms involved. The investigation of gene expression profiles (including time course analysis) that control drug and ion transport using microarrays will be a big improvement on the Caco2 cell line as they will enable assessment of changes in transcription using in vitro models demonstrating transport across the gastrointestinal tract. The proposed research has stemmed from a pilot study conducted at Aston University which involved the investigation of trancriptomic changes that occur during in vitro absorption for a model drug (indomethacin) that is absorbed through carrier mediated transport (Khan et al 2009). The study has shown that higher permeability of drug molecules is due to synergy between solute carrier transporters and the ATP binding cassette transporters system. Previous studies had suggested that permeability of indomethacin is independent of other transporter systems and is influenced by ATP binding cassette transporters system. The current proposal will study the gene expression changes within the transporter network system during the process of permeation using microarray and an established and validated permeation study experimental set up. The study will aim to investigate the correlation between the extent of expression (quantitative analysis) with the actual amount of drug absorbed both during in vitro and in vivo studies. The development of an IVIVC by investigating the underlying genomic changes that occur during drug transport will also result in the establishment of a quality control tool applicable to guiding and optimising the development of drug product and serving as a surrogate for bioavailability studies. Khan et al, (2009) Investigation of the influence of formulation on genomic signature during permeability studies. Journal of Pharmacy and Pharmacology, 61, 1, 69-70