Extracellular vesicles as dynamic surrogates for drug transporter expression. A new tool to personalize pharmacotherapy?

细胞外囊泡作为药物转运蛋白表达的动态替代物。

• 批准号: 509856975
• 负责人: Dr. Juan Pablo Rigalli
• 金额: --
• 依托单位: Abteilung Klinische Pharmakologie und Pharmakoepidemiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2022
• 资助国家: 德国
• 起止时间: 2021-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/509856975?language=en
• 关键词: Extracellular; vesicles; dynamic; surrogates; drug

The P-glycoprotein (P-gp/ABCB1), the multidrug resistance-associated protein 2 (MRP2/ABCC2) and the breast cancer resistance protein (BCRP/ABCG2) belong to the superfamily of ABC transporters (ATP binding cassette) and play a major role in drug pharmacokinetics. They are expressed in organs and tissues of high pharmacological relevance such as the liver, the kidneys, the intestine and the blood-brain barrier. In addition, they account for the resistance towards chemotherapeutic agents in cancer cells. The expression of ABC transporters is highly variable within the population and can be regulated by coadministered drugs, the diet and hormones, among other factors. This way, significant differences in the drug clearance between individuals as well as during the lifespan of an individual can be expected. In general, higher transporter expression leads to higher drug clearance and therapy failure. Lower transporter expression leads to lower drug clearance and toxicity. Thus, the individualized estimation of the expression of ABC transporters could aid to adjust the therapy to avoid cases of over- and under-exposure. So far, repeated extraction of biopsies would be the only option to measure ABC transporter expression in relevant tissues. However, due to its intrinsic invasive character, this cannot be performed routinely.Extracellular vesicles (EVs) are nanoparticles released by all cell types of the organism. The EV cargo consists of cellular proteins, RNA, and lipids, the array of these components representing a fingerprint of the cell of origin. The presence of EVs from a wide range of tissues in the blood, plus the presence of kidney EVs in the urine, would allow for an easy access to these nanoparticles in the clinical practice. The general aim of this project is to investigate the potential of the ABC transporter-cargo in EVs as a dynamic surrogate for the transporter expression in normal and tumoral cells and thus, for their drug excretion capacity. We will develop, optimize and validate an ultrasensitive UPLC-MS/MS assay for the absolute and simultaneous quantification of P-gp, MRP2 and BCRP in EVs and the cells of origin. This assay will be applied to analyze the correlation between the transporter expression in EVs and the expression and activity in the cells both under basal and inducing conditions. In tumor cells, the correlation between the transporter expression in EVs and the resistance to chemotherapeutic agents will also be investigated. Finally, a method to isolate cell-specific EVs from a multicellular model (blood-brain-barrier organoids) will be developed in order to elucidate the potential of EVs to estimate the cell-specific transporter expression in a more complex system. Altogether, the use of EVs to estimate the transporter expression could allow to stratify the patients based on their drug clearance capacity and, thus, adjust pharmacotherapy and, ultimately, prevent situations of toxicity or therapy failure.

P-糖蛋白（P-gp/ABCB 1）、多药耐药相关蛋白2（MRP 2/ABCC 2）和乳腺癌耐药蛋白（BCRP/ABCG 2）属于ABC转运蛋白（ATP结合盒）超家族，在药物药代动力学中发挥重要作用。它们在具有高度药理学相关性的器官和组织中表达，例如肝脏、肾脏、肠和血脑屏障。此外，它们还导致癌细胞对化疗剂的耐药性。ABC转运蛋白的表达在人群中存在很大差异，并且可以通过联合用药、饮食和激素等因素进行调节。这样，可以预期个体之间以及个体生命周期内的药物清除率存在显着差异。通常，较高的转运蛋白表达导致较高的药物清除率和治疗失败。较低的转运蛋白表达导致较低的药物清除率和毒性。因此，ABC转运蛋白表达的个体化估计可以帮助调整治疗，以避免过度暴露和暴露不足的情况。到目前为止，重复提取活检组织将是测量相关组织中ABC转运蛋白表达的唯一选择。细胞外囊泡（Extracellular vesicles，EVs）是由生物体所有类型的细胞释放的纳米颗粒。EV货物由细胞蛋白质、RNA和脂质组成，这些组分的阵列代表了起源细胞的指纹。来自血液中广泛组织的EV的存在，加上尿液中肾脏EV的存在，将允许在临床实践中容易地获得这些纳米颗粒。该项目的总体目标是研究EV中ABC转运蛋白-货物作为正常和肿瘤细胞中转运蛋白表达的动态替代物的潜力，从而研究其药物排泄能力。我们将开发、优化和验证一种超灵敏的UPLC-MS/MS测定法，用于绝对和同时定量EV和来源细胞中的P-gp、MRP 2和BCRP。该测定将用于分析EV中转运蛋白表达与基础和诱导条件下细胞中的表达和活性之间的相关性。在肿瘤细胞中，还将研究EV中的转运蛋白表达与对化疗剂的抗性之间的相关性。最后，将开发一种从多细胞模型（血脑屏障类器官）中分离细胞特异性EV的方法，以阐明EV在更复杂系统中估计细胞特异性转运蛋白表达的潜力。总之，使用EV来估计转运蛋白表达可以允许基于患者的药物清除能力对患者进行分层，从而调整药物治疗，并最终防止毒性或治疗失败的情况。