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

p糖蛋白（P-gp/ABCB1）、多药耐药相关蛋白2 （MRP2/ABCC2）和乳腺癌耐药蛋白（BCRP/ABCG2）属于ABC转运蛋白（ATP结合盒）超家族，在药物药代动力学中起重要作用。它们在肝脏、肾脏、肠道和血脑屏障等具有高度药理意义的器官和组织中表达。此外，它们还解释了癌细胞对化疗药物的耐药性。在人群中，ABC转运蛋白的表达是高度可变的，可以通过共同给药、饮食和激素以及其他因素来调节。通过这种方式，可以预期个体之间以及个体寿命期间药物清除率的显著差异。一般来说，更高的转运蛋白表达导致更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，对ABC转运蛋白表达的个体化估计可以帮助调整治疗，以避免暴露过度和暴露不足的情况。到目前为止，反复提取活检是测量相关组织中ABC转运蛋白表达的唯一选择。然而，由于其固有的侵入性，这不能常规进行。细胞外囊泡（EVs）是由生物体的所有细胞类型释放的纳米颗粒。EV货物由细胞蛋白、RNA和脂质组成，这些成分的阵列代表了起源细胞的指纹。血液组织中广泛存在的ev，加上尿液中存在的肾脏ev，将使这些纳米颗粒在临床实践中很容易获得。该项目的总体目标是研究电动汽车中ABC转运-货物作为正常和肿瘤细胞中转运蛋白表达的动态替代品的潜力，从而研究它们的药物排泄能力。我们将开发、优化和验证一种超灵敏的UPLC-MS/MS检测方法，用于ev和起源细胞中P-gp、MRP2和BCRP的绝对和同时定量。本实验将用于分析在基础和诱导条件下EVs中转运蛋白表达与细胞表达和活性之间的相关性。在肿瘤细胞中，EVs转运蛋白表达与化疗药物耐药性之间的关系也将被研究。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根据患者的药物清除能力对患者进行分层，从而调整药物治疗，并最终防止出现毒性或治疗失败的情况。

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.