斑马鱼(ZF;Daniorerio)幼虫已成为药物代谢研究中很有前途的体内模型。这里,我们着手准备用于集成质谱成像(MSI)的模型,以全面研究ZF幼虫体内药物及其代谢物的空间分布。在我们的试点研究中,总体目标是改进ZF幼虫的MSI方案,我们研究了阿片类拮抗剂纳洛酮的代谢。我们证实纳洛酮的代谢修饰与HepaRG细胞中检测到的代谢物高度一致,人体生物样本,和其他体内模型。特别是,在ZF幼虫模型中检测到所有三种主要的人类代谢产物。接下来,使用LC-HRMS/MS研究了纳洛酮在ZF幼虫的三个身体部位的体内分布,表明阿片拮抗剂主要存在于头部和身体部位,从公布的人类药理学数据中怀疑。具有针对MSI的优化样品制备程序(即,嵌入层组成,冷冻切片,和基质组成和喷涂),我们能够记录纳洛酮及其代谢物在ZF幼虫中的MS图像,提供信息丰富的分布图像。总之,我们证明了所有主要的ADMET(吸收,分布,新陈代谢,排泄,和毒性)参数,作为体内药代动力学研究的一部分,可以在简单且具有成本效益的ZF幼虫模型中进行评估。我们建立的ZF幼虫使用纳洛酮的协议是广泛适用的,特别是对于MSI样品制备,各种类型的化合物,它们将有助于预测和理解人体代谢和药代动力学。
Zebrafish (ZF; Danio rerio) larvae have emerged as a promising in vivo model in drug metabolism studies. Here, we set out to ready this model for integrated mass spectrometry imaging (MSI) to comprehensively study the spatial distribution of drugs and their metabolites inside ZF larvae. In our pilot study with the overall goal to improve MSI protocols for ZF larvae, we investigated the metabolism of the opioid antagonist naloxone. We confirmed that the metabolic modification of naloxone is in high accordance with metabolites detected in HepaRG cells, human biosamples, and other in vivo models. In particular, all three major human metabolites were detected at high abundance in the ZF larvae model. Next, the in vivo distribution of naloxone was investigated in three body sections of ZF larvae using LC-HRMS/MS showing that the opioid antagonist is mainly present in the head and body sections, as suspected from published human pharmacological data. Having optimized sample preparation procedures for MSI (i.e., embedding layer composition, cryosectioning, and matrix composition and spraying), we were able to record MS images of naloxone and its metabolites in ZF larvae, providing highly informative distributional images. In conclusion, we demonstrate that all major ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters, as part of in vivo pharmacokinetic studies, can be assessed in a simple and cost-effective ZF larvae model. Our established protocols for ZF larvae using naloxone are broadly applicable, particularly for MSI sample preparation, to various types of compounds, and they will help to predict and understand human metabolism and pharmacokinetics.