Determination of liver organoid- and spheroid induced drug metabolites using liquid chromatography-mass spectrometry

Abstract

The liver is the main metabolizing organ in the human body, and several in vitro models have been developed to resemble hepatic drug metabolism. However, commonly used in vitro models such as human liver microsomes (HLMs), S9 fraction, and hepatocytes lack the complexity of the corresponding in vivo tissue, which limits the in vivo resemblance. Liver organoids are three-dimensional tissue models typically derived from induced pluripotent stem cells (iPSCs) and are intended to recapitulate physiological functions of the human liver. Liver spheroids are similar 3D culture systems but lack the multicellularity that characterizes the organoids. Few studies have investigated drug metabolism in liver organoids- and spheroids. The aim of this study was, therefore, to explore liver organoid- and spheroid drug metabolism using liquid chromatography-mass spectrometry (LC-MS) as the liver organoids- and spheroids can be more representative to the in vivo situation. Additionally, an LC-MS method was to be established at the Department of Chemistry for drug metabolite detection. Drug incubated samples were analyzed using a validated LC-MS method at the Department of Forensic Sciences or by the LC-MS method established at the Department of Chemistry. Heroin was chosen as the model drug, and heroin metabolism studies were initially carried out in HLMs to establish a standardized conventional approach for later drug metabolism studies in organoids and spheroids. The concentrations of heroin and its phase I metabolites, 6-monoacetylmorphine (6-MAM) and morphine were measured over time (20 minutes). The heroin metabolism method was downscaled by decreasing the HLM concentration (from 2 mg/mL to 0.1 mg/mL) and the heroin concentration (from 1 μM to 0.1 μM). The downscaled method was applied to the S9 fraction and later executed with the addition of exogenous cofactors to look for the phase II metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). With the addition of cofactors, M3G could be quantified in the S9 fraction. Preliminary heroin metabolism studies in iPSC derived liver organoids showed that the organoids metabolized heroin (1 μM) to 6-MAM and morphine. The phase II metabolites M3G and M6G were also identified but below the limit of quantification (LOQ). Both the phase I- and phase II metabolites were detected above LOQ in liver spheroid samples after incubation in 10 M heroin. To sum up, the detection of heroin drug metabolites using LC-MS showed that the liver organoids- and spheroids had heroin metabolizing properties. Additionally, LC-MS proved to be a valuable tool for detection of liver organoid induced drug metabolites.