Direct Quantification of Cytochromes P450 and Drug Transporters—A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates

Abstract

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Quantitative mass spectrometry-based proteomics in the era of model-informed drug development: Applications in translational pharmacology and recommendations for best practice

“…Quantitative proteomic techniques have therefore been implemented by different laboratories worldwide for various pharmacology applications, leading to improved extrapolation of drug pharmacokinetics (Doki et al, 2018;Kumar et al, 2018) and better understanding of the effects various factors, including age (Ladumor et al, 2019;van Groen et al, 2018), ethnicity (Kawakami et al, 2011;Ladumor et al, 2019;Peng, Bacon, Zheng, Guo, & Wang, 2015), and genetics (Bhatt et al, 2019;Peng et al, 2015;Weiß et al, 2018) on the expression of enzymes and transporters.…”

“…The AQUA-MRM approach is the most widely used quantification method in pharmacokinetic research and has been used to quantify various enzymes and transporters in different human tissues. Quantified enzymes include CYP and UGT enzymes in liver (Cieślak et al, 2016;Fallon, Neubert, Hyland, Goosen, & Smith, 2013;Hansen et al, 2019;Ohtsuki et al, 2012;Prasad et al, 2018;Sato, Nagata, Kawamura, Miyashita, & Usui, 2012;Sato et al, 2014;Weiß et al, 2018), intestine (Drozdzik et al, 2018;Gröer et al, 2014;Harbourt et al, 2012;Sato et al, 2014) and Kidney (Harbourt et al, 2012;Knights et al, 2016;Sato et al, 2014). In brain, the AQUA-MRM workflow was used to quantify CYPs, glutathione Stransferases (GSTs) and catechol O-methyltransferase (COMT) (Shawahna et al, 2011).…”

From Endogenous Compounds as Biomarkers to Plasma‐Derived Nanovesicles as Liquid Biopsy; Has the Golden Age of Translational Pharmacokinetics‐Absorption, Distribution, Metabolism, Excretion‐Drug–Drug Interaction Science Finally Arrived?

“…For CYP2D6 (e.g., *4 allele) and CYP2C19 (e.g., *2 allele), clinical genotype‐phenotype (drug probe–based) associations have been investigated extensively, but such clinical studies are rarely supported by tissue biopsy. Typically, CYP2D6 and CYP2C19 quantitation, either by LC‐MS–based proteomics or immunoblotting, leverages commercially available tissue from genotyped organ donors . The availability of tissue‐derived plasma exosomes could certainly facilitate the building of genotype‐expression‐phenotype associations for larger sets of individual subjects.…”

“…As described above, CYP3A4 is complex because of its expression in the gut and liver. Moreover, it is expressed polymorphically, as are other members of the CYP3A subfamily (e.g., CYP3A5 and CYP3A7) . Unfortunately, most CYP3A substrates are not selective for each individual CYP3A form, and so intersubject variability cannot be addressed with standard probe or current biomarker‐based approaches.…”

Profiling of Drug-Metabolizing Enzymes and Transporters in Human Tissue Biopsy Samples: A Review of the Literature

“…It is well established that numerous human CYP forms, such as CYP2D6, CYP2C19, and CYP2C9, are expressed polymorphically, and efforts have focused on relating genotype to its protein expression in the human liver (Weiß et al, 2018). Such efforts are important because the correlation of genotype with plasma-based phenotypic trait measures is not always apparent (Waring, 2020).…”