Proteomic characterisation of drug metabolising enzymes and drug transporters in pig liver.

Liver enzymes and transporters play an essential role in xenobiotic metabolism, distribution and elimination. Pre-clinical safety assessment relies on studies on animal models, including the (mini)pig. The pig shares many anatomical and physiological characteristics with humans, and there is currently a gap in information about porcine metabolism and disposition pathways and their similarities and differences from human ones.Three different sample preparation methods (filter-aided sample preparation (FASP), enhanced FASP (eFASP) and in-solution sample preparation) were used to prepare porcine liver tissue (two samples) for proteomic analysis. The analysis relied on rapid-separation liquid chromatography coupled to Orbitrap mass spectrometry in data-dependent acquisition mode. MASCOT was used for identification and relative label-free quantification was based on spectral counting.The three sample preparation methods provided complementary results, allowing characterisation of approximately 70 pharmacologically relevant proteins. The main quantified proteins included 16 cytochrome P450 (CYP) enzymes, 5 UGT enzymes, and 11 transporters. In addition, 20 Phase I and 14 Phase II enzymes were also characterised. Inter-operator differences were negligible and the pig liver pies for CYP, UGT and efflux transporter proteins were established. Human homologues of the quantified CYP, UGT and transporter proteins were identified.

Proteomic Quantification of Human Blood-Brain Barrier SLC and ABC Transporters in Healthy Individuals and Dementia Patients.

The blood-brain barrier (BBB) maintains brain homeostasis by controlling traffic of molecules from the circulation into the brain. This function is predominantly dependent on proteins expressed at the BBB, especially transporters and tight junction proteins. Alterations to the level and function of BBB proteins can impact the susceptibility of the central nervous system to exposure to xenobiotics in the systemic circulation with potential consequent effects on brain function. In this study, expression profiles of drug transporters and solute carriers in the BBB were assessed in tissues from healthy individuals ( n = 12), Alzheimer's patients ( n = 5), and dementia with Lewy bodies patients ( n = 5), using targeted, accurate mass retention time (AMRT) and global proteomic methods. A total of 53 transporters were quantified, 19 for the first time in the BBB. A further 20 novel transporters were identified but not quantified. The global proteomic method identified another 3333 BBB proteins. Transporter abundances, taken together with the scaling factor, microvessel protein content per unit tissue (BMvPGB also measured here), can be used in quantitative systems pharmacology models predicting drug disposition in the brain and permitting dose adjustment (precision dosing) in special populations of patients, such as those with dementia. Even in this small study, we see differences in transporter profile between healthy and diseased brain tissue.

Identification and quantification of blood-brain barrier transporters in isolated rat brain microvessels.

The blood-brain barrier (BBB) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make-up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15-fold enrichment of endothelial cell marker Glut1 mRNA, whereas markers for other cell types were not enriched. Filter-aided sample preparation was shown to be superior to in-solution sample preparation (10251 peptides vs. 7533 peptides). Label-free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP-binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

Global Proteomic Analysis of Human Liver Microsomes: Rapid Characterization and Quantification of Hepatic Drug-Metabolizing Enzymes.

Many genetic and environmental factors lead to interindividual variations in the metabolism and transport of drugs, profoundly affecting efficacy and toxicity. Precision dosing, that is, targeting drug dose to a well characterized subpopulation, is dependent on quantitative models of the profiles of drug-metabolizing enzymes (DMEs) and transporters within that subpopulation, informed by quantitative proteomics. We report the first use of ion mobility-mass spectrometry for this purpose, allowing rapid, robust, label-free quantification of human liver microsomal (HLM) proteins from distinct individuals. Approximately 1000 proteins were identified and quantified in four samples, including an average of 70 DMEs. Technical and biological variabilities were distinguishable, with technical variability accounting for about 10% of total variability. The biological variation between patients was clearly identified, with samples showing a range of expression profiles for cytochrome P450 and uridine 5'-diphosphoglucuronosyltransferase enzymes. Our results showed excellent agreement with previous data from targeted methods. The label-free method, however, allowed a fuller characterization of the in vitro system, showing, for the first time, that HLMs are significantly heterogeneous. Further, the traditional units of measurement of DMEs (pmol mg-1 HLM protein) are shown to introduce error arising from variability in unrelated, highly abundant proteins. Simulations of this variability suggest that up to 1.7-fold variation in apparent CYP3A4 abundance is artifactual, as are background positive correlations of up to 0.2 (Spearman correlation coefficient) between the abundances of DMEs. We suggest that protein concentrations used in pharmacokinetic predictions and scaling to in vivo clinical situations (physiologically based pharmacokinetics and in vitro-in vivo extrapolation) should be referenced instead to tissue mass.

Choice of LC-MS methods for the absolute quantification of drug-metabolizing enzymes and transporters in human tissue: a comparative cost analysis.

The quantification of drug-metabolizing enzymes and transporters is important for in vitro-in vivo extrapolation (IVIVE) of xenobiotic clearance, which has become an integral part of drug development. There are different mass spectrometry-based techniques used for quantitative proteomics, and as more laboratories are opting for the use of these methods, selecting the most appropriate tool is becoming a concern. For the first time, we attempt to determine the significance of cost of different LC-MS methods of quantitative analysis of these proteins and to present a framework to objectively assess the choice of the techniques. Based on our analysis, quantification using labeled internal standards is more expensive per sample but provides higher quality data than label-free quantification. Quantification using absolute quantification synthetic peptides is the approach of choice for analyzing less than nine proteins, whereas when quantifying a defined set of proteins (10-50), such as enzymes, in a reasonably large number of samples (20-100), the quantification concatemer technique is more economical, followed by label-free quantification. When analyzing proteomes or sub-proteomes (≥500 proteins), label-free quantification is more cost-effective than the use of labeled internal standards. A cost-benefit approach is described to assess the choice of the most appropriate mass spectrometry-based approach for the quantification of proteins relevant to IVIVE.

Translational value of liquid chromatography coupled with tandem mass spectrometry-based quantitative proteomics for in vitro-in vivo extrapolation of drug metabolism and transport and considerations in selecting appropriate techniques.

INTRODUCTION: Drug-metabolizing enzymes and transporters play an important role in drug absorption, distribution, metabolism and excretion and, consequently, they influence drug efficacy and toxicity. Quantification of drug-metabolizing enzymes and transporters in various tissues is therefore essential for comprehensive elucidation of drug absorption, distribution, metabolism and excretion. Recent advances in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) have improved the quantification of pharmacologically relevant proteins. AREAS COVERED: This report presents an overview of mass spectrometry-based methods currently used for the quantification of drug-metabolizing enzymes and drug transporters, mainly focusing on applications and cost associated with various quantitative strategies based on stable isotope-labeled standards (absolute quantification peptide standards, quantification concatemers, protein standards for absolute quantification) and label-free analysis. EXPERT OPINION: In mass spectrometry, there is no simple relationship between signal intensity and analyte concentration. Proteomic strategies are therefore complex and several factors need to be considered when selecting the most appropriate method for an intended application, including the number of proteins and samples. Quantitative strategies require appropriate mass spectrometry platforms, yet choice is often limited by the availability of appropriate instrumentation. Quantitative proteomics research requires specialist practical skills and there is a pressing need to dedicate more effort and investment to training personnel in this area. Large-scale multicenter collaborations are also needed to standardize quantitative strategies in order to improve physiologically based pharmacokinetic models.