Mass Spectrometry-Based Targeted Proteomics as a Tool to Elucidate the Expression and Function of Intestinal Drug Transporters

Drug Metabolism and Disposition

Neuhoff

et al. 2016

Relative expression factors (REFs) are used to scale in vitro transporter kinetic data via in vitro-in vivo extrapolation linked to physiologically based pharmacokinetic (IVIVE-PBPK) models to clinical observations. Primarily two techniques to quantify transporter protein expression are available, immunoblotting and liquid chromatographytandem mass spectrometry. Literature-collated REFs ranged from 0.4 to 5.1 and 1.1 to 90 for intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), respectively. The impact of using human jejunum-Caco-2 REFs for P-gp (REF iP-gp ) and BCRP (REF iBCRP ), generated from the same samples and using different proteomic methodologies from independent laboratories, on PBPK outcomes was assessed. A 5-fold decrease in REF iP-gp for a single oral dose of digoxin resulted in a 1.19-and 1.31-fold higher plasma area under the curve and C max , respectively. All generated REF iP-gp values led to simulated digoxin C max values within observed ranges; however, combining kinetic data generated from a different laboratory with the 5-fold lower REF iP-gp could not recover a digoxin-rifampicin drug-drug interaction, emphasizing the necessity to obtain transporter-specific kinetic estimates and REFs from the same in vitro system. For a theoretical BCRP compound, with absorption taking place primarily in the jejunum, a decrease in the REF iBCRP from 2.22 (University of Manchester) to 1.11 (Bertin Pharma) promoted proximal intestinal absorption while delaying t max 1.44-fold. Laboratory-specific differences in REF may lead to different IVIVE-PBPK outcomes. To understand the mechanisms underlying projected pharmacokinetic liabilities, it is important to assess the potential impact of bias on the generation of REFs on an interindividual basis within a target population.

Section: Methodsmentioning

confidence: 99%

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-glycoprotein and Breast Cancer Resistance Protein: Part II. The Impact of Cross-Laboratory Variations of Intestinal Transporter Relative Expression Factors on Predicted Drug Disposition

Drug Metabolism and Disposition

Neuhoff

et al. 2016

“…This study provides a validated LC-MS/MS method using QconCAT derived proteotypic standard peptides to measure the abundances of 6 key drug transporter proteins in human distal jejunum (n = 3) and distal ileum (n = 1), adding to the limited data currently available that quantifies the absolute abundance of transporter proteins in the human intestine using targeted proteomic techniques [6][7][8]. The advantage that the QconCAT technology provides over conventional QTAP techniques is the ability to use one standard for a host of target proteins, making it more amenable to simultaneous multiplexed quantitative analysis, in addition to the sustainability of the expressed standard and transferability of the QconCAT vector providing an unlimited source of standard peptides in different laboratories, for lower cost when analysing ≥10 proteins [16].…”

Section: Application Of Methodsmentioning

confidence: 99%

“…Recently, studies have employed LC-MS/MS-based quantitative targeted absolute proteomic strategies, to quantify the absolute abundances of transporter proteins in human intestines by an absolute quantification (AQUA) strategy for generating synthetic isotope labelled peptides [6][7][8]. The QconCAT technique is an alternative method for generating proteotypic isotope labelled peptide standards [9].…”

Section: Introductionmentioning

confidence: 99%

Application of an LC–MS/MS method for the simultaneous quantification of human intestinal transporter proteins absolute abundance using a QconCAT technique

Journal of Pharmaceutical and Biomedical Analysis

Russell

et al. 2015

Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments. A quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) together with a quantitative concatenation (QconCAT) strategy to provide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase; Na/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast cancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A (Organic Solute Transporter subunit alpha; OST-α), in human distal jejunum (n=3) and distal ileum (n=1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were optimised to enable quantification of the proteotypic peptides for each transporter. After harvesting enterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were subjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a gravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard (correlation coefficient, R(2)=0.998) and quantification of all target peptides in a pooled intestinal quality control sample (R(2)≥ 0.980) was established. The assay was also assessed for within and between-day precision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs, over 2 days. The methods were applied to obtain the absolute abundances for all targeted proteins. In all samples, Na/K-ATPase, HPT1, P-gp and BCRP were detected above the lower limit of quantitation (i.e., >0.2 fmol/μg membrane protein). MRP2 abundance could be quantified in distal jejunum but not in the distal ileum sample. OST-α was not detected in 2 out of 3 jejunum samples. This study highlights the utility of a QconCAT strategy to quantify absolute transporter abundances in human intestinal tissues.

“…In fact, several laboratories foster links with the forerunning developers of these methodologies, i.e., Tohoku University (Sendai, Japan) and Pfizer Ltd. (Groton, CT). Peptides that are used as surrogates for the quantification of the complete protein are selected on the basis of a variety of criteria Oswald et al, 2013;Prasad and Unadkat, 2014b;Qiu et al, 2014). Consequently, for the frequently quantified transporter-protein P-gp, three peptides are routinely employed to quantify human P-gp (AGAVAEEVLAAIR, NTTGALTTR, FYD-PLAGK; Table 2), highlighting a current lack of consensus among groups.…”

Section: Interlaboratory Methods Differences Between Groups Quantifyinmentioning

confidence: 99%

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells

Drug Metabolism and Disposition

Neuhoff

et al. 2015

Over the last 5 years the quantification of transporter-protein absolute abundances has dramatically increased in parallel to the expanded use of in vitro-in vivo extrapolation (IVIVE) and physiologically based pharmacokinetics (PBPK)-linked models, for decisionmaking in pharmaceutical company drug development pipelines and regulatory submissions. Although several research groups have developed laboratory-specific proteomic workflows, it is unclear if the large range of reported variability is founded on true interindividual variability or experimental variability resulting from sample preparation or the proteomic methodology used. To assess the potential for methodological bias on end-point abundance quantification, two independent laboratories, the University of Manchester (UoM) and Bertin Pharma (BPh), employing different proteomic workflows, quantified the absolute abundances of Na/K-ATPase, P-gp, and breast cancer resistance protein (BCRP) in the same set of biologic samples from human intestinal and Caco-2 cell membranes. Across all samples, P-gp abundances were significantly correlated (P = 0.04, Rs = 0.72) with a 2.4-fold higher abundance (P = 0.001) generated at UoM compared with BPh. There was a systematically higher BCRP abundance in Caco-2 cell samples quantified by BPh compared with UoM, but not in human intestinal samples. Consequently, a similar intestinal relative expression factor (REF), derived from distal jejunum and Caco-2 monolayer samples, between laboratories was found for P-gp. However, a 2-fold higher intestinal REF was generated by UoM (2.22) versus BPh (1.11). We demonstrate that differences in absolute protein abundance are evident between laboratories and they probably result from laboratoryspecific methodologies relating to peptide choice.