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

Abstract

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.

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

“…), optimization and standardization, such as this work, should lead to reliable quantification and subsequently valid pharmacokinetic/pharmacodynamic conclusions (Harwood et al . ; Rostami‐Hodjegan ). Inter‐species differences between rats and humans are highlighted, in line with published literature.…”

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…In contrast, the QconCAT-based dataset showed overall poor correlation with catalytic activity, with moderate correlations observed only for UGTs 1A1, 1A3, and 2B7. Therefore, these intermethodology differences suggest that effective characterization of ADME protein expression levels in different systems should ideally be complemented with specific activity data, preferably generated within the same system or tissue samples, as previously advocated (Harwood et al, 2016b). The abundance data related to UGT1A3 is an interesting case, where the expression data from the two laboratories correlated and both datasets correlated with CDCA glucuronidation activity.…”

“…A recent cross-laboratory study that looked into differences in measured abundances of two intestinal efflux transporters, P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), reported up to three-and fivefold differences in abundance measured in matched human jejunal and Caco-2 samples, respectively (Harwood et al, 2016a). However, the laboratory-specific IVIVE relative expression factors (REFs) generated for these proteins were within twofold for both transporters and did not reflect significant differences in simulated pharmacokinetic outcomes (Harwood et al, 2016b), suggesting that systematic bias in methodology can lead to valid IVIVE conclusions if more suitable study designs are implemented, where specific REF values of ADME proteins are generated in the same or a similar setting. In contrast, another cross-laboratory study that compared quantitative proteomic data of several ADME proteins (P450s, UGTs, and transporters) from six independent laboratories (Wegler et al, 2017) reported large interlaboratory differences that led, in the case of organic anion transporting polypeptide transporters, to significant discrepancies in simulated uptake clearance of atorvastatin.…”

Non-animal approaches for toxicokinetics in risk evaluations of food chemicals

“…Co-culturing with HT29 cells provides a mucus layer and has been suggested to reduce the "tightness" of the tight junctions to better represent the physiology of the small intestine (Pan et al, 2015;Hilgendorf et al, 2000). Variability in expression levels of transporters can be accounted for by applying a correction factor representing the relative expression level of transporters within Caco-2 cells compared with the in vivo situation (Harwood et al, 2016). Addition of vitamin D3 to the culture medium results in increased expression of CYP3A4 (Schmieldlin-Ren et al, 1997).…”

“…Though Caco-2 cells are proficient in the main transporters, including P-glycoprotein (PgP), multidrug resistance protein 2 (MRP2), and breast cancer resistance proteins (BCRP), expression levels of these transporters are generally quite variable (Larregieu and Benet, 2013;Harwood et al, 2013Harwood et al, , 2016. In addition, the under-expression of transporters such as peptide transporter 1 (PEPT1), organic cation transporters (OCTs), and organic anion transporters (OATs), makes the model less suitable for compounds that use these transporters (Larregieu and Benet, 2013).…”