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Quantification of P-Glycoprotein in the Gastrointestinal Tract of Humans and Rodents: Methodology, Gut Region, Sex, and Species Matter

Mai, Y; Dou, L; Yao, Z; Madla, CM; Gavins, FKH; Taherali, F; Yin, H; ... Basit, AW; + view all (2021) Quantification of P-Glycoprotein in the Gastrointestinal Tract of Humans and Rodents: Methodology, Gut Region, Sex, and Species Matter. Molecular Pharmaceutics , 18 pp. 1895-1904. 10.1021/acs.molpharmaceut.0c00574. Green open access

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Abstract

Intestinal efflux transporters affect the gastrointestinal processing of many drugs but further data on their intestinal expression levels are required. Relative mRNA expression and relative and absolute protein expression data of transporters are commonly measured by real-time polymerase chain reaction (RT-PCR), Western blot and mass spectrometry-based targeted proteomics techniques. All of these methods, however, have their own strengths and limitations, and therefore, validation for optimized quantification methods is needed. As such, the identification of the most appropriate technique is necessary to effectively translate preclinical findings to first-in-human trials. In this study, the mRNA expression and protein levels of the efflux transporter P-glycoprotein (P-gp) in jejunal and ileal epithelia of 30 male and female human subjects, and the duodenal, jejunal, ileal and colonic tissues in 48 Wistar rats were quantified using RT-PCR, Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A similar sex difference was observed in the expression of small intestinal P-gp in humans and Wistar rats where P-gp was higher in males than females with an increasing trend from the proximal to the distal parts in both species. A strong positive linear correlation was determined between the Western blot data and LC-MS/MS data in the small intestine of humans (R^{2} = 0.85). Conflicting results, however, were shown in rat small intestinal and colonic P-gp expression between the techniques (R^{2} = 0.29 and 0.05, respectively). In RT-PCR and Western blot, an internal reference protein is experimentally required; here, beta-actin was used which is innately variable along the intestinal tract. Quantification via LC-MS/MS can provide data on P-gp expression without the need for an internal reference protein and consequently, can give higher confidence on the expression levels of P-gp along the intestinal tract. Overall, these findings highlight similar trends between the species and suggest that the Wistar rat is an appropriate preclinical animal model to predict the oral drug absorption of P-gp substrates in the human small intestine.

Type: Article
Title: Quantification of P-Glycoprotein in the Gastrointestinal Tract of Humans and Rodents: Methodology, Gut Region, Sex, and Species Matter
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1021/acs.molpharmaceut.0c00574
Publisher version: https://doi.org/10.1021/acs.molpharmaceut.0c00574
Language: English
Additional information: © 2021 The Authors. Published by American Chemical Society. This is an Open Access article published under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
Keywords: ABCB1, MDR1, gastrointestinal drug bioavailability, multidrug resistance protein, preclinical drug delivery and development, sex differences
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharmaceutics
URI: https://discovery.ucl.ac.uk/id/eprint/10126919
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