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

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.

A Non-Nutritive Feeding Intervention Alters the Expression of Efflux Transporters in the Gastrointestinal Tract

“…The normal meal caused an increase from the fasted state for males (+50%), but not for females, where jejunal MRP2 expression was 4.23 ± 0.57 and 4.28 ± 0.72 ng/mg for the fasted and normal meal interventions, respectively. The fasted and housing food (normal meal) P-gp-related findings were similar to previous findings in our research group by Dou et al using a validated LC-MS/MS method [19], a Western blot method [20] and PCR [16]. Importantly, the inter-individual variations were low in this study, shown by narrow standard deviation in the Supplementary Materials, Tables S1-S3, in comparison to the Western blotting and LC-MS/MS methods [16,19,20].…”

“…The fasted and housing food (normal meal) P-gp-related findings were similar to previous findings in our research group by Dou et al using a validated LC-MS/MS method [19], a Western blot method [20] and PCR [16]. Importantly, the inter-individual variations were low in this study, shown by narrow standard deviation in the Supplementary Materials, Tables S1-S3, in comparison to the Western blotting and LC-MS/MS methods [16,19,20].…”

“…Importantly, there are similarities between the rat's and the human's GI physiology; both show small intestinal transit times of 3-4 h and rates of gastric emptying that are controlled by the energy content of the ingested food [13][14][15]. Our group has previously shown similar sex differences in the GI P-gp expression of fasted male and female rats and humans [16], suggesting that the rat is an appropriate animal model to predict the drug absorption of P-gp substrates in humans.…”

Overview of Drug Transporters in Human Placenta

“…MDR1 recognizes a wide range of lipophilic substrates. In the gastrointestinal mucosa, MDR1 drains substrates to the luminal side, and in cerebrovascular endothelial cells, MDR1 regulates the substrate distribution into the brain tissue [ 6 , 7 , 8 , 9 ]. ABCB4 (MDR3) acts as a flippase for phosphatidylcholine, a phospholipid that forms cell membranes, and changes the composition of the phospholipids that are present in the cell membranes.…”

Progesterone Metabolism by Human and Rat Hepatic and Intestinal Tissue

“…The poor oral bioavailability of progesterone has primarily been attributed to extensive metabolism in the liver [ 7 ]. However, in the current literature, very little information exists on progesterone intestinal metabolism, although extensive enzymatic expression is found throughout the small intestinal mucosa [ 8 , 9 , 10 , 11 , 12 ]. Furthermore, the small intestine contains appreciable levels of bacteria [ 13 , 14 ].…”