The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo
Introduction
P-gp and CYP3A4 are important and extensively studied members of the families of efflux transporters and metabolizing enzymes (Bojcsev et al., 2013, DeGorter et al., 2012, Zhen, 2013). In addition to their respective roles of excretion and metabolism of xenobiotics, they can work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs, due to their co-localization in the intestinal epithelium and the largely overlapping substrate specificities (von Richter et al., 2004, Wacher et al., 1995, Watkins, 1997). This P-gp/CYP3A4 interplay is the most well-known transport-metabolism interplay (Benet, 2009). Because of the broad and overlapping spectrum of inhibitors for both proteins, drug-drug interactions (DDIs) based on this interplay may occur frequently (Shi and Li, 2015, Wacher et al., 1995, Wandel et al., 1999). The studies on the P-gp/CYP3A4 interplay and related DDIs are of clinical importance, as they may result in altered bioavailability and altered intestinal toxicity due to altered intracellular exposure and require investigation in the pre-clinical phase of drug development. For instance, Choi et al. demonstrated a significant interaction between verapamil and atorvastatin in healthy volunteers (Choi et al., 2008). Atorvastatin, dual inhibitor of both P-gp and CYP3A4, enhanced the oral bioavailability of verapamil by inhibiting the P-gp efflux pump, and meanwhile decreased verapamil metabolism, probably due to inhibition of CYP3A4 (Srinivas, 2008). Thus, the studies on the P-gp/CYP3A4 interplay and related DDIs are of clinical importance and require investigation in the pre-clinical phase. Moreover, it is obvious that the effect of P-gp/CYP3A4 interplay and related DDIs highly correlates with the expression levels of P-gp and CYP3A4 in the intestine. Results of mRNA and Western blot analysis show that their expression patterns are very different along the intestinal tract, with colon ≤ duodenum < jejunum < ileum for P-gp (MacLean et al., 2008, Takano et al., 2006) and colon ≤ ileum < jejunum < duodenum for CYP3A4 (Mitschke et al., 2008). The most recent quantification of protein abundance by LC-MS/MS confirmed the different expression of P-gp in human intestinal regions (Drozdzik et al., 2014), while the absolute abundance of CYP3A4 protein in human intestine is not available currently. Notably, the expression of transporters and metabolizing enzymes in in vitro models of the human intestine, such as Caco-2 cell lines, is different from their physiological levels, thus they cannot mimic the physiological situation in different intestinal region (Artursson and Borchardt, 1997). Therefore, a proper model which could directly use the intestinal tissue from human and/or animals, and meanwhile provide efficient screening capacity, is preferable. The precision-cut intestinal slice (PCIS) model has been established to investigate intestinal drug metabolism, toxicity, and more recently transport in both human and animals (Khan et al., 2011, Li et al., 2015, Martignoni et al., 2006, van de Kerkhof et al., 2005, van de Kerkhof et al., 2006), and was recently presented as an adequate model to study transport-metabolism interplay in rat intestine (Li et al., 2016b). The aim of this study was to investigate the P-gp/CYP3A4 interplay and the DDIs based on this interplay to P-gp inhibition in different regions of the human intestine. As species differences with respect to metabolizing enzymes and transporters functions between rats and human are prominent, results as obtained in the previous study with rat intestine (Li et al., 2016b) cannot be directly extrapolated to human. To the best of our knowledge, the consequences of the P-gp/CYP3A4 interplay is still under debate (Benet, 2009, Pang et al., 2009) and have not been studied before in human tissue ex vivo.
Since several drug transporters and metabolizing enzymes are present in PCIS, selective P-gp inhibitors are needed in order to avoid the influence of inhibition of metabolism, as many P-gp inhibitors are also cytochrome P450 inhibitors. CP100356 and PSC833 were chosen as selective P-gp inhibitors, due to their high selectivity for P-gp inhibition (Kalgutkar et al., 2009, Wandel et al., 1999), whereas verapamil and ketoconazole, two well-studied inhibitors of both P-gp and CYP3A4 (Wandel et al., 1999), were employed as dual inhibitors to investigate the consequences of DDIs under non-selective P-gp inhibition, as most of P-gp inhibitive compounds inhibit both P-gp and CYP3A4. Quinidine (Qi), which has been used clinically for > 200 years as antiarrhythmic drug (Grace and Camm, 1998), was chosen as the probe drug for the P-gp/CYP3A4 interplay, because it is a well-known dual substrate of P-gp and CYP3A4 that does not significantly inhibit CYP3A4 (McLaughlin et al., 2005). It was reported to inhibit P-gp but at a relative high concentration with an IC50 of 14.1 μM in MDCKII cells (Keogh and Kunta, 2006), 23.6 μM in rat PCIS (Li et al., 2015) and 35.7 μM in human PCIS (Li et al., 2016a). Furthermore, Qi is extensively metabolized by CYP3A4 into 3-hydroxy-quinidine (3OH-Qi) (Hu et al., 2004), which is considered to be the specific marker reaction for CYP3A4 activity (Nielsen et al., 1999).
Section snippets
Chemicals
Quinidine, verapamil hydrochloride, ketoconazole and agarose (low gelling temperature, type VII-A) were from Sigma-Aldrich (USA). PSC833 and CP100356 were obtained from Tocris Bioscience (UK). Amphotericin B (fungizone)-solution, gentamicin, and William's medium E with glutamax-I (WME) were purchased from Invitrogen (UK). HEPES was from MP Biomedicals (Germany). Antipyrine was purchased from O. P. G. Pharma (the Netherlands). 3-Hydroxy-quinidine was purchased from Toronto Research Chemicals
Viability of the human PCIS
After 3 h of incubation the PCIS from human jejunum, ileum and colon retained > 80% (p > 0.05) of the level of intracellular ATP in fresh slices at 0 h, as shown earlier (Li et al., 2016a), indicating that the PCIS remain viable during incubation. Furthermore, the exposure to the P-gp substrate (Qi, 200 μM) or inhibitors at their highest concentration had no significant influence on the ATP content in PCIS (results not shown). These results indicate that Qi and the P-gp inhibitors did not influence
Discussion
The activity of P-gp and CYP3A4 in the intestine is one of the reasons of the low oral bioavailability of many drugs. In addition, many studies support that P-gp and CYP3A4 act synergistically to reinforce their limiting effect on intestinal absorption and enhancement of intestinal metabolism of their shared substrates, such as Qi (Dufek et al., 2013, Peng et al., 2006, Watkins, 1997). Furthermore, DDIs based on this interplay are expected to occur frequently, because P-gp and CYP3A4 not only
Conclusions
To the best of our knowledge, the present study is the first ex vivo study on P-gp/CYP3A4 interplay and its related DDIs in human intestine. We show that human PCIS are an adequate and efficient ex vivo model to study these effects. The results indicate that the outcome of DDIs based on P-gp/CYP3A4 interplay varies among the different intestinal regions (jejunum, ileum, and colon), probably because of the different abundance of P-gp and CYP3A4. Moreover, the DDI was shown to be dependent on the
Transparency document
Acknowledgements
Ming Li is very grateful to be granted by the China Scholarship Council (CSC) (2011616035) for his PhD scholarship.
The authors thank Annie van Dam (Interfaculty Mass Spectrometry Center, University of Groningen) for the LC-MS/MS measurement of Qi and 3OH-Qi.
The authors thank Wouter Tobias van Haaften, Prof. Dr. Peter Olinga (Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, University of Groningen), Dr. Koert P. de Jong, Dr. Sijbrand Hofker and their colleagues (Department of
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