The goal of the studies outlined in this dissertation was to test the hypothesis that P-gp efflux decreases the rate of CYP3A-mediated metabolism of dual substrates in the intestine in a concentration-dependent manner. As discussed in this chapter, there is some controversy in this area regarding previously reported data and especially the interpretation of the data. This dissertation aimed to examine the problem more broadly by studying the metabolism of P-gp/CYP3A substrates over a wide concentration range, in the presence and absence of P-gp efflux activity. These studies used in vitro models to study the interaction between P-gp and
CYP3A on a more mechanistic level.
Although some in vitro screens are available to study the properties of new drug
entities, these systems determine the susceptibility of a molecule to transport and metabolic processes separately and therefore cannot be used to study transport/metabolism interplay. An in vitro system that could be used to
simultaneously study transport and metabolism would have significant advantages over existing methodologies. Therefore, two in vitro systems that provide the
capability of measuring both transport and metabolism were used to study the P- gp/CYP3A interaction: Caco-2 cells (an in vitro model derived from human colon
carcinoma cells) that express both P-glycoprotein (constitutively) and CYP3A4 (by induction with 1α, dihydroxyvitamin D3) and fresh mouse intestine from P-gp- competent and -deficient mice. The intestinal tissues were used in a side-by-side
diffusion chamber in order to measure metabolism during absorptive transport across the tissues. Although this model has been used for some time, it has rarely been used for metabolic experiments. In fact, most of the studies done in this area of transport/metabolism interaction have involved cell lines only. The diffusion chamber system is more physiologically relevant than a simple model consisting of only one cell type and therefore provides valuable information about the P- gp/CYP3A interaction. However, potential species differences must be kept in mind when using the mouse model. Chemical inhibitors were used to modulate P-gp in the Caco-2 cell system, while genetic knockout was used for the mouse model. Combination of the data from these two systems afforded enhanced interpretive power as well as better predictions of clinical scenarios.
For this dissertation, two drugs, loperamide and terfenadine, were chosen to study the P-gp/CYP3A interaction. Since drugs with high permeability and low solubility (BCS Class II) are more likely to be affected by metabolism and transport, and are therefore more likely to show the interaction between the two processes, both drugs were selected from this class.
First, the dual P-gp/CYP3A substrate loperamide was selected for study due to its proven affinity for CYP3A (Kim et al., 2004) and its susceptibility to P-gp efflux (Wandel et al., 2002). Loperamide is an opioid derivative that is used as an anti- diarrheal agent. Loperamide is poorly absorbed due to high first-pass and transporter effects: after an oral dose in humans only 0.3% of the dose was found in
the plasma (Heykants et al., 1974). The extensive first-pass metabolism of loperamide made it a good candidate for these experiments, since metabolites were readily detectable. Loperamide has been characterized as a P-gp substrate (Bentz et al., 2005). Central nervous system side effects are uncommon due to the low plasma concentrations attained and due to P-gp efflux at the blood-brain barrier. Brain uptake clearances of loperamide were reported to be approximately 10-fold higher in P-gp-deficient mice than in the wild-type (Dagenais et al., 2004). Loperamide has also been shown to alter the disposition of the well-known P-gp substrate ivermectin: intestinal secretion of ivermectin was decreased in the presence of loperamide, implying competition for the P-gp transporter (Lifschitz et al., 2004). In Caco-2 cell studies, the secretory permeability of loperamide has been reported to be 17-fold higher than absorptive permeability (Crowe and Wong, 2004).
Loperamide has two major metabolites, which are formed from subsequent demethylation reactions. The drug is extensively metabolized, which facilitates detection of metabolites. Loperamide has been shown to be metabolized by several CYP isoforms in human liver microsomes, particularly by CYP3A4 and CYP2C8. A few drug-drug interactions have been reported for this compound. One study reported an increase in AUC with coadministration of ritonavir (a CYP3A inhibitor) (Tayrouz et al., 2001), while another showed that plasma concentrations of loperamide were increased by both CYP3A4 inhibitor itraconazole and CYP2C8 inhibitor gemfibrozil after oral dosing in healthy human volunteers (Niemi et al., 2006). However, CYP3A appears to be predominately responsible for its
metabolism, since 1 µM ketoconazole was shown to inhibit the formation of demethyl-loperamide (DLOP) by 90% in human liver microsomes (Kim et al., 2004).
To help determine the parameters that influence the interaction between P-gp and CYP3A in the intestine, the dual P-gp/CYP3A substrate terfenadine (Ling et al., 1995; Raeissi et al., 1999; Polli et al., 2001) was also selected for study. Terfenadine is a second-generation (non-sedating) antihistamine prodrug that was once widely prescribed but has been removed from the market due to safety concerns. It is known to be extensively metabolized by CYP3A during absorption, resulting in a bioavailability of <1% (Raeissi et al., 1999); this high first-pass metabolism makes the drug particularly susceptible to drug-drug interactions, since inhibition of this enzyme has the potential to increase systemic concentrations of the parent drug substantially. The two main metabolites of terfenadine are fexofenadine and azacyclonol.
Terfenadine is also subject to efflux by P-gp, which represents another barrier to its absorption from the intestine (Raeissi et al., 1999; Polli et al., 2001). Therefore, terfenadine is likely to be affected by both metabolic and efflux transporter processes during absorption. This makes it a good candidate for studying the interplay between P-gp and CYP3A. However, the extent to which its absorption is affected by P-gp is less than that for loperamide. Therefore, a different extent of effect of P-gp on its metabolism was expected.
In summary, two in vitro models and two dual P-gp/CYP3A substrates were used
to study the interaction between P-gp and CYP3A in intestine. Pharmacokinetic modeling was also used in order to make predictions about the behavior of dual substrates under differing conditions (i.e., in different regions of the intestine) and also to examine the parameters commonly used to assess the P-gp/CYP3A interaction. This combination of approaches allowed prediction of the in vivo