CYP3A4-Transfected Caco-2 Cells as a Tool for Understanding Biochemical Absorption Barriers: Studies with Sirolimus and Midazolam

Abstract

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-{4- [2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl}-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH Ͼ 4-OH midazolam and 39-O-desmethyl Ͼ 12-OH Ͼ 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 M) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.Oral bioavailability and intestinal drug absorption can be significantly limited by metabolizing enzymes and efflux transporters in the gut (Benet et al., 1996b). The most prevalent oxidative drug-metabolizing enzyme present in the intestine is cytochrome P450 3A4 (CYP3A4). Currently, more than 50% of the drugs on the market metabolized by P450 enzymes are metabolized by CYP3A4 (Benet et al., 1996a). Oral absorption of CYP3A4 substrates can also be limited by the multidrug resistance transporter P-glycoprotein (P-gp), because there is extensive substrate overlap between these two proteins (Wacher et al., 1995). P-gp is an ATP-dependent transporter on the apical plasma membrane of enterocytes that functions to limit the entry of drugs into the cell (Ambudkar et al., 1999). We have previously hypothesized that the interplay between CYP3A4 and P-gp in the intestine can serve to enhance drug metabolism and significantly decrease intestinal drug absorption (Cummins et al., 2002).

Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

“…A pharmacokinetic interaction has already been reported between calcineurin inhibitors and sirolimus (Kaplan et al, 1998;Cattaneo et al, 2004). Its mechanism mainly involves inhibition of the CYP 3A isoenzymes and, to a minor extent, inhibition of P-glycoprotein (Wacher et al, 2002;Cummins et al, 2004). Here, the interaction was investigated using the in vitro metabolic half-life method.…”

“…In renal transplant patients, coadministration of the two drugs resulted in increased sirolimus area under the concentration curve (1.45-fold), through concentration (1.49-fold) and maximal concentration (1.72-fold), compared with staggered administration, with no significant consequence on cyclosporine exposure (Kaplan et al, 1998). This interaction mainly involves CYP 3A inhibition as demonstrated in rats (Wacher et al, 2002) as well as in vitro using CYP 3A4-transfected Caco-2 cells (Cummins et al, 2004). However, there has been no study of the particular involvement of CYP 3A5 in, and none investigating the consequence of its polymorphism on, the cyclosporine/sirolimus interaction.…”

Extensive Intestinal First-Pass Elimination and Predominant Hepatic Distribution of Berberine Explain Its Low Plasma Levels in Rats

“…Furthermore, concurrent intake with quinine (an inhibitor of P-gp and OCT) resulted in a significant reduction in biliary excretion of unbound berberine (Tsai and Tsai, 2004), suggesting that the efflux transporters expressed in the liver may play an important role in biliary excretion of berberine. However, intravenous coadministration with cyclosporine, an inhibitor of P-gp (Cummins et al, 2004), organic anion-transporting polypeptide (Shitara et al, 2009), breast cancer resistance protein (Xia et al, 2007), and CYP3A (Cummins et al, 2004) led to a dramatic decrease in unbound concentrations of berberine in the liver and bile but not in blood (Tsai and Tsai, 2004). Therefore, the role drug transporters may play in the entire first-pass elimination of berberine would be more complex than we assumed, and a knockout mouse model of each transporter (in particular P-gp) will be required to clarify this issue if no highly specific chemical inhibitor of each of the drug transporters is available.…”

The Biotransformation of Prasugrel, a New Thienopyridine Prodrug, by the Human Carboxylesterases 1 and 2

“…3). The active metabolite of prasugrel, R-138727, was not monitored because CYP3A4, which is the primary P450 involved in its formation from the thiolactone, R-95913 (Rehmel et al, 2006), is not routinely expressed in Caco-2 monolayers (Cummins et al, 2004). Figure 3, A and B, shows the apical to basolateral (representing lumen to blood) conversion and transport of the cumulative amounts of prasugrel and R-95913 in the donor and receiver compartments, respectively.…”