In vitro characterization of axitinib interactions with human efflux and hepatic uptake transporters: implications for disposition and drug interactions.

Axitinib is an inhibitor of tyrosine kinase vascular endothelin growth factor receptors 1, 2, and 3. The ATP-binding cassette (ABC) and solute carrier (SLC) transport properties of axitinib were determined in selected cellular systems. Axitinib exhibited high passive permeability in all cell lines evaluated (Papp ≥ 6 × 10(-6) cm/s). Active efflux was observed in Caco-2 cells, and further evaluation in multidrug resistance gene 1 (MDR1) or breast cancer resistance protein (BCRP) transfected Madin-Darby canine kidney cells type 2 (MDCK) cells indicated that axitinib is at most only a weak substrate for P-glycoprotein (P-gp) but not BCRP. Axitinib showed incomplete inhibition of P-gp-mediated transport of digoxin in Caco-2 cells and BCRP transport of topotecan in BCRP-transfected MDCK cells with IC50 values of 3 µM and 4.4 µM, respectively. Axitinib (10 mg) did not pose a risk for systemic drug interactions with P-gp or BCRP per regulatory guidance. A potential risk for drug interactions through inhibition of P-gp and BCRP in the gastrointestinal tract was identified because an axitinib dose of 10 mg divided by 250 mL was greater than 10-fold the IC50 for each transporter. However, a GastroPlus simulation that considered the low solubility of axitinib resulted in lower intestinal concentrations and suggested a low potential for gastrointestinal interactions with P-gp and BCRP substrates. Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 transfected human embryonic kidney 293 (HEK293) cells transported axitinib to a minor extent but uptake into suspended hepatocytes was not inhibited by rifamycin SV suggesting that high passive permeability predominates. Mouse whole-body autoradiography revealed that [(14)C]axitinib-equivalents showed rapid absorption and distribution to all tissues except the brain. This suggests that efflux transport of axitinib may occur at the mouse blood-brain barrier.

Digoxin is not a substrate for organic anion-transporting polypeptide transporters OATP1A2, OATP1B1, OATP1B3, and OATP2B1 but is a substrate for a sodium-dependent transporter expressed in HEK293 cells.

Digoxin, an orally administered cardiac glycoside cardiovascular drug, has a narrow therapeutic window. Circulating digoxin levels (maximal concentration of ∼1.5 ng/ml) require careful monitoring, and the potential for drug-drug interactions (DDI) is a concern. Increases in digoxin plasma exposure caused by inhibition of P-glycoprotein (P-gp) have been reported. Digoxin has also been described as a substrate of various organic anion-transporting polypeptide (OATP) transporters, posing a risk that inhibition of OATPs may result in a clinically relevant DDI similar to what has been observed for P-gp. Although studies in rats have shown that Oatps contribute to the disposition of digoxin, the role of OATPs in the disposition of digoxin in humans has not been clearly defined. Using two methods, Boehringer Ingelheim, GlaxoSmithKline, Pfizer, and Solvo observed that digoxin is not a substrate of OATP1A2, OATP1B1, OATP1B3, and OATP2B1. However, digoxin inhibited the uptake of probe substrates of OATP1B1 (IC(50) of 47 µM), OATP1B3 (IC(50) > 8.1 µM), and OATP2B1 (IC(50) > 300 µM), but not OATP1A2 in transfected cell lines. It is interesting to note that digoxin is a substrate of a sodium-dependent transporter endogenously expressed in HEK293 cells because uptake of digoxin was significantly greater in cells incubated with sodium-fortified media compared with incubations conducted in media in which sodium was absent. Thus, although digoxin is not a substrate for the human OATP transporters evaluated in this study, in addition to P-gp-mediated efflux, its uptake and pharmacokinetic disposition may be partially facilitated by a sodium-dependent transporter.

Development of a new permeability assay using low-efflux MDCKII cells.

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

P-glycoprotein related drug interactions: clinical importance and a consideration of disease states.

IMPORTANCE OF THE FIELD: P-glycoprotein (P-gp) is the most characterized drug transporter in terms of its clinical relevance for pharmacokinetic disposition and interaction with other medicines. Clinically significant P-gp related drug interactions appear restricted to digoxin. P-gp may act as a major barrier to current and effective drug treatment in a number of diseases including cancer, AIDS, Alzheimer's and epilepsy due to its expression in tumors, lymphocytes, cell membranes of brain capillaries and the choroid plexus. AREAS COVERED IN THIS REVIEW: This review summarizes the current understanding of P-gp structure/function, clinical importance of P-gp related drug interactions and the modulatory role this transporter may contribute towards drug efficacy in disease states such as cancer, AIDS, Alzheimer's and epilepsy. WHAT THE READER WILL GAIN: The reader will gain an understanding that the clinical relevance of P-gp in drug interactions is limited. In certain disease states, P-gp in barrier tissues can modulate changes in regional distribution. TAKE HOME MESSAGE: P-gp inhibition in isolation will not result in clinically important alterations in systemic exposure; however, P-gp transport may be of significance in barrier tissues (tumors, lymphocytes, brain) resulting in attenuated efficacy.

Effects of an E-cadherin-derived peptide on the gene expression of Caco-2 cells.

PURPOSE: The goal of this study was to determine the effects of exposure to an HAV peptide (Ac-SHAVSS-NH2) on the protein and gene expression in Caco-2 cells, a model for the intestinal mucosa. METHODS: Caco-2 cells were incubated with either 100 or 500 microM of the hexapeptide then evaluated over a 48-h time period. RESULTS: Cell detachment from the monolayer was seen only after 48 h of exposure to the peptide, with the greatest effects occurring with a peptide concentration of 500 microM. Total protein expression of E-cadherin showed a decrease of nearly 20% at the 24-h time point for each concentration examined, whereas no significant changes were detected at the other time points studied. Short term exposure to a 500 microM solution of Ac-SHAVSS-NH2 caused few changes in gene expression as determined by Affymetrix GeneChip microarrays; however, longer exposure periods produced numerous changes in the treated cells. The variations in mRNA expression indicate that this HAV peptide has an effect in the E-cadherin signaling pathways. The greatest increases in mRNA expression were found in genes regulating excretion or degradation of the peptide. CONCLUSIONS: This work suggests that this HAV peptide produces effects that reach beyond modulation of adhesion.

The effect of bergamottin on diazepam plasma levels and P450 enzymes in beagle dogs.

Bergamottin, a furanocoumarin isolated from grapefruit juice, was investigated for the ability to increase diazepam bioavailability and for its effect on cytochrome P450 (P450) enzymes in the beagle dog liver and intestine. To study the effect of bergamottin on diazepam pharmacokinetics, male beagle dogs were dosed with bergamottin (1 mg/kg) p.o. 0 or 2 h before p.o. diazepam (10 mg). In a second experiment, bergamottin (0.1 mg/kg) was dosed i.v. or p.o. 1 h before p.o. diazepam (10 mg). Plasma samples were collected over 24 h postdose, analyzed by liquid chromatography/mass tandem spectrometry, and diazepam pharmacokinetic parameters were determined. To study the effect of bergamottin on P450 enzymes, beagle dog liver and jejunum was harvested after a 10-day dosing regimen of bergamottin (1 mg/kg) p.o. per day; microsomes were prepared and analyzed for CYP3A12, CYP2B11, CYP1A1/2, and tolbutamide hydroxylase activity. Bergamottin predosing increased the plasma levels of diazepam as observed by C(max) (278.75 ng/ml versus 5.49 ng/ml) and the area under the curve [AUC((0-TLDC))] (247.69 versus 2.79 ng x hr/ml) in bergamottin versus placebo groups, respectively, indicating P450 enzyme inhibition. Diazepam plasma concentrations were increased to a similar level in the presence of i.v. and p.o. administered bergamottin. In hepatic microsomes, bergamottin treatment for 10 days reduced the activity of CYP3A12 by 50% and CYP1A1/2 by 75%. Tolbutamide hydroxylase activity did not change, and CYP2B11 activity was moderately induced. In jejunal microsomes, CYP3A12 activity doubled with bergamottin treatment. CYP2B11, CYP1A1/2 activity and tolbutamide hydroxylation was not detected. In conclusion, bergamottin is both an inhibitor and an inducer of P450 enzymes.