Characterization of efflux transporters involved in distribution and disposition of apixaban.

The studies reported here were conducted to investigate the transport characteristics of apixaban (1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide) and to understand the impact of transporters on apixaban distribution and disposition. In human permeability glycoprotein (P-gp)- and breast cancer resistance protein (BCRP)-cDNA-transfected cell monolayers as well as Caco-2 cell monolayers, the apparent efflux ratio of basolateral-to-apical (PcB-A) versus apical-to-basolateral permeability (PcA-B) of apixaban was >10. The P-gp- and BCRP-facilitated transport of apixaban was concentration- and time-dependent and did not show saturation over a wide range of concentrations (1-100 µM). The efflux transport of apixaban was also demonstrated by the lower mucosal-to-serosal permeability than that of the serosal-to-mucosal direction in isolated rat jejunum segments. Apixaban did not inhibit digoxin transport in Caco-2 cells. Ketoconazole decreased the P-gp-mediated apixaban efflux in Caco-2 and the P-gp-cDNA-transfected cell monolayers, but did not affect the apixaban efflux to a meaningful extent in the BCRP-cDNA-transfected cell monolayers. Coincubation of a P-gp inhibitor (ketoconazole or cyclosporin A) and a BCRP inhibitor (Ko134) provided more complete inhibition of apixaban efflux in Caco-2 cells than separate inhibition by individual inhibitors. Naproxen inhibited apixaban efflux in Caco-2 cells but showed only a minimal effect on apixaban transport in the BCRP-transfected cells. Naproxen was the first nonsteroidal antiinflammatory drug that was demonstrated as a weak P-gp inhibitor. These results demonstrate that apixaban is a substrate for efflux transporters P-gp and BCRP, which can help explain its low brain penetration, and low fetal exposures and high milk excretion in rats.

P-gp inhibition potential in cell-based models: which "calculation" method is the most accurate?

The objective was to directly compare the four different "calculation" methods of assessing P-gp inhibition potential using experimental data obtained from approximately 60 structurally diverse internal research and marketed compounds. Bidirectional studies for digoxin (probe for P-gp substrate) were performed with and without test compounds (at 10 microM). Four different calculation methods were applied to the same dataset (raw bidirectional permeability values) to obtain the "percent inhibition of P-gp" for these compounds using the different methods. Significantly different inhibition potential was obtained with the "exact" same experimental dataset depending on the calculation method used. Subsequently, entirely different conclusions regarding the "inhibition potential" of test compound was reached due to the different calculation methods. Based on the direct comparison of these methods, method no. 3 (i.e., inhibition of B to A permeability of digoxin) is recommended as the calculation method ideal during screening stages due to its high throughput amenability. The methodology is capable of rapidly screening compounds with adequate reliability for early stage drug discovery. Method no. 3 provides an abridged version of a bidirectional study that is fully capable of identifying all non-inhibitors (0-20%), moderate inhibitors (20-60%), and potent inhibitors (>60%) and demonstrates high correlation with method no. 1 (inhibition based on both A to B and B to A permeability of digoxin). Nevertheless, method no. 1 might be appropriate for more detailed mechanistic studies required in late stage discovery and development.

Development of a quantitative LC-MS/MS analytical method coupled with turbulent flow chromatography for digoxin for the in vitro P-gp inhibition assay.

Caco-2 cells, the human colon carcinoma cells, are typically used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential during discovery and development. The P-gp inhibition of test compounds is assessed by performing bi-directional permeability studies with digoxin, a well established P-gp substrate probe. Studies performed with digoxin alone as well as digoxin in presence of test compounds as putative inhibitors constitute the P-gp inhibition assay used to assess the potential liability of discovery compounds. Radiolabeled (3)H-digoxin is commonly used in such studies followed by liquid scintillation counting. This manuscript describes the development of a sensitive, accurate, and reproducible LC-MS/MS method for analysis of digoxin and its internal standard digitoxin using an on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection that is amendable to high throughput with use of 96-well plates. The standard curve for digoxin was linear between 10 nM and 5000 nM with regression coefficient (R(2)) of 0.99. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio (permeability B to A over permeability A to B) greater than 10 for digoxin in Caco-2 cells. Additional evaluations were performed on 13 marketed compounds by conducting inhibition studies in Caco-2 cells using classical P-gp inhibitors (ketoconazole, cyclosporin, verapamil, quinidine, saquinavir etc.) and comparing the results to historical data with (3)H-digoxin studies. Similarly, P-gp inhibition studies with LC-MS/MS analytical method for digoxin were also performed for 21 additional test compounds classified as negative, moderate, and potent P-gp inhibitors spanning multiple chemo types and results compared with the historical P-gp inhibition data from the (3)H-digoxin studies. A very good correlation coefficient (R(2)) of 0.89 between the results from the two analytical methods affords an attractive LC-MS/MS analytical option for labs that need to conduct the P-gp inhibition assay without using radiolabeled compounds.