Materials and Methods

Materials.

Erlotinib (Tarceva) was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada), and flavopiridol [Alvocidib, HMR-1275, (−)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-(4R-(3S-hydroxy-1-methyl) piperidinyl)-4H-1-benzopyran-4-one] was kindly supplied by sanofi-aventis (Bridgewater, NJ). Dantrolene (Dantrium) and mitoxantrone (Novantrone) were purchased from LKT Labs (St. Paul, MN). Quinidine was purchased from Tokyo Kasei (Tokyo, Japan). All other chemicals were commercially available and of reagent grade.

Animals.

Male wild-type FVB, Mdr1a/b(−/−), Bcrp(−/−), and Mdr1a/b(−/−)/Bcrp(−/−) mice, 9 weeks of age, were obtained from Taconic Farms (Germantown, NY). The mice used in the present study were 10 to 18 weeks old and weighed 23 to 36 g. All animals were maintained at a controlled temperature under a 12-h light/dark cycle. Food and water were available ad libitum.

Determination of the Transcellular Transport across Monolayers of Cell Lines Expressing P-gp or Bcrp.

In vitro mouse Bcrp transport experiments were performed as reported previously (Enokizono et al., 2007, 2008). In brief, MDCK II cells were seeded into 24-well Transwell plates (Corning Life Sciences, Lowell, MA) at a density of 1.4 × 10⁵ cells/well and grown for 2 days in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 1% antibiotic–antimycotic solution (Sigma-Aldrich). The cells were infected with recombinant adenovirus harboring green fluorescent protein (GFP) or mouse Bcrp expression vector at a ×200 multiplicity of infection. Details of the construction of these recombinant adenoviruses have been described previously (Kondo et al., 2004). After 2 days in culture, both GFP- and Bcrp-expressing cells (GFP-MDCK and Bcrp-MDCK, respectively) were used for transport studies.

In vitro transport experiments to determine the transport activity by mouse Mdr1a were conducted with Mdr1a-expressing LLC-PK1 cells (L-Mdr1a) that were established previously (Schinkel et al., 1995, 1996). L-Mdr1a and parent LLC-PK1 cells were seeded in 24-well Transwell plates at a density of 3.2 × 10⁵ and 2.1 × 10⁵ cells/well, respectively, and grown in Medium 199 (Invitrogen) with 10% fetal bovine serum and 1% antibiotic–antimycotic solution. Medium was changed on the 2nd day of culture, and cells were subjected to the transport study on the 4th day.

The cells were preincubated in Krebs–Henseleit buffer (118 mM NaCl, 23.8 mM NaHCO₃, 4.83 mM KCl, 0.96 mM KH₂PO₄, 1.20 mM MgSO₄, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl₂, pH 7.4) at 37°C for more than 30 min, and transport experiments were initiated by replacing the medium on one side of the cell monolayer with Krebs–Henseleit buffer containing 3 μM test compounds. At the appropriate times (60, 120, and 180 min), 100-μl aliquots were taken from the opposite side of the cell monolayer and replaced with 100 μl of drug-free buffer. The medium (100 μl) obtained from in vitro transport studies was mixed with 50 μl of acetonitrile for all compounds except mitoxantrone, which was mixed with 50 μl of 20% (w/v) ascorbic acid–saline.

Transport rates were calculated from the slopes of the time profiles of the apical-to-basal and basal-to-apical transport. Flux ratios (Table 1) were obtained by dividing the efflux rates in the basal-to-apical direction by those in the apical-to-basal direction. Flux ratios in transporter-expressing cells were divided by those in control cells to give a corrected flux ratio (CFR), an in vitro index of the P-gp- and Bcrp-mediated efflux.

Determination of the Tissue-to-Plasma Ratio in Mice.

Under urethane anesthesia (1.25 g/kg i.p.), the right jugular vein of the mice was cannulated with a polyethylene tube (PE-10; BD Biosciences, San Jose, CA). Compounds were administered via the cannula by continuous infusion for 120 min. The infusion rates of dantrolene, erlotinib, flavopiridol, mitoxantrone, and quinidine were 2, 4, 32, 8, and 8 μmol/h/kg after priming doses of 1, 2, 8, 2, and 6 μmol/kg, respectively. Blood samples were collected from the left jugular vein at the appropriate time points and centrifuged at 4°C and 9000g for 5 min to obtain plasma. Immediately after the final blood sampling, mice were sacrificed by exsanguination. For mitoxantrone, the plasma samples were transferred to microtubes containing ascorbic acid. For dantrolene, erlotinib, flavopiridol, and quinidine, the brain and testes specimens were homogenized with three volumes of phosphate-buffered saline (pH 7.4), whereas 0.9% saline containing 20% (w/v) ascorbic acid was used to prepare the homogenates containing mitoxantrone, to obtain a 25% homogenate for all compounds. Plasma specimens and tissue homogenates were stored at −80°C until use. Tissue-to-plasma ratios (C_brain/C_plasma and C_testis/C_plasma) were obtained by dividing the drug concentrations in the brain and testes by the plasma concentrations at the last sampling point.

Determination of PS_P-gp and PS_Bcrp.

Adachi et al. (2001) demonstrated previously that the K_p,brain is given by the ratio of permeability surface area (PS) products for the uptake (PS_{brain-to-blood}) and efflux (PS_{blood-to-brain}). The PS_{tissue-to-blood} and PS_{blood-to-tissue} in wild-type mice are given by eqs. 1 and 2, respectively, where PS_b,inf and PS_b,eff represent the PS product for the influx and efflux across the blood-side membrane of the endothelial cells, PS_t,inf and PS_t,eff represent the PS product for the influx and efflux across the tissue-side membrane of the endothelial cells, and PS_Bcrp and PS_P-gp represent the PS product for the efflux mediated by Bcrp and P-gp on the blood-side membrane of the endothelial cells, respectively. Because the K_p value is given by PS_{blood-to-tissue}/PS_{tissue-to-blood}, K_p in wild-type mice [K_p (WT)] is given by eq. 3. The K_p in Mdr1a/1b(−/−), Bcrp(−/−), and Mdr1a/1b(−/−)/Bcrp(−/−) mice are given by eqs. 4, 5, and 6, respectively. The ratios of K_p (R) in Mdr1a/1b(−/−), Bcrp(−/−), and Mdr1a/1b(−/−)/Bcrp(−/−) mice to that in FVB mice are given by eqs. 7, 8, and 9, respectively. The PS_P-gp and PS_Bcrp, intrinsic efflux transport activities at the BBB and BTB relative to passive diffusion, were obtained by using a nonlinear least-squares method by fitting eqs. 7 to 9 to the actual R_{Mdr1a/1b(−/−)}, R_{Bcrp(−/−)}, and R_{Mdr1a/1b(−/−)/Bcrp(−/−)} by using the MULTI program (Yamaoka et al., 1981). The algorithm used for the fitting was the Damping Gauss Newton Method (Yamaoka et al., 1981).

Quantification of Drugs in the Biological Samples.

Plasma samples were diluted with two volumes of phosphate-buffered saline for all compounds except mitoxantrone. Mitoxantrone was mixed with 0.9% saline containing 20% (w/v) ascorbic acid to obtain 33% diluted plasma. The proteins in these diluted plasma and tissue homogenates were precipitated with two volumes of acetonitrile, and the suspensions were centrifuged twice at 4°C and 5000g for 10 min. The supernatant were evaporated, and the remaining residues were reconstituted in mobile phase and subjected to LC-MS/MS or LC-UV analysis. The media (100 μl) mixed with 50 μl of acetonitrile or 20% (w/v) ascorbic acid–saline in in vitro transport studies were centrifuged at 4°C and 5000g for 5 min. The supernatants were also subjected to analysis by LC-MS/MS or LC-UV. All compounds except mitoxantrone were analyzed in a multiple reaction monitoring mode with an API2000 instrument (Applied Biosystems, Foster City, CA) equipped with an Agilent 1100 series LC system (Agilent Technologies, Santa Clara, CA). The transitions (precursor/product) used for quantification of erlotinib, flavopiridol, and quinidine were 394/278, 402/70, and 325/160, respectively, under positive atmospheric pressure chemical ionization mode, and that of dantrolene was 313/214 under negative atmospheric pressure chemical ionization mode. Mitoxantrone was detected by its absorbance at 655 nm by using an Agilent 1100 series diode array detector. All the compounds was separated by using a Capcell Pak C₁₈ MGII column (3 μm, 3-mm i.d. × 35 mm; Shiseido, Kanagawa, Japan) at room temperature at the flow rate of 0.8 ml/min. Mobile phase A was 0.05% formic acid for all compounds except dantrolene, and 10 mM ammonium acetate was used as mobile phase A for dantrolene. Mobile phase B was acetonitrile for all compounds. For the analysis of erlotinib and quinidine, the fraction of mobile phase B was initially 5%, kept at 5% for 0.5 min, linearly increased to 90% over 2.9 min, kept at 90% for an additional 0.1 min, and finally re-equilibrated at 5% for 1.5 min. For the analysis of flavopiridol, the fraction of mobile phase B was initially 5%, kept at 5% for 1 min, linearly increased to 90% over 2.4 min, kept at 90% for an additional 0.1 min, and finally re-equilibrated at 5% for 1.5 min. For the analysis of mitoxantrone, the fraction of mobile phase B was initially 5%, kept at 5% for 1 min, linearly increased to 75% over 3 min, kept at 75% for an additional 0.5 min, and finally re-equilibrated at 5% for 1.5 min. For the analysis of dantrolene, the fraction of mobile phase B was initially 5%, kept at 5% for 0.5 min, linearly increased to 90% over 2.95 min, kept at 90% for an additional 0.05 min, and finally re-equilibrated at 5% for 1.5 min.

Statistical Analysis.

The presented values are all mean ± S.E. For comparison between genotype groups, log-transformed data were processed by using one-way analysis of variance, followed by a Tukey post hoc test. For comparison of the in vitro transcellular transport between transporter-expressing cells and the corresponding control cells, Student's two-tailed t test was used. Differences were considered significant at P < 0.05. All statistical calculations were performed with SAS software (version 9; SAS Institute, Cary, NC).