Article Figures & Data
Figures
- Fig. 1.
Typical observed and predicted cellular accumulation and efflux profiles of [3H]-RSV in two lots of SCRHs, each showing modest (RSD288) or no (RSD294) biliary efflux of RSV. Circles/solid line and triangles/dashed line indicate the observed/predicted [3H]-RSV and [3H]-RSV lactone cellular accumulation (A, B, E, and F) or efflux (C, D, G, and H) of [3H]-RSV into the buffer with Ca2+-containing and Ca2+-free HBSS, respectively. The small difference (RSD288) or lack of difference (RSD294) in the cellular accumulation of [3H]-RSV between Ca2+-containing and Ca2+-free HBSS indicate modest or no biliary efflux of [3H]-RSV. (B, D, F, and H) The [3H]-RSV profiles in the presence of unlabeled 1 mM RSV.
- Fig. 2.
Comparison of in vivo hepatobiliary clearance of RSV predicted from SCRHs with our previous PET imaging study. RSV
in SCRHs was significantly lower than that observed in our PET imaging study (He et al., 2014). In contrast, the in vivo RSV and values were well predicted by SCRHs. The inset shows RSV and values estimated using SCRHs. The data shown are the mean ± S.D. values from three rats (from our previous PET imaging study), with six lots of SCRHs for and or three lots of SCRHs for *P < 0.05, significantly different from our previous PET imaging study. - Fig. 3.
Oatp protein expression in the SD rat liver and SCRHs. The expression of Oatp transporter proteins in SCRHs was significantly lower than that in the rat liver [data are from the study by Wang et al. (2015)]. The data shown are the mean ± S.D. of six SD rat livers and three lots of SCRHs. *P < 0.05, significantly different from SD rat livers.
- Fig. 4.
Comparison of recreated in silico
of RSV with values in SCRHs or from our previous PET imaging study. The recreated in silico RSV or better estimated the observed in SCRHs (A) or in vivo (B). In addition, the recreated in silico , , values of RSV were within 2-fold to 3-fold of values estimated from SCRHs (A) or in vivo (B). , , , , and values indicate the total values estimated from SCRHs not corrected for the Oatp protein expression, the recreated in silico values from Oatp-expressing cells (active) and SCRHs (passive), the recreated in silico values from Oatp-expressing cells (active) and CHO-mock cells (passive), the recreated in silico values from Oatp-expressing cells (active) and HEK293-mock cells (passive), and total values in our previous in vivo PET imaging study, respectively. The data shown are the mean ± S.D. of three lots of SCRHs, three rats (from our previous PET imaging study, He et al. (2014)), and six rat livers (Wang et al., 2015). †P < 0.05 significantly different from . - Fig. 5.
Simulated RSV hepatic concentrations based on hepatobiliary clearances of the drug estimated from Oatp-expressing cells and SCRHs. The mean predicted hepatic concentration of RSV (orange solid line), generated from the estimated
, , and values from SCRHs (see Materials and Methods), fell within the 95% CI of the observed concentrations in our previous in vivo PET imaging study (blue circles and gray dashed lines) (He et al., 2014), suggesting an excellent agreement between the observed and predicted hepatic RSV concentrations.
Tables
- TABLE 1
Estimates of [3H]-RSV hepatobiliary CLs in SCRHs in the presence of unlabeled RSV (0.5 µM or 1 mM)
Data are the mean ± S.D. of six lots (in the presence of 0.5 µM unlabeled RSV) or five lots (in the presence of 1 mM unlabeled RSV) of SCRH.
CL 0.5 µM 1 mM µl/min per milligram protein 27.7 ± 7.18 2.14 ± 0.49* 0.56 ± 0.40 1.86 ± 0.95* 0.32 ± 0.24a 0 (fixed)b 32.1 ± 14.2 142 ± 70 0.097 ± 0.062c (min−1)0.108d ↵a
in the presence of 0.5 µM unlabeled RSV was detected in only three lots of SCRHs.↵b
in the presence of 1 mM unlabeled RSV was virtually zero and therefore could not be estimated. Consequently, this value was fixed as 0.↵c
could be estimated in only five lots of SCRHs.↵d
could be estimated in only two lots of SCRHs; therefore, only the mean values of are shown.↵* P < 0.05 significantly different from the CL in the presence of 0.5 µM CL.
- TABLE 2
Protein expression of oatps and transport-mediated intrinsic
of RSV in CHO-Oatp1a1, HEK293-Oatp1a4, and HEK293-Oatp1b2 cellsData are the mean ± S.D. of triplicate determination. Transporter-mediated uptake CL of RSV was calculated as the ratio of RSV transporter-mediated uptake (uptake in transfected cells minus that in mock cells) over 5 s (Oatp1a1) or 15 s (Oatp1a4 and Oatp1b2) and the total buffer RSV concentration (0.07 µM).
Oatp Isoform Transporter Expression Intrinsic µl/min per mg total protein µl/min per femtomol transporter fmol/µg membrane protein 1a1 6.72 ± 0.63 459 0.113 1a4 2.61 ± 0.25 54.0 0.043 1b2 18.1 ± 1.79 143 0.015 - TABLE 3 values estimated from SCRHs, CHO-mock cells, and HEK293-mock cells
Data shown are the mean ± S.D. of five lots (SCRHs), three independent experiments (CHO-mock cells), or six independent experiments (HEK293-mock cells).
Cells µl/min per milligram protein SCRH 2.14 ± 0.49 CHO-mock 47.6 ± 10.1 HEK293-mock 5.68 ± 0.21
Data Supplement
- Supplemental Data -
Supplemental Figure 1 - Microscopic Images of SCRH
Supplemental Figure 2 - Observed and Predicted Cellular Accumulation and Efflux Profiles of [3H]-RSV in the Remaining Four Lots of SCRH
Supplemental Table 1 - LC Condition for Surrogate Peptide Quantification
Supplemental Table 2 - Surrogate Peptides of Rat Hepatic Oatps and Their MS/MS Parameters
Supplemental Table 3 - Estimates of [3H]-RSV Hepatobiliary Clearances in SCRH in the presence of unlabeled RSV (0.5 μM or 1 mM) and the corresponding Biliary Excretion Index (BEI) of RSV and Taurocholic acid (TCA)
Supplemental Table 4 - The Percentage of Total Radioactivity Associated with RSV and RSV Lactone in SCRH Incubated with 0.5 μM RSV (data are mean ± SD and range of 3 lots of SCRH)
- Supplemental Data -
