TABLE 2

Reconstruction of P-gp activities at the cynomolgus monkey BBB on the basis of the in vitro transport activities and protein expression levels

On the basis of the results shown in Fig. 1, the apical-to-basal (A to B) transport rate, the basal-to-apical (B to A) transport rate, the flux ratio, and the in vitro P-gp efflux ratio across LLC-PK1/cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayers were calculated as described in Materials and Methods. For the quantifications of P-gp expression, cynomolgus monkey P-gp–transfected LLC-PK1 cells were cultured under the same conditions that were used for the transcellular transport experiments, and the expression levels in the transfected cell monolayers were determined using LC-MS/MS–based quantification (triplicate experiments). After the constant infusion of each compound, the cynomolgus monkeys were sacrificed, the cerebrums were collected, and the left cerebrums were used to isolate brain microvessels using the nylon mesh method. P-gp protein expression levels in the brain microvessels of each cynomolgus monkey were determined using LC-MS/MS–based quantification (triplicate experiments for each cynomolgus monkey). Reconstructed Kp,brain ratios were calculated from the in vitro P-gp efflux ratios and the P-gp protein expression levels using eq. 2. Each value represents the mean ± S.E.M. The S.E.M. was calculated according to the law of propagation of error.

CompoundLLC-PK1Cynomolgus Monkey 
P-gp–Transfected LLC-PK1In Vitro 
P-gp Efflux RatioProtein Expression Levels 
of Cynomolgus Monkey P-gpReconstructed Kp,brain Ratio
Transport RateFlux RatioTransport Rate Flux RatioCynomolgus Monkey 
P-gp–Transfected LLC-PK1Brain Microvessels Isolated from Cynomolgus Monkey
A to BB to AA to BB to A
fmol/min per wellfmol/min per wellfmol/μg protein
Indinavir88.2 ± 0.5298 ± 193.37 ± 0.1533.8 ± 1.1320 ± 209.47 ± 0.472.81 ± 0.132.31 ± 0.155.84 ± 0.30a5.58 ± 0.65
Quinidine338 ± 34744 ± 842.21 ± 0.2472.8 ± 8.4786 ± 7910.8 ± 1.24.90 ± 0.532.31 ± 0.155.72 ± 0.30a10.7 ± 1.9
Loperamide79.8 ± 4.160.3 ± 2.60.755 ± 0.03630.0 ± 2.8181 ± 136.04 ± 0.498.00 ± 0.532.31 ± 0.155.05 ± 0.4416.3 ± 2.6
Paclitaxel98.7 ± 6.3425 ± 274.31 ± 0.2833.8 ± 4.2577 ± 3217.1 ± 1.63.96 ± 0.322.31 ± 0.155.21 ± 0.467.68 ± 1.27
Diazepam723 ± 25938 ± 411.30 ± 0.05937 ± 641197 ± 611.28 ± 0.080.984 ± 0.0502.31 ± 0.157.07 ± 0.46a0.951 ± 0.177
Verapamil163 ± 6246 ± 81.51 ± 0.0592.6 ± 3.7394 ± 214.26 ± 0.202.83 ± 0.122.31 ± 0.155.20 ± 0.415.12 ± 0.58
  • a Previously reported in Ito et al. (2011) and cited from that study.