Contribution of hepatic organic anion-transporting polypeptides to docetaxel uptake and clearance

Abstract

The antimicrotubular agent docetaxel is a widely used chemotherapeutic drug for the treatment of multiple solid tumors and is predominantly dependent on hepatic disposition. In this study, we evaluated drug uptake transporters capable of transporting radiolabeled docetaxel. By screening an array of drug uptake transporters in HeLa cells using a recombinant vaccinia-based method, five organic anion-transporting polypeptides (OATP) capable of docetaxel uptake were identified: OATP1A2, OATP1B1, OATP1B3, OATP1C1, and Oatp1b2. Kinetic analysis of docetaxel transport revealed similar kinetic parameters among hepatic OATP1B/1b transporters. An assessment of polymorphisms (SNPs) in SLCO1B1 and SLCO1B3 revealed that a number of OATP1B1 and OATP1B3 variants were associated with impaired docetaxel transport. A Transwell-based vectorial transport assay using MDCKII stable cells showed that docetaxel was transported significantly into the apical compartment of double-transfected (MDCKII-OATP1B1/MDR1 and MDCKII-OATP1B3/MDR1) cells compared with single-transfected (MDCKII-OATP1B1 and MDCKII-OATP1B3) cells (P < 0.05) or control (MDCKII-Co) cells (P < 0.001). In vivo docetaxel transport studies in Slco1b2(-/-) mice showed approximately >5.5-fold higher plasma concentrations (P < 0.01) and approximately 3-fold decreased liver-to-plasma ratio (P < 0.05) of docetaxel compared with wild-type (WT) mice. The plasma clearance of docetaxel in Slco1b2(-/-) mice was 83% lower than WT mice (P < 0.05). In conclusion, this study demonstrates the important roles of OATP1B transporters to the hepatic disposition and clearance of docetaxel, and supporting roles of these transporters for docetaxel pharmacokinetics.

Figure 1

Multiple OATPs transport docetaxel in vitro. A panel of OATP drug uptake transporters was assessed for docetaxel (0.1 μM) transport activity at 10 min. Vector control (pEF6) was used as a reference. Multiple human OATPs, including OATP1A2, OATP1B1, OATP1B3, and OATP1C1 are capable of transporting docetaxel in vitro. Rat Oatp1b2 also transports docetaxel in vitro. Data are expressed as percentage of cellular uptake compared to vector control (mean ± S.E., n = 6). *, p < 0.05; ***, p < 0.001.

Figure 2

Docetaxel transport kinetics. An Eadie-Hofstee plot at a broad range of concentrations (0.1-300 μM) (A) for OATP1B1 showed a high affinity, low capacity component (K_m 0.43 ± 1.32 μM, V_max 37.7 ± 47.2 pmol/mg/min) together with a non-saturable component that is low affinity, high capacity (V—reaction rate; S—substrate concentration). Michaelis-Menten-type nonlinear curve-fitting are demonstrated for OATP1B3 (0.1-50 μM) (B) and Oatp1b2 (0.1-100 μM) (C). Data are expressed as mean ± S.E. (n=8 for OATP1B3 and Oatp1b2; n=12 for OATP1B1).

Figure 3

OATP1B1 and OATP1B3 variants differentially transport docetaxel in vitro. Uptake of radiolabeled docetaxel (0.1 μM) at 20 min by HeLa cells transfected with OATP1B1 or OATP1B3 variants was assessed relative to wild-type OATP1B1*1a and OATP1B3*1, respectively. The commonly occurring SLCO1B1 variants 521T>C (*5) and 388A>G/521T>C (*15) were associated with significantly impaired docetaxel transport. There was no significant differences in transport between the two reference alleles SLCO1B1 388G>A (*1a) and SLCO1B1 388A>G (*1b) (A). Several SLCO1B3 variants, including 699G>A, 1559A>C, 1679T>C, and 334T>G/699G>A, were associated with impaired docetaxel transport (B). Values are expressed as percentage of cellular uptake by OATP1B1*1a or OATP1B3*1 (mean ± SE, n = 6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4

Immunoblot analyses of OATP1B1, OATP1B3 and MDR1 expression in MDCKII-control cells (Co), single-(OATP1B1, OATP1B3, MDR1) and double-(OATP1B1/MDR1, OATP1B3/MDR1) cell lines. Crude cell membrane fractions (A) and biotinylated cell surface proteins (B) were subjected to 10% SDS polyacrylamide gels and then transferred onto nitrocellulose membranes. The blots were probed or re-probed with different antibodies, including OATP1B1, OATP1B3, MDR1, and Na⁺/K⁺-ATPase α antibodies. Whereas OATP1B1 in crude cell membrane fractions showed one glycosylated form 84 kDa and one unglycosylated form 58 kDa, only a glycosylated form 85 kDa of OATP1B1 was detected in cell surface proteins (arrows). OATP1B3 showed one band with approximately 120 kDa and MDR1 one with 170 kDa. Human liver extracts were used as a positive control.

Figure 5

Vectorial transport and intracellular accumulation of radiolabeled docetaxel. [³H]Docetaxel (0.1 μM) was administered to the basal compartment of monolayers of MDCKII-control (Co), single-(MDR1, OATP1B1, OATP1B3) and double-(OATP1B1/MDR1, OATP1B3/MDR1) cell lines. After 0.5, 1, 2, and 3h incubation, translocation of docetaxel into the apical compartment (A, B) and intracellular accumulation of docetaxel (C, D) are shown. Whereas apical docetaxel was significantly higher in MDCKII-OATP1B1/MDR1 and -OATP1B3/MDR1 cells compared to MDCKII-OATP1B1 and -OATP1B3 cells respectively at all time points, intracellular docetaxel was significantly lower in MDCKII-OATP1B1/MDR1 and –OATP1B3/MDR1 cells. Data are expressed as mean ± S.E. (n = 6 for vectorial transport study; n = 4 for intracellular accumulation study). *p < 0.05, **p < 0.01, and ***p < 0.001 versus MDCKII-Co cells; ⁺p < 0.05, ⁺⁺p < 0.01, and ⁺⁺⁺p < 0.001 versus MDCKII-MDR1 cells; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 versus MDCKII-OATP1B1cells; ^$p < 0.05, ^$$p < 0.01, and ^$$$p < 0.001 versus MDCKII-OATP1B3 cells.

Figure 6

Docetaxel disposition studies in Slco1b2^−/− mice. Slco1b2−/− mice (KO) have significantly higher docetaxel plasma AUC than wild-type mice (WT) (A). Measured after a single intravenous dose 1 mg/kg 3H-docetaxel (30 Ci/mmol). Blood was sampled from the saphenous vein of each mouse at 5, 15 and 30 minutes post-injection. Data are expressed as mean ± SD. *, p < 0.05; **, p < 0.01. (n=4 per group). Slco1b2−/− mice have a significantly lower docetaxel liver:plasma ratio compared to wild-type mice (B). Liver-to-plasma ratio comparing wild-type and Slco1b2−/− mice was shown. Data are expressed as mean ± SD. *, p < 0.05; **, p < 0.01. (n= 4/group; 21-24 weeks).