Targeted disruption of murine organic anion-transporting polypeptide 1b2 (Oatp1b2/Slco1b2) significantly alters disposition of prototypical drug substrates pravastatin and rifampin

Abstract

Organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are widely acknowledged as important and rate-limiting to the hepatic uptake of many drugs in clinical use. Accordingly, to better understand the in vivo relevance of OATP1B transporters, targeted disruption of murine Slco1b2 gene was carried out. It is noteworthy that Slco1b2(-/-) mice were fertile, developed normally, and exhibited no overt phenotypic abnormalities. We confirmed the loss of Oatp1b2 expression in liver using real-time polymerase chain reaction, Western Blot analysis, and immunohistochemistry. Expression of Oatp1a4 and Oatp2b1 but not Oatp1a1 was greater in female Slco1b2(-/-) mice, but expression of other non-OATP transporters did not significantly differ between wild-type and Slco1b2(-/-) male mice. Total bilirubin level was elevated by 2-fold in the Slco1b2(-/-) mice despite the fact that liver enzymes ALT and AST were normal. Pharmacological characterization was carried out using two prototypical substrates of human OATP1B1 and -1B3, rifampin and pravastatin. After a single intravenous dose of rifampin (1 mg/kg), a 1.7-fold increase in plasma area under the concentration-time curve (AUC) was observed, whereas the liver-to-plasma ratio was reduced by 5-fold, and nearly 8-fold when assessed at steady-state conditions after 24 h of continuous subcutaneous infusion in Slco1b2(-/-) mice. Likewise, continuous subcutaneous infusion at low (8 microg/h) or high (32 microg/h) dose rates of pravastatin resulted in a 4-fold lower liver-plasma ratio in the in Slco1b2(-/-) mice. This is the first report of altered drug disposition profile in the Slco1b2 knockout mice and suggests the utility of this model for understanding the in vivo role of hepatic OATP transporters in drug disposition.

Figure 1

The Slco1b2-knockout mouse was generated by excision of exon 10 to 12 of the Slco1b2 gene locus by standard targeting methods. The targeting vector consisted of 5′ and 3′ sequence homology arms flanking the neomycin selection cassette (A). For negative selection with gancyclovir, a thymidine kinase cassette was included in the targeting vector (A). Digest of the wildtype locus with BamHI results in a 11.0 kb fragment detected by Southern blotting using a 3′ probe for detection whereas successful targeting with deletion of exon 10 to 12 resulted in a 6.9 kb fragment after digest with BamHI and XhoI (B). For genotyping of the resulting mice, PCRs detecting a fragment of the Slco1b2 gene locus or neomycin were performed (C).

Figure 2

Detection of Oatp1b2 by Western Blot analysis revealed the absence of Oatp1b2 expression in Slco1b2^−/− mice (A). Immunohistochemical staining of Oatp1b2 (red) in mouse liver showed expression of the transporter in the basal membrane of hepatocytes (B). In animals with targeted disruption of Slco1b2, expression of the transporter was not detectible (C). mRNA expression was assessed by real-time RT-PCR and showed a significant reduction of Slco1b2 in knock-out animals (D).

Figure 3

Hepatic mRNA expression of Slco1a1 (Oatp1a1) , Slco1a4 (Oatp1a4) and Slco2b1 (Oatp2b1) as determined by real-time PCR comparing wildtype and Slco1b2^−/− animals. * p <0.05.

Figure 4

Rifampin concentration in plasma (○) and liver (Δ) of wildtype (A) and Slco1b2^−/− (B) mice (n=5−6/time point) after a single intravenous dose of rifampin (1mg/kg). Steady-state rifampin concentration in liver (C) and plasma (D) in animals treated with continuous subcutaneous infusion of rifampin at a rate of 8 μg/hr over 24 hrs using Alzet Mini-osmotic pumps (n=4). Panel E shows the calculated liver to plasma ratio comparing wildtype and Slco1b2^−/− animals. Data are expressed as mean ± SD.

Figure 5

Steady-state concentrations of pravastatin comparing wildtype (○) and Slco1b2^−/− (•) animals. The concentration of pravastatin was determined in plasma (A & D) and liver (B & E) following 24 hours subcutaneous infusion of low dose rate (8 μg/hr) (n=4) or high dose rate (32 μg/hr) (n=5) pravastatin using Alzet Mini-osmotic pumps. The liver-to-plasma ratios are shown in panels C & F.