Insights Into Atorvastatin Pharmacokinetics in Rats Reveal Regulation of CYP3A1 by Humanization of SLCO2B1

Abstract

The organic anion transporting polypeptide (OATP) 2B1 is expressed in pharmacokinetically relevant organs such as the liver, the kidney, and the small intestine and it is known to transport a broad range of substrates including statins. In pharmacokinetic studies applying two rat models-one deficient for rat Oatp2b1 and the other expressing the human transporter-we investigated the disposition of atorvastatin, a known OATP2B1 substrate, and observed a reduced exposure and an increased clearance of this OATP2B1 substrate in humanized rats compared to knockout animals. Atorvastatin is predominantly cleared via the bile and due to the clearance increase, we assessed hepatic accumulation of this OATP2B1 substrate. Blood and organs were collected 1 h after administration of the HMG-CoA reductase inhibitor via tail vein injection. Sample analysis revealed lower drug concentrations in serum but also 40% lower hepatic atorvastatin levels in SLCO2B1-humanized rats compared to knockout animals, despite the expected role of OATP2B1 in facilitating hepatocellular uptake. Expression analysis of other drug transporters involved in hepatocellular handling of atorvastatin in treatment-naive rats revealed no differences in the sinusoidal uptake transporter rOatp1b2 and the canalicular efflux transporters rBcrp and rMdr1a between genotypes. However, transcript and protein analysis of rCyp3a1 in liver tissues showed approximately 3-fold higher levels in SLCO2B1-humanized rats compared to rSlco2b1-knockout animals. The increase in rCyp3a1 levels likely reflects an enhanced metabolic rate leading to lower hepatic atorvastatin content. Our finding suggests that humanization of OATP2B1 might influence hepatic disposition of rCyp3a1 substrates.

FIGURE 1

Western blot analysis of liver tissue and atorvastatin levels in serum after 1 h of i.v. injection. (A) Detection of rat and human OATP2B1 in the enriched membrane fraction of liver from wild‐type, rSlco2b1 ^−/− and humanized (SLCO2B1 ^+/+ ) rats by Western blot analysis (representative image). Na⁺/K⁺‐ATPase served as loading control. (B) Atorvastatin levels in serum 1 h after intravenous administration determined by LC–MS/MS. Data are shown as boxplots (n = 7 animals per genotype; Kruskal–Wallis with uncorrected Dunn's test).

FIGURE 2

Atorvastatin tissue content 1 h after intravenous injection. Levels of the HMG‐CoA reductase inhibitor were assessed in liver tissue (A) and small intestine (SI) (B). Normalization of liver to serum concentrations (C) and small intestine to serum concentrations (D) in the respective animal. Data are shown as boxplots (n = 7 animals per genotype; *Kruskal–Wallis with uncorrected Dunn's test).

FIGURE 3

Protein expression of rat Bcrp (rBcrp) and Mdr1 (rMdr1) in liver tissue of wild‐type, rSlco2b1 ^−/− , and SLCO2B1 ^+/+ rats. (A) Western blot analysis detecting rBcrp, and rMdr1 in liver tissue lysates. (B) Protein abundance of rBcrp1 or rMdr1 as the result of the densitometric analysis, where the density of the band for the efflux transporter is normalized to that of rat Gapdh (rGapdh). (C) Absolute quantification of rOatp1b2, rMdr1, and rBcrp by targeted proteomic analysis in membrane fraction, which was isolated from liver tissue using the ProteoExtract Native Membrane Protein Extraction Kit. (D) Localization of both rBcrp and rMdr1 in liver tissue section detected by immunohistochemistry (black scale indicates 100 μM) – arrows indicated the detection of the drug transporters by immunostaining.

FIGURE 4

Expression of rat Cyp3a1 (rCyp3a1) in liver tissue of wild‐type, rSlco2b1 ^−/− , and SLCO2B1 ^+/+ rats. (A) Western blot analysis to detect rCyp3a1 abundance in hepatic tissue lysates, where rGapdh served as a loading control. (B) Densitometric analysis of Western blot bands allows relative quantification of protein abundance between genotypes. (C) Localization of the drug metabolizing enzymes rCyp3a1 in liver tissue slices of 5 μm thickness (black bar corresponds 100 μm) – arrows indicated the detection of the drug transporters by immunostaining.