Hepatocellular Shuttling and Recirculation of Sorafenib-Glucuronide Is Dependent on Abcc2, Abcc3, and Oatp1a/1b

Abstract

Recently, an efficient liver detoxification process dubbed "hepatocyte hopping" was proposed on the basis of findings with the endogenous compound, bilirubin glucuronide. According to this model, hepatocytic bilirubin glucuronide can follow a liver-to-blood shuttling loop via Abcc3 transporter-mediated efflux and subsequent Oatp1a/1b-mediated liver uptake. We hypothesized that glucuronide conjugates of xenobiotics, such as the anticancer drug sorafenib, can also undergo hepatocyte hopping. Using transporter-deficient mouse models, we show here that sorafenib-glucuronide can be extruded from hepatocytes into the bile by Abcc2 or back into the systemic circulation by Abcc3, and that it can be taken up efficiently again into neighboring hepatocytes by Oatp1a/1b. We further demonstrate that sorafenib-glucuronide excreted into the gut lumen can be cleaved by microbial enzymes to sorafenib, which is then reabsorbed, supporting its persistence in the systemic circulation. Our results suggest broad relevance of a hepatocyte shuttling process known as "hepatocyte hopping"-a novel concept in clinical pharmacology-for detoxification of targeted cancer drugs that undergo hepatic glucuronidation, such as sorafenib.

Figure 1

Transport of sorafenib and SG by ABC transporters. Transport of (A) sorafenib (10 µM) or (B) SG (10 µM) in transporter-expressing inside-out vesicles using a 5-min incubation period in the presence or absence of ATP (4 mM) or rifampin (rif; 100 µM). Mouse and rat transporters are designated by the prefix “m” or “r”, respectively. The human transporters are shown in capitals. Data represent the mean ± SE of difference between ATP- and AMP-dependent transport (both expressed in pmol/min/mg) after normalization for non-specific transport observed in control vesicles, from 3–4 independent experiments (3–18 replicates). Asterisks above bars indicate significant differences in uptake between vesicles expressing the indicated transporter and control vesicles: *, P<0.05;***, P<0.0005; ****, P<0.0001. Square brackets: *, P,0.05 and **, P<0.005 for differences in uptake by ABCC2, ABCC3 or ABCC4 with or without rifampin. (C) ABCC2-, (D) ABCC3-, or (E) ABCC4-expressing or control vesicles were incubated with increasing concentrations of SG for 5 minutes in the presence of ATP (4 mM).

Figure 2

Pharmacokinetics of sorafenib and SG in wildtype and Abcc2(−/−) mice. (A and D) Plasma concentration-time profiles and (B and E) liver-to-plasma ratio of sorafenib and SG, respectively, in female wildtype and Abcc2(−/−) mice. Sorafenib was administered orally at a dose of 10 mg/kg. Livers were taken at 2 and 7.5 h after sorafenib administration (n = 4 per group). Concentrations in liver (C_L) were normalized to corresponding concentrations in plasma (C_p). (C and F) Bile-to-plasma concentration ratios of sorafenib and SG, respectively, in wildtype and Abcc2(−/−) mice. Sorafenib (10 mg/kg) was administered orally 30 min before the start of bile collection (N=3 wildtype; N=2 Abcc2(−/−) mice). Bile was collected for 2 hours. Data represent the mean ± SE.

Figure 3

Pharmacokinetics of sorafenib in wildtype and Abcc2(−/−) rats. Plasma concentration-time profiles of (A) sorafenib and (B) sorafenib N-oxide in wildtype and Abcc2(−/−) rats after oral administration of sorafenib at 10 mg/kg (n = 8 per group). SG concentrations were below the limit of quantitation (BLQ). Data represent the mean ± SE. (C) Biliary excretion of sorafenib and SG in wildtype and Abcc2(−/−) rats. Sorafenib (10 mg/kg) was administered orally 2 hours 25 min before bile collection (n = 2 per group). Bile was collected in 15-min fractions for 45 min. The results show collection at 2 hours 40 min after sorafenib administration (from 2 hours 25 min to 2 hours 40 min after sorafenib). (D) Ex vivo metabolism of sorafenib in liver microsomes of wildtype (WT) mice and rats. Liver microsomes (1 mg/ml) were incubated with 10 µM sorafenib for 60 min. Data represent the mean ± SE from 1–2 independent experiments (3–6 replicates). BLQ, below the analytical assay limit of SG quantitation (<0.26 pmol/min/mg).

Figure 4

Pharmacokinetics of SG in wildtype, Abcc3(−/−) mice, Oatp1a/1b(−/−), and combination Abcc2(−/−) and Abcc3(−/−) mice. Concentration-time profiles of SG in plasma (A, D), liver (B, E), and liver-to-plasma ratios (C, F) in wildtype and transporter knockout mice. Sorafenib 10 mg/kg was administered orally (n ≥ 4 per group). Data represent the mean ± SE. Asterisks indicate a significant difference in SG liver concentrations and liver to plasma concentration ratios between transporter knockout and wildtype mice: *, P<0.05; **, P<0.005.

Figure 5

Sorafenib formation from SG by mouse intestinal contents. (A) Formation of sorafenib upon ex vivo incubation of FVB mouse cecal contents with SG (2 µM) with or without heat pre-treatment (65°C) or (B) after treatment of mice with saline or oral neomycin 200 mg/kg given twice daily for 5 days (n ≥ 4 per group). Data were normalized to SG concentration at t=0 and represent the mean ± SE. (C) Sorafenib plasma concentrations in mice pre-treated with oral neomycin (200 mg/kg) given twice daily for 5 days. On the day of blood sample collection, SG (10 mg/kg) was administered orally. Sorafenib was not detected in plasma until 8 hours after administration of SG. All animals were female FVB mice. Data represent the mean ± SE.

Figure 6

Hepatocyte hopping and recirculation of SG. After oral administration, sorafenib enters the hepatocytes by incompletely defined transporters mechanisms, including OATP1B-type carriers and OCT1, and undergoes CYP3A4 mediated metabolism to sorafenib-N-oxide (s-N-oxide) and conjugation by UGT1A9 to form SG. After conjugation, SG is extensively secreted into the bile by a process that is mainly mediated by ABCC2. Under physiological conditions, a fraction of the intracellular SG is secreted by ABCC3 and at least one other transporter back to the blood, from where it can be taken up again into downstream hepatocytes via OATP1B1-type carriers (Oatp1a and Oatp1b in mice). This secretion-and-reuptake loop may prevent the saturation of ABCC2-mediated biliary excretion in the upstream hepatocytes, thereby ensuring efficient biliary elimination and hepatocyte detoxification. Once secreted into bile, SG enters the intestinal lumen where it serves as a substrate for as yet unknown bacterial β-glucuronidases (β-GLU) that produce sorafenib, which is subsequently undergoing intestinal absorption and re-enters the systemic circulation.