OATP1B1 polymorphism as a determinant of erythromycin disposition

Abstract

Previous studies have demonstrated that the pharmacokinetic profile of erythromycin, a probe for CYP3A4 activity, is affected by inhibitors or inducers of hepatic solute carriers. We hypothesized that these interactions are mediated by OATP1B1 (gene symbol, SLCO1B1), a polypeptide expressed on the basolateral surface of hepatocytes. Using stably transfected Flp-In T-Rex293 cells, erythromycin was found to be a substrate for OATP1B1*1A (wild type) with a Michaelis-Menten constant of ~13 µmol/l, and that its transport was reduced by ~50% in cells expressing OATP1B1*5 (V174A). Deficiency of the ortholog transporter Oatp1b2 in mice was associated with a 52% decrease in the metabolic rate of erythromycin (P = 0.000043). In line with these observations, in humans the c.521T>C variant in SLCO1B1 (rs4149056), encoding OATP1B1*5, was associated with a decline in erythromycin metabolism (P = 0.0072). These results suggest that impairment of OATP1B1 function can alter erythromycin metabolism, independent of changes in CYP3A4 activity.

Figure 1

In vitro transport studies of erythromycin. (A) Transport of [6,7-³H(N)]estradiol-17β-Dglucuronide (E2-17β-glu), a positive control, by human OATP1B1 and mouse Oatp1b2 was evaluated with constructs transfected into mammalian cells (E2-17β-glu concentration, 0.1 µM; 15-min incubations) in the absence or presence of rifampin (200 µM; 15-min pre-incubation). (B) Transport of [¹⁴C-N-methyl]erythromycin (erythromycin) by human OATP1B1 and mouse Oatp1b2 was evaluated in transfected into cells (erythromycin concentration, 0.1 µM; 15-min incubations) in the absence or presence of rifampin (200 µM; 15-min pre-incubation). (C) Characterization of erythromycin concentration-dependent transport by human OATP1B1. (D) Transport of E2-17β-glu (0.1 µM; 15-min incubations) and erythromycin (concentration, 0.1 µM; 60-min incubations) was evaluated in Flp-In T-Rex293 cells transfected with OATP1B1*1A (wildtype), OATP1B1*1B [c.388A>G (N130D); rs2306283], OATP1B1*5 [c.521T>C (V174A); rs4149056], or OATP1B1*15 (N130D, V174A) . All data represent the mean of at least 6 observations, and are expressed as the average percent of uptake values in cells transfected with an empty vector (VC). Error bars represent the standard error. The star (*) denotes a significant difference from each corresponding VC (P < 0.05).

Figure 2

Theoretical influence of Oatp1b2-knockout on the metabolism of [¹⁴C-Nmethyl] erythromycin (¹⁴C-ER) in mice following an erythromycin breath test.

Figure 3

Loss of Oatp1b2 in mice alters erythromycin metabolism without impacting Cyp3a function. (A) Time course of cumulative exhaled ⁴CO₂ was evaluated in wildtype mice and Oatp1b2-deficient [Oatp1b2(−/−)] mice using 12 animals per group, representing triplicate observations from an experiment performed on 4 separate occasions. (B) Pairwise comparison of select genes encoding transporter or enzymes of known or suspected relevance to erythromycin was evaluated at baseline in livers of wildtype mice and Oatp1b2(−/−) mice using 5 animals per group. (C/D) Protein expression for Cyp3a11, normalized to β-actin in livers of wildtype mice and Oatp1b2(−/−) was evaluated using Western blotting on samples from 4 animals per group. (E) Hepatic microsomal Cyp3a activity in the same livers, as determined by midazolam hydroxylation. All data represent mean (bars) and SEM (error bars).

Figure 4

Erythromycin metabolism as a function of SLCO1B1 genotype. (A) Data were obtained in 91 predominantly white cancer patients undergoing an erythromycin breath test (ERMBT). The same information is shown after eliminating subjects expressing CYP3A5 (B), and those individuals with either the SLCO1B3 334TT (C) or the ABCC2 -24TT genotype (D). Each symbol represents an individual patient, and horizontal lines indicate median values. The P-value denotes a statistical comparison of the ERMBT results in the different genotype group, and a star (*) denotes a significant difference from each corresponding group with the reference SLCO1B1 genotype (P < 0.05).