Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia

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Abstract

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug–drug and drug–endogenous substance interactions.

Introduction

Hyperbilirubinemia, or in its most severe form, jaundice, is not an uncommon finding among drug-induced hepatic side effects [1]. Drug-induced hyperbilirubinemia appears to be mediated by both drug factors (drug identity, exposure, etc.) and patient factors (patient susceptibility, genetics, etc.). Hyperbilirubinemia develops in up to 25% of patients receiving indinavir, an HIV protease inhibitor. However, hyperbilirubinemia was not attributed to saquinavir, another HIV protease inhibitor [2]. Other drugs capable of inducing hyperbilirubinemia include immune suppressant cyclosporin A [3], [4], and antituberculosis drug rifamycin SV [5]. It is particularly interesting to us that all of these drugs can cause unconjugated hyperbilirubinemia, with cyclosporin A capable of causing both unconjugated and conjugated hyperbilirubinemia [3], [4].

Bilirubin, the breakdown product of heme from red blood cells, is cleared from the systemic circulation by the liver. Once inside the liver, it is enzymatically conjugated with glucuronic acid to bilirubin-glucuronide and bilirubin-diglucuronide. These conjugated bilirubin-glucuronides are then excreted into bile by a hepatobiliary transporter, MRP2 [6]. As a compensatory pathway, the glucuronidated bilirubin may also be secreted back to sinusoidal blood by MRP3 under conditions of impaired biliary excretion [6], [7], [8]. The organic anion transporting polypeptide C (a.k.a. OATP2, LST-1; formally known as solute carrier family 21 member A6, or SLC21A6) has recently been named OATP1B1 under the new nomenclature [9]. This protein has been shown to be able to transport unconjugated bilirubin into the liver [10], [11]. The OATP1B1-mediated uptake of bilirubin can be inhibited by estradiol 17-β-d-glucuronide and bromosulfophthalein (BSP), and vice versa [10]. In addition, the OATP1B1 transport efficiency of bilirubin, as expressed by Vmax/Km, was reportedly eight times higher than another hepatic uptake transporter OATP1B3 [11].

We, therefore, hypothesized that inhibition of human OATP1B1 by drugs is an important mechanism of unconjugated hyperbilirubinemia. We studied the interaction of various drugs with the OATP1B1 in stably transfected HEK 293 cells. In this paper, we report that these drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 that can be inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support that inhibition of OATP1B1 by drugs can cause transient unconjugated hyperbilirubinemia. This mechanism of elevated bilirubin is drug concentration-related and reversible, and therefore can explain the benign reversible patterns of hyperbilirubinemia caused by these drugs.

Section snippets

Cells and chemicals

Human embryonic kidney (HEK 293) cells transfected with OATP1B1 were obtained from Prof. Dietrich Keppler (DKFZ-Heidelberg, Germany) [12]. [3H]Estradiol 17β-d-glucuronide (44 Ci/mmol >97% purity) was obtained from Perkin-Elmer Life Sciences (Boston, MA). Sodium N-butyrate was purchased from ICN Biochemicals (Aurora, OH). Saquinavir and Indinavir were obtained from Pfizer chemical sample bank. All other reagents were purchased from Sigma and were of the highest grade available.

Inhibition of OATP1B1-mediated uptake of estradiol 17β-d-glucuronide

Transfected HEK 293

Cyclosporin A, indinavir, and rifamycin SV inhibited OATP1B1 uptake

Because OATP1B1 has been identified to be a more efficient bilirubin uptake transporter in adult human liver than OATP1B3 [11], we investigated the relationship of OATP1B1 inhibition by known therapeutic drugs with various incidences of reversible unconjugated hyperbilirubinemia. Specifically, the effects of cyclosporin A, saquinavir, indinavir, and rifamycin SV on OATP1B1-mediated uptake were examined.

The uptake of 2 μM estradiol 17β-d-glucuronide was measured in HEK-OATP1B1 and HEK-Control

Discussion

Our study indicates that the liver organic anion uptake transporter, OATP1B1, can be a vulnerable target of drug-induced side effects. Inhibition of OATP1B1 may be an important mechanism of drug-induced unconjugated hyperbilirubinemia. Our conclusion is based on the following experimental data: (a) lipophilic anionic drugs from different therapeutic families (HIV protease inhibitor, immune suppressant, anti-infective) are capable of inhibiting the human OATP1B1 with sub-micromolar Ki values;

Acknowledgement

The authors gratefully acknowledge Dr. Dietrich Keppler (Heidelberg, Germany) for providing the HEK-OATP1B1 cells used in this study.

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