(T1130-05-32) Global Identification of OATP1B1-Associated Proteins and Implications of Calcineurin Inhibition in Time-Dependent OATP1B1 Inhibition

Purpose: The organic anion transporting polypeptide (OATP) 1B1 is an important determinant of transporter-mediated drug-drug interactions (DDIs). Time-dependent inhibition of OATP1B1 is highlighted in the US FDA final guidance for in vitro DDI studies due to the clinical significance of an exemplar drug, the immunosuppressant calcineurin inhibitor (CNI) cyclosporine A (CsA), in causing OATP-mediated DDIs. The mechanism underlying such time-dependent inhibition is unknown. The CNIs CsA and tacrolimus inhibit activity of the protein phosphatase calcineurin. Mechanistically, this inhibition occurs via binding of the CNIs to intracellular immunophilin proteins, CsA to cyclophilins and tacrolimus to FK506-binding proteins (FKBPs), to form inhibitory ternary complexes with calcineurin. OATP1B1 is a phosphoprotein; calcineurin, which is known to modulate the function of many phosphoproteins, has not been investigated in the context of time-dependent inhibition of OATP1B1. The current study is designed to identify the OATP1B1-associated proteins and to determine the effects of calcineurin inhibition on time-dependent inhibition of OATP1B1.

Methods: OATP1B1-associated proteins were identified by immunoprecipitation (IP) followed by liquid chromatography with tandem mass spectrometry. FLAG-tagged OATP1B1 proteins were purified by FLAG-IP from OATP1B1-overexpressing human embryonic kidney (HEK) 293 stable cell lines. FLAG-IP in HEK293-mock cells served as the negative control. The preincubation effects of CsA and tacrolimus – and of SCY-635, an inactive analog of CsA lacking calcineurin inhibition activity – were determined in OATP1B1-expressing HEK293 stable cell line using [³H]-estradiol 17 β-D-glucuronide (E₂17βG) (1 µM, 2 min) as the probe substrate.

Results: A total of 846 proteins were identified as specifically associated with OATP1B1, including the tacrolimus-binding proteins FKBP5 and FKBP8. The CsA-binding proteins cyclophilin A and cyclophilin B were also identified in the immunocomplexes with FLAG-OATP1B1. Preincubation with CsA (0.001-10 µM) rapidly reduced OATP1B1-mediated transport as early as 10 min, even after washing, yielding preincubation IC₅₀ values of 0.30 ± 0.04, 0.17 ± 0.04, and 0.11 ± 0.04 µM (best fit values ± associated SE, N=3 in triplicate) after preincubation for 10, 30, and 60 min, respectively. The IC₅₀ values against OATP1B1 after SCY-635 (0.001-10 µM) preincubation for 10 and 30 min were greater than 10 and 3 µM, respectively, and could not be estimated due to the minimal inhibition. Preincubation with SCY-635 for 60 min yielded IC₅₀ of 2.7 ± 0.1 µM against OATP1B1, which is ~24.5 fold greater than that by CsA. Preincubation (10-60 min) with tacrolimus rapidly achieved maximum inhibitory effects on OATP1B1-mediated transport as early as after 10 min preincubation at concentrations of either 1 or 10 µM, significantly reducing substrate transport to 0.70 ± 0.03 and 0.18 ± 0.03 fold of control at 1 and 10 µM, respectively (Mean ± SE, N=3 in triplicate).

Conclusion: We report the novel finding that the intracellular receptors of the CNIs CsA and tacrolimus are in complex with OATP1B1. For the first time, we report that the CNI tacrolimus causes time-dependent inhibition of OATP1B1 in vitro and that the calcineurin-inhibition activity of CsA is likely involved in its time-dependent inhibition of OATP1B1. The current study provides novel insight into the underlying mechanism of time-dependent inhibition of OATP1B1.

Acknowledgements: JP, RK, and AC contributed equally. Supported by NIH R01 GM146956-01 (WY)