Fusidic Acid Inhibits Hepatic Transporters and Metabolic Enzymes: Potential Cause of Clinical Drug-Drug Interaction Observed with Statin Coadministration

Abstract

Fusidic acid (FA), which was approved in the 1960s in many European and Asian countries, has gained renewed interest due to its continued effectiveness against methicillin-resistant Staphylococcus aureus As rhabdomyolysis has been reported upon coadministration of FA with statins, we aimed to elucidate the underlying molecular mechanisms that contribute to FA-statin drug-drug interactions. Because of the association between rhabdomyolysis and increased exposure to statins, we investigated if cytochrome P450 (CYP) enzymes and transporters involved in the disposition of various statins are inhibited by FA. FA was found to inhibit BCRP and OATP1B1 but not P-gp. In overexpressing cell systems, FA inhibited BCRP-mediated efflux (50% inhibitory concentration [IC50], ∼50 to 110 μM) and OATP1B1-mediated uptake (IC50, ∼4 to 35 μM) of statins at clinically relevant concentrations achievable in the intestine and liver (based on a 550-mg oral dose of FA, the expected maximum theoretical gastrointestinal concentration is ∼4 mM, and the maximum total or unbound concentration in the inlet to the liver was reported to be up to 223 μM or 11 μM, respectively, upon multiple dosing). Similarly, FA inhibited metabolism of statins in human liver microsomes (IC50, ∼17 to 195 μM). These data suggest that FA inhibits at least 3 major dispositional pathways (BCRP, OATP1B1, and CYP3A) and thus affects the clearance of several statins. We confirmed that FA is eliminated via phase 1 metabolism (primarily via CYP3A); however, there is also some phase 2 metabolism (mediated primarily by UGT1A1). Taken together, these data provide evidence for molecular mechanisms that may explain the occurrence of rhabdomyolysis when FA is administered with statins.

FIG 1

Transcellular transport of prototypical substrates of BCRP and P-gp was measured using monolayers of MDCK cells transfected with the ABCG2 (MDCK-BCRP) and P-gp (MDCK-MDR1) genes, respectively, or of MDCK-WT control cells seeded on transwell membranes. Prazosin (2 μM), a substrate of BCRP (A), and labetalol (2 μM), a substrate of P-gp (B), were incubated with the respective cell lines for 2 h, and the transport in the apical to basal (P_app, _{A → B}) and basal to apical (P_app, _{B → A}) directions was measured in the absence or presence of P-gp, the BCRP inhibitor CsA (10 μM), and/or the test compound FA at various concentrations (10 μM to 250 μM). The efflux ratio (ER) was calculated as follows: ER = P_app, _{B → A}/P_app, _{A → B}. Data are expressed as means ± standard deviations for at least 4 biological replicates. The statistical significance of ERs obtained in the absence versus presence of CsA or FA was determined by Student's t test (*, P < 0.05; **, P < 0.005).

FIG 2

Transcellular bidirectional transport of atorvastatin (A), fluvastatin (B), pitavastatin (C), and rosuvastatin (D) was measured following incubation of each statin (2 μM) in the absence or presence of various concentrations of FA for 2 h, using MDCK-ABCG2 cell monolayers. The efflux ratio was calculated as described in the legend to Fig. 1. Data are expressed as means ± standard deviations for at least 4 biological replicates. The IC₅₀ for vectorial transport was calculated by fitting a nonlinear regression model to the data on efflux ratio and FA concentration by using GraphPad Prism 5. Calculated fitting curves were overlaid.

FIG 3

Intracellular uptake of various statins. Atorvastatin (A), fluvastatin (B), pitavastatin (C), simvastatin (D), rosuvastatin (E), and cerivastatin (F) levels were measured in the TransportoCells system (Corning's proprietary cell system overexpressing OATP1B1) by incubating the test compound (1 μM) in the absence or presence of various concentrations of FA (3 μM to 1,000 μM) for 5 min. Uptake in the presence of inhibitor was normalized to the amount of test compound accumulated in the absence of inhibitor. Data are expressed as means ± standard deviations for at least 4 biological replicates. The IC₅₀ for OATP1B1 was calculated by fitting a nonlinear regression model to percent uptake and FA concentration by using GraphPad Prism 5. Calculated fitting curves were overlaid.

FIG 4

Hepatic uptake of [³H]E₃S, [³H]propranolol, and [³H]rosuvastatin was measured using an oil spin accumulation assay with fresh human hepatocytes. Uptake of [³H]E₃S (2 μM), a marker substrate for active uptake (A), or [³H]propranolol (2 μM), a marker substrate for passive permeability (B), was measured in the absence (squares) or presence (triangles) of the OATP inhibitor CsA (10 μM) or with a test concentration (100 μM) of FA (circles) at 37°C (open symbols) or 4°C (closed symbols) and at various time points (10 s to 40 s). Subsequently, to calculate the IC₅₀, uptake of [³H]E₃S (C) or [¹⁴C]rosuvastatin (D) in the absence and presence of various concentrations of FA was measured at various time points (10 s to 40 s). The uptake at the highest accumulation time point (40 s) in the presence of various concentrations of FA was then normalized to uptake in the absence of FA for both [³H]E₃S (E) and [¹⁴C]rosuvastatin (F) and then expressed as % uptake. The IC₅₀ for uptake was calculated by fitting a nonlinear regression model to the % uptake and FA concentration by using GraphPad Prism 5. Calculated fitting curves were overlaid. RSV, rosuvastatin.

FIG 5

Reaction phenotyping was performed to ascertain the pathways involved in the metabolism of fusidic acid (FA). The amount of FA remaining was determined at various time points for incubation mixtures containing either recombinant human P450 (A) or UGT (B) isoforms. Data are presented as percentages of FA remaining at the end of 30 min of incubation, corrected for control incubation in the absence of NADPH.

FIG 6

Inhibition of P450-mediated metabolism of atorvastatin (A), cerivastatin (B), fluvastatin (C), and simvastatin (D) was studied in human liver microsomes in the presence of various concentrations of FA (0 μM to 1,000 μM). Intrinsic clearance was estimated using the slope of the curve for parent disappearance as described in Materials and Methods. The IC₅₀s were calculated by fitting a nonlinear regression model to the intrinsic clearance values and the FA concentration by using GraphPad Prism 5. Calculated fitting curves were overlaid.

FIG 7

To determine the specific P450 isozymes involved in the interaction, FA-mediated inhibition of metabolism was studied with 2 μM atorvastatin (A), cerivastatin (B), and rosuvastatin (C), using a recombinant CYP3A4 system in the presence of various concentrations of FA (0 μM to 1,000 μM). Intrinsic clearance was estimated using the slope of the curve for parent disappearance as described in Materials and Methods. The IC₅₀s were calculated by fitting a nonlinear regression model to the intrinsic clearance values and the FA concentration by using GraphPad Prism 5. Calculated fitting curves were overlaid for atorvastatin and cerivastatin only.