Validation of Pharmacological Protocols for Targeted Inhibition of Canalicular MRP2 Activity in Hepatocytes Using [99mTc]mebrofenin Imaging in Rats

Abstract

The multidrug resistance-associated protein 2 (MRP2) mediates the biliary excretion of drugs and metabolites. [99mTc]mebrofenin may be employed as a probe for hepatic MRP2 activity because its biliary excretion is predominantly mediated by this transporter. As the liver uptake of [99mTc]mebrofenin depends on organic anion-transporting polypeptide (OATP) activity, a safe protocol for targeted inhibition of hepatic MRP2 is needed to study the intrinsic role of each transporter system. Diltiazem (DTZ) and cyclosporin A (CsA) were first confirmed to be potent MRP2 inhibitors in vitro. Dynamic acquisitions were performed in rats (n = 5–6 per group) to assess the kinetics of [99mTc]mebrofenin in the liver, intestine and heart-blood pool after increasing doses of inhibitors. Their impact on hepatic blood flow was assessed using Doppler ultrasound (n = 4). DTZ (s.c., 10 mg/kg) and low-dose CsA (i.v., 0.01 mg/kg) selectively decreased the transfer of [99mTc]mebrofenin from the liver to the bile (k3). Higher doses of DTZ and CsA did not further decrease k3 but dose-dependently decreased the uptake (k1) and backflux (k2) rate constants between blood and liver. High dose of DTZ (i.v., 3 mg/kg) but not CsA (i.v., 5 mg/kg) significantly decreased the blood flow in the portal vein and hepatic artery. Targeted pharmacological inhibition of hepatic MRP2 activity can be achieved in vivo without impacting OATP activity and liver blood flow. Clinical studies are warranted to validate [99mTc]mebrofenin in combination with low-dose CsA as a novel substrate/inhibitor pair to untangle the role of OATP and MRP2 activity in liver diseases.

Repurposing 99mTc-Mebrofenin as a Probe for Molecular Imaging of Hepatocyte Transporters

“…Pharmacokinetic modeling revealed a dose-dependent inhibition of the sinusoidal efflux (liver-to-blood) of 99m Tc-mebrofenin by CsA, consistent with the expression of MRP3 at this specific interface (17,21). In rats, the extent of MRP3mediated sinusoidal and MRP2-mediated canalicular efflux transport of 99m Tc-mebrofenin was similar but remained much lower than the OATP-mediated sinusoidal uptake, thus providing information regarding the relative importance of carrier-mediated processes in hepatocytes (21). Imaging probes for quantitative determination of MRP2 and MRP3 function in hepatocytes are still not available (6).…”

“…This finding illustrates that dramatic changes in the liver exposure to drugs can be observed, which may potentially lead to hepatotoxicity, with a nondetectable impact on plasma pharmacokinetics. Pharmacokinetic modeling revealed a dose-dependent inhibition of the sinusoidal efflux (liver-to-blood) of 99m Tc-mebrofenin by CsA, consistent with the expression of MRP3 at this specific interface (17,21). In rats, the extent of MRP3mediated sinusoidal and MRP2-mediated canalicular efflux transport of 99m Tc-mebrofenin was similar but remained much lower than the OATP-mediated sinusoidal uptake, thus providing information regarding the relative importance of carrier-mediated processes in hepatocytes (21).…”

“…Cyclosporin A (CsA), known to inhibit several efflux transporters including MRP2, did not further decrease the biliary excretion of 99m Tc-mebrofenin in MRP2-deficient rats, suggesting a limited role of other ABC transporters at the liver-bile interface (16). We recently investigated the impact of increasing doses of CsA on the liver kinetics of 99m Tc-mebrofenin, revealing that CsA was more potent in inhibiting MRP2 than OATP (21). Low-dose CsA (0.01 mg/kg administered intravenously) completely inhibited the MRP2-mediated biliary excretion of 99m Tc-mebrofenin without affecting the OATPmediated sinusoidal uptake.…”

“…2) (31). CsA is a potent inhibitor of the OATP-and MRP2mediated transport of 99m Tc-mebrofenin (21). A study performed on patients undergoing liver transplantation showed that hepatic extraction of 99m Tc-mebrofenin was consistently lower in CsAtreated patients than in tacrolimus-treated patients, despite similar liver function assessed using biochemistry assays (32).…”

Imaging-Based Characterization of a Slco2b1(-/-) Mouse Model Using [11C]Erlotinib and [99mTc]Mebrofenin as Probe Substrates

“…Images were analyzed with PMOD ® software (version 3.9, PMOD Technologies LLC, Zürich, Switzerland) as described in a previous study in rats [ 27 ]. Regions of interest were manually drawn over the liver, intestine (assumed to represent excreted bile) and whole heart (image-derived blood curve).…”