Hepatobiliary transport mechanisms are crucial for the excretion of substrate toxic compounds. Drugs can inhibit these transporters, which can lead to drug-drug interactions causing toxicity. Therefore, it is important to assess this early during the development of new drug candidates. The aim of the current study is the (radio)synthesis, in vitro and in vivo evaluation of a technetium labeled chenodeoxycholic and cholic acid analogue: [(99m)Tc]-DTPA-CDCA and [(99m)]Tc-DTPA-CA, respectively, as biomarker for disturbed transporter functionality.
[99mTc]-DTPA-CDCA([(99m)Tc]-3a) and [99mTc]-DTPA-CA ([(99m)Tc]-3b) were synthesized and evaluated in vitro and in vivo. Uptake of both tracers was investigated in NTCP, OCT1, OATP1B1, OATP1B3 transfected cell lines. Km and Vmax values were determined and compared to [(99m)Tc]-mebrofenin ([(99m)Tc]-MEB). Efflux was investigated by means of CTRL, MRP2 and BSEP transfected inside-out vesicles. Metabolite analysis was performed using pooled human liver S9. Wild type (n=3) and rifampicin treated (n=3) mice were intravenously injected with 37MBq of tracer. After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of heart, liver, gallbladder and intestines were obtained.
We demonstrated that OATP1B1 and OATP1B3 are the involved uptake transporters of both compounds. Both tracers show a higher affinity compared to [(99m)Tc]-MEB, but are in a similar range as endogenous bile acids for OATP1B1 and OATP1B3. [(99m)Tc]-3a shows higher affinities compared to [(99m)Tc]-3b. Vmax values were lower compared to [(99m)Tc]-MEB, but in the same range as endogenous bile acids. MRP2 was identified as efflux transporter. Less than 7% of both radiotracers was metabolized in the liver. In vitro results were confirmed by in vivo results. Uptake in the liver and efflux to gallbladder + intestines and urinary bladder of both tracers was observed. Transport was inhibited by rifampicin.
The involved transporters were identified; both tracers are taken up in the hepatocytes by OATP1B1 andOATP1B3 with Km and Vmax values in the same range as endogenous bile acids and are secreted into bile canaliculi via MRP2. Dynamic small-animal SPECT imaging can be a useful noninvasive method of visualizing and quantifying hepatobiliary transporter functionality and disturbances thereof in vivo, which could predict drug pharmacokinetics.

OBJECTIVE: Demonstration of feasibility and protocol optimization for the combined use of gadofosveset trisodium with gadoxetic acid for delayed T1-weighted liver MRI.
METHODS: Eleven healthy volunteers underwent hepatobiliary phase imaging at 3 Tesla (T) using gadoxetic acid. Multiple breathheld T1-weighted three-dimensional spoiled gradient echo sequences were performed at varying flip angles before and after injection of gadofosveset. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were measured to determine optimal T1-weighting. Examples of three patients with focal liver lesions were acquired.
RESULTS: The addition of gadofosveset to the hepatobiliary phase of gadoxetic acid renders vessels isointense to liver tissue at low flip angles due to increased vessel SNR (P < 0.001). The lowest CNR of liver relative to portal vein (CNR = 15; 95% confidence interval [CI]: -14-44) was observed at a 10º flip angle. The highest CNR of liver relative to muscle (CNR = 214; 95% CI: 191-237) was observed at a 20º flip angle. The combined enhancement leads to homogenously enhanced liver tissue and liver vasculature. Cysts were detected in three volunteers and metastases were detected in two patients. In these anecdotal cases the cysts and metastases stood out as conspicuous focal hypointensities on combined gadoxetic acid and gadofosveset enhanced images.
CONCLUSIONS: Combined gadoxetic acid and gadofosveset enhanced liver MRI is feasible, with low flip angles minimizing contrast between vessels and liver. Further clinical studies are needed to confirm that low flip angles provide an optimal combination of sensitivity and specificity for lesion detection in patients.