Stef De Lombaerde

Synthesis, in vitro and in vivo evaluation of 3β-[18F]fluorocholic acid for the detection of drug-induced cholestasis in mice

Introduction Drug-induced cholestasis is a liver disorder that might be caused by interference of drugs with the hepatobiliary bile acid transporters. It is important to identify this interference early on in drug development. In this work, Positron Emission Tomography (PET)-imaging with a 18F labeled bile acid analogue was introduced to detect disturbed hepatobiliary transport of bile acids. Methods 3β-[18F]fluorocholic acid ([18F]FCA) was prepared by nucleophilic substitution of a mesylated precursor with [18F]fluoride, followed by deprotection with sodium hydroxide. Transport of [18F]FCA was assessed in vitro using CHO-NTCP, HEK-OATP1B1, HEK-OATP1B3 transfected cells and BSEP & MRP2 membrane vesicles. Investigation of [18F]FCA metabolites was performed with primary mouse hepatocytes. Hepatobiliary transport of [18F]FCA was evaluated in vivo in wild-type, rifampicin and bosentan pretreated FVB-mice by dynamic μPET scanning. Results Radiosynthesis of [18F]FCA was achieved in a moderate radiochemical yield (8.11 ± 1.94%; non-decay corrected; n = 10) and high radiochemical purity (>99%). FCA was transported by the basolateral bile acid uptake transporters NTCP, OATP1B1 and OATP1B3. For canalicular efflux, BSEP and MRP2 are the relevant bile acid transporters. [18F]FCA was found to be metabolically stable. In vivo, [18F]FCA showed fast hepatic uptake (4.5 ± 0.5 min to reach 71.8 ± 1.2% maximum % ID) and subsequent efflux to the gallbladder and intestines (93.3 ± 6.0% ID after 1 hour). Hepatobiliary transport of [18F]FCA was significantly inhibited by both rifampicin and bosentan. Conclusion A 18F labeled bile acid analogue, [18F]FCA, has been developed that shows transport by NTCP, OATP, MRP2 and BSEP. [18F]FCA can be used as a probe to monitor disturbed hepatobiliary transport in vivo and accumulation of bile acids in blood and liver during drug development.

Synthesis, in vitro and in vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered) hepatic transporter function.

INTRODUCTION 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. METHODS [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. RESULTS 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. CONCLUSION 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.

Performance evaluation of the LightPath imaging system for intra-operative Cerenkov luminescence imaging

The performances of an intra-operative optical imaging system for Cerenkov luminescence imaging of resected tumor specimens were evaluated with phantom studies. The spatial resolution, the linearity of the measured signal with the activity concentration and the minimum detectable activity concentration were considered. A high linearity was observed over a broad range of activity concentration (R2⩾0.99 down to ∼40 kBq/ml of 18F-FDG). For 18F-FDG activity distributions 2 mm deep in biological tissue, the measured detection limit was 8 kBq/ml and a spatial resolution of 2.5 mm was obtained. The detection limit of the imaging system is comparable with clinical activity concentrations in tumor specimens, and the spatial resolution is compatible with clinical requirements.

Evaluating hepatobiliary transport with 18F-labeled bile acids : the effect of radiolabel position and bile acid structure on radiosynthesis and in vitro and in vivo performance

Introduction An in vivo determination of bile acid hepatobiliary transport efficiency can be of use in liver disease and preclinical drug development. Given the increased interest in bile acid Positron Emission Tomography- (PET-) imaging, a further understanding of the impact of 18-fluorine substitution on bile acid handling in vitro and in vivo can be of significance. Methods A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3-, 7-, or 12-OH function is substituted with a fluorine atom (3α-[18F]FCA; 7β-[18F]FCA; 12β-[18F]FCA); a glycocholic and chenodeoxycholic acid analogue, substituted on the 3-position (3β-[18F]FGCA and 3β-[18F]FCDCA, resp.). Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines and efflux by BSEP in membrane vesicles. PET-scans with the tracers were performed in wild-type mice (n = 3 per group): hepatobiliary transport was monitored and compared to a reference tracer, namely, 3β-[18F]FCA. Results Compounds 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA were synthesized in moderate radiochemical yields (4–10% n.d.c.) and high radiochemical purity (>99%); 7β-[18F]FCA and 12β-[18F]FCA could not be synthesized and included further in this study. In vitro evaluation showed that 3α-FCA, 3β-FGCA, and 3β-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1, and BSEP. In vivo, 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3β-[18F]FCA epimers. Conjugation of 3β-[18F]FCA with glycine had no significant effect in vivo. Compound 3β-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines. Conclusion A set of 18F labeled bile acids was synthesized that are substrates of the bile acid transporters in vitro and in vivo and can serve as PET-biomarkers for hepatobiliary transport of bile acids.