A novel trifluorinated cholic acid derivative, CA-lys-TFA, was designed and synthesized for use as a tool to measure bile acid transport noninvasively using magnetic resonance imaging (MRI). In the present study, the in vivo performance of CA-lys-TFA for measuring bile acid transport by MRI was investigated in mice. Gallbladder CA-lys-TFA content was quantified using MRI and liquid chromatography/tandem mass spectrometry. Results in wild-type (WT) C57BL/6J mice were compared to those in mice lacking expression of Asbt, the ileal bile acid transporter. 19F signals emanating from the gallbladders of WT mice 7 h after oral gavage with 150 mg/kg CA-lys-TFA were reproducibly detected by MRI. Asbt-deficient mice administered the same dose had undetectable 19F signals by MRI, and gallbladder bile CA-lys-TFA levels were 30-fold lower compared to WT animals. To our knowledge, this represents the first report of in vivo imaging of an orally absorbed drug using 19F MRI. Fluorinated bile acid analogues have potential as tools to measure and detect abnormal bile acid transport by MRI.

Purpose Our objectives were to synthesize a trifluorinated bile acid that can be used for 19F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and 19F-MRI signal, and assess its ability to concentrate in the gallbladder of C57BL/6 mice. Methods Target compound CA-lys-TFA was synthesized and tested for affinity toward the apical sodium dependent bile acid transporter (hASBT) and the Na+/taurocholate cotransporting polypeptide (hNTCP). In a pilot study, fasted mice were gavaged with vehicle control, 150 mg/kg or 300 mg/kg CA-lys-TFA. CA-lys-TFA in gallbladder, liver and plasma at t=5 h was quantified. Additionally, a 24-h time course (24 mice across eight time points) was studied using 50 mg/kg CA-lys-TFA. Results CA-lys-TFA was a potent substrate of hASBT (Kt=39.4 μM, normalized Vmax=0.853) and hNTCP (Kt=8.99 μM, normalized Vmax=0.281). 19F MRI phantom imaging showed linear signal-concentration dependence. In vivo studies showed that rapid accumulation of CA-lys-TFA in the gallbladder was maximal within 4-7 h. Conclusion These findings suggest that CA-lys-TFA, a fluorinated non-radioactive bile acid analogue, has potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport.

Previously, we developed a trifluorinated bile acid, CA-lys-TFA, with the objective of noninvasively assessing bile acid transport in vivo using 19F magnetic resonance imaging (MRI). CA-lys-TFA was successfully imaged in the mouse gallbladder, but was susceptible to deconjugation in vitro by choloylglycine hydrolase (CGH), a bacterial bile acid deconjugating enzyme found in the terminal ileum and colon. The objective of the present study was to develop a novel trifluorinated bile acid resistant to deconjugation by CGH. CA-sar-TFMA was designed, synthesized, and tested for in vitro transport properties, stability, imaging properties, and its ability to differentially accumulate in the gallbladders of normal mice, compared with mice with known impaired bile acid transport (deficient in the apical sodium-dependent bile acid transporter, ASBT). CA-sar-TFMA was a potent inhibitor and substrate of ASBT and the Na+/taurocholate cotransporting polypeptide. Stability was favorable in all conditions tested, including the presence of CGH. CA-sar-TFMA was successfully imaged and accumulated at 16.1-fold higher concentrations in gallbladders from wild-type mice compared with those from Asbt-deficient mice. Our results support the potential of using MRI with CA-sar-TFMA as a noninvasive method to assess bile acid transport in vivo.

Floxuridine is often used to treat metastatic liver disease and is given as an infusion directly into the hepatic artery to increase the amount of intact drug that reaches the liver. The objective of this work was to design and synthesize prodrugs of floxuridine through conjugation to chenodeoxycholic acid (CDCA) to target the liver via the bile acid liver uptake transporter Na+/taurocholate cotransporting polypeptide (NTCP, SLC10A1). Two isomeric prodrugs of floxuridine were synthesized: floxuridine 3′ glutamic acid-CDCA and floxuridine 5′-glutamic acid-CDCA. Both were potent inhibitors and substrates of NTCP. Floxuridine 3′ glutamic acid-CDCA showed Ki = 6.86 ± 1.37 μM, Km = 10.7 ± 2.1 μM, and passive permeability = 0.663 (± 0.121) x 10−7 cm/s while floxuridine 5′-glutamic acid-CDCA showed Ki = 0.397 ± 0.038 μM, Km = 40.4 ± 15.2 μM, and passive permeability = 1.72 (± 0.18) x 10−7 cm/s. Floxuridine itself had a higher passively permeability of 7.54 (± 0.45) x 10−7 cm/s in the same cell line, indicating that both prodrugs have the potential for lower non-specific effects than the drug alone. Prodrugs were stable in rat plasma (t = 3h), but quickly released in rat liver s9 fraction, suggesting future in vivo evaluation.

The aim was to elucidate how steps in drug translocation by a solute carrier transporter impact Michaelis-Menten parameters Km, Ki, and Vmax. The first objective was to derive a model for carrier-mediated substrate translocation and perform sensitivity analysis with regard to the impact of individual microrate constants on Km, Ki, and Vmax. The second objective was to compare underpinning microrate constants between compounds translocated by the same transporter. Equations for Km, Ki, and Vmax were derived from a six-state model involving unidirectonal transporter flipping and reconfiguration. This unidirectional model is applicable to co-transporter type solute carriers, like the apical sodium-dependent bile acid transporter (ASBT) and the proton-coupled peptide cotransporter (PEPT1). Sensitivity analysis identified the microrate constants that impacted Km, Ki, and Vmax. Compound comparison using the six-state model employed regression to identify microrate constant values that can explain observed Km and Vmax values. Results yielded some expected findings, as well as some unanticipated effects of microrate constants on Km, Ki, and Vmax. Km and Ki were found to be equal for inhibitors that are also substrates. Additionally, microrate constant values for certain steps in transporter functioning influenced Km and Vmax to be low or high.