In vivo performance of a novel fluorinated magnetic resonance imaging agent for functional analysis of bile acid transport.

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. (19)F 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 (19)F 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 (19)F MRI. Fluorinated bile acid analogues have potential as tools to measure and detect abnormal bile acid transport by MRI.

Design and characterization of a novel fluorinated magnetic resonance imaging agent for functional analysis of bile Acid transporter activity.

PURPOSE: To synthesize a trifluorinated bile acid that can be used for (19)F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and (19)F-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). (19)F 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. CONCLUSIONS: 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.

Common Deficiencies of in vitro Binding Bioequivalence (BE) Studies Submitted in Abbreviated New Drug Applications (ANDAs).

There are several drug products that bind phosphate or bile acid in the gastrointestinal (GI) tract to exert their therapeutic efficacy. In vitro binding studies are used to assess bioequivalence (BE) of these products. The objective of this study is to identify the common deficiencies in Abbreviated New Drug Applications (ANDAs) for these products. Deficiencies were compiled from ANDAs containing in vitro binding BE studies. The deficiencies were classified into eight categories: Pre-Study Method Validation, During-Study Sample Analysis, Study Design, Study Procedure, Dissolution/Disintegration, Analytical Site Inspection, Data Submission, and Formulations. Within each category, additional subcategories were defined to characterize the deficiencies. A total of 712 deficiencies from 95 ANDAs for 11 drug products were identified and included in the analysis. The four categories with the most deficiencies were During-Study Sample Analysis (27.8%), Pre-Study Method Validation (17.3%), Data Submission (16.7%), and Study Design (15.7%). For the During-Study Sample Analysis category, failure to submit complete raw data or analytical runs ranked as the top deficiency (32.8%). For the Study Design category, using an unacceptable alternate study design (26.8%) was the most common deficiency. Within this category, other commonly occurring deficiencies included incorrect/insufficient number of absorbent concentrations, failure to pre-treat drug product with acid, insufficient number of replicates in study, incorrect calculation of k1 and k2 values, incorrect dosage form or pooled samples used in the study, and incorrect pH of study medium. The review and approval of these products may be accelerated if these common deficiencies are addressed in the original ANDA submissions.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Synthesis and in vitro evaluation of bile acid prodrugs of floxuridine to target the liver.

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)×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)×10(-7) cm/s. Floxuridine itself had a higher passively permeability of 7.54(±0.45)×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=3 h), but quickly released in rat liver s9 fraction, suggesting future in vivo evaluation.

Mechanistic interpretation of conventional Michaelis-Menten parameters in a transporter system.

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 unidirectional 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.

Design and evaluation of a novel trifluorinated imaging agent for assessment of bile acid transport using fluorine magnetic resonance imaging.

Previously, we developed a trifluorinated bile acid, CA-lys-TFA, with the objective of noninvasively assessing bile acid transport in vivo using (19) F 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.

Phrygian Cap Appearance of a Mouse Gallbladder on Magnetic Resonance Imaging.

We used live-animal magnetic resonance imaging (MRI) to examine the gallbladders of male mice. These healthy mice were fasted overnight before the study and anesthetized in an animal chamber, with a gas mixture of oxygen and isoflurane for small animal MRI. In the course of these live-animal MRI studies, we observed a Phrygian cap appearance to the gallbladder of one healthy-appearing 6-week-old male mouse, similar to that of the human gallbladder described in many reports. After euthanasia for measurement of bile content, this mouse's gallbladder appeared anatomically normal. To our knowledge, this is the first report of a Phrygian cap appearance of the murine gallbladder.