291 Development and Testing of a Novel Fluorinated Bile Acid for Detection of Bile Acid Malabsorption by Magnetic Resonance Imaging (MRI)

Abstract

Bile Acid Malabsorption (BAM) resulting from bile acid overproduction or reduced transport can increase fecal bile acids and cause chronic diarrhea. BAM is thought to account for up to 30% of patients diagnosed with irritable bowel syndrome (IBS-D). Current methods of diagnosing BAM are limited; it is likely under-diagnosed. Our objective was to develop a non-invasive method of detecting BAM by synthesizing and testing a multi-fluorinated bile acid (MFBA) that can be used for fluorine magnetic resonance imaging (MRI) of bile acid enterohepatic circulation. A prototype MFBA was synthesized by conjugating trifluoroacetyl lysine to cholic acid (19F-CA-lys). This MFBA was tested for transport and inhibition of the apical sodium dependent bile acid transporter (ASBT) using stably transfected MDCK cell monolayers and of Na+/taurocholate cotransporting polypeptide (NTCP) using stably transfected HEK cell monolayers. In Vitro, 19F-CA-lys was a potent inhibitor and substrate of both ASBT (Ki = 20.0±3.9 μM, Kt = 39.4±23.8 μM, normalized Vmax =0.853 ± 0.197) and NTCP (Ki = 2.93±0.60 μM, Kt = 8.99 ± 2.79 μM, normalized Vmax = 0.281±0.052). Stability of 19F-CA-lys (5 μM) was measured after treatment with Caco-2 cell homogenates, rat plasma, rat liver microsomal S9 pools, simulated intestinal fluid with pancreatic enzymes, 1 M HCl, buffers at pH 7.4 and 6.8, and choloylglycine hydrolase (CGH) (N=3 for each condition). Except for CGH, 19F-CA-lys was stable in each condition. After 1 h in CGH, 60.4±4.0% 19F-CA-lys remained compared to 2.4±0.5% naturally-occurring taurocholic acid (P<0.005). To test In Vivo stability and utility of 19F-CA-lys for MRI, we examined its distribution in 8 male C57BL/6 mice (average weight, 25.7 g). After overnight fasting, mice were gavaged with 1:1 polyethylene glycol (PEG):water vehicle control (2 mice), 150 mg/ kg 19F-CA-lys (3 mice) and 300 mg/kg 19F-CA-lys (3 mice). Four hours after dosing, 19FCA-lys in gallbladder, liver and plasma was measured by LC/MS. 19F-CA-lys was not detected in control mice receiving vehicle alone. With 150 and 300 mg/kg 19F-CA-lys, 4 h gallbladder 19F-CA-lys concentrations were 27.0±2.5 and 36.8±8.8 μmol/g, liver concentrations were 0.079±0.035 and 0.057±0.010 μmol/g, and plasma concentrations were 0.444±0.170 and 0.418 ± 0.015 μM, respectively. We calculated that at both doses, 10.8±1.9% (95% CI, 5.915.8%) of administered 19F-CA-lys was present in the gallbladder. We conclude that 19FCA-lys, a novel MFBA, is stable In Vitro and In Vivo, and accumulates in the gallbladder at concentrations that will provide a 19F signal in excess of that we previously determined to be the minimum detectable by 19F MRI (Drug Metab. Disp. 39:736, 2011). These findings suggest that non-radioactive MFBAs have excellent potential to measure In Vivo bile acid transport and serve as clinical imaging tools to diagnosis BAM.