Attenuated Accumulation of Novel Fluorine (19F)-Labeled Bile Acid Analogues in Gallbladders of Fibroblast Growth Factor-15 (FGF15)-Deficient Mice

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00454

Our work has focused on defining the utility of fluorine (¹⁹F)-labeled bile acid analogues and magnetic resonance imaging (MRI) to identify altered bile acid transport in vivo. In the current study, we explored the ability of this approach to d

Full text of DOI:10.1021/acs.molpharmaceut.8b00454

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Article
Attenuated Accumulation of Novel Fluorine (19F)-Labeled Bile Acid Analogues
in Gallbladders of Fibroblast Growth Factor-15 (FGF15)-Deficient Mice
Melissa Metry, Jessica Felton, Kunrong Cheng, Su Xu, Yong
Ai, Fengtian Xue, Jean-Pierre Raufman, and James E Polli
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00454 • Publication Date (Web): 24 Sep 2018
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Attenuated Accumulation of Novel Fluorine (19F)-Labeled Bile Acid Analogues in
Gallbladders of Fibroblast Growth Factor-15 (FGF15)-Deficient Mice
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Melissa Metry†, Jessica Felton‡, Kunrong Cheng§, Su Xu∥, Yong Ai†, Fengtian Xue†, JeanꢀPierre
Raufman§, and James E. Polli† *
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†
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland, 21201, United States
‡
Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland,
21201, United States
§
VA Maryland Healthcare System, and the Department of Medicine, Division of
Gastroenterology & Hepatology, and the Marlene and Stewart Greenebaum Comprehensive
Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, 21201, United
States
∥ Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of
Medicine, Baltimore, Maryland, 21201, United States
KEYWORDS: Bile acid synthesis, fluorine MRI, enterohepatic circulation, FGF15/19, bile acid
diarrhea, imaging methods
*Address correspondence to:
James E. Polli, Ph.D.
20 Penn Street, N623
Baltimore, MD 21201
Tel: 410ꢀ706ꢀ8292;
Fax: 410ꢀ706ꢀ5017;
Email: jpolli@rx.umaryland.edu
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ABSTRACT
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Our work has focused on defining the utility of fluorine (19F)ꢀlabeled bile acid analogues
and magnetic resonance imaging (MRI) to identify altered bile acid transport in vivo. In the
current study, we explored the ability of this approach to differentiate fibroblast growth factorꢀ15
(FGF15)ꢀdeficient from wildꢀtype (WT) mice, a potential diagnostic test for bile acid diarrhea, a
commonly misdiagnosed disorder. FGF15 is the murine homologue of human FGF19, an
intestinal hormone whose deficiency is an underappreciated cause of bile acid diarrhea. In a pilot
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and three subsequent pharmacokinetic studies, we treated mice with two Fꢀlabeled bile acid
analogues, CAꢀlysꢀTFA and CAꢀsarꢀTFMA. After oral dosing, we quantified 19Fꢀlabeled bile
acid analogue levels in the gallbladder, liver, small and large intestine, and plasma using liquid
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chromatography mass spectrometry (LCꢀMS/MS). Both Fꢀbile acid analogues concentrated in
the gallbladders of FGF15ꢀdeficient and WT mice, attaining peak concentrations at
approximately 8.5 h after oral dosing. However, analogue levels in gallbladders of FGF15ꢀ
deficient mice were severalꢀfold less compared to those in WT mice. Liveꢀanimal 19FꢀMRI
provided agreement with our LCꢀMS/MSꢀbased measures; we detected robust CAꢀlysꢀTFA 19F
signals in gallbladders of WT mice, but no signals in FGF15ꢀdeficient mice. Our finding that 19Fꢀ
MRI differentiates FGF15ꢀdeficient from WT mice provides additional proofꢀofꢀconcept for the
development of 19Fꢀbile acid analogues and 19F MRI as a clinical test to diagnose bile acid
diarrhea due to FGF19 deficiency and other disorders.
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INTRODUCTION
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The past 20 years have witnessed a great expansion in our understanding of the role bile
acids play in both normal physiology and disease, as well as the complex processes that maintain
bile acid homeostasis.1 Despite these advances, there remains a paucity of tools to examine bile
acid transport in vivo. To address this limitation, we investigated the potential of using bile acid
analogues labeled with naturally occurring fluorine, 19F, and in vivo magnetic resonance imaging
(MRI) to visualize bile acids in anatomical compartments, primarily the gallbladder.2ꢀ4 The work
described herein focuses on the potential utility of 19Fꢀbile acid MRI to diagnose bile acid
diarrhea, a condition frequently misdiagnosed as diarrheaꢀpredominant irritable bowel syndrome
(IBSꢀD).5
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In humans, a gut hormone, fibroblast growth factorꢀ19 (FGF19), governs intestinal
feedback regulation of hepatic bile acid synthesis. FGF15 is the murine homologue of FGF19;
the designation FGF19/15 refers to processes common to both the human and murine hormones.
Bile acids actively absorbed by ileal enterocytes via the apical sodiumꢀdependent bile acid
transporter (ASBT, encoded by SLC10A2), bind and activate the nuclear farnesoid X receptor
(FXR) which, amongst many actions, regulates the production and release of FGF19/15.
FGF19/15 circulates to the liver where its interaction with the FGF receptorꢀ4 complex on
hepatocytes results in inhibition of hepatocyte 7ꢀalphaꢀhydroxylase (CYP7A1), the rateꢀlimiting
enzyme that catalyzes cholesterol hydrolysis and the formation of primary bile acids.
In FGF19/15 deficiency, the absence of feedback regulation of bile acid synthesis results
in their excess production, reduced gallbladder filling, and increased levels of bile acids in the
distal small intestinal that may overwhelm ASBT transport capacity, thereby increasing bile acid
spillage into the colon. Increased fecal bile acid levels in the colon are associated with several
harmful effects, including inflammation, altered water and electrolyte secretion, and stimulated
motility that may result in diarrhea. Estimates suggest as many as oneꢀthird of individuals
diagnosed with IBSꢀD, a common disorder, actually have bile acid diarrhea due to FGF19
deficiency.5
For more than 30 years, the most common approach available to diagnose bile acid
diarrhea in Europe and Canada has been 75Selenium homotaurocholic acid retention testing
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(SeHCAT). Although relatively easy to perform, this test requires ingestion of a radiolabeled
compound, a nuclear medicine laboratory, and is neither FDAꢀapproved nor available for use in
the United States.6 Another method, direct measurement of fecal bile acids, is expensive,
unavailable in most hospitals and clinics, requires a research laboratory, and performed rarely.
The recently developed plasma 7αꢀhydroxyꢀ4ꢀcholesteneꢀ3ꢀone (C4) assay is less sensitive and
specific than SeHCAT testing and is not widely available. Measuring serum FGF19 levels is
possible, but requires additional validation before clinical use.7 In the United States, the most
common approach to diagnosing bile acid diarrhea remains the exclusion of other causes of
diarrhea and a therapeutic trial of a bile acid binder such as cholestyramine or colesevalam.
Although decreased diarrhea following treatment with these agents is considered indirect
evidence of bile acid diarrhea, this approach is nonꢀspecific.8
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The initial concept driving the current line of investigation was to use MRI, a technique
that does not expose individuals to ionizing radiation, to ‘see’ the transport of 19Fꢀlabeled bile
acid analogues that mimic native bile acid transport in both in vitro and in vivo studies.2ꢀ4,9 We
chose to image the gallbladder, an organ that concentrates bile acids into the millimolar range,
orders of magnitude greater than bile acid concentrations observed in any other organ. In mice
gavaged with these fluorinated bile acid probes, we consistently detected a strong 19FꢀMRI signal
in gallbladders of wildꢀtype (WT) mice; 19FꢀMRI signals were not detected in gallbladders of
ASBTꢀdeficient mice.2,3 With regard to the present work, we reasoned FGF15ꢀdeficient mice
would also have a diminished 19FꢀMRI signal in the gallbladder. We anticipated overꢀproduction
of native bile acids and their spillage into the gut would dilute 19Fꢀlabeled bile acid analogues
and increase competition for transport by ASBT. These factors were likely to reduce intestinal
uptake, enterohepatic circulation, and gallbladder accumulation of 19Fꢀlabeled bile acid probes.
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In the present study, we investigated the potential utility of Fꢀlabeled bile acids as tools
to diagnose bile acid diarrhea resulting from FGF19/15 deficiency. We used biochemical
quantification and in vivo 19FꢀMRI imaging to compare the accumulation of two previously
characterized 19Fꢀbile acid analogue probes, trifluoroacetyl Lꢀlysine and trifluoroꢀNꢀmethylꢀ
acetamide conjugates of the native human primary bile acid cholic acid (i.e. CAꢀlysꢀTFA and
CAꢀsarꢀTFMA), in gallbladders from WT and FGF15ꢀdeficient mice.
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EXPERIMENTAL SECTION
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Materials
Cholic acid, Dulbecco’s phosphateꢀbuffered saline (DPBS), and poly(ethylene glycol)
(PEG) 400 were purchased from Sigma Aldrich (St. Louis, Missouri). Compounds 1, 3, and 5
were commercially available. We obtained all chemicals from commercial suppliers and used
them without further purification.
Synthesis of CA-lys-TFA (Compound 4)
We synthesized CAꢀlysꢀTFA, as well as CAꢀsarꢀTFMA (below), using an approach
modified from that described previously (Supporting Information Synthesis of CAꢀlysꢀTFA and
CAꢀsarꢀTFMA).2,4 Briefly, we synthesized target compound 4 as depicted in Figure 1. Activation
of cholic acid 1 using HATU yielded the intermediate ester 2. Coupling of compound 2 with
trifluoroacetyl Lꢀlysine 3 provided the final product 4, CAꢀlysꢀTFA, in good yield.
Synthesis of CA-sar-TFMA (Compound 10)
The synthesis of compound 10 began with NꢀBocꢀethylenediamine 5 (Figure 2).
Compound 5 was allowed to react with benzyl bromoacetate to yield compound 6 in high yield.
Next, we removed the Bocꢀprotecting group of compound 6 using TFA to generate amine 7,
which was treated with trifluoroacetic anhydride (TFAA) to give compound 8 in high yield.
Compound 8 was coupled to compound 2 to obtain the benzyl ester 9 in high yield. Finally, the
benzylꢀprotecting group was removed by catalytic hydrogenation to yield product 10, CAꢀsarꢀ
TFMA, in good yield.
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Figure 1. Synthesis of Compound 4 (CAꢀlysꢀTFA). Reagents and conditions: (a) HATU, Et3N,
DMF, RT, 4 h; (b) DMF, RT, 12 h, 80% for two steps.
Figure 2. Synthesis of Compound 10 (CAꢀsarꢀTFMA). Reagents and conditions: (a) benzyl
bromoacetate, NaOH, DMF, RT, 12 h, 78%; (b) TFA, DCM, RT, 1 h; (c) trifluoroacetic
anhydride, DCM, RT, 12 h, 83% for two steps; (d) 2, DMF, RT, 12 h; (e) Pd/C, H2, EtOH, RT,
12 h, 81% for two steps.
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Chemical Analysis
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We visualized analytical thin layer chromatography by ultraviolet light at 254 nM. We
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recorded H NMR and 19F NMR spectra on a Varian (400 MHz) spectrometer. Mass spectra
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were recorded using electrospray as the ionization technique. Per NMR, the purity of the final
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products 4 and 10 were > 95% (Supporting Information Figures S1ꢀS8).
Animals
All animal studies were approved by the Institutional Animal Care and Use Committee of
the University of Maryland School of Medicine and the Research and Development Committee
at the VA Maryland Health Care System. We conducted animal studies in accordance with the
Guide for the Care and Use of Laboratory Animals prepared by the U.S. National Academy of
Sciences. Male FGF15ꢀdeficient and WT mice (on the same 25% C57BL/6J; 75% 129SvJ mixed
genetic background) were kindly provided by Dr. Grace Guo (Rutgers University, Ernest Mario
School of Pharmacy, Piscataway, NJ).10 We housed mice with free access to water and standard
mouse chow in a pathogenꢀfree environment with a 12:12ꢀh light/dark cycle for 1 week prior to
experiments. After a fast of at least 6 h, mice underwent oral gavage with CAꢀsarꢀTFMA and/or
CAꢀlysꢀTFA in vehicle [60% PEG 400 and 40% DPBS, 8.33 µL per g mouse body weight] and
subsequently anesthetized with ketamine/xylazine at different time points. Doses of imaging
agents reflect their slight difference in molecular weight and provide the same molar dose in
micromole/kg mouse body weight (1.24 mmol/kg for 75 mg/kg CAꢀsarꢀTFMA and 78.5 mg/kg
CAꢀlysꢀTFA; 2.48 mmol/kg for 150 mg/kg CAꢀlysꢀTFA).
Oral Disposition Characterization
We characterized the oral disposition of the two bile acid analogues via destructive
sampling in Fgf15ꢀ/ꢀ and WT mice through a series of experiments. In a pilot study, four Fgf15ꢀ/ꢀ
and four WT mice were concomitantly gavaged with 75 mg/kg CAꢀsarꢀTFMA and 78.5 mg/kg
CAꢀlysꢀTFA. At 7, 10, 24, and 72 h post dosing, we euthanized mice and harvested the
gallbladder, liver, and blood. We collected blood into heparinized tubes that we then centrifuged
at 12,000g for 10 min. We analyzed supernatants using LCꢀMS/MS. The liver and gallbladder
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were homogenized on ice with a glass tissue homogenizer (Duall sizeꢀ21; Kimble Chase Life
Science, Vineland, New Jersey). We extracted CAꢀsarꢀTFMA and CAꢀlysꢀTFA with 75% ACN
in water (800 µL for liver and 300 µL for gallbladder). We centrifuged extracts at 12,000g for 10
min before analysis by LC/MS/MS.
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Favorable gallbladder results from the pilot study led to pharmacokinetic study 1,
wherein five Fgf15ꢀ/ꢀ and five WT mice were concomitantly gavaged with 7.5 mg/kg CAꢀsarꢀ
TFMA and 7.85 mg/kg CAꢀlysꢀTFA. At 7, 8.5, 10, 16, and 24 h post gavage, we euthanized
mice and harvested and processed gallbladders as above. In pharmacokinetic study 2, nine
Fgf15ꢀ/ꢀ and nine wildꢀtype mice were concomitantly gavaged with 7.5, 22.5, or 75 mg/kg CAꢀ
sarꢀTFMA and 7.85, 23.5, or 78.5 mg/kg CAꢀlysꢀTFA, respectively. We euthanized mice 8.5 h
post gavage, and harvested and processed gallbladders as above. In pharmacokinetic study 3,
nine Fgf15ꢀ/ꢀ and nine WT mice were concomitantly gavaged in triplicate with 75 mg/kg CAꢀsarꢀ
TFMA and 78.5 mg/kg CAꢀlysꢀTFA. We euthanized mice at 7, 8.5, and 10 h post dosing and
harvested the gallbladder, small intestine, cut into three equal segments, cecum, and colon. We
extracted CAꢀsarꢀTFMA and CAꢀlysꢀTFA with 75% ACN in water (300 µL for gallbladder, 450
µL for small intestine segments, 650 µL for cecum, and 600 µL for colon). We centrifuged
extracts at 12,000g for 10 min before LCꢀMS/MS analysis.
In Vitro Stability of CA-lys-TFA and CA-sar-TFMA in Mouse Stool
We evaluated the ability of mouse stool to hydrolyze CAꢀlysꢀTFA and CAꢀsarꢀTFMA in
vitro. We collected and pooled stool pellets from multiple Fgf15ꢀ/ꢀ and WT mice maintained in
metabolic cages to prevent coprophagia. We removed debris and then lyophilized stool for 2 h
using a Thermo Scientific Savant SpeedVac concentrator (Thermo Fisher Scientific, Waltham,
MA). Then, we ground stool into a fine powder using a mortar and pestle. We incubated 50ꢀmg
aliquots of stool with CAꢀsarꢀTFMA or CAꢀlysꢀTFA in a water bath maintained at 37ºC for 0, 2,
4, 8, and 24 h. We quenched samples with ACN, centrifuged at 10,000g for 10 min, and stored
them at ꢀ80ºC until analysis by LCꢀMS/MS.
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In Vitro Stability of CA-sar-TFMA and CA-lys-TFA in Gallbladder Homogenate
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To prepare gallbladder homogenates, we collected, pooled, and homogenized in DPBS
the gallbladders of four WT mice that had not been gavaged with probes. Aliquots of gallbladder
homogenate were prepared in triplicate with CAꢀsarꢀTFMA and CAꢀlysꢀTFA and incubated in a
water bath maintained at 37ºC for 0, 20, 40, 60, 90, 120, 240, 480, and 1440 min. Samples were
quenched with ACN, centrifuged at 12,000g for 10 min, and stored at ꢀ80ºC until analysis by
LCꢀMS/MS.
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Murine Gallbladder Imaging
After fasting and gavage as described above, we anesthetized mice with
ketamine/xylazine injected through an intraperitoneal (i.p.) catheter and imaged them by 1H MRI
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and F MRI ~8.5 h after CAꢀlysꢀTFA dosing (1.5 h total F acquisition time). Approximately
every 30 min throughout imaging, we infused maintenance doses of ketamine/xylazine through
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the i.p. catheter. The mice remained in the MRI machine for 2 h, for system calibration and H
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and F MRI acquisition time. On each occasion, we imaged a 30ꢀmM phantom of CAꢀlysꢀTFA
dissolved in methanol in a short glass NMR tube (5ꢀmm diameter) alongside mice.
Magnetic Resonance Imaging
All in vivo MRI data were acquired using identical parameters, as described previously3
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(i.e., phantom and animal experiments used identical 19F and H parameters). Briefly, we
performed MRI experiments using a Bruker BioSpec 70/30USR Avance III 7T horizontal bore
MR scanner (Bruker Biospin MRI GmbH, Ettlingen, Germany) with a BGA12S gradient system
and used Bruker ParaVision 5.1 software for acquisition and processing. The coil was a Bruker
40ꢀmm 19F/1H dualꢀtuned linear volume coil that transmitted and received radiofrequency signals
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at 300.28 MHz for H and 282.55 MHz for 19F nuclei. Multislice H MR images used an
acquisition time of 6 min and 10 s using a RARE (rapid acquisition with relaxation
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enhancement) sequence in the cross view of the phantom or animal body. H MRI employed
repetition time 1850 ms, RARE factor 8, field of view 4 × 4 cm2, echo time 8.58 ms, slice
thickness 1.0 mm, matrix size 200 x 200, number of averages 8, and inꢀplane resolution 200 x
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200 µm2 with 20 slices. 19F images were acquired with a FLASH (fast low angle shot) sequence
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in an identical region to H MRI. F acquisition time was 1 h and 30 min. Parameters were the
same as 1H MRI, but with repetition time 220 ms, echo time 3.078 ms, inꢀplane resolution 1.25 ×
1.25 mm2, matrix size 32 × 32, slice thickness 4.0 mm, number of averages 768, and flip angle =
30º. We optimized the flip angle using the T1 relaxation time of the phantom. The phantom was
a 5ꢀmm diameter shortened glass NMR tube containing 30 mM CAꢀlysꢀTFA dissolved in
methanol.
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We calculated CAꢀlysꢀTFA concentrations in the mouse gallbladder by comparing the
mean signal intensity in the region of interest (ROI) identified in the gallbladder to the mean
signal intensity in the phantom ROI imaged next to the mouse body. In each case, we drew the
ROI to exclude the edges of the phantom and gallbladder, so that signal intensity calculation did
not suffer from a partial volume edge effect due to the image resolution. Mean signal intensity
was calculated with Bruker ParaVision 5.1. The limit of quantification of 19F signal for this
method was previously determined to be 6.82 mM, which corresponds to the noise magnitude of
an ROI on the image periphery plus 2.5ꢀtimes its standard deviation.3 Using this method, there is
greater than 99% confidence that voxels with concentrations above this limit represent real 19F
signal, and not noise.11
We used Medical Image Processing, Analysis and Visualization software (MIPAV
v7.0.1; CIT, NIH, Bethesda, Maryland) to generate a color 19F MR image of the mice and
adjacent phantom. The image threshold used was 0.7 on a scale where the strongest signal (in
red) was 1.0.
LC-MS/MS Analysis
We determined concentrations of CAꢀsarꢀTFMA, CAꢀlysꢀTFA, and cholic acid by
LC/MS/MS using a Waters Acquity UPLC system with triple quadrupole detector (Waters
Corporation, Milford, Massachusetts, USA). We used a Waters Acquity UPLC ethylene bridged
hybrid C8 1.7 ꢁm 2.1 × 50 mm column with a flow rate of 0.4 mL/min. The gradient was as
follows (expressed as % ACN in water, all mobile phases include 0.1% formic acid): 50% from 0
to 0.5 min, then increased to 95% until 3 min, then decreased to 50% at 3.01 min and held at
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50% until 4 min. Injection volume was 10 ꢁL. For CAꢀlysꢀTFA, cone voltage was 65 V, dwell
time 0.100 s and collision energy 43 V. We used negative electrospray ionization with a multiple
reaction monitoring method for the transition 631.15 to 240.92 Da. The method was linear over a
range of 5 to 2000 nM (r2 ≥ 0.97). For CAꢀsarꢀTFMA, cone voltage was 65 V, dwell time 0.100 s
and collision energy 36 V. We used negative electrospray ionization with a multiple reaction
monitoring method for the transition 603.53 to 213.12 Da. The method was linear over a range of
5 to 2000 nM (r2 ≥ 0.99). For cholic acid, cone voltage was 66 V, dwell time 0.025 s and
collision energy 5 V. We used negative electrospray ionization with a single reaction monitoring
method at 407.50 Da. The method was linear over a range of 0.5 to 10000 µM (r 2 ≥ 0.96).
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Statistical Analysis
Error shown in the text and figures is standard error of the mean. We used Student’s
paired tꢀtest to evaluate stability data. We used Student’s unpaired tꢀtest to evaluate
concentration differences of CAꢀsarꢀTFMA and CAꢀlysꢀTFA in FGF15ꢀdeficient versus WT
mouse. We considered a p value < 0.05 significant.
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Oral Disposition Characterization
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We calculated the concentrations of CAꢀlysꢀTFA and CAꢀsarꢀTFMA in the gallbladder
and liver by assuming an organ density of 1 g/mL. Gallbladder concentrations of 19F at 10 h were
above the 19F MRI limit of detection, as previously defined, and liver and plasma concentrations
were below the limit of detection.3 The gavage concentration of 75 mg/kg CAꢀsarꢀTFMA and
78.5 mg/kg CAꢀlysꢀTFA showed high accumulation in the WT mouse gallbladder at 10 h after
oral dosing with a >20 fold difference compared to the gallbladder of the FGF15ꢀdeficient mouse
(Figure 3). In contrast, CAꢀsarꢀTFMA and CAꢀlysꢀTFA concentrations in the liver and plasma
were at least 1000ꢀfold lower on average than in the gallbladder; note the µM scales for liver and
plasma vs mM scale for gallbladder. The doseꢀresponse curves showed a linear relationship for
both CAꢀlysꢀTFA and CAꢀsarꢀTFMA (Figure 4).
Given the results from the pilot study, 8.5 h was included as an additional time point to
better elucidate the peak concentration. Peak concentration occurred in gallbladders of mice at
8.5 h for both compounds at the higher gavage concentration of 75 mg/kg CAꢀsarꢀTFMA and
78.5 mg/kg CAꢀlysꢀTFA when tested in triplicate (Figure 5AꢀB). Differences at the 8.5ꢀh time
point were statistically significant and showed a >2 fold difference when comparing WT to
Fgf15ꢀ/ꢀ mice (p = 0.04 and p = 0.03, CAꢀsarꢀTFMA and CAꢀlysꢀTFA, respectively). Gallbladder
concentrations in WT mice at 8.5 h were above the 19F MRI limit of detection, whereas all
concentrations in the small intestines, cecum, and colon were below the limit of detection (Figure
5CꢀG). Concentrations of CAꢀsarꢀTFMA and CAꢀlysꢀTFA between WT and Fgf15ꢀ/ꢀ mice in the
small intestines, cecum, and colon were not statistically different.
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Figure 3. Concentration versus time for CAꢀlysꢀTFA (circles) and CAꢀsarꢀTFMA (squares) in
the gallbladder (A), liver (B), and plasma (C) harvested from WT (closed symbols) and FGF15ꢀ
deficient (open symbols) mice after concomitant oral administration of 75 mg/kg CAꢀsarꢀTFMA
and 78.5 mg/kg CAꢀlysꢀTFA. 19Fꢀlabeled bile acid analogues were simultaneously coꢀ
administered to the same animal. Gavage with the two 19Fꢀlabeled bile acid analogues resulted in
much higher concentrations in the gallbladder than the liver or plasma. In gallbladder and liver,
bile acid analogue exposure was greater in WT mice than FGF15ꢀdeficient mice. Each data point
reflects n = 1 mouse from destructive sampling.
Figure 4. Doseꢀresponse curve in the gallbladder of WT (closed) and FGF15ꢀdeficient (open
symbols) mice after concomitant oral administration of CAꢀlysꢀTFA (circles) and CAꢀsarꢀTFMA
(squares). Larger doses resulted in higher gallbladder concentrations of 19Fꢀlabeled bile acid
analogues. Each data point reflects n = 3 mice.
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Figure 5. Concentration versus time of CAꢀlysꢀTFA (circles) and CAꢀsarꢀTFMA (squares) in the
gallbladder (A, B), small intestine (C, D, E), cecum (F), and colon (G) of WT (closed symbols)
and FGF15ꢀdeficient (open symbols) mice after concomitant oral administration of 75 mg/kg
CAꢀsarꢀTFMA and 78.5 mg/kg CAꢀlysꢀTFA. All panels use the same symbols. The only
statistical difference in 19Fꢀlabeled bile acid analogue concentration between FGF15ꢀdeficient
and WT mice was in the gallbladder at 8.5 h (A, p = 0.04 and B, p = 0.03). Each data point
reflects n = 3 mice.
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In Vitro Stability of CA-sar-TFMA and CA-lys-TFA in Mouse Stool
Incubation of CAꢀsarꢀTFMA and CAꢀlysꢀTFA in stool showed no significant degradation
at the longest time tested (p > 0.05, Figure 6).
Stool
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Figure 6. Percent remaining versus time of CAꢀlysꢀTFA (circles) and CAꢀsarꢀTFMA (squares)
in stool samples of WT (closed symbols) and FGF15ꢀdeficient (open symbols) mice performed
in triplicate. Each 19Fꢀlabeled bile acid analogue exhibited favorable stability over 24 h in stool.
There was about a 15% loss of each 19Fꢀlabeled bile acid analogue over 24 h.
In Vitro Stability of CA-sar-TFMA and CA-lys-TFA in Gallbladder Homogenate
Incubation of CAꢀsarꢀTFMA and CAꢀlysꢀTFA in gallbladder homogenates showed
statistically significant degradation at the longest duration tested (p < 0.01). Cholic acid also
showed statistically significant degradation (p < 0.01). However, degradation of CAꢀsarꢀTFMA
and CAꢀlysꢀTFA was slightly more than that of cholic acid, which exhibited about a 12% loss at
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the longest duration tested (Figure 7).
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Figure 7. Percent remaining versus time of CAꢀlysꢀTFA (circles), CAꢀsarꢀTFMA (squares), and
cholic acid (triangles) in pooled gallbladder homogenate prepared in triplicate from four WT
mice. The two 19Fꢀlabeled bile acid analogues exhibited favorable stability over 24 h in WT
gallbladder homogenate. There was about a 25% decrease in each bile acid analogue over 24 h.
For reference, there was a 12% loss of cholic acid over 24 h.
Murine Gallbladder Imaging
Based on LCꢀMS/MS results of the peak accumulation occurring at 8.5 h, mice
underwent MRI at 8.5 h after oral gavage of 150 mg/kg CAꢀlysꢀTFA – we imaged mice once
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followed by euthanasia. We visualized strong F MRI signals emanating from the gallbladders
1
of all WT mice tested (Figure 8A). Overlay of images obtained by H and 19F MRI signal
acquisition confirmed 19F signals emanated from the gallbladder (Figure 8A). CAꢀlysꢀTFA
concentration in the gallbladder, calculated by normalizing the average intensity of the
gallbladder ROI to that within the phantom, was determined to be 27 mM.
As predicted from our LCꢀMS/MS data, MRI of three consecutive Fgf15ꢀ/ꢀ mice revealed
no detectable signal (Figure 8B); as a control, we observed a robust 19F MRI signal emanating
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from phantoms placed next to these mice.
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Figure 8. 19F MRI in WT and FGF15ꢀdeficient mice gavaged with 150 mg/kg CAꢀlysꢀTFA. We
detected a 19F signal only in gallbladders from WT mice. Arrowheads (blue) and arrows (yellow)
indicate 19F signals emanating from phantoms and gallbladder, respectively. This imaging result,
representative of findings in three separate WT and FGF15ꢀdeficient mice, is consistent with the
LC/MS/MSꢀbased observations.
DISCUSSION
Performance of Bile Acid Analogues
To address the unmet need for a safe and accurate method of diagnosing bile acid
diarrhea due to FGF19 deficiency in countries like the United State where clinicians do not have
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access to SeHCAT testing, we evaluated a novel noninvasive approach using in vivo F MRI.
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We tested the ability of gavage with a Fꢀbile acid analogue, CAꢀlysꢀTFA, to detect abnormal
bile acid transport in FGF15ꢀdeficient mice, an animal model of human FGF19 deficiency. We,
and others, have shown that, like humans with FGF19 deficiency, FGF15ꢀdeficient mice have
increased fecal bile acid excretion.12ꢀ15 We detected low quantities of intact CAꢀlysꢀTFA and
CAꢀsarꢀTFMA in the stool, with more in stool from FGF15ꢀdeficient mice than from wildꢀtype
mice (Supporting Information Figure S9).
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In the present communication, our LCꢀMS/MS data show that, in both WT and Fgf15ꢀ/ꢀ
mice, each 19Fꢀbile acid analogue accumulated in the gallbladder and, at much lower
concentrations, in the liver, plasma, small intestine, and colon (Figures 3 and 5). Notably, across
three doses, we observed that FGF15 deficiency reduced the concentrations of 19Fꢀlabeled bile
acid analogues in gallbladder bile (Figure 4). In WT mice, average gallbladder bile CAꢀlysꢀTFA
and CAꢀsarꢀTFMA concentrations exceeded our 19F MRI limit of detection (i.e. above 2.27 mM,
as there are three equivalent fluorine atoms in each structure).3 In contrast, the concentrations of
these molecules measured by LCꢀMS/MS in tissues from FGF15ꢀdeficient mice were generally
below the 19F MRI limit of detection (Figures 3 ꢀ 5). The lack of 19F MRI signals from
gallbladders of FGF15ꢀdeficient mice 8.5 h after gavage with 150 mg/kg CAꢀlysꢀTFA
substantiated this finding; gallbladders of comparably treated WT mice provided strong imaging
signals (see representative examples in Figure 8).
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Interestingly, CAꢀlysꢀTFA and CAꢀsarꢀTFMA provided similar in vivo pharmacokinetic
distribution profiles, although prior in vitro studies suggested greater stability against
cholylglycine hydrolase for CAꢀsarꢀTFMA compared to CAꢀlysꢀTFA.2,4 To address the similar
in vivo stabilities of CAꢀlysꢀTFA and CAꢀsarꢀTFMA observed here, we assessed the stability of
these molecules in murine stool and gallbladder homogenates. Our finding of similar stability of
CAꢀlysꢀTFA and CAꢀsarꢀTFMA in stool (Figure 6) and gallbladder homogenates (Figure 7) is
consistent with the compounds’ similar in vivo stability. In mice, cholylglycine hydrolase does
not appear to be a large determinant of CAꢀsarꢀTFMA or CAꢀlysꢀTFA stability in vivo. Overall,
neither CAꢀsarꢀTFMA nor CAꢀlysꢀTFA is preferred over the other. These observations highlight
the importance of confirming in vitro observations using in vivo models.
Potential Use of Fluorinated Bile Acid Analogues
Previously, we synthesized CAꢀlysꢀTFA and CAꢀsarꢀTFMA and evaluated the in vitro
stability and affinity of these molecules for ASBT and Na+/taurocholate coꢀtransporting
polypeptide (NTCP, SLC10A1).2,4 In addition, to assess their ability to concentrate in the
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gallbladder, the Fꢀbile acid analogues’ F MRI signals were measured in the gallbladders of
WT and ASBTꢀdeficient mice in vivo.2ꢀ4 In the present study, we evaluated CAꢀlysꢀTFA and
CAꢀsarꢀTFMA in FGF15ꢀdeficient mice.
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In humans, reduced levels of FGF19 impair bile acid homeostasis within the
enterohepatic circulation. Specifically, the liver continues to synthesize bile acids despite the
presence of abundant bile acids in the terminal ileum. The loss of feedback repression of bile
acid synthesis by FGF19 results in excess spillage of bile acids into the colon that simulates
electrolyte and water secretion, inflammation, and motility, thus leading to diarrhea. Bile acid
uptake from the distal small intestine by ASBT is unimpaired, but the excess bile acids in the
ileum overwhelm ASBT transport capacity, causing diarrhea.16 Approximately one of three
individuals diagnosed with IBSꢀD may actually have bile acid diarrhea.5 Distinguishing these
conditions is important; incorrectly attributing bile acid diarrhea to IBS may result in prolonged
treatment with costly, ineffective drugs that may have substantial side effects. The resulting
therapeutic failure may have a negative impact on the quality of life.
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Conceptually, the clinical use of 19F bile acid analogueꢀMRI to diagnose bile acid
diarrhea is attractive; the test would not require phlebotomy or exposure to ionizing radiation.
Nonetheless, there are limitations to our work and to the translational potential of this test. We
employed FGF15ꢀdeficient mice, which excrete excess bile acids, but do not exhibit diarrhea.12,15
Thus, although these mice mimic the physiology of FGF19 deficiency, the clinical phenotype is
different; this difference may reflect differences between murine and human colon physiology,
diet, and other factors. Additionally, we needed high doses to detect analogues in mouse
gallbladder via MRI, employing a 1.5ꢀh acquisition time. The necessary dose and acquisition
time for use in humans would require clinical testing. Since 19F bile acid analogueꢀMRI requires
the presence of a functional gallbladder, this test would not work in experimental animals
lacking a gallbladder (e.g. rats) or in humans after cholecystectomy. Finally, a major obstacle to
such a diagnostic approach is the current paucity of clinical MRI facilities equipped with the
appropriate hardware (coil) and software needed to detect fluorine signals.
Overall, our findings support the concept that increased fecal bile acids in FGF15/19
deficiency result from a reduction in the fraction of intestinal bile acids transported by ASBT.
Moreover, 19Fꢀbile acid MRI shows potential as a novel test for diagnosing bile acid diarrhea
resulting from FGF19 deficiency. Nonetheless, we must address and overcome several
limitations and obstacles before 19Fꢀbile acid MRI can transition to clinical practice.
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SUPPORTING INFORMATION
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Synthesis of CAꢀlysꢀTFA and CAꢀsarꢀTFMA, HꢀNMR of compound 4, 19FꢀNMR of compound
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4, HRMS of compound 4, HꢀNMR of compound 6, HꢀNMR of compound 8, HꢀNMR of
compound 10, 19FꢀNMR of compound 10, HRMS of compound 10, and mass of CAꢀlysꢀTFA
and CAꢀsarꢀTFMA in stool samples per 24ꢀh stool collection
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ACKNOWLEDGEMENTS
We thank Dr. Grace Guo, Ernest Mario School of Pharmacy, Rutgers University, for providing
FGF15ꢀdeficient and WT control mice. We also thank Drs. Alan F. Hofmann, Paul A. Dawson,
and Michael Camilleri for their helpful discussions and guidance. This work was supported by
Food and Drug Administration [U01FD005946], National Institutes of Health, National Institute
of Diabetes and Digestive and Kidney Diseases [Grants R21 DK093406 and T32 DK067872],
and a Merit Review Award [Grant BX002129] from the United States Department of Veterans
Affairs Biomedical Laboratory Research and Development Program. The contents do not
represent the views of the U.S. Department of Veterans Affairs or the United States Government.
ABBREVIATIONS USED
ASBT, the apical sodium dependent bile acid transporter; C4, 7αꢀhydroxyꢀ4ꢀcholesteneꢀ3ꢀ
one; CA, cholic acid; DPBS, Dulbecco’s phosphate buffered saline; EtOAc, ethyl acetate;
FGF/Fgf, fibroblast growth factor; FLASH, fast low angle shot; IBSꢀD, diarrhea predominantꢀ
irritable bowel syndrome; IP, intraperitoneal; LCꢀMS/MS, liquid chromatography/ tandem mass
spectrometry; MRI, magnetic resonance imaging; NTCP, the Na+/taurocholate coꢀtransporting
polypeptide; PEG, polyethylene glycol; RARE, rapid acquisition with relaxation
enhancement; rf, radio frequency; ROI, region of interest; SeHCAT, 75Selenium
homotaurocholic acid retention test; TFAA, trifluoroacetic anhydride; WT, wild type
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REFERENCES
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Molecular Pharmaceutics
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19F MRI was only detected in WT mice and not in FGF15-deficient mice gavaged with CA-lys-TFA.
139x64mm (96 x 96 DPI)
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