UGT-dependent regioselective glucuronidation of ursodeoxycholic acid and obeticholic acid and selective transport of the consequent acyl glucuronides by OATP1B1 and 1B3

Article abstract of DOI:10.1016/j.cbi.2019.108745

Ursodeoxycholic acid (UDCA) is a major effective constituent of bear bile powder, which is widely used as function food in China and is documented in the Chinese pharmacopoeia as a traditional Chinese medicine. UDCA has been developed as the only accepted therapy by the US FDA for primary biliary cholangitis. Recently, the US FDA granted accelerated approval to obeticholic acid (OCA), a semisynthetic bile acid derivative from chenodeoxycholic acid, for primary biliary cholangitis. However, some perplexing toxicities of UDCA have been reported in the clinic. The present work aimed to investigate the difference between UDCA and OCA in regard to potential metabolic activation through acyl glucuronidation and hepatic accumulation of consequent acyl glucuronides. Our results demonstrated that the metabolic fates of UDCA and OCA were similar. Both UDCA and OCA were predominantly metabolically activated by conjugation to the acyl glucuronide in human liver microsomes. UGT1A3 played a predominant role in the carboxyl glucuronidation of both UDCA and OCA, while UGT2B7 played a major role in their hydroxyl glucuronidation. Further uptake studies revealed that OATP1B1- and 1B3-transfected cells could selectively uptake UDCA acyl glucuronide, but not UDCA, OCA, and OCA acyl glucuronide. In summary, the liver disposition of OCA is different from that of UDCA due to hepatic uptake, and liver accumulation of UDCA acyl glucuronide might be related to the perplexing toxicities of UDCA.

Full text of DOI:10.1016/j.cbi.2019.108745

Chemico-Biological Interactions 310 (2019) 108745
Contents lists available at ScienceDirect
Chemico-Biological Interactions
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UGT-dependent regioselective glucuronidation of ursodeoxycholic acid and
obeticholic acid and selective transport of the consequent acyl glucuronides
by OATP1B1 and 1B3
Dandan Zhoua,1, Linghua Kong , Yiguo Jiang , Cheng Wang , Yao Ni , Yedong Wang ,
a,1
b
b
a
a
a,**
a,*
Hongjian Zhang , Jianqing Ruan
College of Pharmaceutical Sciences, Soochow University, Suzhou, China
a
b
Department of Pharmacy, Suzhou Science & Technology Town Hospital, Suzhou, China
A B S T R A C T
Ursodeoxycholic acid (UDCA) is a major effective constituent of bear bile powder, which is widely used as function food in China and is documented in the Chinese
pharmacopoeia as a traditional Chinese medicine. UDCA has been developed as the only accepted therapy by the US FDA for primary biliary cholangitis. Recently,
the US FDA granted accelerated approval to obeticholic acid (OCA), a semisynthetic bile acid derivative from chenodeoxycholic acid, for primary biliary cholangitis.
However, some perplexing toxicities of UDCA have been reported in the clinic. The present work aimed to investigate the difference between UDCA and OCA in
regard to potential metabolic activation through acyl glucuronidation and hepatic accumulation of consequent acyl glucuronides. Our results demonstrated that the
metabolic fates of UDCA and OCA were similar. Both UDCA and OCA were predominantly metabolically activated by conjugation to the acyl glucuronide in human
liver microsomes. UGT1A3 played a predominant role in the carboxyl glucuronidation of both UDCA and OCA, while UGT2B7 played a major role in their hydroxyl
glucuronidation. Further uptake studies revealed that OATP1B1- and 1B3-transfected cells could selectively uptake UDCA acyl glucuronide, but not UDCA, OCA, and
OCA acyl glucuronide. In summary, the liver disposition of OCA is different from that of UDCA due to hepatic uptake, and liver accumulation of UDCA acyl
glucuronide might be related to the perplexing toxicities of UDCA.
1
. Introduction
perplexing toxicities of UDCA have been reported and include fever,
hepatitis, cholangitis, vanishing bile duct syndrome, liver cell failure,
severe watery diarrhea, pneumonia, interstitial lung disease, convul-
sions and mutagenic effects [5]. The toxicities of UDCA remain con-
troversial, and the molecular mechanism of UDCA toxicity has not been
well defined yet.
In the liver, UDCA undergoes extensive amino acid conjugation,
mainly with glycine and taurine, by amino acid N-acyltransferase
(BAAT) to form glycoursodeoxycholic acid and tauroursodeoxycholic
acid [6]. In addition to amino acid conjugates, glucuronides of UDCA
have been found in the urine of jaundiced patients and exist as not only
hydroxyl glucuronide [7] but also acyl glucuronide at the C-24 car-
boxylic acid [8,9]. Extensive glucuronidation of bile acids might occur
when the administered natural bile acids exceed the amidation capacity
of the hepatocytes [10,11].
Bear bile powder is a well-known traditional Chinese medicine
documented in the Chinese pharmacopoeia to treat various hepato-
biliary disorders. Bear bile is also widely used as function food in China
for detoxification and improvement of eyesight for centuries.
Ursodeoxycholic acid (UDCA, Fig. 1A) was first identified from the bile
of bear in 1902 and was further developed as a modern drug [1]. UDCA
is the only accepted therapy by the US FDA for primary biliary cho-
langitis, a chronic cholestatic liver disease characterized by the de-
struction of small intrahepatic bile ducts [2,3]. In May 2016, the US
FDA granted accelerated approval to obeticholic acid (OCA), a semi-
synthetic bile acid derivative, for the treatment of primary biliary
cholangitis in combination with UDCA in adults with an inadequate
response to UDCA, or as monotherapy in adults who cannot tolerate
UDCA [4].
Organic anion transporting polypeptides (OATPs) are a family of
important transporters that play a significant role in the trans-mem-
brane transport of endogenous molecules, food constituents, drugs and
toxins [12]. OATP1B1 and 1B3 belong to the OATP family and are
In addition to primary biliary cholangitis, UDCA was approved by
the US FDA for cholesterol gall stone dissolution and was investigated
in a wide range of hepatic and extra-hepatic diseases. However, some
*
Corresponding author. Soochow University, College of Pharmaceutical Sciences, Suzhou, 215123, China.
*
*
Corresponding author. Soochow University, College of Pharmaceutical Sciences, Suzhou, 215123, China.
E-mail addresses: zhanghongjian@suda.edu.cn (H. Zhang), ruanjianqing@suda.edu.cn (J. Ruan).
Zhou Dandan and Kong Linghua equally contribute the work and share the first authorship.
1
https://doi.org/10.1016/j.cbi.2019.108745
Received 21 November 2018; Received in revised form 25 June 2019; Accepted 8 July 2019
Available online 09 July 2019
0009-2797/ © 2019 Elsevier B.V. All rights reserved.

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
Fig. 1. Chemical structure of UDCA and OCA (A); synthetic method of UDCA acyl glucuronide (B) and UDCA 3-O-hydroxyl glucuronide (C).
exclusively expressed on the basolateral membrane of hepatocytes in
normal human liver [13]. It was reported that the uptake of UDCA
glycine and taurine conjugates by human hepatocytes was mediated by
OATP1B1 and 1B3 [14]. However, the role of OATP1B1 and 1B3 in the
transport of UDCA and OCA and their glucuronidation metabolites re-
mains unknown.
Acyl glucuronide conjugates are a series of well-studied reactive
metabolites, capable of undergoing molecular acyl migration and
covalent binding to proteins/DNAs, which may be associated with the
perplexing toxicities of many carboxylic acid-containing drugs.
Although the conjugation pathways of UDCA have been studied inter-
mittently since the 1980s, the glucuronidation pathways, especially
acyl glucuronidation, have not been well defined. Furthermore, as a
new drug, no study has reported on OCA glucuronidation. Therefore,
the present study comparatively investigated the glucuronidation of
UDCA and OCA in vitro to explore the potential metabolic activation of
UDCA and OCA. The regioselectivity of UDCA and OCA glucuronidation
by different human uridine 5′-diphospho-glucuronosyltransferases
(UGTs) was also examined to identify the major UGT(s) responsible for
the glucuronidation of UDCA and OCA. In addition, the potential role of
2

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
OATP1B1 and 1B3 in the hepatic uptake of UDCA, OCA, and their
glucuronidation metabolites was also studied.
glucuronides were confirmed by LC-MS and proton NMR. Column
chromatography was performed using silica gel (100–200 mesh;
Qingdao Marine Chemical Inc., Qingdao, China). H NMR and C NMR
spectra were obtained on 400 MHz (Varian) spectrometers. Chemical
shifts were given in ppm using tetramethylsilane as the internal stan-
dard.
1
13
2. Materials and methods
2.1. Materials
UDCA was obtained from Meryer Chemical Technology Co., Ltd.
2.4. Glucuronidation of UDCA and OCA in human liver microsomes
(
Shanghai, China). OCA was obtained from Shandong Kangmei
Pharmaceutical Technology Co., Ltd. (Shandong, China). Lapatinib was
purchased from Chembest Research Laboratories Co., Ltd. (Shanghai,
China). Steviol acyl glucuronide was chosen as the internal standard
The formation of UDCA and OCA glucuronides was examined in
HLMs. To obtain the kinetic parameters for UDCA and OCA glucur-
onidation, different concentrations of UDCA and OCA were incubated
(
IS), which was synthesized according to the procedures previously
2
with HLMs in 100 mM phosphate buffer (pH 7.4) with 5 mM MgCl ,
described [15]. Human liver microsomes (HLMs, pooled from 20 dif-
ferent organ donors) and recombinant human UGT isoforms (UGT1A1,
UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10,
UGT2B4, UGT2B7, UGT2B15 and UGT2B17) were purchased from BD
Gentest (Woburn, MA). Alamethicin, L-glutathione reduced, D-saccharic
acid 1,4-lactone monohydrate, and formic acid were obtained from
Sigma (St. Louis, MO). Uridine diphosphate glucuronic acid (UDPGA)
was purchased from Roche (Basel, Switzerland). Dimethylsulfoxide,
disodium hydrogen phosphate, and sodium dihydrogen phosphate were
purchased from Sinopharm Chemical Reagent co., Ltd. (Shanghai,
China). Acetonitrile of HPLC (high performance liquid chromato-
graphy) grade was purchased from Merck (Darmstadt, Germany). Ul-
trawater was purified by Hitech Laboratory water purification systems
25 μg/mL of alamethicin, and 5 mM D-saccharic acid 1, 4-lactone. The
reaction was initiated by the addition of 2 mM UDPGA and was in-
cubated at 37 °C for 120 min. The incubation time was set as 120 min
according to the linear formation of UDCA and OCA glucuronides. The
final volume of the incubation mixture was 100 μL. The reaction was
terminated by the addition of 200 μL of cold acetonitrile containing IS.
2.5. Glucuronidation of UDCA and OCA by recombinant human UGT
isoforms
To identify human UGT isoform(s) involved in the glucuronidation
of UDCA and OCA, 12 recombinant UGT isoforms (UGT1A1, 1A3, 1A4,
1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) were incubated
with UDCA and OCA. OCA and UDCA (1 μM) were incubated with
(
Shanghai, China).
5
2
mM MgCl , 0.025 mg/mL of alamethicin, 5 mM saccharolactone and
2
.2. Synthesis of UDCA and OCA glucuronides
0.1 mg/mL of UGTs in 100 mM potassium phosphate buffer (pH 7.4).
The reaction was initiated by adding 2 mM uridine dipho-
sphoglucuronic acid and incubating for 120 min at 37 °C and then was
terminated by adding 200 μl of cold acetonitrile containing 100 nM
SVAG (steviol acyl glucuronide). All reactions were conducted in tri-
plicate.
The synthetic route of UDCA acyl glucuronide is outlined in Fig. 1B.
After reaction with 3-bromoprop-1-ene, allyl glucuronate 2 was syn-
thetized from glucuronide. Condensation of UDCA 1 with allyl glucur-
onate 2 yielded the allyl glucuronate conjugate 3. To remove the allyl
3 4
protection, compound 3 was treated with Pd (PPh ) and morpholine.
The deprotected product was precipitated from the solvent, which was
2.6. Kinetics of UDCA and OCA glucuronidation in pooled HLMs and
recombinant human UGTs
filtered and acidified with Amberlyst A-15 (H+) to yield the free UDCA
1
acyl glucuronide 4. UDCA acyl glucuronide H NMR (400 MHz, CDCl
3
)
analysis revealed the following: δ 0.62 (s, 3H, 18-CH
J = 5.6 Hz, 3H, H-21), 1.13 (s, 3H, 19-CH ), 2.97 (m, 1H, H-7), 4.45
m, 1H, H-3), 11.95 (brs, 1H, -COOH).
The synthetic route of OCA acyl glucuronide is the same as that of
3
), 0.93 (d,
UDCA ranging from 1 μM to 400 μM was incubated with selected
UGT isoforms (UGT1A3, 1A7, 1A8, and 2B7). Meanwhile, OCA ranging
from 1 μM to 400 μM was incubated with different UGT isoforms
(UGT1A1, 1A3, 1A4, and 2B7). The reaction mixtures consisted of se-
lected UGTs (0.1 mg/mL), 100 mM phosphate buffer (pH 7.4), 5 mM
MgCl2, 5 mM D-saccharic acid 1, 4-lactone, and 25 μg/mL of ala-
methicin. The reactions were initiated by the addition of 2 mM UDPGA,
incubated at 37 °C for 2 h, and terminated by adding 200 μl of cold
acetonitrile containing 100 nM SVAG. Each experiment was conducted
in triplicate.
3
(
UDCA acyl glucuronide. After completion of the synthetic process, OCA
1
acyl glucuronide was obtained as a white solid (40 mg). H NMR
3
(
3
3
400 MHz, CDCl
3
+CD OD) analysis revealed the following: δ 0.61 (s,
H, 18-CH
H, 21-CH
The synthetic route of UDCA hydroxyl glucuronide is presented in
3
), 0.80–0.83 (m, 6H, 20-CH
3 3
, 19-CH ), 0.95 (d, J = 6.43 Hz,
3
), 3.42 (m, 1H, 3-CH), 3.72 ppm (s, 1H, 7-CH).
Fig. 1C. To obtain UDCA methyl ester 5, a mixture of UDCA and PTSA
in MeOH was stirred for 4 h. Methyl 1-O-arylcarbonyl-2,3,4-tri-acetyl-
beta-D-glucopyranuronate (compound 6) was produced by reaction of
2.7. Inhibition of UDCA and OCA glucuronidation in UGT1A3 by lapatinib
1
,2,3,4-tetra-O-acetyl-beta-D-glucuronate methyl ester with morpholine
The inhibition effect of lapatinib from 0.01 μM to 500 μM on
UGT1A3-mediated glucuronidation of UDCA and OCA was investigated
under the conditions described above. After 120 min of incubation, the
reaction was terminated, and samples were prepared and analyzed by
LC-MS/MS. Each experiment was conducted in triplicate.
in anhydrous. Trichloroacetyl glucuronic methyl ester 7 was obtained
through stirring compound 6 and 2,2,2-trichloroacetonitrile in DCM,
followed by the addition of DBU. Condensation of UDCA methyl ester 5
with trichloroacetyl glucuronic methyl ester 7 yielded the hydroxyl
glucuronate conjugate of UDCA methyl ester 8. To remove the methyl
protection, compound 8 was treated with MeONa and LiOH. UDCA 3-O-
2.8. Uptake of UDCA and UDCA-G1 in hOATP1B1-, 1B3- and NTCP-
transfected cells
1
hydroxyl glucuronide H NMR (400 MHz, CDCl
3
) analysis revealed the
), 0.93 (d, J = 5.6 Hz, 3H, H-21), 1.13
), 3.37 (m, 1H, H-7),4.45 (m, 1H,H-3), 11.95 (brs, 1H,
following: δ 0.59 (s, 3H, 18-CH
s, 3H, 19-CH
COOH).
3
(
3
Cellular uptake of UDCA, OCA, UDCA-G1 and OCA-G1 was in-
vestigated using CHO cells stably transfected with hOATP1B1, 1B3 and
MDKII cells for NTCP transporters. All CHO cell lines were maintained
-
2.3. NMR analysis of UDCA and OCA acyl or hydroxyl glucuronide
2
at 37 °C with 5% CO and 95% humidity in low-glucose Dulbecco's
modified Eagle's medium (Hyclone, USA) containing 10% fetal bovine
serum (Gibco, USA), 100 U/mL of penicillin and 100 μg/mL of
The purity and structural integrity of the synthesized UDCA
3

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
streptomycin (Hyclone, USA), while MDCKII cell lines were cultured in
high-glucose DMEM (Hyclone, USA).
V = Vmax/(1 + Km/[S] + [S]/Ki)
(3)
4
where V is the velocity of metabolite formation, Vmax is the maximum
velocity, Km is the Michaelis constant defined as the substrate con-
centration at which the reaction rate is half of Vmax, [S] is the substrate
concentration, Ki is the inhibition constant, and CLint is the intrinsic
clearance of liver.
Next, 8 × 10 cells/well were seeded in 24-well culture plates and
were grown in medium for four days. The cells were induced with 5 mM
sodium butyrate 24 h before the uptake experiments. Uptake experi-
ments were initiated by the addition of 500 μL of UDCA, OCA, UDCA-
G1 and OCA-G1 with a defined concentration (10 μM) in HBSS. The
cells were then incubated with the test solutions at 37 °C for 2 min.
Rosuvastatin (5 μM) was chosen as the prototypical substrate of
hOATP1B1 and 1B3, and taurocholate (10 μM) was chosen as the sub-
strate of NTCP. Rifampicin (100 μM) was chosen as the typical inhibitor
for hOATP1B1 and 1B3. All the uptake experiments were terminated by
aspirating the incubation solution containing test compounds and/or
inhibitors and washing the cells with ice-cold HBSS three times. To
release compounds taken up in the cells, the cells were repeatedly
frozen in a refrigerator at −80 °C and thawed at room temperature
three times. Samples were then prepared and analyzed by LC-MS/MS.
Additionally, the protein concentration was determined using the BCA
Protein Assay Kit (TaKaRa, Japan).
The value of Ki was calculated using the Enzyme Kinetics Modules
of substrate inhibition Sigma Plot 12.3 based on the Dixon equations
[
16].
GraphPad Prism 5.0 was used to calculate the IC50 using equation
Y = 100/(1 + 10∧((X-LogIC50))).
The data reported represented the average of two or three separate
experiments.
3. Results
3.1. Identification of the glucuronidation metabolites of UDCA and OCA in
human liver microsomes
When UDCA was incubated with HLMs in the presence of UDPGA,
three peaks with the same ion transition of 567.4 → 391.3 were ob-
served in all incubations (Fig. 2A). These three metabolites were ten-
tatively identified as the monoglucuronides of UDCA, corresponding to
two monoglucuronides of two hydroxyl groups and one mono-
glucuronide of acyl group. Comparison with the standard compounds
synthesized, the peaks appearing at 3.32 min (UDCA-G1) and at 3.5 min
2
.9. Sample preparation
All the glucuronidation reactions were terminated by the addition of
00 μL of ice-cold acetonitrile containing 0.1% formic acid and IS. The
2
mixtures were vortexed immediately, followed by centrifugation at
1
LC–MS/MS.
3000 rpm for 10 min. Aliquots of the supernatants were subjected to
(
UDCA-G2) showed the same retention times and MS spectra as UDCA
acyl glucuronide and UDCA 3-O-hydroxyl glucuronide standards, re-
spectively. As a result, UDCA-G1 and UDCA-G2 were unambiguously
identified as UDCA acyl glucuronide and UDCA 3-O-hydroxyl glucur-
onide, respectively. UDCA-G3 with a retention time of 3.77 min was
tentatively assigned as another UDCA hydroxyl glucuronide. Among
these three metabolites, UDCA acyl glucuronide showed a much greater
peak area than the other two hydroxyl glucuronides, indicating that
HLMs preferentially catalyze glucuronidation of UDCA at the 24-COOH
group, rather than at the 3-OH or 6-OH groups.
2
.10. LC–MS/MS analysis
All samples were analyzed by an LC–MS/MS system consisting of an
API 4000 Qtrap mass spectrometer equipped with a turbo-V ionization
source (Applied Biosystems, Foster City, CA, USA), two LC-20AD pumps
with a CBM-20A controller, a DGU-20A solvent degasser, and a SIL-20A
autosampler (Shimadzu, Columbia, MD, USA). A Phenomenex Synergi
Hydro-RP 80 Å column (75 × 4.6 mm; 4 μm) was used to achieve HPLC
separation. The column temperature was held at 40 °C.
When OCA was incubated with HLMs in the presence of UDPGA,
two additional peaks with the ion transition of 595.5 → 419.4 were
observed in all incubations (Fig. 2B), which were absent in controls.
Both peaks (named as OCA-G1 and OCA-G2) showed the molecular ion
at m/z 595, 176 mass units higher than that of OCA (m/z 419). Con-
sequently, the two metabolites were tentatively identified as the
monoglucuronides of OCA. Compared with the standard compounds
synthesized, OCA-G1 showed the same retention time and mass spectra
as those of OCA acyl glucuronide. Therefore, the structure of OCA-G1
was unambiguously identified as OCA acyl glucuronide, and the other
monoglucuronide of OCA with a retention time of 4.32 min (OCA-G2)
was tentatively assigned as OCA hydroxyl glucuronide. The peak area of
OCA-G1 was much higher than that of OCA-G2, demonstrating that
OCA was predominantly transformed to OCA acyl glucuronide rather
than to OCA hydroxyl glucuronide.
For MS/MS quantitation, the API 4000 Qtrap mass spectrometer
was operated in the ESI negative mode with multiple reaction-mon-
itoring (MRM). The MS/MS parameters were set as follows: curtain gas,
30 psi; nebulizer gas (GS1), 55 psi; turbo gas (GS2), 55 psi; ion spray
voltage, 4500 V; ion source temperature, 450 °C. For the selected ion
transitions, the declustering potential (DP) and the collision energy
(
CE) were optimized. The ion transitions monitored were as follows:
OCA, 419.1 → 401.4; OCA acyl glucuronide, 595.5 → 419.4; UDCA,
91.4 → 391.4; UDCA acyl glucuronide, 567.4 → 391.3; UDCA hy-
3
droxyl glucuronide, 567.4 → 567.4; the internal standard steviol acyl
glucuronide, 493.3 → 317.1. UDCA and OCA acyl glucuronide were
quantified by standard calibration curves from 1 to 5000 nM and
1
–10000 nM, respectively. The quantitative method displayed good
sensitivity and reproducibility. The data were collected and processed
using AB Sciex Analyst 1.5.2 data collection and integration software.
3.2. Kinetics of UDCA glucuronidation in human liver microsomes
2.11. Data analysis
By incubation with HLMs within the tested substrate concentration
All the data were presented as the means ± SD. Apparent enzyme
range (1–400 μM), OCA and UDCA were mainly metabolized into
UDCA-G1 and OCA-G1, respectively. As shown in Fig. 3A, the formation
of UDCA-G1 by HLMs followed substrate inhibition kinetics with a Km
of 66.75 ± 20.94 μM and Vmax of 18.33 ± 3.34 pmol/min/mg pro-
tein, respectively, and a Ki value at 585.1 ± 292.1 μM much higher
than the Km, indicating minor inhibition of UDCA on HLMs. The for-
mation of OCA-G1 by HLMs followed typical Michaelis-Menten kinetics
with the Km of 79.4 ± 7.14 μM and Vmax of 18.04 ± 0.58 pmol/min/
mg protein, respectively. The apparent intrinsic clearance of UDCA in
HLMs through acyl glucuronidation was 0.29 ± 0.02 μL/min/mg
kinetics by UGTs was obtained by fitting the experimental data with the
Michaelis–Menten (eq (1)) model or substrate inhibition (eq (3)) with
nonlinear regression analysis using GraphPad Prism 5.0 (GraphPad
Software, CA). Assuming a well-stirred model, the in vitro intrinsic
hepatic clearance (CLint) was then calculated with eq (2).
The Michaelis–Menten equation:
V = (Vmax × [S])/(Km + [S])
CLint = Vmax/Km
(1)
(2)
4

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
Fig. 2. Representative LC-MS/MS chromatograms of samples obtained from the incubation of 100 μM UDCA (A) and 50 μM OCA (B) with HLMs for 120 min. UDCA-G
and OCA-G represent different monoglucuronides of UDCA and OCA, respectively.
protein, and the clearance of OCA in HLMs through acyl glucuronida-
3.4. Kinetics of UDCA and OCA acyl glucuronidation by human UGTs
tion was 0.23 ± 0.04 μL/min/mg protein.
The formation of UDCA-G1 mediated by UGT 1A3, 1A8, 1A9 and
2
B7 fits substrate inhibition kinetics better than the Michaelis–Menten
model, as shown in Fig. 5A–D. UGT 1A3 exhibited the highest Vmax
(168.7 ± 16.26 pmol/min/mg protein) and low Km
10.43 ± 2.56 μM) on UDCA-G1 formation. Consequently, UGT 1A3
showed the highest efficiency in catalyzing 24-COOH glucuronidation
(CLint 16.18 ± 0.86 μl/min/mg protein, 55 times that of HLMs), fol-
lowed by UGT 1A8 (4.10 ± 0.77 μL/min/mg protein). UDCA-G1 for-
mation by UGT 1A9 and 2B7 showed similar Vmax and Km values, and,
consequently, similar CLint to those of HLMs (Table 1). In addition, UGT
1A8 showed the highest affinity towards UDCA acyl glucuronidation
with the lowest Km at 4.10 ± 1.19 μM. However, the Ki value in UGT
1A8 was 27.6 ± 7.6 μM, indicating a high substrate inhibition of
UDCA on UGT 1A8.
3
.3. Identification of UGT isoforms involved in the glucuronidation of
UDCA and OCA
(
The relative activities of 12 recombinant human UGT isoforms (UGT
A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17)
1
were determined in terms of the formation of UDCA and OCA glucur-
onides. For UDCA, the acyl glucuronidation was mediated pre-
dominantly by UGT 1A3, followed by 1A8, 1A9 and UGT2B7 (Fig. 4A).
The hydroxyl glucuronidation of UDCA, including both 3-OH and 6-OH,
was mainly mediated by UGT 2B7, as well as 1A3, 1A4 and 2B17
(
Fig. 4B and C). OCA acyl glucuronide (OCA-G1) formation was pre-
dominantly catalyzed by UGT1A3, followed by UGT1A1 (Fig. 4D).
Different from OCA-G1 formation, the OCA-G2 formation was almost
solely mediated by UGT1A4 (Fig. 4E).
Within the substrate concentration range tested, only UGT 1A1 and
1A3 catalyzed OCA-1G formation from OCA. As shown in Fig. 5E and F,
the acyl glucuronidation of OCA by UGT 1A1 and 1A3 followed typical
5

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
OATP1B1 and OATP1B3. Coincubation of rifampicin (characteristic
inhibitor for both OATP1B1 and 1B3) with UDCA-G1 significantly de-
creased the uptake of UDCA-G1 by OATP1B1- and 1B3-transfected cells
to the level of control cells. However, OATP1B1- and 1B3-expressing
cells did not transport OCA or OCA-G1, indicating that both OCA and
OCA-G1 were not the substrates of OATP1B1 and 1B3.
Fig. 7C and D shows the uptake of prototypical NTCP substrate
taurocholate (TCA) into MDCKII-NTCP cells compared with the re-
spective vector control cells. However, no significant difference was
found between the uptake of OCA, UDCA-G1 and OCA-G1 by NTCP-
expressing cells and that by the control cells. Uptake of UDCA into
MDCKII-NTCP cells was higher than that into the control cells, with an
uptake ratio of 1.68-fold (Fig. 7D).
4. Discussion
Bear bile powder is a well-known traditional Chinese health food
and medicine and has been widely used in detoxification, pain relief
and, particularly, various liver diseases for thousands of years [17]. It
has been documented that bear bile possesses anti-allergic, anti-in-
flammatory, and analgesic properties [18]. Bile acids were regarded as
the effective constituents of bear bile, among which UDCA has a high
content [19]. In modern medicine, UDCA has been approved by the US
FDA as the only pharmacological treatment for primary biliary cho-
langitis until OCA was licensed in Europe for patients not responsive to
UDCA [20].
Previous studies have shown that UDCA could increase bile flow,
change the hydrophobicity index of the bile acid pool, and control the
levels of serum bilirubin and hepatic transaminases [21–23], indicating
that UDCA would be safe and beneficial. However, some “un-
anticipated” toxicities of UDCA were reported in recent years. In the
treatment of primary sclerosing cholangitis with UDCA, more than
twice the number of patients died, developed varices, or became eli-
gible for liver transplantation in the UDCA group than in the placebo
group [24]. In a study of neonatal and infancy cholestasis, UDCA
treatment increased the risk of failure of resolution of cholestasis, life-
threatening complications, liver cell failure, and death [25]. Moreover,
the incidence of hepatocellular carcinoma increased after UDCA treat-
ment in patients with primary biliary cholangitis, especially in the
group of non-responders to UDCA [26]. The safety of UDCA remains
controversial, and the molecular mechanism for UDCA toxicity is un-
clear.
UDCA conjugation with glycine and taurine at the C-24 carboxyl
group by BAAT has been extensively studied [27]. However, studies of
the UDCA glucuronidation pathway are rather limited. Endogenous bile
acids are more easily conjugated with amino acids than with a glu-
curonosyl group, but exogenously administered UDCA might undergo
extensive glucuronidation due to its high dose. In addition, genetic
polymorphisms of BAAT had been reported in Japanese [27] and
French Caucasian individuals [28] that might cause significant inter-
individual variation in the amino acid conjugation of UDCA [29].
Consequently, the glucuronidation pathway might be the major meta-
bolism pathway of UDCA in some individuals with BAAT gene defi-
ciency. In human urine, both UDCA hydroxyl glucuronide and acyl
glucuronide were found [8]. However, the UGTs responsible for the
different glucuronidation pathways, especially acyl glucuronidation,
are still unknown. In the present study, for the first time, our results
proved the UGT-dependent regioselective glucuronidation of UDCA
towards hydroxyl and carboxyl groups. UGT1A3 showed a predominant
role in the carboxyl glucuronidation of UDCA, while UGT2B7 exhibited
the highest glucuronidation activity towards both 3-OH and 6-OH of
UDCA.
Fig. 3. Effects of the UDCA and OCA concentrations on the formation of UDCA-
G1 (A) and OCA-G1 (B) in HLMs. Graph A and B were plotted using substrate
inhibition kinetic and typical Michaelis-Menten kinetic equation, respectively.
Michaelis–Menten and substrate inhibition kinetics, respectively. UGT
1A3 displayed a high capacity and affinity (Vmax 138.7 ± 9.42 pmol/
min/mg protein, Km 5.08 ± 0.73 μM) in converting OCA to OCA-G1,
resulting in a high CLint (27.20 ± 1.34 μL/min/mg protein, 118 times
that of HLMs), while UGT1A1 exhibited a much lower capacity (Vmax
32.43 ± 8.95 pmol/min/mg
protein)
and
affinity
(Km
8
4.11 ± 51.9 μM), resulting in a lower CLint (0.38 ± 0.05 μL/min/mg
protein).
3.5. Inhibition of UDCA and OCA glucuronidation mediated by UGT1A3
UGT 1A3 plays a significant role in the 24-COOH glucuronidation of
both UDCA and OCA. The effect of characteristic inhibitor (lapatinib) of
UGT1A3 on UDCA-G1 and OCA-G1 formation was investigated at a
series of concentrations from 0.01 μM to 500 μM. As shown in Fig. 6, a
potent inhibitory effect of lapatinib toward UDCA and OCA glucur-
onidation mediated by UGT1A3 was observed, with IC50 values of
1.76 ± 0.10 μM and 2.14 ± 0.30 μM, respectively.
3.6. Uptake of UDCA, UDCA-G1, OCA and OCA-G1 into OATP1B1-, 1B3-
and NTCP-transfected cells
As shown in Fig. 7A and B, the uptake values of rosuvastatin (known
substrate for OATP1B1 and 1B3) in OATP1B1- and OATP1B3-expres-
sing CHO cells were approximately 6 and 4 times those in vector-
transfected control cells, showing good function of OATP1B1 and 1B3
in the transfected cells. The uptake of UDCA by OATP1B1- and 1B3-
transfected cells was < 2-folds that by control cells. By contrast, the
uptake of UDCA-G1 into OATP1B1 and 1B3 cells was > 7-fold that into
control cells, indicating that UDCA-G1 was the substrate for both
As a new drug, OCA is modified from chenodeoxycholic acid
(CDCA) and exhibited much greater potency for farnesoid X receptor
activation than CDCA, resulting in reduced bile acid synthesis and
improved choleresis [30,31]. The metabolism study of OCA was rather
6

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
Fig. 4. Formation of UDCA-G1 (A), UDCA-G2 (B), UDCA-G3 (C), OCA-G1 (D) and OCA-G2 (E) by the incubation of UDCA and OCA with recombinant UGTs for
20 min.
1
limited compared with that of CDCA, and no study was reported on the
glucuronidation of OCA. It was demonstrated that CDCA-glucuronide is
the most abundant bile acid glucuronide in human serum and urine,
which mainly existed as CDCA acyl glucuronide [32,33]. Our study
showed that OCA also underwent glucuronidation in HLMs, and the
main glucuronide formed was OCA acyl glucuronide. UGT1A3 showed
the highest activity and affinity towards OCA acyl glucuronide forma-
tion, a finding that was consistent with the result of the study showing
CDCA glucuronidation was mainly catalyzed by UGT1A3 [33]. UGT1A1
also played an important role in OCA carboxyl glucuronidation,
7

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
Fig. 5. Effects of the UDCA and OCA concentrations on the formation of UDCA acyl glucuronide by human UGT1A1 (A), UGT1A3 (B), UGT1A9 (C), UGT 2B7 (D), and
OCA acyl glucuronidation by human UGT1A3 (E), UGT1A1 (F). All the graphs were plotted using substrate inhibition kinetic equation except graph F using
Michaelis-Menten kinetic equation.
Table 1
Kinetics parameters of UDCA and OCA acyl glucuronidation in HLMs and recombinant human UGTs.
Vmax (pmol/min/mg protein)
Km (μM)
Ki(μM)
CLint (μL/min/mg protein)
UDCA-G1
OCA-G1
HLM
18.33 ± 3.34
168.7 ± 16.26
16.38 ± 2.44
13.83 ± 0.86
17.03 ± 2.15
18.04 ± 0.58
138.70 ± 9.42
32.43 ± 8.95
66.75 ± 20.94
10.43 ± 2.56
4.10 ± 1.19
28.47 ± 3.68
28.94 ± 6.58
79.4 ± 7.14
5.08 ± 0.73
84.11 ± 51.9
585.1 ± 292.1
536.0 ± 191.5
27.6 ± 7.6
603.9 ± 123.3
213.8 ± 56.3
–
0.29 ± 0.02
16.18 ± 0.86
4.10 ± 0.77
0.48 ± 0.05
0.58 ± 0.005
0.23 ± 0.04
27.20 ± 1.34
0.38 ± 0.05
UGT1A3
UGT1A8
UGT1A9
UGT2B7
HLM
UGT1A3
UGT1A1
57.8 ± 8.1
–
8

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
On the other hand, acyl glucuronide protein adducts can act as haptens
and initiate immune reactions that might lead to immune-mediated
toxicity (hypersensitivity reactions) [41]. In our study, we proved that
UDCA and OCA were mainly metabolized into acyl glucuronide by
UGT1A3. The acyl glucuronide formation of UDCA might be related to
the perplexing toxicity of UDCA observed in the clinic, although most
studies have focused on the direct toxicity of parent UDCA.
After formation in the hepatocytes, UDCA and OCA glucuronides
might be exported to the blood by MRP3 or excreted into bile via efflux
transporters. The potential mechanism involved in the efflux of UDCA
and OCA has not been revealed in the present study and need further
investigation. OATP1B1 and OATP1B3 are specifically expressed in the
human liver, where most drug metabolism occurs [42]. Our results
demonstrated that OATP1B1 and 1B3 transport UDCA acyl glucuronide,
but not the parent UDCA, indicating that hepatocytes selectively uptake
UDCA acyl glucuronide rather than UDCA from the blood. It was re-
ported that UDCA could be transported significantly by NTCP, but not
by OATP1B1 and OATP1B3, in the transfected cells [43,44], but the
uptake ratios were only slightly higher than 100% (less than 1.5-fold), a
finding that is consistent with our results that OATP1B1 and 1B3 were
not involved in UDCA transport. Consequently, UDCA acyl glucuronide
would be accumulated in the liver by uptake through OATP1B1 and
1B3, which could lead to high exposure of UDCA acyl glucuronide in
the human liver. Due to the reactivity of acyl glucuronide, the accu-
mulation of UDCA acyl glucuronide in the liver might be related to the
liver toxicity observed in the clinic. Our result also proved that, in
contrast to UDCA acyl glucuronide, OCA acyl glucuronide was not ac-
tively transported by OATP1B1 and 1B3, which would not result in the
accumulation of OCA acyl glucuronide in hepatocytes. Consequently,
OCA might be a safer drug than UDCA for the treatment of primary
biliary cholangitis.
It has been uncovered that nuclear receptor FXR could directly
transactivate genes encoding UGTs and indirectly negatively regulate
OATP1Bs [45–47]. Our result demonstrated that UGT1A3 and UGT2B7
are mainly responsible for the glucuronidation of UDCA and OCA and
OATP1Bs are involved in the absorption of UDCA glucuronide. It is
possible that the expression of UGTs and OATP1Bs may be changed
over time after dosing of UDCA and OCA repeatedly. Consequently,
when OCA and UDCA were prescribed clinically for long time usage,
physicians need to take into consideration thedose adjustment.
In conclusion, the present study investigated the roles of human
UGTs and OATP1B1 and 1B3 in the disposition of UDCA and OCA. Both
UDCA and OCA underwent UGT-dependent regioselective glucur-
onidation with a positional preference at the carboxyl group. The acyl
glucuronidation of UDCA and OCA was predominantly mediated by
UGT1A3. The hepatic transporters OATP1B1 and 1B3 selectively
transport UDCA acyl glucuronide, but not UDCA, OCA, or OCA acyl
glucuronide. The acyl glucuronidation of UDCA and further accumu-
lation of UDCA acyl glucuronide in the liver might be related to the
perplexing toxicity caused by UDCA. The liver disposition of OCA acyl
glucuronide is different from that of UDCA, which might make OCA a
safer drug for the treatment of primary biliary cholangitis.
Fig. 6. Inhibition of UDCA (A) and OCA (B) glucuronidation by lapatinib in
human UGT1A3. The activity remaining is calculated by regarding the gen-
eration rate of UDCA-G1or OCA-G1 without inhibitor as 100%.
especially at high concentrations.
UGT1A3, which is mainly expressed in human liver and intestine,
plays an important role in UDCA acyl glucuronidation [34]. UGT1A1,
1A3, 1A4, 1A6 and 1A9 are the major UGTs of the UGT1A family ex-
pressed in human liver. The relative expression of hepatic UGT1A3
ranged from 0.2 to 2.4% with significant inter-individual variability
[
35,36], approximately one-fifth of UGT1A1 and 1A9 expressed in the
liver. Our result demonstrated that the apparent clearance of UDCA by
UGT1A3 was more than 30 times that by UGT1A9. According to our
kinetics results, UGT1A3 is the major enzyme responsible for UDCA
acyl glucuronidation, although it is not highly expressed in the liver.
Similarly, the apparent clearance of OCA by UGT1A3 was approxi-
mately 70 times that by UGT1A1. Thus, UGT1A3 is the major enzyme
responsible for both UDCA and OCA acyl glucuronidation.
The extrahepatic UGTs also play important roles in OCA and UDCA
glucuronidation. The mRNA expression of UGT1A3 and 2B7 were also
detected in the human intestine, but much lower than those in the liver
Conflicts of interest
There are no conflicts to declare.
Declaration of interests
[
37]. In addition, Western blot analysis showed that UGT2B7 was
faintly detectable in intestinal microsomes [38], indicating that the
glucuronidation of OCA and UDCA mainly occurs in the liver. As a
result, the present study mainly focused on the investigation of hepatic
glucuronidation of OCA and UDCA.
Acyl glucuronide conjugates are reactive metabolites capable of
covalent binding to proteins and DNAs [39,40]. Covalent binding to
critical proteins, DNAs or some regulatory pathway components by acyl
glucuronides might lead to cellular necrosis and result in direct toxicity.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science
9

D. Zhou, et al.
Chemico-Biological Interactions 310 (2019) 108745
Fig. 7. Uptake of UDCA, UDCA-G1, OCA and OCA-G1 into CHO-OATP1B1 cells (A), CHO-OATP1B3 cells (B), and MDCKII-NTCP cells (C). Control experiments are
shown using rosuvastatin as the prototypical OATP1B1 and 1B3 substrate and TCA as the prototypical NTCP substrate. Rifampin (100 μM) was chosen as the
prototypical OATP1B1 and 1B3 inhibitor.
Foundation of China (Nos. 81473278 and 81703596) and Jiangsu
Province Science Foundation for Youths (BK20150349).
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