Toxicology and Applied Pharmacology 424 (2021) 115595

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/taap

Comparison of the drug-drug interactions potential of ibrutinib and

acalabrutinib via inhibition of UDP-glucuronosyltransferase
Xiaoyu Wang a, Zhe Wang a, Xiaoyu Fan a, Mingrui Yan a, Lili Jiang a, Yangliu Xia a, Jun Cao b, **,
Yong Liu a, *
a
School of Life and Pharmaceutical Sciences, Dalian University of Technology, Panjin 124221, China
b
Department of Occupational and Environmental Health, Dalian Medical University, Dalian 116044, China

A R T I C L E I N F O A B S T R A C T

Keywords: Ibrutinib and acalabrutinib are two Bruton's tyrosine kinase (BTK) inhibitors which have gained Food and Drug
Ibrutinib Administration (FDA) approval for the treatment of various B cell malignancies. Herein, we investigated the
Acalabrutinib effects of the two drugs on UDP-glucuronosyltransferase (UGT) activities to evaluate their potential risk for drug-
UDP-glucuronosyltransferase
drug interactions (DDIs) via UGT inhibition. Our data indicated that ibrutinib exerted broad inhibition on most of
Drug-drug interaction
UGTs, including a potent competitive inhibition against UGT1A1 with a Ki value of 0.90 ± 0.03 μM, a
noncompetitive inhibition against UGT1A3 and UGT1A7 with Ki values of 0.88 ± 0.03 μM and 2.52 ± 0.23 μM,
respectively, while acalabrutinib only exhibited weak UGT inhibition towards all tested UGT isoforms. DDI risk
prediction suggested that the inhibition against UGT1A1 and UGT1A3 by ibrutinib might bring a potential DDIs
risk, while acalabrutinib was unlikely to trigger clinically significant UGT-mediated DDIs due to its weak effects.
Our study raises an alarm bell about potential DDI risk associated with ibrutinib, however, the extrapolation
from in vitro data to in vivo drug interactions should be taken with caution, and additional systemic study is
needed.

1. Introduction
Ibrutinib and acalabrutinib are two oral small-molecule Bruton's
tyrosine kinase (BTK) inhibitors approved for the treatment of adult
patients with chronic lymphocytic leukemia (CLL) and mantle cell
lymphoma (MCL) by blocking B-cell receptor signaling pathway to
induce tumor cell death (Advani et al., 2013; Byrd et al., 2016). Phase III
trials of both two drugs are currently underway worldwide to evaluate
their treatment effects and give more combinations of drugs, meaning
that drug-drug interactions (DDIs) may first be encountered during
clinical development (Cameron and Sanford, 2014; Markham and
Dhillon, 2018). Previous study has suggested that co-administration
acalabrutinib with itraconazole, a cytochrome P450 (CYP) 3A inhibi­
tor, will be expected to increase its plasma concentrations which may
result in toxicity (Zhou et al., 2019). Co-treatment with ketoconazole (a
known CYP3A inhibitor) will increase the Cmax and AUC of ibrutinib by
29- and 24-fold, respectively, so that lead to potential adverse events at
therapeutic dose (de Zwart et al., 2016). Therefore, DDIs based on drug
metabolizing enzyme remain a problem of considerable clinical
significance.
In addition to giving focus on CYP enzyme driving DDIs, indeed,
UDP-glycosyltransferase (UGT) inhibition could also be implicated in
hepatotoxicity and associated with clinically DDIs (Meech et al., 2019).
UGT is an important class of phase II metabolizing enzyme superfamily
which plays a critical role in the elimination of numerous exogenous
chemicals and endogenous substances and in the pathological proced­
ures of some diseases (Zhou et al., 2013). Particularly, UGT1A1, for
which participates in bilirubin glucuronidation and has a high level of
polymorphism, is a major concern. Several tyrosine kinase inhibitors
(TKIs) have been observed as potent UGT inhibitors implying a risk for
adverse DDIs in clinic. For example, concomitant administration of
gefitinib (700 mg/day) and irinotecan may increase the SN-38 AUC by

* Correspondence to: Y. Liu, School of Life and Pharmaceutical Sciences, Dalian University of Technology, 2 Dagong Road, Liaodongwan New District, Panjin
124221, China.
** Correspondence to: J. Cao, Department of Occupational and Environmental Health, Dalian Medical University, No. 9 W. Lvshun South Road, Dalian 116044,
China.
E-mail addresses: caojunly@163.com (J. Cao), yliu@dlut.edu.cn (Y. Liu).

https://doi.org/10.1016/j.taap.2021.115595
Received 31 January 2021; Received in revised form 18 May 2021; Accepted 21 May 2021
Available online 24 May 2021
0041-008X/© 2021 Elsevier Inc. All rights reserved.
X. Wang et al.
Fig. 1. Chemical structures of ibrutinib and acalabrutinib.
49% via UGT1A1 inhibition (Li et al., 2015). Regorafenib and sorafenib
can potently inhibit UGT1A1 activity, which contributed significantly to
the hyperbilirubinemia observed in patients (Miners et al., 2017).
However, the issue of prediction of DDIs risk by acalabrutinib and
ibrutinib via UGT inhibition is far from exhaustive.
Here, we aimed to evaluate the inhibitory effects of two BTK in­
hibitors ibrutinib and acalabrutinib towards the activity of 12 human
recombinant UGT isoforms and identify the potential DDIs in vivo based
on in vitro-in vivo extrapolation (IVIVE). These results provide a new
sight for explanation of the toxicity of BTK-TKIs and might be helpful to
develop new clinical combination therapies.
2. Materials and methods
2.1. Chemical reagents
Ibrutinib, acalabrutinib, and trifluoperazine (TFP) were obtained
from Selleck Chemicals (Houston, TX, USA), umbelliferone, 4-methyl­
umbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-
MUG), and uridine 5′ -diphosphoglucuronic acid trisodium salt (UDPGA)
were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tris-HCl,
MgCl2 were purchased from Aladdin (Shanghai, China). All other
chemicals used were of HPLC grade commercially available.
Recombinant UGT supersomes (UGT1A1, UGT1A3, UGT1A4,
UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7,
UGT2B15, and UGT2B17) were purchased from Corning Gentest
(Tewksbury, MA, USA).
2.2. Inhibition of recombinant human UGT enzymes by BTK inhibitors
The incubation systems were conducted in a final volume of 200 μL
in 50 mM Tris-HCl buffer (pH 7.4) with 10 mM MgCl2, 5 mM UDPGA,
UGTs and 4-MU. Ibrutinib and acalabrutinib (structures are shown in
Fig. 1) were dissolved in DMSO. The linear initial velocity enzymatic
conditions were used as described previously (Wang et al., 2020). Re­
actions were pre-incubated for 5 min at 37 ◦ C and initiated by addition
of 20 μL UDPGA. Enzymatic reactions were terminated by adding equal
volume of ice-cold acetonitrile containing 1 μM umbelliferone as an
internal standard. Samples were centrifuged at 20,050g for 20 min at
4 ◦ C to remove the protein.
Detection of 4-MUG was performed by ultra-performance liquid
chromatography-tandem mass spectrometry (UPLC-MS/MS) system
consisting of ACQUITY UPLC module (Waters, Milford, MA) and Qtrap
6500 (SCIEX, San Jose, CA, USA). Aliquot 2 μL of each sample was
injected on a BEH C18 column (50 × 2.1 mm, 1.7 μm). The mobile phase
2
Toxicology and Applied Pharmacology 424 (2021) 115595
was consisted of solvent A (0.1% formic acid in water) and solvent B
(0.1% formic acid in acetonitrile) with a flow rate of 0.3 mL/min. The
gradient was used as follows: 5% (v/v) B at 0–2 min, 5%–50% (v/v) B at
2–2.4 min, 50% (v/v) B at 2.4–3 min, 50%–5% (v/v) B at 3–4 min, 5%
(v/v) B at 4–5 min. The MRM m/z transitions monitored were 353.3/
177.2 for 4-MUG and 163.1/107.1 for internal standard in positive ion
mode which were identified to be optimal for quantitative analysis.
Calibration standard of 4-MUG was prepared at concentrations of
0.125–15 μM and the accuracy was within 10%.
In addition, trifluoperazine (TFP) was used as the selective substrate
in the UGT1A4 inhibition studies (Uchaipichat et al., 2006) and the
supernatant of reactions were separated by HPLC system (Waters, Mil­
ford, MA) with a Tnature C18 column (250 × 4.6 mm, 5 μm). Mobile
phase was containing 0.5% formic acid in water (A) and acetonitrile (B).
A gradient program was as follows: 29–48% (v/v) B at 0–7 min, 48%–
29% (v/v) B at 7.0–7.5 min, 29% (v/v) B at 7.5–9 min. The flow rate was
maintained at 1 mL/min and analytes were quantified with UV detection
at 254 nm. Umbelliferone (18 μM) was used as the internal standard.
Calibration standard of TFP was prepared at concentrations of 0.025–10
μM since no TFP-G standards were available (Seo et al., 2014).
2.3. Determination of IC50 and Ki
The same incubation condition as for the preliminary inhibition
study was used for each UGT isoform. The relative formation rate of 4-
MUG or TFP-G in the presence vs. absence of inhibitors was estimated.
For UGT isoforms whose activity is inhibited by >50% by individual
BTK inhibitors, half maximal inhibitory concentration (IC50) values for
TKIs were determined. Subsequent inhibition kinetic experiments were
performed for the lower IC50 values groups. The most appropriate in­
hibition type and Ki value were ascertained from comparison of
goodness-of-fit parameters using equations for competitive inhibition
(eq. 1), noncompetitive inhibition (eq. 2), uncompetitive (eq. 3), or
mixed inhibition (eq. 4).
v = (Vmax S)/(Km (1 + I/Ki ) + S ) (1)
v = (Vmax S)/(Km + S)(1 + I/Ki ) (2)
v = (Vmax S)/(Km + (1 + I/Ki )S ) (3)
v = (Vmax S)/[Km (1 + I/Ki ) + S(1 + I/αK i ) ] (4)
where v is the velocity of the reaction, Vmax is the maximum velocity, S
and I are the substrate and inhibitor concentrations, respectively. Km is
Michaelis constant (the substrate concentration at half of the Vmax of the
reaction), Ki is inhibition constant. α reflects the effect of inhibitor on the
affinity of the enzyme for its substrate.
2.4. Predicted concentrations of TKIs at UGT active site
For IVIVE, the inhibitor concentrations ([I]) after oral administration
can be calculated using the following equation: average systemic plasma
concentration ([I]av) (eq.5), maximum systemic plasma concentration
([I]max) (eq.6), and maximum hepatic input concentration ([I]in) (eq.7)
to evaluate the highest risk (Ito et al., 2004).
/
[I]av = (D/τ) (CL/F) (5)
( )/
[I]max = [I]av κτ (1 − exp( − κτ) ) (6)
/
[I]in = [I]av + ka Fa D Qh (7)
where D and τ are oral dose and dosing interval, respectively, CL/F is
apparent (oral) drug clearance, k is the elimination rate constant which
is expressed as k = 0.693/t1/2 (t1/2, half time), ka is the absorption rate
constant, Fa is the fraction absorbed from gut into the portal vein, and Qh
X. Wang et al.
Fig. 2. The inhibition of ibrutinib and acalabrutinib towards human recombinant UGT isoforms. TFP was used as substrate for the inhibition studies with UGT1A4, 4-
MU was used as the substrate for all other UGT isoforms. Each bar represents the mean ± standard error of duplicate experiments.
is the hepatic blood flow rate, assumed to be 1450 mL/min (Davies and
Morris, 1993). The parameters of CL/F, t1/2, ka and Fa obtained from
previous publications for ibrutinib (Pharmacyclics Inc, 2013; de Jong
et al., 2015; Marostica et al., 2015) and acalabrutinib (Markham and
Dhillon, 2018; Zhou et al., 2019) were shown in Table 2.
2.5. Prediction of in vivo drug–drug interactions magnitude
The potential DDIs risk arising from inhibition of a drug metabolizing
enzyme can be determined from the Ki value generated in vitro using the
following equations (eq.8) for drugs with negligible renal clearance
(Miners et al., 2010).
AUCi /AUC = 1/(fm /(1 + [I]/Ki ) + (1 − fm )
where the victim drug is metabolized by a single enzyme or has a high
hepatic clearance (fm assumed as 1), eq. (8) simplifies to
AUCi /AUC = 1 + [I]/Ki
where AUCi/AUC is the ratio of areas under the plasma concentration
against time of the victim drug in the presence and absence of the in­
hibitor and fm is the fraction of UGT substrates metabolized by the
Toxicology and Applied Pharmacology 424 (2021) 115595
inhibited enzyme which ranged of 0.1–1. Here, [I]in was used to predict
the highest potential risk from the perspective of pharmacokinetics
theory (Oda et al., 2015). As for UGT1A7, only expressed in extrahepatic
tissues (Strassburg et al., 1997), [I]max was used to represent the in­
hibitor concentrations at UGT enzyme active site.
In the present analysis, the DDIs potential for ibrutinib and acalab­
rutinib were evaluated by calculating the AUC ratios. The FDA draft
guidance on drug interaction studies proposes that a 25% increase in the
AUC of victim drugs, which is normally considered to be bioequivalent,
should not merit full consideration for clinically relevant DDIs (Yu and
Tweedie, 2013). As such, the AUC ratio cut-off value of 1.25 was used
according to the FDA Guidance (USFDA, 2020) and interactions with
AUC ratio changes more than 5-fold, 2-to 5-fold, or 1.25-to 2-fold were
(8) considered strong, moderate, or weak inhibition drug interactions,
respectively (Yu et al., 2019).
3. Results
(9)
3.1. Inhibition of UGT activities by ibrutinib and acalabrutinib
The inhibitory effects of ibrutinib and acalabrutinib on UGT activ­
ities were shown in Fig. 2. Ibrutinib (100 μM) inhibited almost all the
3
X. Wang et al. Toxicology and Applied Pharmacology 424 (2021) 115595

Fig. 3. Concentration-dependent inhibition of ibrutinib in recombinant human UGTs.

Fig. 4. Concentration-dependent inhibition of acalabrutinib in recombinant human UGT1A1, UGT1A3, UGT1A7, and UGT2B15.

UGTs in reducing the substrate glucuronidation more than 70%. The

Table 1
inhibition of acalabrutinib was observed against UGT1A1, 1A3, 1A7,
The IC50 of ibrutinib, acalabrutinib for the inhibition against UGT activities.
and 2B15 with corresponding residual activities were 48.68%, 28.57%,
UGTs Ibrutinib (μM) Acalabrutinib (μM) 28.13%, and 11.8%, respectively. To further investigate the inhibitory
UGT1A1 0.70 ± 0.05 17.89 ± 0.72 potentials of both two BTK-inhibitors, IC50 values were further deter­
UGT1A3 0.79 ± 0.05 8.54 ± 0.75 mined and corresponding inhibition curves of ibrutinib and acalabruti­
UGT1A4 20.26 ± 0.41 > 100 nib were shown in Figs. 3 and 4, respectively. As shown in Table 1,
UGT1A6 24.92 ± 0.93 > 100
UGT1A7 1.05 ± 0.01 27.62 ± 2.08
ibrutinib potently inhibited the enzyme activities of UGT1A1 (IC50 0.70
UGT1A8 16.19 ± 0.71 > 100 ± 0.05 μM), UGT1A3 (IC50 0.79 ± 0.05 μM), and UGT1A7 (IC50 1.05 ±
UGT1A9 11.69 ± 0.48 > 100 0.01 μM) (Fig. 3), and moderately inhibited of UGT1A9 (IC50 11.69 ±
UGT1A10 13.65 ± 0.11 > 100 0.48 μM), and UGT2B7 (6.94 ± 0.08 μM), weakly inhibited of other UGT
UGT2B4 80.84 ± 4.07 > 100
isoforms which IC50 values ranged from 13.65 to 80.84 μM. Meanwhile,
UGT2B7 6.94 ± 0.08 > 100
UGT2B15 21.84 ± 0.53 19.84 ± 0.13 acalabrutinib demonstrated moderate or slight inhibition of UGT1A1,
UGT2B17 26.08 ± 4.56 > 100 1A3, 1A7 and UGT2B15 activities with IC50 values ranged from 8.54 to
27.62 μM, since these values are more than an order of magnitude higher
Data represent the mean ± standard error of duplicates experiments.
than the reported Cmax (0.69 μM) (Markham and Dhillon, 2018), and
hence Ki was not determined but estimated based on the equation Ki =
1/2 × IC50, assuming competitive inhibition (Haupt et al., 2015).

4
X. Wang et al. Toxicology and Applied Pharmacology 424 (2021) 115595

Fig. 5. Representative Lineweaver-Burk plots and Dixon plots for the inhibition of.
4-MU glucuronidation by ibrutinib in human recombinant UGT1A1(A and B), UGT1A3(C and D), UGT1A7(E and F). All data points shown represent the mean ±
standard error of duplicate measurements.

5
X. Wang et al. Toxicology and Applied Pharmacology 424 (2021) 115595

Table 2 with Ki values of 0.88 ± 0.03 and 2.52 ± 0.23 μM, respectively.
Calculation of possible concentrations of ibrutinib and acalabrutinib.
Drugs Ibrutinib Acalabrutinib
3.3. Quantitative prediction of DDIs potential risk
Dose (mg) 560 200
Dosing Interval (hr) 24 12
Cmax (μM) 0.29 0.69 The calculated concentrations of [I]in and [I]max of ibrutinib and
Absorption Rate Constant (ka, h− 1) 0.46 1.65 acalabrutinib were shown in Table 2. Then the prediction results for
Plasma Unbound Fraction (fu,p) 2.70% 2.50% each TKI were calculated by eq. (8) and reported as isolines plot in
t1/2 (h) 4 1.57
Figs. 6 and 7, respectively. Values in the line represented the AUC ratio
CL/F (L/h) 1060 159
Oral bioavailability (F) 67% 25%
(AUCi/AUC) and an isoline was joining equal values of AUC ratio
Calculated concentrations Iav (μM) 0.05 0.23 generated from the corresponding oral dose and fm.
Imax (μM) 0.21 1.20 As shown in Fig. 6A, when the oral dose of ibrutinib is 560 mg/day,
Iin (μM) 2.05 1.17 co-administered substrate is mainly metabolized by UGT1A1 (fm > 0.8),
the predicted AUC ratio is approximately 2.40, which means that an
3.2. Inhibition kinetics in recombinant human UGTs by ibrutinib increase in the AUC of substrate of more than 140% might be observed
when substrates are co-administrated with ibrutinib. Also, when the
Kinetic experiments were performed to further characterize the in­ dose is more than 560 mg/day and fm is 1, the predicted AUC ratio is
hibition of UGT1A1, UGT1A3 and UGT1A7 activities by ibrutinib 3.05 (Fig. 6B), which means that the AUC of substrate totally metabo­
(Fig. 5). Nonlinear regression analysis indicated that 4-MU glucur­ lized by UGT1A3 will increase more than 2-fold in vivo. Specifically,
onidation in UGT1A1 was greatly inhibited by ibrutinib in a competitive when the dose of ibrutinib was 280 mg/day and fm was more than 0.4,
manner with a Ki value of 0.90 ± 0.03 μM, whereas inhibition of the AUC ratio of victim drug cleared by UGT1A1 or UGT1A3 were
UGT1A3 and UGT1A7 followed noncompetitive inhibition mechanism increased by at least 1.34, which indicated a clinically relevant DDI
potential for both UGT isoforms substrate. However, even when

Fig. 6. Isolines plots for relationship of AUC ratio against oral dose of ibrutinib and fm by UGT1A1 (A), UGT1A3 (B), and UGT1A7 (C) for DDI study. An isoline was
joining equal points, values in each line represent the AUC ratio (AUCi/AUC) calculated by the eq. (8) according to the corresponding oral dose of ibrutinib and fm.

6
X. Wang et al.
Fig. 7. Isolines plots for relationship of AUC ratio against oral dose of acalabrutinib and fm by UGT1A1 (A), UGT1A3 (B), and UGT1A7 (C) for DDI study. An isoline
was joining equal points, values in each line represent the AUC ratio (AUCi/AUC) calculated by the eq. (8) according to the corresponding oral dose of acalabrutinib
and fm.
administered at the highest dose (700 mg/day) and an fm is 1, the AUC
ratio is 1.10 (less than the cut-off value 1.25) for the substrates metab­
olized by UGT1A7 (Fig. 6C), indicating minor clinical impacts on the
pharmacokinetics of UGT1A7 substrates. Consequently, ibrutinib is
likely to result into the potential for clinically significant DDIs when co-
administered with UGT1A1, or UGT1A3 substrates through inhibition of
glucuronidation.
In this study, we assumed the Ki values of UGT1A1,1A3, and 1A7 by
acalabrutinib were 4.27, 8.56, and 13.81 μM, respectively. As shown in
Fig. 7, when administered acalabrutinib at the recommended dose of
200 mg/day and fm is 1, the AUC ratio of victim drugs cleared by
UGT1A1,1A3 and 1A7 are increased by 1.28, 1.14 and 1.09-fold,
respectively, which indicated that acalabrutinib could not bring the
evident changes in the AUC of the victim drugs.
4. Discussion
The advent of BTK inhibitors ibrutinib and acalabrutinib represents a
major breakthrough in the treatment of CLL and other B cell malig­
nancies (Sibaud et al., 2020). However, the coadministration with a
range of concomitant treatments is expected to under the high risks of
DDIs. As these kinds of events are by nature essentially unprevented,
7
Toxicology and Applied Pharmacology 424 (2021) 115595
there is an urgent need for carefully evaluation of the clinical DDIs po­
tential based on methodology and modeling methods in vitro to recog­
nize whether adjust dose regiments (Tornio et al., 2019). It has been
reported that strong inhibitors or inducers of CYP3A may have a rele­
vant effect on the PK of both two drugs and lead to a series of adverse
events, the effects on human UGTs still need further discussed. Previous
study revealed the inhibitory effects of ibrutinib on UGT enzymes and
gave the prediction of DDIs risk of UGT1A1, 1A4 and 1A9 (Korpra­
sertthaworn et al., 2019). In the current study, our data offered evidence
that potent inhibition against UGT1A1 and UGT1A3 by ibrutinib might
increase the risk of clinically significant DDIs when UGT1A1, or
UGT1A3 substrates were co-administered. Acalabrutinib was unlikely to
trigger clinically significant DDIs through UGT inhibition due to its weak
effects.
UGT1A1 is the only enzyme that metabolized bilirubin and facilitates
its elimination from the body (Bosma et al., 1994). Inhibition of UGT1A1
by ibrutinib may cause hyperbilirubinemia in vivo. This result might
provide a better understanding about ibrutinib-induced acute liver
injury which initially characterized by marked elevations of bilirubin
levels (Nandikolla et al., 2017; Tafesh et al., 2019). Indeed, several TKIs
such as erlotinib, nilotinib, pazopanib, vemurafenib with potent inhib­
itory effect on UGT1A1 have been reported to have a high incidence of
X. Wang et al.
hyperbilirubinemia (Qosa et al., 2018). Particularly, pazopanib inhibi­
ted UGT1A1-mediated SN-38 (active metabolite of irinotecan) glucur­
onidation and increased SN-38 AUC in recombinant human UGT1A1
Supersomes by a factor of 2.2 (Iwase et al., 2019). These results were
comparable to the ~90% increase in the AUC of SN-38 clinically
observed in patients who received 800 mg pazopanib once daily (Ben­
nouna et al., 2015). The prediction from in vitro data demonstrated that
atazanavir (a potent UGT1A1 inhibitor) increased in molidustat AUC of
2.9-fold. In the clinical DDI study, a 2-fold increase of AUC for moli­
dustat was also observed when pre- and co-treatment with atazanavir
which helps to confirm the assumptions made from the in vitro-in vivo
correlation(van der Mey et al., 2021). In addition, common genetic
polymorphisms of UGT1A1*28 will lead to lower drug clearance and
have an increased risk of DDIs. Therefore, co-administered of antineo­
plastic agents with a narrow therapeutic index such as etoposide and
irinotecan could result in excessive cytotoxic drugs exposure even a
slight increase of AUC(Wen et al., 2007) (Ichikawa et al., 2008). Taken
together, more attention should be paid when ibrutinib is co-
administered with UGT1A1 substrates which may cause a buildup of
endogenous substances or toxic drug levels.
UGT1A3 is mainly responsible for metabolizing nonsteroidal anti-
inflammatory drugs (NSAIDs) and some important endogenous sub­
stances, such as bile acids, estrogens (Lepine et al., 2004; Kuehl et al.,
2005). Inhibition of UGT1A3 might affect the conjugation of cheno­
deoxycholic acid and thus to cholestasis (Trottier et al., 2006). Prior
studies have noted that cholestatic features were shown in patients with
severe acute liver injury after ibrutinib treatment (Tafesh et al., 2019).
Additionally, UGT1A3 activity is subject to about 60-fold inter-
individual variability, implying increased clinical risks such as signifi­
cant variations of plasma concentrations and subsequent toxicity (Izu­
kawa et al., 2009). Evidence suggested that the observed increase in
atorvastatin lactonization in carriers of one or two UGT1A3*2 alleles,
was the result of the strong increase in UGT1A3 expression. Also, data
demonstrated that UGT1A3*2 genotype has a very substantial increase
in mRNA (5-fold) and protein (7.3-fold) in the human liver (Riedmaier
et al., 2010).Therefore, it is conceivable that individuals with
UGT1A3*2/*2 may endure the toxic side effects caused by atorvastatin
lactone. In addition, UGT1A3 has been reported to catalyze the glu­
curonidation of ibuprofen and ketoprofen (Sakaguchi et al., 2004), so it
represents bleeding risk whilst taking ibrutinib with anti-inflammatory
drugs. Although the level of UGT1A3 expression in human liver is
much lower than that of other UGT1A isoforms(Green et al., 1998), the
co-administrated ibrutinib with xenobiotic/drug mainly cleared by
UGT1A3 needs to be taken additional caution.
Since UGT1A7 is an important extra-hepatic UGT isoform that in­
fluences the disposition of drugs within the gut (Court et al., 2012) and
plays a prominent role in the detoxification of tobacco carcinogens
benzo(α)pyrene (Fang et al., 2002). Previous study has described a
striking correlation between the UGT1A7 genotypes and the occurrence
of irinotecan-induced toxicity that inhibition of UGT1A7-medicated
glucuronidation pathway in the gut may predispose for irinotecan-
induced diarrhea (Carlini et al., 2005). Additionally, it was reported
that variations in the UGT1A7 gene that reduce UGT1A7 activity may
increase the risk of smoking-related orolaryngeal cancer (Zheng et al.,
2001). A meta-analysis also demonstrated that individuals with
UGT1A7*3 allele (intermediate, and low activity UGT1A7 genotypes)
were involved in a high risk of cancer (Lu et al., 2011). It can be
anticipated that inhibition of UGT1A7 and UGT1A7*3 genotype in vivo
deserved more attention. Although the degree of DDIs risk via UGT1A7
inhibition is low in our prediction, the potent inhibition of ibrutinib on
UGT1A7 in the gut is still noteworthy.
There are several limitations to this study. First, DDIs risk based-on
IVIVE approach would be regarded as an initial discriminating screen,
therefore, prediction from in vitro data to in vivo drug interactions must
be taken with caution (Ito et al., 2004). Moreover, variable factors such
as transporters, various metabolic enzymes, the extrinsic factors (diet,
8
Toxicology and Applied Pharmacology 424 (2021) 115595
smoking) and diseases, will need consideration to progress the predic­
tion towards a quantitative basis (Rostami-Hodjegan and Tucker, 2007).
Thus, assessing DDIs involving UGT inhibition remains challenging.
However, this research was a preliminary study. Further mechanistic
and clinical studies still need to be performed to gain more accurate
evaluation of DDIs potential associated with ibrutinib and acalabrutinib.
In conclusion, results from this research provide essential informa­
tion about the inhibition of ibrutinib and acalabrutinib towards UGTs
isoforms and evaluate the possible DDIs risk. The present findings raise
an alarm bell about potential DDI risk associated with ibrutinib, how­
ever, the extrapolation from in vitro data to in vivo drug interactions
should be taken with caution, and additional systemic study is needed.
Declaration of Competing Interest
The authors declare that there is no conflict of interests regarding the
publication of this article.
Acknowledgements
This study was financially supported by the National Key Research
and Development Program of China (2017YFC1702006), the Funda­
mental Research Funds for the Central Universities (DUT21LK11).
References
Advani, R.H., Buggy, J.J., Sharman, J.P., Smith, S.M., Boyd, T.E., Grant, B., Kolibaba, K.
S., Furman, R.R., Rodriguez, S., Chang, B.Y., Sukbuntherng, J., Izumi, R., Hamdy, A.,
Hedrick, E., Fowler, N.H., 2013. Bruton tyrosine kinase inhibitor ibrutinib (PCI-
32765) has significant activity in patients with relapsed/refractory B-cell
malignancies. J. Clin. Oncol. 31, 88–94.
Bennouna, J., Deslandres, M., Senellart, H., de Labareyre, C., Ruiz-Soto, R., Wixon, C.,
Botbyl, J., Suttle, A.B., Delord, J.P., 2015. A phase I open-label study of the safety,
tolerability, and pharmacokinetics of pazopanib in combination with irinotecan and
cetuximab for relapsed or refractory metastatic colorectal cancer. Investig. New
Drugs 33, 138–147.
Bosma, P.J., Seppen, J., Goldhoorn, B., Bakker, C., Oude Elferink, R.P., Chowdhury, J.R.,
Chowdhury, N.R., Jansen, P.L., 1994. Bilirubin UDP-glucuronosyltransferase 1 is the
only relevant bilirubin glucuronidating isoform in man. J. Biol. Chem. 269,
17960–17964.
Byrd, J.C., Harrington, B., O’Brien, S., Jones, J.A., Schuh, A., Devereux, S., Chaves, J.,
Wierda, W.G., Awan, F.T., Brown, J.R., Hillmen, P., Stephens, D.M., Ghia, P.,
Barrientos, J.C., Pagel, J.M., Woyach, J., Johnson, D., Huang, J., Wang, X.,
Kaptein, A., Lannutti, B.J., Covey, T., Fardis, M., McGreivy, J., Hamdy, A.,
Rothbaum, W., Izumi, R., Diacovo, T.G., Johnson, A.J., Furman, R.R., 2016.
Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia. N. Engl. J. Med.
374, 323–332.
Cameron, F., Sanford, M., 2014. Ibrutinib: first global approval. Drugs 74, 263–271.
Carlini, L.E., Meropol, N.J., Bever, J., Andria, M.L., Hill, T., Gold, P., Rogatko, A.,
Wang, H., Blanchard, R.L., 2005. UGT1A7 and UGT1A9 polymorphisms predict
response and toxicity in colorectal cancer patients treated with capecitabine/
irinotecan. Clin. Cancer Res. 11, 1226–1236.
Court, M.H., Zhang, X., Ding, X., Yee, K.K., Hesse, L.M., Finel, M., 2012. Quantitative
distribution of mRNAs encoding the 19 human UDP-glucuronosyltransferase
enzymes in 26 adult and 3 fetal tissues. Xenobiotica 42, 266–277.
Davies, B., Morris, T., 1993. Physiological parameters in laboratory animals and humans.
Pharm. Res. 10, 1093–1095.
de Jong, J., Sukbuntherng, J., Skee, D., Murphy, J., O’Brien, S., Byrd, J.C., James, D.,
Hellemans, P., Loury, D.J., Jiao, J., Chauhan, V., Mannaert, E., 2015. The effect of
food on the pharmacokinetics of oral ibrutinib in healthy participants and patients
with chronic lymphocytic leukemia. Cancer Chemother. Pharmacol. 75, 907–916.
de Zwart, L., Snoeys, J., De Jong, J., Sukbuntherng, J., Mannaert, E., Monshouwer, M.,
2016. Ibrutinib dosing strategies based on interaction potential of CYP3A4
perpetrators using physiologically based pharmacokinetic modeling. Clin.
Pharmacol. Ther. 100, 548–557.
Fang, J.L., Beland, F.A., Doerge, D.R., Wiener, D., Guillemette, C., Marques, M.M.,
Lazarus, P., 2002. Characterization of benzo(a)pyrene-trans-7,8-dihydrodiol
glucuronidation by human tissue microsomes and overexpressed UDP-
glucuronosyltransferase enzymes. Cancer Res. 62, 1978–1986.
Green, M.D., King, C.D., Mojarrabi, B., Mackenzie, P.I., Tephly, T.R., 1998.
Glucuronidation of amines and other xenobiotics catalyzed by expressed human
UDP-glucuronosyltransferase 1A3. Drug Metab. Dispos. 26, 507–512.
Haupt, L.J., Kazmi, F., Ogilvie, B.W., Buckley, D.B., Smith, B.D., Leatherman, S., Paris, B.,
Parkinson, O., Parkinson, A., 2015. The reliability of estimating Ki values for direct,
reversible inhibition of cytochrome P450 enzymes from corresponding IC50 values:
a retrospective analysis of 343 experiments. Drug Metab. Dispos. 43, 1744–1750.
Ichikawa, W., Araki, K., Fujita, K., Yamamoto, W., Endo, H., Nagashima, F., Tanaka, R.,
Miya, T., Kodama, K., Sunakawa, Y., Narabayashi, M., Ando, Y., Akiyama, Y.,
X. Wang et al.
Kawara, K., Sasaki, Y., 2008. Re: UGT1A1*28 genotype and irinotecan-induced
neutropenia: dose matters. J. Natl. Cancer Inst. 100, 224–225 (author reply 225).
Ito, K., Brown, H.S., Houston, J.B., 2004. Database analyses for the prediction of in vivo
drug-drug interactions from in vitro data. Br. J. Clin. Pharmacol. 57, 473–486.
Iwase, M., Fujita, K.I., Nishimura, Y., Seba, N., Masuo, Y., Ishida, H., Kato, Y., Kiuchi, Y.,
2019. Pazopanib interacts with irinotecan by inhibiting UGT1A1-mediated
glucuronidation, but not OATP1B1-mediated hepatic uptake, of an active metabolite
SN-38. Cancer Chemother. Pharmacol. 83, 993–998.
Izukawa, T., Nakajima, M., Fujiwara, R., Yamanaka, H., Fukami, T., Takamiya, M.,
Aoki, Y., Ikushiro, S., Sakaki, T., Yokoi, T., 2009. Quantitative analysis of UDP-
glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers.
Drug Metab. Dispos. 37, 1759–1768.
Korprasertthaworn, P., Chau, N., Nair, P.C., Rowland, A., Miners, J.O., 2019. Inhibition
of human UDP-glucuronosyltransferase (UGT) enzymes by kinase inhibitors: effects
of dabrafenib, ibrutinib, nintedanib, trametinib and BIBF 1202. Biochem.
Pharmacol. 169, 113616.
Kuehl, G.E., Lampe, J.W., Potter, J.D., Bigler, J., 2005. Glucuronidation of nonsteroidal
anti-inflammatory drugs: identifying the enzymes responsible in human liver
microsomes. Drug Metab. Dispos. 33, 1027–1035.
Lepine, J., Bernard, O., Plante, M., Tetu, B., Pelletier, G., Labrie, F., Belanger, A.,
Guillemette, C., 2004. Specificity and regioselectivity of the conjugation of estradiol,
estrone, and their catecholestrogen and methoxyestrogen metabolites by human
uridine diphospho-glucuronosyltransferases expressed in endometrium. J. Clin.
Endocrinol. Metab. 89, 5222–5232.
Li, W., Xing, Y., Liu, Y., 2015. Inhibition of SN-38 glucuronidation by gefitinib and its
metabolite. Cancer Chemother. Pharmacol. 75, 1253–1260.
Lu, P.H., Chen, M.B., Wu, X.Y., Gu, J.H., Liu, Y., Gu, R.M., 2011. Genetic polymorphisms
of UGT1A7 and cancer risk: evidence from 21 case-control studies. Cancer Investig.
29, 645–654.
Markham, A., Dhillon, S., 2018. Acalabrutinib: first global approval. Drugs 78, 139–145.
Marostica, E., Sukbuntherng, J., Loury, D., de Jong, J., de Trixhe, X.W., Vermeulen, A.,
De Nicolao, G., O’Brien, S., Byrd, J.C., Advani, R., McGreivy, J., Poggesi, I., 2015.
Population pharmacokinetic model of ibrutinib, a Bruton tyrosine kinase inhibitor,
in patients with B cell malignancies. Cancer Chemother. Pharmacol. 75, 111–121.
Meech, R., Hu, D.G., McKinnon, R.A., Mubarokah, S.N., Haines, A.Z., Nair, P.C.,
Rowland, A., Mackenzie, P.I., 2019. The UDP-glycosyltransferase (UGT) superfamily:
new members, new functions, and novel paradigms. Physiol. Rev. 99, 1153–1222.
Miners, J.O., Mackenzie, P.I., Knights, K.M., 2010. The prediction of drug-
glucuronidation parameters in humans: UDP-glucuronosyltransferase enzyme-
selective substrate and inhibitor probes for reaction phenotyping and in vitro-in vivo
extrapolation of drug clearance and drug-drug interaction potential. Drug Metab.
Rev. 42, 196–208.
Miners, J.O., Chau, N., Rowland, A., Burns, K., McKinnon, R.A., Mackenzie, P.I.,
Tucker, G.T., Knights, K.M., Kichenadasse, G., 2017. Inhibition of human UDP-
glucuronosyltransferase enzymes by lapatinib, pazopanib, regorafenib and sorafenib:
implications for hyperbilirubinemia. Biochem. Pharmacol. 129, 85–95.
Nandikolla, A.G., Derman, O., Nautsch, D., Liu, Q., Massoumi, H., Venugopal, S.,
Braunschweig, I., Janakiram, M., 2017. Ibrutinib-induced severe liver injury. Clin.
Case Rep. 5, 735–738.
Oda, S., Fujiwara, R., Kutsuno, Y., Fukami, T., Itoh, T., Yokoi, T., Nakajima, M., 2015.
Targeted screen for human UDP-glucuronosyltransferases inhibitors and the
evaluation of potential drug-drug interactions with zafirlukast. Drug Metab. Dispos.
43, 812–818.
Pharmacyclics Inc, 2013. Imbruvica™ (ibrutinib) Capsules for Oral Use; US Prescribing
Information. http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/20
5552s000lbl.pdf. (Accessed 10 December 2013).
Qosa, H., Avaritt, B.R., Hartman, N.R., Volpe, D.A., 2018. In vitro UGT1A1 inhibition by
tyrosine kinase inhibitors and association with drug-induced hyperbilirubinemia.
Cancer Chemother. Pharmacol. 82, 795–802.
Riedmaier, S., Klein, K., Hofmann, U., Keskitalo, J.E., Neuvonen, P.J., Schwab, M.,
Niemi, M., Zanger, U.M., 2010. UDP-glucuronosyltransferase (UGT) polymorphisms
Toxicology and Applied Pharmacology 424 (2021) 115595
affect atorvastatin lactonization in vitro and in vivo. Clin. Pharmacol. Ther. 87,
65–73.
Rostami-Hodjegan, A., Tucker, G.T., 2007. Simulation and prediction of in vivo drug
metabolism in human populations from in vitro data. Nat. Rev. Drug Discov. 6,
140–148.
Sakaguchi, K., Green, M., Stock, N., Reger, T.S., Zunic, J., King, C., 2004.
Glucuronidation of carboxylic acid containing compounds by UDP-
glucuronosyltransferase isoforms. Arch. Biochem. Biophys. 424, 219–225.
Seo, K.A., Kim, H.J., Jeong, E.S., Abdalla, N., Choi, C.S., Kim, D.H., Shin, J.G., 2014. In
vitro assay of six UDP-glucuronosyltransferase isoforms in human liver microsomes,
using cocktails of probe substrates and liquid chromatography-tandem mass
spectrometry. Drug Metab. Dispos. 42, 1803–1810.
Sibaud, V., Beylot-Barry, M., Protin, C., Vigarios, E., Recher, C., Ysebaert, L., 2020.
Dermatological toxicities of Bruton’s tyrosine kinase inhibitors. Am. J. Clin.
Dermatol. 21, 799–812.
Strassburg, C.P., Oldhafer, K., Manns, M.P., Tukey, R.H., 1997. Differential expression of
the UGT1A locus in human liver, biliary, and gastric tissue: identification of UGT1A7
and UGT1A10 transcripts in extrahepatic tissue. Mol. Pharmacol. 52, 212–220.
Tafesh, Z.H., Coleman, M., Fulmer, C., Nagler, J., 2019. Severe hepatotoxicity due to
ibrutinib with a review of published cases. Case Rep. Gastroenterol. 13, 357–363.
Tornio, A., Filppula, A.M., Niemi, M., Backman, J.T., 2019. Clinical studies on drug-drug
interactions involving metabolism and transport: methodology, pitfalls, and
interpretation. Clin. Pharmacol. Ther. 105, 1345–1361.
Trottier, J., Verreault, M., Grepper, S., Monte, D., Belanger, J., Kaeding, J., Caron, P.,
Inaba, T.T., Barbier, O., 2006. Human UDP-glucuronosyltransferase (UGT)1A3
enzyme conjugates chenodeoxycholic acid in the liver. Hepatology 44, 1158–1170.
Uchaipichat, V., Mackenzie, P.I., Elliot, D.J., Miners, J.O., 2006. Selectivity of substrate
(trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid,
hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for
human udp-glucuronosyltransferases. Drug Metab. Dispos. 34, 449–456.
USFDA, 2020. Guidance for Industry: In Vitro Drug Interaction Studies—Cytochrome
P450 Enzyme-and Transporter-Mediated Drug Interactions (Draft Guidance). Center
for Drug Evaluation and Research. FDA, Silver Spring, MD.
van der Mey, D., Gerisch, M., Jungmann, N.A., Kaiser, A., Yoshikawa, K., Schulz, S.,
Radtke, M., Lentini, S., 2021. Drug-drug interaction of atazanavir on UGT1A1-
mediated glucuronidation of molidustat in human. Basic Clin. Pharmacol. Toxicol.
128, 511–524.
Wang, Z., Wang, X., Jia, Y., Yin, H., Feng, Y., Jiang, L., Cao, J., Liu, Y., 2020. Inhibition of
human UDP-glucuronosyltransferase enzymes by midostaurin and ruxolitinib:
implications for drug-drug interactions. Biopharm. Drug Dispos. 41, 231–238.
Wen, Z., Tallman, M.N., Ali, S.Y., Smith, P.C., 2007. UDP-glucuronosyltransferase 1A1 is
the principal enzyme responsible for etoposide glucuronidation in human liver and
intestinal microsomes: structural characterization of phenolic and alcoholic
glucuronides of etoposide and estimation of enzyme kinetics. Drug Metab. Dispos.
35, 371–380.
Yu, H., Tweedie, D., 2013. A perspective on the contribution of metabolites to drug-drug
interaction potential: the need to consider both circulating levels and inhibition
potency. Drug Metab. Dispos. 41, 536–540.
Yu, J., Petrie, I.D., Levy, R.H., Ragueneau-Majlessi, I., 2019. Mechanisms and clinical
significance of pharmacokinetic-based drug-drug interactions with drugs approved
by the U.S. Food and Drug Administration in 2017. Drug Metab. Dispos. 47,
135–144.
Zheng, Z., Park, J.Y., Guillemette, C., Schantz, S.P., Lazarus, P., 2001. Tobacco
carcinogen-detoxifying enzyme UGT1A7 and its association with orolaryngeal
cancer risk. J. Natl. Cancer Inst. 93, 1411–1418.
Zhou, X., Xie, Y., Qi, Q., Cheng, X., Liu, F., Liao, K., Wang, G., Hao, H., 2013. Disturbance
of hepatic and intestinal UDP-glucuronosyltransferase in rats with trinitrobenzene
sulfonic acid-induced colitis. Drug Metab. Pharmacokinet. 28, 305–313.
Zhou, D., Podoll, T., Xu, Y., Moorthy, G., Vishwanathan, K., Ware, J., Slatter, J.G., Al-
Huniti, N., 2019. Evaluation of the drug-drug interaction potential of acalabrutinib
and its active metabolite, ACP-5862, using a physiologically-based pharmacokinetic
modeling approach. CPT Pharmacometrics Syst. Pharmacol. 8, 489–499.