In Vitro Assessment of Potential for CYP-Inhibition-Based Drug–Drug Interaction Between Vonoprazan and Clopidogrel

Article abstract of DOI:10.1007/s13318-018-0521-7

Background and Objectives: It was recently proposed that CYP-mediated drug–drug interactions (DDIs) of vonoprazan with clopidogrel and prasugrel can attenuate the antiplatelet actions of the latter two drugs. Clopidogrel is metabolized to the pharmacologically active metabolite H4 and its isomers by multiple CYPs, including CYP2C19 and CYP3A4. Therefore, to investigate the possibility of CYP-based DDIs, in vitro metabolic inhibition studies using CYP probe substrates or radiolabeled clopidogrel and human liver microsomes (HLMs) were conducted in this work. Methods: Reversible inhibition studies focusing on the effects of vonoprazan on CYP marker activities and the formation of the [¹⁴C]clopidogrel metabolite H4 were conducted with and without pre-incubation using HLMs. Time-dependent inhibition (TDI) kinetics were also measured. Results: It was found that vonoprazan is not a significant direct inhibitor of any CYP isoforms (IC₅₀ ≥ 16?μM), but shows the potential for TDI of CYP2B6, CYP2C19, and CYP3A4/5. This TDI was weaker than the inhibition induced by the corresponding reference inhibitors ticlopidine, esomeprazole, and verapamil, based on the measured potencies (k_inact/K_I ratio and the R2 value). In a more direct in vitro experiment, vonoprazan levels of up to 10?μM (a 100-fold higher concentration than the plasma C_max of 75.9?nM after taking 20?mg once daily for 7?days) did not suppress the formation of the active metabolite H4 or other oxidative metabolites of [¹⁴C]clopidogrel in a reversible or time-dependent manner. Additionally, an assessment of clinical trials and post-marketing data suggested no evidence of a DDI between vonoprazan and clopidogrel. Conclusions: The body of evidence shows that the pharmacodynamic DDI reported between vonoprazan and clopidogrel is unlikely to be caused by the inhibition of CYP2B6, CYP2C19, or CYP3A4/5 by vonoprazan.

Full text of DOI:10.1007/s13318-018-0521-7

European Journal of Drug Metabolism and Pharmacokinetics
https://doi.org/10.1007/s13318-018-0521-7
ORIGINAL RESEARCH ARTICLE
In Vitro Assessment of Potential for CYP‑Inhibition‑Based Drug–Drug
Interaction Between Vonoprazan and Clopidogrel
Mitsuhiro Nishihara1 · Hitomi Yamasaki2 · Richard Czerniak3 · Helen Jenkins4
© Springer Nature Switzerland AG 2018
Abstract
Background and Objectives It was recently proposed that CYP-mediated drug–drug interactions (DDIs) of vonoprazan with
clopidogrel and prasugrel can attenuate the antiplatelet actions of the latter two drugs. Clopidogrel is metabolized to the
pharmacologically active metabolite H4 and its isomers by multiple CYPs, including CYP2C19 and CYP3A4. Therefore,
to investigate the possibility of CYP-based DDIs, in vitro metabolic inhibition studies using CYP probe substrates or radi-
olabeled clopidogrel and human liver microsomes (HLMs) were conducted in this work.
Methods Reversible inhibition studies focusing on the efects of vonoprazan on CYP marker activities and the formation of
the [14C]clopidogrel metabolite H4 were conducted with and without pre-incubation using HLMs. Time-dependent inhibi-
tion (TDI) kinetics were also measured.
Results It was found that vonoprazan is not a signifcant direct inhibitor of any CYP isoforms (IC50 ≥16 μM), but shows the
potential for TDI of CYP2B6, CYP2C19, and CYP3A4/5. This TDI was weaker than the inhibition induced by the corre-
sponding reference inhibitors ticlopidine, esomeprazole, and verapamil, based on the measured potencies (kinact/KI ratio and
the R2 value). In a more direct in vitro experiment, vonoprazan levels of up to 10 µM (a 100-fold higher concentration than
the plasma Cmax of 75.9 nM after taking 20 mg once daily for 7 days) did not suppress the formation of the active metabolite
H4 or other oxidative metabolites of [14C]clopidogrel in a reversible or time-dependent manner. Additionally, an assessment
of clinical trials and post-marketing data suggested no evidence of a DDI between vonoprazan and clopidogrel.
Conclusions The body of evidence shows that the pharmacodynamic DDI reported between vonoprazan and clopidogrel is
unlikely to be caused by the inhibition of CYP2B6, CYP2C19, or CYP3A4/5 by vonoprazan.
Key Points
Vonoprazan is a potassium-competitive acid blocker
drug approved in Japan for multiple indications.
It causes weak time-dependent inhibition of CYP2B6,
CYP2C19, and CYP3A4/5, but it is not expected to cause
CYP-based interactions at clinical doses and concentrations.
Vonoprazan did not exhibit signifcant inhibitory
efects on the formation of the active metabolite of
[14C]clopidogrel, as IC50 >10 μM, which is>100× the
* Mitsuhiro Nishihara
mitsuhiro.nishihara@takeda.com
plasma Cmax of vonoprazan in a clinical setting. Thus,
1
the recently reported pharmacodynamic interaction
between vonoprazan and clopidogrel is not likely to be
CYP mediated, and the reason for the pharmacodynamic
interaction is unknown at this time.
Global Drug Metabolism and Pharmacokinetics, Takeda
Pharmaceuticals International Co., 35 Landsdowne Street,
Cambridge, MA 02139, USA
2
3
4
Drug Disposition and Analysis, Research Division, Axcelead
Drug Discovery Partners, Inc., Fujisawa, Kanagawa, Japan
An assessment of clinical trials and post-marketing
data suggested no evidence of drug–drug interactions
between vonoprazan and clopidogrel.
Quantitative Clinical Pharmacology, Takeda Pharmaceuticals
International Co., Cambridge, MA, USA
d3 Medicine, A Certara Company, London, UK
Vol.:(0123456789)

M. Nishihara et al.
The objective of the present study was to investigate the
potential for DDI between vonoprazan and clopidogrel from
the point of view of their metabolism. The ability of vono-
prazan to inhibit the major CYP enzymes (namely CYP1A2,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and
CYP3A4/5) in vitro in a direct (reversible) or time-depend-
ent (irreversible) manner was investigated using pooled
human liver microsomes (HLMs) with or without pre-incu-
bation to determine the potential of vonoprazan to cause
DDIs with other concomitantly administered CYP-substrate
drugs. In a more direct experiment, the inhibitory efect of
vonoprazan on the in vitro metabolism of [14C]clopidogrel
in HLMs was investigated. A partial summary of these stud-
ies was communicated recently in a Letter to the Editor in
Clinical Pharmacology and Therapeutics [12].
1 Introduction
Vonoprazan fumarate (“vonoprazan”), 1-[5-(2-fuorophenyl)-
1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethan-
amine monofumarate, is an orally active potassium-com-
petitive acid blocker (P-CAB) that has been approved in
Japan for the treatment of gastroesophageal refux disease,
peptic ulcer, gastric ulcer, erosive esophagitis, and refux
esophagitis, and for use as an adjunct in Helicobacter pylori
eradication [1, 2]. Nonclinical studies have established that
vonoprazan has a rapid, potent, stable, and long-lasting acid-
inhibitory efect resulting from the reversible inhibition of
the gastric proton pump via competition with K+ binding on
the luminal surface of H+,K+-ATPase [3, 4]. These fndings
were also confrmed clinically, as vonoprazan caused rapid
and profound suppression of gastric acid secretion in healthy
male subjects over a 24-h period [5, 6].
2 Materials and Methods
The biotransformation of vonoprazan has been investi-
gated in nonclinical [7, 8] and clinical studies [9], and four
major metabolites, M-I, M-II, M-III, and M-IV-Sul, have
been identifed. None show pharmacological activity. Pre-
vious in vitro studies indicated that multiple metabolizing
enzymes, including cytochrome P450 (CYP) 3A4, CYP2B6,
CYP2C19, CYP2D6, and a non-CYP enzyme, sulfotrans-
ferase (SULT) 2A1, are involved in the primary metabolism
of vonoprazan. CYP3A4 predominantly contributes to the
metabolism of vonoprazan to the metabolites M-I, M-III, and
N-demethylated vonoprazan (a presumed metabolite), while
SULT2A1 transforms vonoprazan to vonoprazan N-sulfate,
and CYP2C9 mediates the formation of M-IV-Sul from
vonoprazan N-sulfate [8]. The contribution of CYP2C19 to
the metabolism of vonoprazan was found to be small, so the
infuence of CYP2C19 genotype status on the pharmacoki-
netics of vonoprazan is considered to be minimal [10]. The
involvement of multiple metabolic pathways could also help
minimize the efects of coadministered CYP inhibitors or
inducers on the pharmacokinetics of vonoprazan.
2.1 Chemicals
Vonoprazan fumarate (vonoprazan) was synthesized by
Takeda Pharmaceutical Company Limited (Tokyo, Japan).
Phenacetin, acetaminophen, α-naphthoflavone, furafyl-
line, ticlopidine hydrochloride, diclofenac sodium salt,
4′-hydroxydiclofenac, tranylcypromine hydrochloride,
1′-hydroxybufuralol maleate, quinidine, 1′-hydroxymida-
zolam, testosterone, 6β-hydroxytestosterone, verapamil
hydrochloride, and esomeprazole magnesium hydrate
were obtained from Merck KGaA (Darmstadt, Germany).
Bupropion hydrochloride, hydroxybupropion, gemfbrozil
1-O-β-glucuronide, (S)-mephenytoin, 4′-hydroxymephe-
nytoin, bufuralol hydrochloride, paroxetine hydrochloride,
2-oxo-clopidogrel hydrochloride (mixture of diastereom-
ers), and clopidogrel carboxylic acid hydrochloride were
from Toronto Research Chemicals Inc. (North York, ON,
USA). Paclitaxel, midazolam, and clopidogrel sulfate were
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
6α-Hydroxypaclitaxel was from Corning (Corning, NY,
USA). Montelukast sodium was from LKT Laboratories
(St. Paul, MN, USA). Sulfaphenazole was from Santa Cruz
Biotechnology (Dallas, TX, USA). Tienilic acid was from
Cayman Chemical (Ann Arbor, MI, USA). Ketoconazole
was from Tokyo Chemical Industry (Tokyo, Japan). [14C]
clopidogrel was from Moravek, Inc. (Brea, CA, USA).
The other reagents used in the study were commercially
available in analytical grade.
An assessment of clinical trials and post-marketing data
suggested that, as yet, there is no evidence of a drug–drug
interaction (DDI) between vonoprazan and clopidogrel or
prasugrel (data on fle). However, in a recent article, Kagami
et al. reported that vonoprazan was an inhibitor of not only
CYP3A4 but also CYP2C19, and suggested that a CYP-
mediated DDI between vonoprazan and clopidogrel or pras-
ugrel attenuated their antiplatelet function [11]. However,
no evidence of any pharmacokinetic changes was provided,
and the concentrations of clopidogrel, prasugrel, and their
active thiol metabolites (H4 and R-138727 [(2Z)-{1-[(1RS)-
2-cyclopropyl-1-(2-fuorophenyl)-2-oxoethyl]-4-sulfanyl-
piperidin-3-ylidene}ethanoic acid], respectively) were not
measured to obtain direct evidence of metabolism/PK-based
DDI in that study.
2.2 Biological Materials
The pooled human liver microsomes (HLMs) from 50
donors of mixed gender (lot nos. 1210153 and 1410013)

Potential for CYP-Based Drug–Drug Interaction Between Vonoprazan and Clopidogrel
were commercially available from Sekisui Xenotech, LLC
(Kansas City, KS, USA).
2.4 Time‑Dependent Inhibition Kinetics for CYP2B6,
CYP2C19, and CYP3A4/5
2.3 CYP Inhibition Study With or Without
Pre‑incubation
The time-dependent inhibition study was conducted by the
dilution method to determine the kinetic parameters, the
maximum inactivation rate constant (kinact), and the inacti-
vator concentration for half the inactivation rate kinact (KI) for
CYP2B6, CYP2C19, and CYP3A4/5 [13]. The frst incuba-
tion mixtures consisted of 0.1 M Na2HPO4–KH2PO4 phos-
phate bufer (pH 7.4), 0.2 mg protein/mL HLMs, 0.05 mM
EDTA, the NADPH-generating system, and various con-
centrations of vonoprazan or a positive control substance
(ticlopidine for CYP2B6, esomeprazole for CYP2C19, or
verapamil for CYP3A4/5) in a fnal volume of 1000 μL.
After a pre-warming period of 5 min at 37 °C, the reaction
was initiated by adding the NADPH-generating system and
conducted for 2, 5, 10, 15, or 30 min (pre-incubation step).
After each pre-incubation time, a 100-μL aliquot was
taken from the frst incubation mixture and added to the sec-
ond incubation mixture containing 0.1 M Na2HPO4–KH2PO4
phosphate bufer (pH7.4), and the NADPH-generating sys-
tem, which was pre-warmed for 5 min, and then the substrate
was added immediately to initiate the reaction. The fnal
volume was 1000 μL. After incubation at 37 °C for 20 min
(CYP2B6), 30 min (CYP2C19), or 5 min (CYP3A4/5), the
reaction was terminated by adding 1 mL of acetonitrile.
The concentrations and identities of the marker substrates
and positive control inhibitors as well as the incubation
times for each CYP reaction are shown in Table 1.
For the direct inhibition study, the standard incubation
mixtures consisted of 0.1 M Na2HPO4–KH2PO4 phosphate
bufer (pH 7.4), 0.2 mg protein/mL HLMs, 0.05 mM EDTA,
the NADPH-generating system (5 mM MgCl2, 5 mM glu-
cose 6-phosphate, 0.5 mM β-NADP+, and 1.0 unit/mL glu-
cose-6-phosphate dehydrogenase), and a marker substrate
in the presence or absence of inhibitor (vonoprazan or a
positive control substance) in a fnal volume of 1000 μL. The
concentrations of vonoprazan in the incubation mixture were
set at 1, 3, 10, or 30 μM based on the maximum solubility in
this reaction solution. After a pre-warming period of 5 min
at 37 °C, the reaction was initiated by adding the NADPH-
generating system and conducted at 37 °C. The reaction was
terminated by adding 1 mL of acetonitrile.
For the time-dependent inhibition study, the incubation
mixtures were pre-warmed at 37 °C for 5 min without the
NADPH-generating system and the marker substrate. The
NADPH-generating system was then added, and the incuba-
tion mixtures were pre-incubated at 37 °C for 30 min. After
that, the reaction was initiated by adding the marker substrate
and conducted at 37 °C. The fnal concentrations of the incu-
bation mixtures and the incubation times were the same as in
the direct inhibition study. After the incubation, the reaction
was terminated by adding 1 mL of acetonitrile. All incuba-
tions in a series of in vitro studies including the following
studies were carried out in duplicate, and no signifcant dif-
ference between the duplicate samples was observed.
2.5 Inhibition of [14C]Clopidogrel Metabolism
by Vonoprazan
The incubation mixtures consisted of 50 mM
KH2PO4–K2HPO4 phosphate bufer (pH 7.4), 1 mg protein/
mL HLMs, 5 mM glutathione (GSH), the NADPH-gener-
ating system, and 10 μM [14C]clopidogrel in the presence
or absence of inhibitor (vonoprazan or esomeprazole) in a
Table 1 Summary of experimental conditions and analytical methods for direct and time-dependent CYP-inhibition assays
Enzyme
Substrate
Conc. (μM) Incuba- Direct inhibitor
Time-dependent
inhibitor (conc. in
μM)
Metabolite
Mass transition
tion
(conc. in μM)
monitored (m/z)
(min)
CYP1A2
Phenacetin
30
30
α-Naphthofavone
Furafylline (0.3)
Ticlopidine (0.1)
Acetaminophen
152→110
(0.2)
CYP2B6
CYP2C8
Bupropion
Paclitaxel
100
10
30
20
Ticlopidine (1)
Montelukast (1)
Hydroxybupropion
256→167
871→525
Gemfbrozil 1-O-β- 6α-Hydroxypaclitaxel
glucuronide (10)
CYP2C9
CYP2C19 (S)-Mephenytoin 20
Diclofenac
5
5
30
Sulfaphenazole (5)
Tienilic acid (1)
S-fuoxetine (10)
4′-Hydroxydiclofenac
4′-Hydroxymephenytoin 235→150
312→231
Tranylcypromine
(25)
CYP2D6
CYP3A4/5 Midazolam
Testosterone
Bufuralol
10
5
50
20
5
10
Quinidine (1)
Ketoconazole (0.5)
Paroxetine (0.3)
Verapamil (10)
1′-Hydroxybufuralol
1′-Hydroxymidazolam
6β-Hydroxytestosterone 305→269
278→186
342→324
conc. concentration

M. Nishihara et al.
fnal volume of 300 μL. After pre-incubating for 0 min or
30 min at 37 °C, the reaction was initiated by adding [14C]
clopidogrel solution and conducted for 30 min at 37 °C.
The reaction was terminated by adding 150 μL of acetoni-
trile. Each analytical sample was centrifuged at 1500×g for
10 min at 4 °C to obtain the supernatant, which was used as
the HPLC sample.
2.7 Data Analysis
All calculations were performed with Microsoft Excel
2003 or 2010 (Microsoft Corp., Redmond, WA, USA)
except for the calculations described below. IC50, KI, and
kinact values were determined by nonlinear least-squares
regression analysis using WNL5 Classic Modeling (phar-
macodynamic model 103—simple Emax inhibitory efect
model or pharmacodynamic model 102—simple Emax
model) along with Phoenix WinNonlin 6.2 or 6.3 (Cer-
tara LP, Princeton, NJ, USA) using the following equation:
2.6 Analytical Procedures
For the CYP inhibition and kinetic studies, a 100-μL ali-
quot of methanol/ultrapure water (1:1, v/v) and a 100-μL
aliquot of each internal standard solution were added to the
reaction mixture after adding the acetonitrile. Deuterium-
labeled metabolites were used for internal standards. Each
analytical sample was centrifuged at 1600×g for 10 min at
4 °C to obtain the supernatant. Undiluted supernatant or
supernatant diluted with ultrapure water (analytical samples)
were analyzed using validated high-performance liquid chro-
matography (HPLC)–tandem mass spectrometry (LC-MS/
MS) methods.
k
inact × [I]
kobs = kobs [I = 0]
+
,
(1)
KI + [I]
where kobs [I=0] is the apparent inactivation rate constant
(kobs) at 0 μM of inhibitor and [I] is the mean concentration
of the inhibitor.
In the inhibition study using [14C]clopidogrel with
HLMs, “diastereomers of 2-oxo-clopidogrel” and “H4 and
its isomers” were evaluated as individual groups due to the
difculties involved with separating the diastereomer peaks
and the isomer peaks in the radiochromatogram. The relative
activity was calculated as the formation ratios of “diastere-
omers of 2-oxo-clopidogrel” and “H4 and its isomers” in the
sample with the inhibitor when that in the control sample
with 0 and 30 minutes pre-incubation is regarded as 100%.
The samples were injected into a Quattro micro tandem
mass spectrometer (Waters Corp., Milford, MA, USA)
equipped with an Alliance 2695 or 2795 liquid chromato-
graph (Waters Corp.), as well as other equivalent LC–MS/
MS systems that use validated analytical methods. A Sym-
metry C18 column (2.1 × 100 mm; 3.5 μm, Waters Corp.)
with a column temperature of 40 °C was used for chroma-
tographic separation, and elution was carried out at a fow
rate of 0.25 mL/min using a linear gradient for the mobile
phase, which consisted of a mixture of A (acetic acid/ace-
tonitrile, 1:1000, v/v) and B (acetic acid/ultrapure water,
1:1000, v/v). The eluate was introduced into the MS/MS
system in the electrospray, positive-ion mode. Transitions
shown in Table 1 were used in the selected reaction-mon-
itoring method to quantify the specifc metabolites.
[14C]clopidogrel and its metabolites in the in vitro incu-
bation mixture were analyzed by HPLC (Shimadzu Corp.,
Kyoto, Japan) with an on-line fow scintillation analyzer
(PerkinElmer Inc., Waltham, MA, USA). The HPLC condi-
tions were as follows. The column was a CAPCELL PAK
C18 MGII (5 μm, 250 × 4.6 mm I.D.; Shiseido Co., Ltd.,
Tokyo, Japan). The column temperature and the fow rate
were 40 °C and 1.0 mL/min, respectively. Mobile phase
A was 10 mM ammonium formate/acetonitrile/formic
acid (900:100:2, v/v), and mobile phase B was 10 mM
ammonium formate/acetonitrile/formic acid (100:900:2,
v/v/v). The time program for the gradient elution was as
follows: the concentration of mobile phase (B) was linearly
increased from 15 to 100% over a period of 20 min, then
held at 100% for 5 min, and then cycled back to the initial
conditions.
3 Results
3.1 Direct Inhibition of CYP Activities
by Vonoprazan
The reversible inhibitory effects of vonoprazan on the
marker activities of seven CYP isoforms are presented in
Table 2. Vonoprazan reversibly inhibited the activities of
CYP2B6 and CYP3A4/5 (midazolam 1′-hydroxylation) with
IC50 values of 16 μM and 29 μM, respectively. At 30 μM, it
also suppressed the activities of CYP2C19, CYP2D6, and
CYP3A4/5 (testosterone 6β-hydroxylation) to 64.3%, 61.3%,
and 61.3%, respectively, of the control activities; IC50 val-
ues were > 30 μM. Vonoprazan did not substantially inhibit
other CYP activities at concentrations of up to 30 μM.
3.2 Time‑Dependent Inhibition of CYP Activities
by Vonoprazan
The time-dependent inhibitory efects of vonoprazan on
the marker activities of the seven CYP isoforms are also
presented in Table 2. In the study with pre-incubation,
vonoprazan inhibited the activities of CYP2B6, CYP2C19,
CYP3A4/5 (midazolam 1′-hydroxylation), and CYP3A4/5
(testosterone 6β-hydroxylation), with IC50 values of

Potential for CYP-Based Drug–Drug Interaction Between Vonoprazan and Clopidogrel
Table 2 IC50 values of vonoprazan for marker enzyme activities in
k
inact × 50 × I
, u
, u
max
human liver microsomes
kobs
=
(4)
KI + 50 × I
max
Enzyme
Marker reaction
IC50 (μM)
Without
With pre-
I
, u = C
× fu,p(unbound plasma protein binding ratio)
max
max
pre-incuba- incubation
tion
(5)
where KI is IC50/2 and the nonspecifc binding to micro-
somes is 1.0 when calculated using a physicochemical prop-
erty, kobs is the apparent inactivation rate constant of CYP as
calculated using Eq. 4, and the values of kdeg for CYP2B6,
CYP2C19, and hepatic CYP3A4/5 are 0.00036 min−1,
0.00045 min−1, and 0.00032 min−1, respectively [19, 20].
Imax,u is the unbound inhibitor concentration in plasma as
calculated using Eq. 5. The value of Cmax was 0.0759 μM for
vonoprazan 20 mg after 7 days of repeat dosing [5], 2.74 μM
for ticlopidine 250 mg after a single dose [21], 2.65 μM for
esomeprazole 20 mg after 5 days of repeat dosing [22], and
0.139 μM for verapamil 80 mg after a single dose [23],
while the value of fu,p was 0.135 for vonoprazan [7], 0.02
for ticlopidine [24], 0.03 for esomeprazole [14], and 0.069
for verapamil [25].
CYP1A2
CYP2B6
CYP2C8
CYP2C9
Phenacetin O-deethylation
Bupropion hydroxylation
Paclitaxel 6α-hydroxylation
Diclofenac 4′-hydroxylation
> 30
16
> 30
> 30
> 30
2.6
> 30
> 30
13
CYP2C19 (S)-Mephenytoin 4′-hydroxy- > 30
lation
CYP2D6
Bufuralol 1′-hydroxylation
> 30
29
> 30
10
9.8
CYP3A4/5 Midazolam 1′-hydroxylation
CYP3A4/5 Testosterone 6β-hydroxylation > 30
The inhibitory efects of vonoprazan were investigated with or with-
out pre-incubation for 30 min in the presence of the NADPH-gener-
ating system. Each value represents the mean of duplicate determi-
nations. IC50 half-maximal inhibitory concentration, CYP cytochrome
P450
Since the IC50 values for the direct inhibition of CYP2B6
and CYP3A4/5 were 16 and 29 μM, respectively, the calcu-
lated R1 values were less than the guidance criteria (1.02)
for these CYPs. The R2 values are also shown in Table 3.
2.6 μM, 13 μM, 10 μM, and 9.8 μM, respectively. Vono-
prazan did not show substantial inhibition of other CYP
activities at concentrations of up to 30 μM. Due to the
large shifts in IC50 for vonoprazan with respect to the
activities of CYP2B6, CYP2C19, and CYP3A4/5 upon
pre-incubation, the TDI kinetics were assessed (Fig. 1).
The TDI parameters (kinact, KI, kinact/KI) for vonoprazan
and the positive controls ticlopidine, esomeprazole, and
verapamil—the known time-dependent inactivators of
CYP2B6, CYP2C19, and CYP3A4/5, respectively [14,
15]—are shown in Table 3.
3.4 Inhibitory Efects of Vonoprazan
and Esomeprazole on the Metabolism of [14C]
Clopidogrel in Human Liver Microsomes
The inhibitory efects of vonoprazan and esomeprazole on
the metabolism of [14C]clopidogrel in vitro were exam-
ined at concentrations of 0.03, 0.1, 0.3, 1, 3, and 10 μM
(vonoprazan) or 1, 3, 10, and 30 μM (esomeprazole) with
or without pre-incubation for 30 min with HLMs in the
presence of the NADPH-generating system, and the results
are shown in Table 4. The experimental samples were ana-
lyzed promptly after being prepared. For all samples, the
recoveries with the pretreatment were 100 10%, and no
signifcant diference was observed between the duplicate
samples and during a series of sequential analyses.
3.3 Evaluation of the Drug–Drug Interaction Based
on Guidance and Guidelines
Furthermore, to evaluate the degree of drug–drug interac-
tion, R1 and R2 values (the predicted ratio of the inhibited
drug’s area under the curve (AUC) in the presence and
absence of an inhibitor for basic models of direct inhi-
bition and TDI, respectively) were calculated as shown
below (these equations are also used in the U.S. Food and
Administration (FDA) draft guidance [16], the European
Medicines Agency (EMA) guideline [17], and the Phar-
maceuticals and Medical Devices Agency (PMDA) draft
guideline [18]):
Clopidogrel was metabolized to a variety of metabo-
lites in the presence of the NADPH-generating system and
GSH. Seven of the metabolites were characterized by LC/
MS analysis (data not shown). The metabolites identifed
were clopidogrel carboxylic acid, two diastereomers of
2-oxo-clopidogrel, and the active metabolite H4 and its
three isomers (H1, H2, and H3) (data not shown). The
clopidogrel carboxylic acid was formed independently of
NADPH. The diastereomers of 2-oxo-clopidogrel as well
as H4 and its isomers could not be fully resolved in the
radiochromatogram. Therefore, “diastereomers of 2-oxo-
clopidogrel” and “H4 and its isomers” were evaluated as
I
, u
max
KI
R1 = 1 +
(2)
kobs + kdeg
R2 =
(3)
kdeg

M. Nishihara et al.
Fig. 1 Time-dependent
inhibition (TDI) kinetics for
the activities of CYP2B6,
CYP2C19, and CYP3A4/5
inhibited by vonoprazan in
human liver microsomes. The
left panels show the time- and
concentration-dependent inhibi-
tion of the activities of CYP2B6
(a), CYP2C19 (b), and
CYP3A4/5 (c) by vonoprazan
in HLMs, and the right panels
show the corresponding plots of
kobs (the apparent inactivation
rate constant of CYP) against
the vonoprazan concentration,
which were used to estimate the
TDI kinetic parameters. Each
value shown is the mean of
duplicate determinations
Table 3 Time-dependent
inhibitory parameters for
vonoprazan and the positive
controls in relation to the
activities of CYP2B6,
Inhibitor
kinact (min−1
)
KI (μM)
kinact/KI
R2
(min−1 × μM−1
)
CYP2B6
Vonoprazan
Ticlopidine
Vonoprazan
Esomeprazole
Vonoprazan
Verapamil
0.0115
0.127
0.0182
0.0547
0.0161
0.0441
3.50
0.201
3.67
2.58
1.22
1.60
0.00329
0.632
0.00496
0.0212
0.0132
0.0276
5.08
330
5.95
74.7
15.9
32.8
CYP2C19, and CYP3A4/5
CYP2C19
CYP3A4/5
kinact and KI are the maximum inactivation rate constant and the inactivator concentration for half kinact in
the kinetic analysis, respectively. Each value represents the mean of duplicate determinations. R2 is the pre-
dicted ratio of the inhibited drug’s area under the curve (AUC) in the presence and absence of an inhibitor
for basic models of enzyme TDI [16–18]

Potential for CYP-Based Drug–Drug Interaction Between Vonoprazan and Clopidogrel
Table 4 Inhibitory efects of vonoprazan and esomeprazole on the metabolism of [14C]clopidogrel in human liver microsomes in vitro
Conc. (μM) Without pre-incubation
With pre-incubation
Formation of diastereomers Formation of H4 and its Formation of diastereomers Formation of H4 and its
of 2-oxo-clopidogrel (% w.r.t. isomers (% w.r.t. control) of 2-oxo-clopidogrel (% w.r.t. isomers (% w.r.t. control)
control)
control)
Control
Vonoprazan
0
100.0
99.0
100.0
100.0
100.0
98.5
97.1
86.8
83.8
98.5
97.1
86.8
70.6
100.0
100.0
99.1
100.0
103.8
101.9
105.8
94.2
82.7
73.1
105.8
100.0
90.4
0.03
0.1
0.3
1
3
10
1
3
10
30
102.1
105.2
106.2
105.2
109.3
99.0
103.1
107.2
115.5
102.7
108.0
107.1
103.6
104.5
107.1
107.1
111.6
Esomeprazole
71.2
[14C]clopidogrel (10 μM) and the inhibitor were incubated with human liver microsomes (1 mg protein/mL) in the presence of the NADPH-gen-
erating system and GSH at 37 °C for 30 min with or without pre-incubation of the inhibitor and microsomes for 30 min. Each value represents
the mean of duplicate determinations. Diastereomers of 2-oxo-clopidogrel as well as H4 and its isomers are metabolites of clopidogrel. % w.r.t.
control is % with respect to the metabolite formation in the control sample
Conc. concentration
two individual groups for quantitation purposes. The for-
mation of each diastereomer of 2-oxo-clopidogrel as well
as H1, H2, H3, and H4 in the mass chromatogram did not
change in the presence of vonoprazan or esomeprazole.
In the study without pre-incubation, vonoprazan and
esomeprazole did not exhibit a signifcant inhibitory efect
on the formation of H4 and its isomers from [14C]clopi-
dogrel; IC50 values were greater than 10 μM and 30 μM,
the highest tested concentrations (these concentrations
suppressed activity to ≥83.8% and≥70.6% of the control
activity, respectively). Neither vonoprazan nor esomeprazole
showed substantial inhibition of the formation of diastere-
omers of 2-oxo-clopidogrel.
esomeprazole showed substantial inhibition of the formation
of diastereomers of 2-oxo-clopidogrel.
4 Discussion
Clopidogrel is a thienopyridine prodrug that is metabo-
lized into active thiols through two CYP-dependent steps.
In the frst step, it is converted into a sulfenic acid; it is
then reduced to the fnal thiol derivative (H4) [26, 27],
the pharmacologically active metabolite, which is the only
isomer responsible for antiplatelet activity in humans [28].
The major metabolic pathways of clopidogrel are shown
in Fig. 2 [27, 29, 30]. About 90% of the absorbed clopi-
dogrel is hydrolyzed by esterases into an inactive form,
clopidogrel carboxylic acid, and the remaining 10% is oxi-
dized initially by CYP1A2, CYP2B6, and CYP2C19 into
the intermediate metabolite 2-oxo-clopidogrel, and then to
the thiolactone sulfoxide by CYP2C19 and CYP3A4 and,
to a lesser extent, by CYP2B6 and CYP2C9. The active
metabolite, H4, is fnally formed in a reducing step [27,
31, 32]. In another study, CYP3A4 was found to be the
main CYP isoform involved in the formation of H4 from
2-oxo-clopidogrel (approximately 50% of the H4 is gen-
erated by CYP3A4) [33]. The inhibitory efects of vono-
prazan on these major CYP isoforms were investigated in
this study.
In the study with pre-incubation, vonoprazan or esome-
prazole again did not exhibit a signifcant inhibitory efect
on the formation of H4 and its isomers; IC50 values were
greater than 10 μmol/L and 30 μmol/L, the highest tested
concentrations (these concentrations suppressed activity to
≥73.1% and≥71.2% of the control activity, respectively).
The inhibitory efect of vonoprazan at the tested concen-
trations on the formation of H4 and its isomers was neg-
ligible and similar to that seen without pre-incubation of
vonoprazan, except at a concentration of 10 μM, in which
case the inhibitory efect with pre-incubation was somewhat
higher (26.9% reduction in activity) than it was without
pre-incubation (16.2% reduction in activity). However, the
IC50 value of vonoprazan with or without pre-incubation
was greater than 10 μM, and the time-dependent inhibitory
efect of vonoprazan on the formation of H4 and its iso-
mers was considered to be weak. Neither vonoprazan nor
Upon studying the direct inhibition of the major CYPs
by vonoprazan in HLMs, we found that vonoprazan
directly inhibited CYP2B6, CYP2C19, CYP2D6, and

M. Nishihara et al.
CYP3A4/5 (midazolam 1′-hydroxylation and testosterone
6β-hydroxylation) in a concentration-dependent manner with
IC50 values of 16,>30,>30, 29, and>30 μM, respectively.
In healthy volunteers, following oral administration of vono-
prazan at a dose of 20 mg (the maximum clinical dose) once
daily for 7 days, the maximum plasma concentration (Cmax) of
vonoprazan was observed to be low, 26.2 ng/mL (76 nM) [5].
Considering the fu,p value of 0.135 [7], the calculated R1 values
for these CYPs were less than the guidance criterion (1.02),
indicating that interaction due to direct inhibition is unlikely.
The potential of vonoprazan to exert TDI of the seven
major CYP isoforms was also evaluated using the IC50 shift
approach [34]. After a 30-min pre-incubation period, vono-
prazan showed increased inhibition of the metabolism of
bupropion, (S)-mephenytoin, midazolam, and testosterone,
which suggests that it has TDI potential towards CYP2B6,
CYP2C19, and CYP3A4/5, and has the potential to afect
the metabolism of clopidogrel. Therefore, the TDI potency
of vonoprazan towards CYP2B6, CYP2C19, and CYP3A4/5
was examined in more detail by measuring kinact and KI
Fig. 2 Metabolic pathways of
clopidogrel to pharmacologi-
cally active metabolites. Aster-
isk indicates the radiolabeled
position. CYP cytochrome P450

Potential for CYP-Based Drug–Drug Interaction Between Vonoprazan and Clopidogrel
and comparing the results with those obtained for positive
Furthermore, the inhibitory efect of vonoprazan on the
controls (ticlopidine, esomeprazole, and verapamil). The
results, including the R2 values calculated according to
the latest DDI guidance or the guidelines from the FDA,
EMA, or PMDA are presented in Table 3. The R2 values
of vonoprazan were more than the guidance criteria (1.25)
for CYP2B6, CYP2C19, and CYP3A4/5, but were 65-fold,
13-fold, and 2.1-fold lower than those of ticlopidine, esome-
prazole, and verapamil, respectively. These results indi-
cate that vonoprazan is capable of the TDI of CYP2B6,
CYP2C19, and CYP3A4/5, but its potency in this regard
is lower than those of the corresponding positive controls
[13]. In actuality, the infuence of the positive controls on the
pharmacokinetics of coadministrated drugs has been found
to be limited in clinical studies, and led to just a few fold
change in exposure at therapeutic doses in most cases; ticlo-
pidine, esomeprazole, and verapamil (positive controls for
assessing CYP2B6, CYP2C19, and CYP3A4/5 activity,
respectively) were reported to increase the mean area under
the plasma concentration vs time curve (AUC) of bupropion
(a typical CYP2B6 substrate) by 61% [35], that of diaze-
pam (a typical CYP2C19 substrate) by 81% [36], and that
of midazolam (a typical CYP3A4/5 substrate) by 191% [37],
respectively. Since vonoprazan is a 2- to 65-fold weaker
inhibitor than these positive controls, the TDI of CYP2B6,
CYP2C19, or CYP3A4/5 by vonoprazan is unlikely to cause
a clinically signifcant DDI.
in vitro metabolism of [14C]clopidogrel with HLMs was
studied to obtain direct evidence for a DDI between vono-
prazan and clopidogrel. The inhibitory efect of esomepra-
zole was also examined as a reference. Both compounds
inhibited the formation of H4 and its isomers in a concen-
tration-dependent manner, albeit weakly, even at the maxi-
mum tested concentration of 10 μM for vonoprazan and
30 μM for esomeprazole. The formation of diastereomers of
2-oxo-clopidogrel was not inhibited by either vonoprazan or
esomeprazole (Table 4). These results imply that CYP3A4
is heavily involved in the formation of H4 from 2-oxo-clopi-
dogrel, but not in the formation of 2-oxo-clopidogrel from
clopidogrel.
In the repeated-dose study of the efects of vonoprazan
10 mg for 7 days and esomeprazole 20 mg for 5 days, Cmax
was found to be 35.3 nM [5] and 2.65 μM [22], respectively.
Since vonoprazan did not appear to inhibit H4 formation
even at concentrations more than 100-fold the plasma Cmax
,
its inhibitory efects on the metabolism of clopidogrel are
likely to be very weak at clinical doses. Meanwhile, the
plasma Cmax of esomeprazole was comparable to the con-
centration tested in this study, so esomeprazole would be
expected to show more inhibition of clopidogrel activation
than vonoprazan would. However, the opposite pharmaco-
dynamic fndings have been reported by Kagami et al. Since
their report did not include any pharmacokinetic data from
that clinical study, the reason for the attenuation of clopi-
dogrel activity in their study is unknown, but it is unlikely
to be related to the inhibition of these CYPs by vonoprazan.
Additionally, since vonoprazan has inhibitory efects on
CYP2B6, CYP2C19, and CYP3A4/5, which are also the
enzymes involved in the metabolism of vonoprazan itself
[8], autoinhibition would be expected to result in the accu-
mulation of vonoprazan on repeat dosing. However, our
clinical data showed only minor accumulation (accumula-
tion index<1.2) following 10-40 mg QD dosing for 7 days
[5]. In addition, no clinically signifcant mutual pharmacoki-
netic interactions have been reported between clarithromycin
(a substrate and inhibitor of CYP3A4/5) and vonoprazan
following triple therapy with vonoprazan–amoxicil-
lin–clarithromycin in a repeat-dose setting [38]. It is known
that amoxicillin is not a CYP3A4/5 inhibitor [39]. In the
present study, exposure to vonoprazan and clarithromycin
was observed to increase moderately while exposure to the
major metabolites of vonoprazan decreased, whereas expo-
sure to hydroxyclarithromycin—a metabolite generated by
CYP3A4/5 [40]—actually increased. In other words, while
the metabolism of vonoprazan was inhibited by clarithromy-
cin (a CYP3A4/5 inhibitor), vonoprazan did not inhibit the
metabolism of clarithromycin by CYP3A4/5. These results
suggest that vonoprazan is not a CYP3A4/5 inhibitor in vivo.
The cause of the increase in exposure to clarithromycin was
not elucidated.
5 Conclusions
A DDI mediated by CYP metabolism is not expected to be a
major contributor to the attenuation of the antiplatelet activ-
ity of clopidogrel upon coadministration with vonoprazan.
Taken together, these fndings suggest that the observed
pharmacodynamic drug interaction of clopidogrel is
unlikely to be due to the inhibition of CYP2B6, CYP2C19,
or CYP3A4/5 by vonoprazan, meaning that further clinical
assessment is needed.
Acknowledgements The authors would like to thank Suresh K. Balani
of Global Drug Metabolism and Pharmacokinetics, the Global Vono-
prazan Project Team members at Takeda, Hideki Hirabayashi of Drug
Metabolism and Pharmacokinetics Research Laboratories, and Junzo
Takahashi for their contributions to these studies.
Author contributions MN and HY mainly wrote the manuscript. MN,
HY, RC and HJ designed the research and analyzed the data. HY per-
formed the research.

M. Nishihara et al.
J. Safety, tolerability, pharmacokinetics, and pharmacodynam-
ics of single rising TAK-438 (vonoprazan) doses in healthy
male Japanese/non-Japanese subjects. Clin Transl Gastroenterol.
2015;6:e94.
Compliance with Ethical Standards
Conflict of interest All the authors are employees of or have retired
from working for Takeda Pharmaceutical Company Limited. The draft
manuscript was prepared by Axcelead. The authors declare no other
conficts of interest.
11. Kagami T, Yamade M, Suzuki T, Uotani T, Hamaya Y, Iwaizumi
M, Osawa S, Sugimoto K, Umemura K, Miyajima H, Furuta T.
Comparative study of efects of vonoprazan and esomeprazole
on antiplatelet function of clopidogrel or prasugrel in relation to
CYP2C19 genotype. Clin Pharmacol Ther. 2018;103(5):906–13.
12. Nishihara M, Czerniak R. CYP-mediated drug–drug interac-
tion is not a major determinant of attenuation of anti-platelet
function of clopidogrel by vonoprazan. Clin Pharmacol Ther.
2018;104(1):31–2.
Ethical approval All studies were performed according to the applica-
ble institutional guidelines.
Funding All studies reported here were supported and conducted by
Takeda Pharmaceutical Company Limited.
13. Obach RS, Walsky RL, Venkatakrishnan K. Mechanism-
based inactivation of human cytochrome P450 enzymes and
the prediction of drug–drug interactions. Drug Metab Dispos.
2007;35(2):246–55.
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