In vitro evaluations for pharmacokinetic drug-drug interactions of a novel serotonin-dopamine activity modulator, brexpiprazole

Article abstract of DOI:10.1080/00498254.2021.1897898

Brexpiprazole, a serotonin-dopamine activity modulator, is indicated for the treatment of schizophrenia and also adjunctive therapy to antidepressants for the treatment of Major Depressive Disorder. To determine the drug–drug interaction risk for cytochrome P450, and SLC and ABC transporters, brexpiprazole and its metabolite, DM-3411 were assessed in this in?vitro investigation. Brexpiprazole exhibited weak inhibitory effects (IC₅₀ >13 μmol/L) on CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activities, but had moderate inhibitor activity on CYP2B6 (IC₅₀ 8.19 μmol/L). The ratio of systemic unbound concentration (3.8 nmol/L) to the K_i value was sufficiently low. DM-3411 had comparable inhibitory potentials with brexpiprazole only for CYP2D6 and CYP3A4. The mRNA expressions of CYP1A2, CYP2B6 and CYP3A4 were not changed by the exposure of brexpiprazole to human hepatocytes. Brexpiprazole and DM-3411 exhibited weak or no inhibitory effects for hepatic and renal transporters (OATPs, OATs, OCTs, MATE1, and BSEP), except for MATE-2K (0.156 μmol/L of DM-3411), even for which the ratio to systemic unbound concentration (5.3 nmol/L) was sufficiently low. Brexpiprazole effected the functions of P-gp and BCRP with IC₅₀ values of 6.31 and 1.16 μmol/L, respectively, however, the pharmacokinetic alteration was not observed in the clinical concomitant study on P-gp and BCRP substrates. These in?vitro data suggest that brexpiprazole is unlikely to cause clinically relevant drug interactions resulting from the effects on CYPs or transporters mediating the absorption, metabolism, and/or disposition of co-administered drugs.

Full text of DOI:10.1080/00498254.2021.1897898

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ixen20
In vitro evaluations for pharmacokinetic drug-
drug interactions of a novel serotonin-dopamine
activity modulator, brexpiprazole
Hiroyuki Sasabe, Toshihisa Koga, Masayuki Furukawa, Masayuki Matsunaga,
Katsunori Sasahara, Kenta Hashizume, Yoshihiro Oozone, Immaculate
Amunom, Mikako Torii, Ken Umehara, Eiji Kashiyama & Kenji Takeuchi
To cite this article: Hiroyuki Sasabe, Toshihisa Koga, Masayuki Furukawa, Masayuki Matsunaga,
Katsunori Sasahara, Kenta Hashizume, Yoshihiro Oozone, Immaculate Amunom, Mikako Torii, Ken
Umehara, Eiji Kashiyama & Kenji Takeuchi (2021) Inꢀvitro evaluations for pharmacokinetic drug-
drug interactions of a novel serotonin-dopamine activity modulator, brexpiprazole, Xenobiotica,
51:5, 522-535, DOI: 10.1080/00498254.2021.1897898
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XENOBIOTICA
2021, VOL. 51, NO. 5, 522–535
https://doi.org/10.1080/00498254.2021.1897898
RESEARCH ARTICLE
In vitro evaluations for pharmacokinetic drug-drug interactions of a novel
serotonin-dopamine activity modulator, brexpiprazole
Hiroyuki Sasabea, Toshihisa Kogaa, Masayuki Furukawaa, Masayuki Matsunagaa, Katsunori Sasaharaa,
Kenta Hashizumeb, Yoshihiro Oozoneb, Immaculate Amunomc, Mikako Toriid, Ken Umeharaa,
Eiji Kashiyamaa and Kenji Takeuchia
b
aDrug metabolism and Pharmacokinetics, Tokushima Research Institute, Otsuka Pharmaceutical Co Ltd., Tokushima, Japan; ADME & Tox.
d
Research Institute, Sekisui Medical Co., Ltd., Naka-gun, Japan;cSekisui XenoTech LLC, Kansas City, KS, USA; Kashima Laboratory, LSIM Safety
Institute Corporation, Kamisu-shi, Japan
ABSTRACT
ARTICLE HISTORY
Received 25 December 2020
Revised 26 February 2021
Accepted 27 February 2021
1. Brexpiprazole, a serotonin-dopamine activity modulator, is indicated for the treatment of schizo-
phrenia and also adjunctive therapy to antidepressants for the treatment of Major Depressive
Disorder. To determine the drug–drug interaction risk for cytochrome P450, and SLC and ABC
transporters, brexpiprazole and its metabolite, DM-3411 were assessed in this in vitro investigation.
2. Brexpiprazole exhibited weak inhibitory effects (IC50 >13 lmol/L) on CYP2C9, CYP2C19, CYP2D6
and CYP3A4 activities, but had moderate inhibitor activity on CYP2B6 (IC50 8.19 lmol/L). The ratio
of systemic unbound concentration (3.8 nmol/L) to the Ki value was sufficiently low. DM-3411 had
comparable inhibitory potentials with brexpiprazole only for CYP2D6 and CYP3A4. The mRNA
expressions of CYP1A2, CYP2B6 and CYP3A4 were not changed by the exposure of brexpiprazole
to human hepatocytes.
KEYWORDS
Brexpiprazole; OPC-34712;
REXULTI; CYPs; ABC
transporter; SLC transporter;
drug–drug interaction
3. Brexpiprazole and DM-3411 exhibited weak or no inhibitory effects for hepatic and renal trans-
porters (OATPs, OATs, OCTs, MATE1, and BSEP), except for MATE-2K (0.156lmol/L of DM-3411),
even for which the ratio to systemic unbound concentration (5.3 nmol/L) was sufficiently low.
4. Brexpiprazole effected the functions of P-gp and BCRP with IC50 values of 6.31 and 1.16 lmol/L,
respectively, however, the pharmacokinetic alteration was not observed in the clinical concomi-
tant study on P-gp and BCRP substrates.
5. These in vitro data suggest that brexpiprazole is unlikely to cause clinically relevant drug interac-
tions resulting from the effects on CYPs or transporters mediating the absorption, metabolism,
and/or disposition of co-administered drugs.
Introduction
beneficial therapeutic response. However, any adverse reactions
rather than expected effects are occasionally induced when 2 or
more medicines are administered together in patients.
Paroxetine and fluoxetine are well-known antidepressant agents
on the market which are CYP2D6 inhibitors (Richelson 1997).
Since brexpiprazole is mainly metabolised by CYP2D6 and
CYP3A4 (Sasabe et al. 2021), any combined application with
antidepressant agents such as these and strong CYP3A4 inhibi-
tors must be performed carefully. In general, most antidepres-
sant and antipsychotic agents are metabolised by several
cytochrome P450s (Richelson 1997), so this poses significant risk
for drug interactions as the treatment of depression requires
long-term management sometimes with different medication in
order to effectively manage and optimise a patient’s clinical
response (Hicks et al. 2015, Hoffelt and Gross 2016). In such situ-
ation of managed treatment with medication, especially, the
introduction of new agents to the area of psychotropic drug
R
Brexpiprazole (REXULTIV; OPC-34712) is a serotonin–dop-
amine activity modulator that acts as a partial agonist at
serotonin 5-HT1A and dopamine D2 receptors and a potent
antagonist at serotonin 5-HT2A receptors and noradrenaline
a1B/a2C receptors (Maeda et al. 2014a, 2014b). It has some
indications for the treatment of schizophrenia and major
depressive disorder (MDD) as an adjunctive therapy to anti-
depressants in the United States, Canada, South America,
and some of the Asian countries. It has also been approved
for schizophrenia in Australia, as well as several European,
and Asian countries including Japan.
In clinical setting, antipsychotic drugs are often administered
concomitantly with other central nervous system (CNS) drugs to
improve the overall treatment response. In patients with MDD
who had experienced an inadequate response to antidepressant
treatment, the add-on application of brexpiprazole has shown a treatment can generate unexpected drug ꢀ drug interactions
CONTACT Hiroyuki Sasabe
Sasabe.Hiroyuki@otsuka.jp
Drug metabolism and Pharmacokinetics, Tokushima Research Institute, Otsuka Pharmaceutical Co Ltd.,
463-10, Kagasuno, Kawauchi-cho, Tokushima, 771-0192 Japan
Supplemental data for this article can be accessed here.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

XENOBIOTICA
523
(DDIs). As described above, DDIs can involve multiple mecha- brexpiprazole. Therefore, in vitro inhibition studies of DM-
nisms and the effects on the metabolic pathways (e.g. effects 3411 were performed on the activities of CYPs and transport-
on CYP enzymes) are common sources of clinically significant ers alone, and substrate studies of DM-3411 were not consid-
adverse events. Thereby, in the present study, we investigated
the potential metabolic effects of brexpiprazole as an inhibitor
and inducer of various cytochrome P450 isozymes through a
series of in vitro tests using human liver microsomes and hepa-
tocytes, as well as human CYP-recombinant microsomes.
ered necessary to evaluate DDI risk, taking into account the
evaluation of the contribution of the metabolite to efficacy.
Taking these points into consideration, the results of this
in vitro investigations will be in useful in fundamentally under-
standing the DDI potential of brexpiprazole particularly when it
is concomitantly administered with several medicines for other
diseases and other CNS medications like antidepressants.
In addition to CYPs regulating the metabolism, multitude
drug efflux transporters and uptake transporters regulate the
absorption, distribution, and excretion of drugs in the intestinal
epithelium, liver, kidney, vascular endothelium, and brain in
which they are expressed (Giacomini et al. 2010). ABC efflux
transporters, P-glycoprotein (MDR1/ABCB1, P-gp), and breast
cancer resistance protein (BCRP/ABCG2) limit drug absorption
through the intestinal epithelium and distribution through the
blood–brain barrier into the brain. Moreover, a bile salt export
pump (BSEP/ABCB11) can accelerate the biliary excretion of bile
salts from the inside of hepatocytes. SLC transporters, organic
anion transporting polypeptides (OATPs), organic anion trans-
porters (OATs), and organic cation transporters (OCTs) function
in the uptake of organic anionic and cationic compounds into
hepatocytes and kidney cells on the plasma membrane.
Multidrug and toxin extrusion proteins (MATEs) may accelerate
urinary and biliary excretions of organic cationic compounds in
the apical membrane side of hepatocytes and kidney cells,
respectively. These transporters are major determinants not
only of the dispositional fate of drugs, but also their organ/total
clearances. Therefore, these transporter-mediated functions can
result in DDIs and can be common sources of significant
Materials and methods
Chemicals and reagents
Brexpiprazole (7-f4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]
butyloxygquinolin-2(1H)-one) (Figure 1) and its sulfoxide,
DM-3411, were synthesised by Otsuka Pharmaceutical Co.,
Ltd (Tokyo, Japan). [quinoline ring-4-14C]Brexpiprazole
(abbreviated as q-[14C]brexpiprazole) and [benzothiophene-
3-14C]brexpiprazole (b-[14C]brexpiprazole) were synthesised
by Daiichi Pure Chemicals Co, Ltd (Naka-gun, Ibaraki, Japan)
(Figure 1). Specific radioactivity was 5.07 MBq/mg and 4.92
MBq/mg, respectively. b-Nicotinamide adenine dinucleotide
reduced form (b-NADPH) and b-nicotinamide adenine
dinucleotide phosphate reduced form (b-NADH) were pur-
chased from Sigma-Aldrich Co. (St. Louis, MO). Monosodium
b-NADPþ oxidised form, disodium glucose 6-phosphate and
glucose 6-phosphate dehydrogenase were purchased from
Oriental Yeast Co., Ltd (Tokyo, Japan).
[3H]Digoxin, [3H]prazosin, [14C]mannitol, [6,7-3H(N)] oes-
trone 3-sulfate sodium salt, ([3H]E3S), [6,7-3H(N)]oestradiol
17b-D-glucuronide ([3H]E217bG) and [3H(G)]taurocholic acid
([3H]TCA) were purchased from PerkinElmer Inc. (Waltham,
MA). p-[glycyl-2-3H]Aminohippuric acid ([3H]PAH) and
[biguanidine-14C]metformin hydrochloride ([14C]metformin)
were purchased from American Radiolabeled Chemicals (St.
Louis, MO) and Moravek Biochemicals (Brea, CA), respectively.
Other chemicals were obtained from the following sources.
Digoxin, PAH, E3S sodium salt, and bovine serum albumin
(BSA) were purchased from Sigma-Aldrich. E217bG sodium
salt and Ko143 were purchased from Santa Cruz
Biotechnology (Dallas, TX) and Tocris Bioscience (Bristol, UK),
€
adverse events, similar to the effects of CYPs (Konig et al. 2013,
Yu et al. 2018). For reference, these transporters are defined in
the DDI guidelines of Food and Drug Administration
(U.S. Department of Health and Human Services 2020),
European Medicines Agency (EMA 2012), and Pharmaceuticals
and Medical Devices Agency (PMDA 2019) as important drug
transporters to evaluate whether an investigational drug is a
potent substrate or inhibitor. Therefore, we conducted a series
of in vitro tests evaluating the effect of brexpiprazole on the
transport function of a number of substrates using the cell cul-
ture systems and membrane vesicles expressing transporters.
The pharmacokinetics of brexpiprazole were investigated
in healthy volunteers and patients and were reported
(Garnock-Jones 2016, Ishigooka et al. 2018), and and at the
steady state during multiple once-daily oral dosing of brexpi-
prazole in Japanese patients with schizophrenia, its main
metabolite, DM-3411 (Figure 1), represents 45%–70% of brex-
piprazole exposure in plasma (Ishigooka et al. 2018). The
potential inhibitory effects of the metabolite on the common
drug metabolising enzymes and transporters should be con-
sidered. In addition, if the main metabolite is active and sig-
nificantly contributes to pharmacological effects in vivo,
altered exposure to this metabolite should be considered in
terms of DDI risk. DM-3411 has substantially less potency in
terms of pharmacologic activity compared with brexpipra-
zole, and does not appear to penetrate the brain (Sasabe
et al. 2021). Consequently, it was concluded that DM-3411
does not to contribute to the therapeutic effects of
(A)
*
**
(B)
Figure 1. Chemical structures of brexpiprazole and its metabolite, DM-3411.
(A) [14C]Brexpiprazole; (B) DM-3411. Labelled position of q-[14C]brexpiprazole;
ꢁꢁ
ꢁ
labelled position of b-[14C]brexpiprazole. q-[14C]Brexpiprazole and b-
14C]brexpiprazole were separately synthesised, being labelled at each position.
[

524
H. SASABE ET AL.
respectively. TCA sodium salt was purchased from Wako Pure
Chemical Industries Ltd (Tokyo, Japan). Paclitaxel and
S-mephenytoin was purchased from Ultrafine Chemicals
(Manchester, UK) and Toronto Research Chemicals Inc. (North
York, ON, Canada), respectively. Other CYP’s substrates, inhib-
itors, inducers and transporter’s substrates and inhibitors
were purchased from Sigma-Aldrich or Wako Pure Chemical
Industries Ltd. All reagents and solvents were of analytical or
HPLC grade, the highest grade or equivalent.
HEK293 cells expressing OATP, OCT, and MATE
Human embryonic kidney (HEK293) cells transfected with a
vector containing human OATP1B1/SLC21A6, OATP1B3/
SLC21A8, OCT1/SLC22A1, OCT2/SLC22A2 or MATE1/SLC47A1
cDNA (Accession number: NM006446, NM019844, NM003057,
NM003058, and NM018242, respectively), and control HEK293
cells were developed at Sekisui Medical Co., Ltd. Transient
MATE2-K/SLC47A2-expressing cells and its control cells were
prepared with vector alone and vector containing human
MATE2-K cDNA (Accession number: NM001099646). Prior to
the experiments, the cells were cultured in 75-cm2 bottom
flasks and subjected to passage every 3–4 days. The
OATP1B1- and OATP1B3-expressing and control cells were
seeded at 3.0 ꢃ 105 cells/well in 24-well plates coated with
collagen I (BD Falcon) and then incubated in a CO2 incubator
for 1 day. The cells were further incubated for 1 day in 0.5 mL
of the same medium plus 10 mmol/L butyric acid. The OCT1-,
OCT2-, and MATE1-expressing and control cells were seeded
at 2.5 ꢃ 105 cells/well in the same medium on 24-well plates
coated with collagen I and incubated for 2 days.
For transient expression of MATE2-K and its control cells,
HEK293 cells were seeded at a density of 3.5 ꢃ 105 cells/well
in 12-well plates coated with collagen I and incubated for
1 day. The vector containing human MATE2-K cDNA, or vec-
tor alone, was introduced into the HEK293 cells, and the cells
were further incubated for 2 days. The medium was com-
posed of Dulbecco’s Modified Eagle Medium containing 9%
FBS, antibiotic-antimycotic and 2 mmol/L L-glutamine.
Human liver microsomes and human hepatocytes
Pooled human liver microsomes (20 mg/mL) for CYP inhib-
ition experiments were obtained from BD Gentest (Franklin
Lakes, NJ) or Xenotec LLC. (Lenexa, KS) and were stored at
ꢀ70 ꢂC or below. Fresh human hepatocytes for CYP induc-
tion were isolated from 3 samples of non-transplantable
human liver tissue (3 males) and were cultured according to
the methods by Quistorff (1990). Cryopreserved human hepa-
tocytes (1 male and 2 females) for CYP2B6 induction experi-
ment were purchased from Celsis In Vitro Technologies, Inc.
(Chicago, IL)
LLC-PK1 cells expressing MDR1 or BCRP
Porcine kidney epithelial LLC-PK1 cells transfected with vec-
tors containing human MDR1 cDNA and control LLC-PK1 cells
transfected with vector only were used under a licence from
BD Biosciences (Franklin Lakes, NJ). LLC-PK1 cells transfected
with vectors containing human BCRP cDNA (Accession num-
ber: NM004827) were developed at the ADME-Tox. Research
Institute, Sekisui Medical Co., Ltd. Prior to the experiments,
the cells were cultured in 75 cm2 bottom flasks and sub-
jected to passage every 3–5 days. Cells expressing MDR1 (or
empty vector) were seeded at 4 ꢃ 104 cells/insert, and cells
expressing BCRP (or empty vector) were seeded at 2.5 ꢃ 104
cells/insert in 24-well transwell plates (0.3 cm2 PET porous fil-
ter culture inserts of 3 mm pore size for MDR1-expressing
cells or 0.4 mm pore size for BCRP-expressing cells, Cell
Culture Insert 24-Well Companion Plate; BD Falcon, Bedford,
MA). Seeded MDR1-expressing LLC-PK1 cells were incubated
in a CO2 incubator (37 ꢂC, 5% CO2) for 7–9 days and BCRP-
expressing cells for 8 days to prepare cell monolayers for the
determination of cellular transport activity. The medium in
flasks and plates was changed every 2 or 3 days. The
medium for passage in flasks was composed of Medium 199
containing 9% foetal bovine serum (FBS), 50 lg/mL gentami-
cin, and 100 lg/mL hygromycin B. The medium used during
preparation of a monolayer for proliferation in 24-well plates
was composed of Medium 199 containing FBS and gentami-
cin. The electrical resistances across the monolayer were
measured prior to preincubation and were 486–591 Xcm2
for MDR1-expressing cells and 159–348 Xcm2 for BCRP-
expressing and control cells, which were within the accept-
able range (100–800 Xcm2).
Renal proximal tubule (S2) cells expressing OAT
Cells derived from the second segment of the proximal
tubule of transgenic mice harbouring the temperature-sensi-
tive simian virus 40 large T-antigen gene (here termed S2
cells) and transfected with a vector, including human OAT1/
SLC22A6 or OAT3/SLC22A8 cDNA (Accession number:
NM004790 and NM004254, respectively), and control S2 cells
transfected with a vector alone, were established by Fuji
Biomedix Co., Ltd (Tokyo, Japan), and the ownership was
succeeded by Sekisui Medical Co., Ltd.
Prior to the experiments, OAT1- and OAT3-expressing S2 cells
and the corresponding control cells were cultured in 75-cm2
bottom flasks and subjected to passage every 2 or 3 days. The
cells were seeded at 2.5 or 4 ꢃ 105 cells/well in 24-well plates
coated with collagen I (Corning, Tewksbury, MA) and incubated
in a CO2 incubator (33 ꢂC, 5% CO2) for 2 days. The passage and
plating medium was composed of RITC80-7 containing 5% FBS,
10 lg/mL EGF, 0.08 unit/mL insulin and 10 mg/mL transferrin.
We used a number of different cell types in this study
because of the development history in the facility where
these experiments were performed. In the process of devel-
oping transporter-expressing cells, we first used HEK293 cells,
which are widely used when creating recombinants, and as a
result, we were able to construct an expression system for
SLC transporters. We subsequently found that S2 cells for
OATs transporters were more resistant to toxicity in culture.
On the other hand, since the ABC transporters of P-gp and

XENOBIOTICA
525
BCRP reflect bidirectional transports through the monolayer reactions were started by substrates for each CYP to the 10-
membrane, it is necessary to express the directional trans- fold diluted reaction mixtures and were subsequently incu-
porters on a monolayer membrane. The established LLC-PK bated. The changing of the metabolic activity by brexpiprazole
cells having polarity and the established HEK293 cells and S2 in the presence of b-NADPH through the preincubation and
cells were suitable for the transport tests, depending on the metabolic incubation was compared with that of the group for
compared results of the transports of the typical substrates which b-NADPH was present only during the metabolic incuba-
to the corresponding control cells.
tion (Venkatakrishnan et al. 2007).
BSEP-expressing vesicles
mRNA assay and microsomal metabolic experiments for
CYP induction
BSEP-expressing and control vesicles (expression system of
the insect cells infected with baculovirus) were obtained Three lots of fresh human cultured hepatocytes were used
from GenoMembrane Co Ltd. (Yokohama, Kanagawa, Japan) for the assessment of relative mRNA levels and metabolic
and stored at ꢀ70 ꢂC until use.
activities of CYP1A2, CYP2C9, and CYP3A4. Hepatocytes were
seeded at approximately 1.3 ꢃ 106 viable cells/mL on colla-
gen coated 60 mm culture dishes and were placed in a
humidified chamber (37 ꢂC, 5% CO2). After seeding, hepato-
cyte culture was performed according to the methods
described in the previous report (Shimokawa et al. 2014).
After the adaptation period, hepatocyte cultures were then
treated daily for 3 consecutive days with 1 of the 3 concen-
trations of brexpiprazole (0.1, 1, or 10 lmol/L), omeprazole
(100 lmol/L, CYP1A2 positive inducer), or rifampin (10 lmol/
L, CYP2C9 and CYP3A4 positive inducer) dissolved in 0.1% (v/
v) DMSO (vehicle). After the last treatment with test com-
pounds, total RNA was extracted from the hepatocytes using
TRIzol (Invitrogen, Carlsbad, CA) and purified using the
RNeasy Mini Kit (Qiagen, Valencia CA). Subsequently, cDNA
was prepared from mRNA using High-Capacity cDNA Reverse
Transcription Kit (Applied Biosynterms, Foster City, CA) and
amplification was conducted using TaqMan Gene Expression
Master Mix (Applied Biosynterms) and the real-time polymer-
ase chain reaction (PCR) sequence detection system (7300,
Applied Biosynterms). For CYP2B6, the relative mRNA levels
were determined using cultured human hepatocytes pre-
pared from the cryopreserved hepatocytes, after 2-consecu-
tive-day treatment days with brexpiprazole (0.05, 0.5, or
5 lmol/L) or phenobarbital (750 lmol/L, CYP2B6 positive
inducer). The maximum incubation concentration of brexpi-
prazole was set to 5 or 10 mmol/L (for CYP2B6 and for other
CYPs, respectively) which was sufficiently more than the
required three orders of magnitude above the Cmax,u
(0.0038 mmol/L) (Ishigooka et al. 2018). The details of PCR
analysis were according to the methods described by
Shimokawa et al. (2014). Microsomal samples were prepared
from the human hepatocytes based on the method
described by Madan et al. (1999). For each hepatocyte prep-
aration, cell suspensions scraped from culture dishes were
sonicated. Sonicated lysates were centrifuged at 7000 to
9000ꢃg at 4 ꢂC for 20 min, after which the supernatant frac-
tion (S9) was removed and centrifuged at 80 000 to
90 000ꢃg at 4 ꢂC for 60 min. The resulting microsomal pellets
were resuspended in 250 mmol/L sucrose aqueous solution.
Microsomal metabolic experiments for CYP inhibition
The CYP enzymatic reactions and their substrates and inhibi-
tors were described as below.
CYPs
Metabolic reactions
Substrate
Inhibitors
Furafylline
8-Methoxypsoralen
Orphenadrine
Quercetin
CYP1A2 Phenacetin O-deethylation Phenacetin 50
CYP2A6 Coumarin 7-hydroxylation Coumarin 5
CYP2B6 Bupropion hydroxylation
Bupropion 50
CYP2C8 Paclitaxel 6a-hydroxylation Paclitaxel 10
CYP2C9 Diclofenac 40-hydroxylation Diclofenac 5
Sulfaphenazole
CYP2C19 S-Mephenytoin
S-Mephenytoin 100 Tranylcypromine
40-hydroxylation
CYP2D6 Bufuralol 10-hydroxylation
CYP2E1 Chlorzoxazone
6-hydroxylation
( )-Bufuralol 20
Chlorzoxazone 50 Diethyldithiocarbamate
Quinidine
CYP3A4 Midazolam 10-hydroxylation Midazolam 100
Ketoconazole
Testosterone
Testosterone 100
6b-hydroxylation
The reaction mixture consisted of 100 mmol/L phosphate
buffered saline (pH 7.4), 2.5 mmol/L b-NADPH, 2.5 mmol/L
b-NADH, human liver microsome, and substrate and test com-
pound as inhibitor at the designated concentration. The details
of the experimental condition in the metabolic reaction are pre-
sented in Supplementary Table 1. The reaction mixture was
preincubated without both b-NADPH and b-NADH at 37 ꢂC for
5 min. After the addition of b-NADPH and b-NADH, the reac-
tions were carried out at 37 ꢂC at the designated time. In the
case of mechanism-based inhibitors, furafylline (CYP1A2), 8-
methoxypsoralen (CYP2A6), orphenadrine (CYP2B6), and diethyl-
dithiocarbamate (CYP2E1), the reaction mixture was preincu-
bated with b-NADPH and b-NADH and without the substrate at
37 ꢂC for 15 min. After the addition of substrate, the reactions
were carried out at 37ꢂC for the designated time. The reaction
was stopped by the addition of 0.5 mL of IS solution (200 nmol/
L of chlorpropamide in acetonitrile), and then the mixture was
centrifuged at 15 000 rpm for 10 min at 4 ꢂC (Himac CF15D,
Hitachi Koki Co., Ltd. Tokyo, Japan). The resultant supernatant
fraction was diluted with distilled water at a ratio of 1:4, and
then analysed using the LC-ESI-MS/MS system. All reactions
were carried out in duplicate. To evaluate the mechanism-
based inactivation of brexpiprazole, after human liver micro-
Transwell transport experiments
somes were incubated with brexpiprazole at 100 mmol/L in the In transwell plates, the apical-side volume of MDR1-express-
presence of b-NADPH at 37 ꢂC for 30 min, the metabolic ing cell monolayers was maintained at 100 mL and the basal-

526
H. SASABE ET AL.
side volume at 600 mL. The apical-side volume of BCRP-
expressing cell monolayers was maintained at 250 mL and the
basal volume at 900 mL. Prior to flux assays, the medium sur-
rounding both the apical and basal sides was removed and
replaced with equal volumes of fresh 1% bovine serum albu-
min containing Hanks’ balanced salt solution (pH ¼ 7.4) (1%
BSA-HBSS, for BCRP) or 4% BSA-HBSS (for BDR1) containing
0.2% DMSO (vehicle) or inhibitor solution, and the cells were
preincubated at 37 ꢂC for 1 h.
For the assessment of drug transport from the apical to
basal sides, the apical-side medium was replaced with 1% or
4% BSA-HBSS containing the test substance as substrate/
inhibitor or with [14C]mannitol solution (indicator for the
tight junction), and the basal side was replaced with 1% or
4% BSA-HBSS containing 0.2% DMSO or inhibitor solution as
appropriate. Conversely, for the assessment of transport from
the basal to apical sides, the apical-side medium was
replaced with fresh 1% or 4% BSA-HBSS containing 0.2%
DMSO or inhibitor solution as appropriate, and the basal side
medium was replaced with the test substance as substrate/
inhibitor or [14C]mannitol solution. Then, the cells were incu-
bated at 37 ꢂC at the designated times (0.25, 0.5 and 1 h for
[3H]prazosin and 0.5, 1, and 2 h or 1, 2, and 4 h for
[14C]brexpiprazole and [3H]digoxin). Incubation was con-
ducted in triplicates. At the designated time, 70 mL (for MDR)
or 100 mL (for BCRP) of the sample in the basal or apical
sides was collected for the assessment of transport from
apical to basal or basal to apical sides, respectively. The
same volume of 1% or 4% BSA-HBSS containing 0.2% DMSO
or inhibitor solution was quickly added to compensate for
the sampled volume.
Vesicular uptake experiment
Prior to incubation, glass fibre filters (MF-membrane filters
#HAWP02500, Merck Millipore, Burlington, MA) were washed
once with an ice-cold stop solution of 10 mmol/L Tris/HEPES,
100 mmol/L KNO3, and 50 mmol/L sucrose. Culture wells
were preincubated at 37 ꢂC for 5 min in the reaction buffer
consisting of Tris/HEPES, KNO3, 4 mmol/L Mg-ATP, and
sucrose plus substrate or inhibitor (45 mL mixture). Then, a
5 mL aliquot of BSEP-expressing or control vesicle solution
was added to start the uptake reaction, and the reaction
mixture was allowed to proceed at 37 ꢂC for 2 min. All incu-
bations were conducted in triplicate. At the designed time,
an ice-cold stop solution (200 mL) was added to terminate
the reaction. An aliquot (200 mL) of each mixture was passed
through the glass fibre filter by aspiration. Loaded filters
were washed twice with 5 mL ice-cold stop solution, trans-
ferred to a vial and mixed with 10 mL scintillator cocktail
(Insta-Gel Plus, PerkinElmer Inc.) for the measurement of the
intravesicular radioactivity. An aliquot (20 mL) of the remain-
ing reaction mixture was mixed with 10 mL of scintillator
cocktail for the measurement of the remaining extravesicular
radioactivity. The details of the experimental condition in the
uptake and transcellular transport assay are presented in
Supplementary Table 2.
Protein content assay
The protein content in the cell lysate was assayed using the
BCA Protein Assay Kit (Thermo Fisher Scientific, Tokyo,
Japan). Briefly, 20 mL of each cell lysate sample or BSA cali-
bration solution (0, 0.05, 0.1, 0.25, 0.5 and 0.75 mg protein/
mL) was allocated to 96-well plates, and 200 mL of reagent
mixture from the reagent kit was added to each well.
Mixtures were incubated at 37 ꢂC for 30 min, and absorbance
For experiments using radiolabelled substrate and refer-
ence compounds, scintillator cocktail (10 mL, Hionic-Fluor,
PerkinElmer Inc.) was added to the collected samples.
Permeability was calculated based on the transported
amount and the concentration observed before incubation.
at 562 nm was measured on
a
microplate reader
(SPECTRAmaxTM190, Molecular Devices Corp., San Jose, CA).
Incubation was performed in duplicate.
Cellular uptake experiments
LC-MS/MS analysis
The medium in each seeded well was replaced by fresh
HBSS (1 mL), and the cells were preincubated at 37 ꢂC for
15 min. Uptake was started by replacing of albumin-free
HBSS (300 mL) containing 0.2% DMSO with the substrate solu-
tion or substrate solution containing inhibitor. The uptake
reaction was performed in triplicate at 37 ꢂC for each desig-
nated time (0.5, 1, 2, 5 and 10 min). The substrate solution
was then removed, and the cells were washed once with
1 mL of ice-cold PBS containing 0.2% BSA and twice with
1 mL of ice-cold PBS. The intracellular fraction was isolated as
described below. After PBS was removed, 0.1 mol/L NaOH
(0.5 mL) was added to each well and mixed thoroughly by
pipetting to dissolve the cells. An aliquot (0.3 mL) of the cell
lysate was mixed with the scintillator cocktail (10 mL, Hionic-
Fluor). Protein content was also determined in the aliquot.
The uptake volume was calculated based on the amount of
drug in the lysate and the concentration observed
before incubation.
LC-MS/MS analysis of the produced metabolites for micro-
somal CYP activity was conducted using an API4000 MS sys-
tem with a Shimadzu LC20A HPLC system (Shimadzu Co.,
Ltd., Kyoto, Japan). Chromatographic separation was
achieved on a Cadenza CD-C18 (3 lm, 2.0 mmID ꢃ 50 mm,
Imataki Corp, Kyoto, Japan) using mixtures of 2 solvents, A
[0.1% (v/v) acetic acid aqueous solution] and B [0.1% (v/v)
acetic acid acetonitrile solution], at a constant flow rate of
0.2 mL/min. The elution steps were as follows: under the
equilibration of 10% B flow, a linear gradient from 10% to
80% B was operated for 2.5 min after sample injection.
Equilibration was achieved for 4 min after returning to the
initial condition. The column effluent was delivered to the
ESI ion source to detect in the positive multiple reaction
monitoring mode except for chlorzoxazone (negative mode).
The calibration of metabolites exhibited good linearity over
the range 50–5000 nmol/L for acetaminophen (phenacetin’

XENOBIOTICA
527
Net transport ratiopresence
metabolite) and 10–1000 nmol/L for others, using weighted
(1/concentration2) least-squares regression. The lower limit of
qualification (LLOQ) defined as the lowest measurable con-
centration was 10 nmol/L except for acetaminophen, with an
LLOQ of 50 nmol/L. The sensitivity of the LC-MS/MS analysis
was sufficient to see microsomal CYP activities and related
inhibition. Acceptance of analytical data was subject to the
criterion that the analytical data of quality check samples
must be within 85% to 115% of the nominal concentration.
of inhibitor
of inhibitor
% of control ¼
ꢃ 100
Net transport ratioabsence
In the uptake inhibition experiments, the remaining activ-
ity was calculated according to the following equation:
% of control
Uptake volumepresence
ðexpressing cellsÞ
ꢀ
absence of inhibitor
¼
ð
Þ
Uptake volumepresence
control cells
ꢀ
absence of inhibitor
ꢃ 100
Measurement of radioactivity
The activity of each CYP enzymatic reaction and perme-
ability were compared with those of the control, being
expressed as the percentage of the remaining control activ-
ity. IC50 values were calculated using WinNonlin (Ver 5.2 or
6.1, Pharsight Co., Mountain View, CA). In the inhibition
experiments, IC50 was calculated by the Emax equation:
The radioactivity in each sample was measured for 2 min
using liquid scintillation counting (LSC, 2500TR, 3100TR, or
1900CA, PerkinElmer Inc., LSC-6101 Aloka, Tokyo, Japan). The
counting efficiency was corrected by the external standard
source method. The detection limit of radioactivity was
defined as the background radioactivity.
!
c
Imax ꢃ ½Iꢄ
% of control ¼ 1 ꢀ
ꢃ 100
c
c
½Iꢄ þ IC50
Calculations of parameters
where Imax denotes the maximum inhibitory effect, [I]
denotes the inhibitor concentration, and c denotes the Hill
coefficient. When percentage of control at the maximum
concentration tested was higher than 50.0%, IC50 was indi-
cated as greater than the maximum concentration. For the Ki
determination of brexpiprazole, the rates of metabolite for-
mation following co-incubation of 3 concentrations (approxi-
mately 1/2 Km, Km, and 2 Km) of the substrate with 0, 3, 10,
and 30 lmol/L of brexpiprazole were determined. The calcu-
lation of the Ki value was performed using the Dixon plot.
Inactivation kinetic parameters were determined using the
nonlinear regression of the data to the following expression:
In the monolayer experiments using MDR1-expressing and BCRP-
expressing LLC-PK1 cells, the permeation volume and clearance
were calculated from the determined permeated amounts across
transporter-expressing or control cell monolayers and the initial
concentration, according to the following equations:
Permeation volume ðlL=wellÞ
Permeated amount ðamount=wellÞ
¼
Initial concentration ðamount=lLÞ
and
Permeation clearance ratio
Permeation clearancebasal
Permeation clearanceapical
ðlL=well=hÞ
ðlL=well=hÞ
to apical
to basal
kinact ꢃ ½Iꢄ
kobs ¼ kobs½Iꢄ¼0 þ
KI þ ½Iꢄ
¼
where permeation clearance was determined by a linear
regression of permeation volume and incubation time.
Further, a ratio of permeation clearance for transporter-
expressing cells to the control cells was defined as a net
transport ratio. In the uptake experiments using HEK293 or
S2 cells expressing OATs, OCTs, OATPs, and MATEs, the
uptake volume was calculated from the amount of substrate
in the cell lysate and the initial substrate concentration
according to the following equation:
in which [I] denotes the concentrations of brexpiprazole in
the inactivation preincubations, kobs denotes the negative
values of the slopes of the natural logarithm of the percent-
age of the remaining activity versus inactivation incubation
time at various [I], kobs[I]¼0 denotes the apparent inactivation
rate constant measured in the absence of brexpiprazole, kin-
denotes the limit maximum inactivation rate constant as
act
[I]!1, and KI denotes the brexpiprazole concentration yield-
ing kobs at the sum of kobs[I]¼0 and 0.5 times kinact
.
Uptake amount ðamount=wellÞ
Uptakevolume ðlL=mg proteinÞ ¼
Initial concentration ðamount=lLÞ ꢃ Protein content ðmg protein=wellÞ
Statistical analysis
Further, the ratio of the uptake volume for transporter-
expressing cells to the control cells was defined as the
uptake ratio. In the vesicle experiment, the uptake amount
and protein content were expressed per tube.
In the inhibition of monolayer permeation, the remaining
activity (% of control) was determined as the ratio of the net
transport in the presence and absence of an inhibitor as follows:
The statistical significance of differences for the test com-
pound and positive control compound for inhibition studies
was determined by the Williams test (lower-tailed) and
unpaired t-test (Student’s or Welch), respectively, using
Statistical Analysis System (SAS) software (Release 9.4, SAS
Institute, Cary, NC). For induction study, The ANOVA was

528
H. SASABE ET AL.
followed by the Dunnett’s post hoc test to identify the group produced a 9.12-fold increase in CYP1A2 mRNA levels, but
this increase was less than 3% of that caused by omeprazole.
The mRNA content increase (1.77-fold) in CYP3A4 at
10 lmol/L was statistically significant from the control, but,
the difference was under 2-fold that is the DDI guideline’s
criterion for the clinical assessment of induction (Table 2). In
addition, the metabolic activities mediated by CYP1A2,
CYP2C9, and CYP3A4 were not significantly changed.
means that were significantly different from the controls
(p < 0.05). Analyses were performed using Sigma Stat
Statistical Analysis System (version 2.03, Systat Software, Inc.,
Point Richmond, CA).
Results
Inhibition of CYP-mediated metabolic activity by
brexpiprazole and DM-3411
Transport of brexpiprazole across P-gp/MDR1- or BCRP-
expressing cell monolayers
Brexpiprazole exhibited several weak inhibitory effects on
CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (Table 1).
The inhibition of brexpiprazole on CYP2B6 was the strongest
among these CYPs, having an IC50 value of 8.19 lmol/L (Ki
value ¼ 5.01 lmol/L). Brexpiprazole exhibited no effects on
the activities of CYP1A2, CYP2A6, CYP2C8, and CYP2E1. DM-
3411 exhibited weak inhibitory effects on CYP2D6 and
CYP3A4 (only midazolam 10-hydroxylation), with similar IC50
values to those of brexpiprazole. For the effects of brexpipra-
zole, the preincubation of microsomes and brexpiprazole
with b-NADPH make the 2 activities by CYP3A4 decreased
over 20%. And when the preincubation time and the con-
centration of brexpiprazole varied for 0, 5, 10, 20, and
30 min, and at 0, 3, 10, 30, and 100 lmol/L, respectively in
the inactivation experiment, KI and Kobs for inactivation were
obtained, both in the midazolam 10-hydroxylation and testos-
terone 6b-hydroxylation (Table 1).
The permeation clearances of [14C]brexpiprazole at 1 lmol/L
across P-gp/MDR1-expressing LLC-PK1 cell monolayers were
2.87 0.08 mL/well/h (mean SD, n ¼ 3) from the apical to
basal sides and 4.83 0.16 mL/well/h from the basal to apical
sides, and the permeation clearance ratio was 1.7 (Figure
2(A)). This was only slightly higher than that across control
LLC-PK1 cell monolayers, in which the clearances were
2.85 0.32 mL/well/h from the apical to basal sides and
2.68 0.17 mL/well/h from the basal to apical sides, with a
clearance ratio of 0.9. The ratio of the permeation clearance
for transporter-expressing cells to the control cells (net trans-
port ratio) was 1.9 under 2 that is criterion for assessment
of transporter’s substrate. The permeation clearances of
[14C]brexpiprazole across BCRP-expressing LLC-PK1 cell
monolayers were 9.47 1.94 mL/well/h from the apical
to basal sides and 20.0 0.9 mL/well/h from the basal to
apical sides, and the permeation clearance ratio was 2.1
(Figure 2(C)). Meanwhile, the permeation clearances across
the control cell monolayers were 13.4 2.7 mL/well/h from
the apical to basal sides and 17.4 0.4 mL/well/h from the
apical to basal sides, and a permeation clearance ratio was
Induction of CYPs by brexpiprazole
At concentrations less 10 lmol/L, brexpiprazole did not affect
the mRNA contents of CYP2B6 and CYP2C9 in human cul-
tured hepatocytes. However, at 10 lmol/L, brexpiprazole 1.3 (Figure 2(C)). These 2 permeation clearance values
Table 1. Inhibitory effects of brexpiprazole and its metabolite, DM-3411, on CYP-mediated metabolic activities in human liver microsomes.
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
w/o
preincubation
IC50 [SE]
with preincubation
w/o preincubation
w/o preincubation
IC50 [SE]
KI [SE]
kinact [SE]
CYPs
(lmol/L)
Decrease from w/o (%)
(lmol/L)
(minꢀ1
)
(lmol/L)
Cinlet max,u
,
/IC50
C
max,u/IC50
Cmax,u/IC50
ꢁ
ꢁ
ꢁ
ꢁꢁ
ꢁꢁꢁ
CYP1A2
CYP2A6
CYP2B6
>100
>100
1.2
4.0
ꢀ2.6
–
–
–
–
>100
>100
>100
–
–
–
–
–
–
–
–
8.19 [0.90]
0.0012
0.00046
ꢁꢁꢁꢁ
(5.01)
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP3A4#
>100
22.2 [6.1]
39.8 [4.5]
13.4 [2.4]
>100
ꢀ4.5
–
–
–
–
–
–
–
–
–
–
>100
>100
>100
15.3 [1.0]
>100
–
–
–
–
–
5.1
0.00044
0.00024
0.00072
0.00017
0.00010
0.00028
ꢀ4.6
0.9
–
0.00034
29.9 [6.4]
40.8 [11.2]
30.5
41.4
32.1 [43.1]
4.7 [3.8]
0.0196 [0.0079]
0.0221
25.8 [0.62]
>100
0.00033
0.00024
0.00013
0.00009
0.00020
–
[0.0035]
The test compound was incubated at 1, 3, 10, 30, and 100 lmol/L with human microsomes in the presence of b-NADPH/b-NADH. In the case of the w/o prein-
cubation, the incubation for metabolic reaction was started by the addition of b-NADPH/b-NADH after preincubation of the test compound, substrate and
human microsomes. In the case of the with preincubation, the incubation for metabolic reaction was started by the addition of substrate after preincubation of
the test compound, human microsomes, and b-NADPH/b-NADH. The IC50 and the estimated standard error (SE) were obtained by the nonlinear regression of
the mean remaining activity (% of control, triplicate determinations) from the control (0 lmol/L) to the Emax model equation, using WinNonlin (Ver. 5.2 or 6.1).
ꢁ
The pharmacokinetic parameters when brexpiprazole was administered to Japanese patients at 4 mg for 14 days were used to calculate the ratios for drug–-
drug interaction (Ishigooka et al. 2018). The Cmax,u/IC50 value was calculated using free fraction (fu: 0.01 for brexpiprazole, 0.035 for DM-3411) and plasma Cmax
(0.38 mmol/L for brexpiprazole and 0.15 mmol/L for DM-3411). Cinlet,max,u was calculated using the equation fu ꢃ [Cmax þ (ka ꢃ dose/Qh)], where the absorption
rate constant (ka: 0.1 minꢀ1, regulatory maximum) was obtained from plasma concentration profiles, and hepatic blood flow (Qh) of 23.4 mL/min/kg was used.
#
Upper: Midazolam 1’-hydroxylation, Lower: Testosterone 6b-hydroxylation.
>100: Inhibitory effects less than 50% at 100 lmol/L of the test compound.
ꢁꢁ
ꢁꢁꢁ
–: not tested,
ꢁꢁꢁꢁ
The value in the parenthesis indicates the Ki value.

XENOBIOTICA
529
Table 2. Effects of brexpiprazole on CYP mRNA expression and CYP-mediated metabolic activities in human primary cultured hepatocytes.
Fold increase
CYP2C9
CYP1A2
Phenacetin
CYP3A4
Testosterone
CYP2B6
Concentration (lmol/L)
mRNA
O-dealkylation
mRNA
Diclofenac 4’-hydroxylation
mRNA
6b-hydroxyation
mRNA
0 (Control)
0.1 (0.05)
1.00
1.00 0.73
0.914 0.103
0.874 0.160
1.24 0.28
18.8 14.6#
1.68 0.57
–
1.00
1.00 0.49
0.940 0.052
0.999 0.070
1.06 0.08
1.72 0.78#
2.75 1.01#
–
1.00
1.00 0.59
1.08 0.16
1.19 0.04
1.56 0.61
2.55 1.31
8.83 5.67#
–
1.00
ꢁ
1.34 0.57
1.61 0.72
9.12 6.33
372 293#
1.74 0.94
–
1.09 0.20
1.23 0.28
1.03 0.24
1.86 0.18#
2.99 0.20#
–
1.06 0.19
1.15 0.24
1.77 0.38¶
2.90 0.91
10.3 2.8#
–
1.02 0.02
1.18 0.05
1.14 0.30
ꢁ
1 (0.5)
10 (5)
ꢁ
ꢁꢁ
–
100 (Omeprazol)
10 (Rifampin)
–
750 (Phenobarbital)
7.20 0.48
The test compound was incubated with human hepatocytes (1.3 ꢃ 106 viable cells/well) for 48 h. The fold increase represents the ratio of mRNA expression or
metabolic activity in the presence of inducer to that in the absence of inducer (0.1% (v/v) DMSO).
The value represents the mean standard deviation (SD) of 3 hepatocyte lots.
ꢁ
ꢁꢁ
The value in parenthesis indicates the concentration of brexpiprazole in the experiment involving mRNA expression of CYP2B6. –: not tested.
¶Significantly different from the vehicle control as a result of one-way analysis of variance (p < 0.05) with the positive control groups (omeprazole and rifampin)
excluded from the statistical analysis using Sigma Stat Statistical Analysis System (version 2.03).
#Significantly different from the vehicle control as a result of one-way analysis of variance (p < 0.05) with all treatment groups included in the statis-
tical analysis.
Figure 2. Time profiles for the transcellular transports of [14C]brexpiprazole and [3H]digoxin or [3H]prazosin across MDR1-expressing or BCRP-expressing LLC-PK1
cells. The experiment was started by the addition of substrate to one compartment: (A) [14C]brexpiprazole (1 mmol/L) and (B) [3H]digoxin (1 mmol/L) across the
MDR1-expressing LLC-PK1 cells in 4% BSA-HBSS; (C) brexpiprazole (1 mmol/L) and (D) [3H]prazosin (1 mmol/L) across the BCRP-expressing LLC-PK1 cells in 1% BSA-
HBSS. Circle symbols: basal to apical flux; triangle symbols: apical to basal flux; closed symbols: the transporter-expressing LLC-PK1 cells; open symbols: control
LLC-PK1 cells. Data are expressed as mean standard deviation (SD) of triplicate determinations.
were close each other, and the net transport ratio was 1.6, with [3H]digoxin and [3H]prazosin in the monolayer
which was lower than 2. The permeation clearance ratio of flux experiments, brexpiprazole was not transported in sig-
the known P-gp substrate, [3H]digoxin, was significantly nificant amounts by P-gp/MDR1 and BCRP, indicating that
larger than that in the control cells (Figure 2(B)), and the net brexpiprazole is not a substrate for the two efflux transport-
transport ratio was 20.0. The net transport ratio of BCRP sub- ers; indeed if it was a substrate, it would be a weak
strate, [3H]prazosin, was 5.6 (Figure 2(D)). Thus, compared and poor substrate.

530
H. SASABE ET AL.
Figure 3. Time profiles for the uptake of [14C]brexpiprazole and [3H]E217bG into OATP1B1 or OATP1B3-expressing cells and for the uptake of [14C]brexpiprazole
and [14C]metformin into OCT1-expressing HEK293 cells. The experiment was started by the addition of substrate: (A) (C) [14C]brexpiprazole (1 mmol/L), (B)
[3H]E217bG (0.05 mmol/L), and (D) [14C]metformin (10 mmol/L). Closed circle symbols: OATP1B1-expressing cells; closed triangle symbols: OATP1B3-expressing cells;
open circle symbol: control HEK293 cells in (A). Open bar: control HEK293 cells; dotted bar: OATP1B1-expressing cells; hatched bar: OATP1B3-expressing cells in (B).
Closed circle symbols: OCT1-expressing cells; open circle symbol: control HEK293 cells in (C). Open bar: Control HEK293 cells; dotted bar: OCT1-expressing cells in
(D). The uptake volume of [3H]E217bG and [14C]metformin are at 2 and 5 min, respectively. Data are expressed as mean SD of triplicate determinations.
(1.04 0.05 lL/mg protein into the control cell), indicating
substantially larger uptake than for control cells and func-
tional uptake activity (Figure 3(D)). These results suggested
that brexpiprazole is not a substrate of hepatic uptake trans-
porters, OATP1B1, OATP1B3, and OCT1.
Uptake transport of brexpiprazole by OATP- or OCT1-
expressing cells
The uptake volume of brexpiprazole was 665 22 lL/mg pro-
tein at 10 min for OATP1B1-expressing HEK293 cells and
496 38 lL/mg protein for OATP1B3-expressing cells (Figure
3(A)). The uptake volume of brexpiprazole into the common
control cells was 455 62 lL/mg protein (Figure 3(A)). The
uptakes ratios of brexpiprazole for the OATP1B1- and
OATP1B3-expressing cells were similar to the control cells
throughout the uptake incubation time. Conversely, the
known OATP substrate, [3H]E217bG, showed substantial
Inhibition of P-gp/MDR1 and BCRP-mediated transports
by brexpiprazole and DM-3411
Brexpiprazole and DM-3411 had influences on the net efflux
ratio of [3H]digoxin across the P-gp/MDR1-expressing cell
monolayers with similar IC50 values of 6.31 and 7.84 mmol/L,
respectively (Tables 3 and 7). Compared with P-gp/MDR1,
both compounds had more influence on the net efflux ratio
of [3H]prazosin across the BCRP expressing cells with IC50 val-
ues of 1.16 and 3.04 mmol/L, respectively (Tables 3 and 7).
uptakes into the
2
expressing cells, 86.0 2.3 and
14.7 0.9 lL/mg
protein for 2 min, respectively
(0.355 0.125 lL/mg protein into the control cells), and indi-
cated functional uptake activity (Figure 3(B)).
The uptake volumes of brexpiprazole for OCT1-expressing
HEK293 cells and the control were 427 28 and 515 12 lL/
mg protein at 10 min, indicating slightly lower uptake than
for control cells (Figure 3(C)). Conversely, the uptake volume
Inhibition of OATP-, OAT-, OCT-, and MATE-mediated
transports by brexpiprazole and DM-3411
of the known OCT1 substrate, [14C]metformin, into the OCT1- Brexpiprazole and DM-3411 reduced the uptake of
expressing cells was 4.70 0.08 lL/mg protein for 5 min [3H]E217bG by the OATP1B1 expressing cells with similar IC50

XENOBIOTICA
531
Table 3. Inhibitory effects of brexpiprazole and its metabolite, DM-3411, on MDR1-mediated [3H]digoxin transport and BCRP-medi-
ated [3H]prazosin transport.
MDR1-mediated [3H]digoxin transport
(Ratio of basal and apical transports)
BCRP-mediated [3H]prazosin transport
(Ratio of basal and apical transports)
Concentration
(lmol/L)
% of control
9.0/1.7
0 (Control)
Inhibitor
15.4/1.0
Brexpiprazole
33.0/1.0
DM-3411
Brexpiprazole
DM-3411
0.1
0.3
1
3
10
30
96.1
119.5
135.7
78.6¶
24.7¶¶
15.6¶¶
11.7##
–
90.0
86.8
111.3
103.8
81.1
58.5¶¶
77.9¶¶
67.3¶¶
50.3¶¶
33.9¶¶
6.1#
98.1
62.3¶¶
39.6¶¶
30.2¶¶
20.8¶¶
62.3¶¶
47.2¶¶
28.3¶¶
ꢁ
–
30 (Verapamil)
1 (Ko123)
–
17.0##
The values at 0 lmol/L represent the ratio of basal to apical transport against apical to basal transport (ratio for transporter-express-
ing LLC-PK1 cells/ratio for non-transporter-expressing LLC-PK1 cells). The % of control represents the net transport ratio in the pres-
ence of inhibitor to that in the absence of inhibitor (concentration 0 lmol/L).
The statistical significance of differences for the test compound and positive control compound for inhibition studies was deter-
mined by the Williams test (lower-tailed) and unpaired t-test (Student’s or Welch), respectively, using SAS software (Release 9.4).
¶p < 0.05, ¶¶P < 0.01, p < 0.05, ##p < 0.01.
Each value represents the ratio of mean permeability rates obtained from triplicate determinations. –: not tested.
#
ꢁ
Table 4. Inhibitory effects of brexpiprazole and its metabolites, DM-3411, on OATP-mediated [3H]E217bG uptakes, OAT1-mediated [3H]PAH uptake, and OAT3-
mediated [3H]E3S uptake.
Uptake volume into OATP1B1-
expressing cells
Uptake volume into OATP1B3-
expressing cells
Uptake volume into OAT1-
expressing cells
Uptake volume into OAT3-
expressing cells
Concentration
(lmol/L)
(lL/mg protein/2 min)
(lL/mg protein/2 min)
(lL/mg protein/2 min)
(lL/mg protein/2 min)
81.4 8.8
(0.27 0.06)
101
(0.32 0.07)
6
17.3 1.6
(0.27 0.06)
15.4 2.0
(0.32 0.07)
43.9 2.5
(0.65 0.18)
41.7 2.2
(1.10 0.70)
34.2 2.5
(2.59 0.26)
41.1 3.8
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
(2.49 0.37)
% of control
0 (Control)
Inhibitor
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
0.1
0.3
1
3
10
30
108.0
115.1
115.7
84.7¶
62.7¶¶
37.8¶¶
5.9##
–
90.9
112.8
104.9
93.7
90.5
119.4
103.6
120.0
111.0
98.4
109.2
122.7
121.2
126.9
122.5
89.7
95.5
99.4
91.9
95.2
83.6
95.2
92.6
99.1
88.9
98.6
85.9¶¶
63.7¶¶
–
103.3
105.3
129.7
130.2
116.9
102.7
–
77.7¶
80.2¶
78.0¶
87.9¶
66.5¶¶
48.7¶¶
–
58.5¶¶
29.0¶¶
5.1##
–
1.6##
–
4.8##
–
ꢁꢁ
–
10 (Rifampin)
100 (Probenecid)
7.8##
5.5##
15.4##
12.7##
OATP1B1- and OATP1B3-mediated uptake volumes of [3H]E217bG are the values in transporter-expressing HEK293 cells minus the corresponding values in non-
expressing control HEK293 cells. OAT1- and OAT2-mediated uptake volumes of [3H]PAH and [3H]E3S are the values in transporter-expressing S2 cells minus the
corresponding values in non-expressing control S2 cells. The % of control represents the ratio of the uptake volume in the presence of inhibitor to that in the
absence of inhibitor (concentration 0 lmol/L).
The statistical significance of differences for the test compound and positive control compound for inhibition studies was determined by the Williams test
(lower-tailed) and unpaired t-test (Student’s or Welch), respectively, using SAS software (Release 9.4). ¶p < 0.05, ¶¶P < 0.01, ##p < 0.01.
The uptake volume and the % of control are expressed as mean SD or mean of triplicate determinations.
ꢁ
The value in the parenthesis represents the uptake volume into non-expressing cells, indicating large transporter-mediated uptake by a comparison of trans-
porters-expressing and non-expressing cells. –: not tested.
ꢁꢁ
values of 8.39 and 9.13 mmol/L, respectively, whereas brexpi-
prazole and DM-3411 at 30 mmol/L had no effect on the
Inhibition of BSEP vesicular transports by brexpiprazole
and DM-3411
[3H]E217bG uptakes by OATP1B3 (Tables
4
and 7).
Brexpiprazole and DM-3411 at 30 mmol/L slightly inhibited
the BSEP-mediated uptake of [3H]TCA into vesicles which
was measured by subtracting the uptake into control vesicles
from that into BSEP-expressing vesicles (Table 6 and 7).
Brexpiprazole did not affect the [3H]PAH and [3H]E3S uptakes
by OAT1- and OAT3-expressing S2 cells, respectively, whereas
DM-3411 diminished their uptake slightly (Table 4).
Brexpiprazole and DM-3411 inhibited the uptakes of
[14C]metformin by OCT1-, OCT2-, MATE1-, or MATE2-K-
expressing HEK293 cells (Table 5). The IC50 values for brexpi-
prazole and DM-3411 were similar with the exception of
MATE2-K, in which the IC50 value of DM-3411 was 10-fold
lower than that of brexpiprazole (0.154 mmol/L for DM-3411
and 1.57 mmol/L for brexpiprazole, Table 7).
Discussion
Brexpiprazole is mainly eliminated through metabolism by
CYP3A4 and CYP2D6 (Sasabe et al. 2021). When brexpipra-
zole was concomitantly administered with strong inhibitors,

532
H. SASABE ET AL.
Table 5. Inhibitory effects of brexpiprazole and its metabolites, DM-3411, on OCT-mediated [14C]metformin uptakes, MATE-mediated [14C]metformin uptakes.
Uptake volume into
OCT1-expressing cells
(lL/mg protein/5 min)
Uptake volume into
OCT2-expressing cells
(lL/mg protein/2 min)
Uptake volume into
MATE1-expressing cells
(lL/mg protein/5 min)
Uptake volume into
MATE2-K-expressing cells
(lL/mg protein/5 min)
Concentration
(lmol/L)
4.91 0.64
(1.18 0.03)
7.45 0.50
(1.83 0.13)
76.2 1.0
(0.64 0.06)
68.4 2.0
(0.67 0.12)
18.3 0.9
(1.88 0.22)
20.8 2.1
(1.57 0.24)
10.9 0.9
(1.06 0.04)
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
% of control
0 (Control)
Inhibitor
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
0.1
0.3
1
3
10
30
87.4
101.6
102.1
82.2
79.7
96.5
103.7
102.8
96.0
99.6
77.9¶¶
80.4¶¶
55.0¶¶
35.3¶¶
17.0¶¶
7.3¶¶
–
83.8¶¶
75.4¶¶
47.4¶¶
39.6¶¶
11.6¶¶
5.1¶¶
–
59.0¶¶
39.3¶¶
16.6¶¶
12.4¶¶
4.9¶¶
2.2¶¶
–
73.7¶
74.8¶
84.4¶
71.1¶¶
18.4¶¶
ꢀ6.0##
105.6
99.7
78.4¶¶
71.1¶¶
45.9¶¶
32.3¶¶
13.2¶¶
110.7
82.5
82.6¶¶
40.3¶¶
35.0¶¶
1.2##
50.0¶¶
39.1¶¶
1.1##
27.9¶¶
0.8##
ꢁꢁ
–
100 (Quinidine)
10
15.6##
14.1##
100 (Cimetidine)
10.7##
OCT1-, OCT2-, MATE1-, and MATE2-K-mediated uptake volumes of [14C]metformin are the value in transporter-expressing HEK293 cells minus the corresponding
values in non-expressing control HEK293 cells. The % of control represents the ratio of the uptake volume in the presence of inhibitor to that in the absence of
inhibitor (concentration, 0 lmol/L).
The statistical significance of differences for the test compound and positive control compound for inhibition studies was determined by the Williams test
(lower-tailed) and unpaired t-test (Student’s or Welch), respectively, using SAS software (Release 9.4).¶p < 0.05,¶¶P < 0.01,##p < 0.01.
The uptake volume and the % of control are expressed as mean SD or mean of triplicate determinations.
ꢁ
The value in the parenthesis represents the uptake volume into non-expressing cells, indicating large transporter-mediated uptake by a comparison of trans-
porter-expressing and non-expressing cells. –: not tested.
ꢁꢁ
brexpiprazole and DM-3411 exhibited weak inhibitory effects
on CYP2B6, CYP2D6, CYP2C9, CYP2C19, and CYP3A4 with
IC50 values sufficiently higher than the unbound Cmax in
human (Table 1). For each these Cmax,u/IC50 ratios were less
than 0.02 which represents the regulatory cut-off value for
potential DDI risk (EMA 2012, PMDA 2019, U.S. Department
of Health and Human Services 2020). It is important to note
that the fu value is 0.01, as in humans brexpiprazole is highly
protein bound (99.8%) (Sasabe et al. 2021). Moreover, brexpi-
prazole has a weak potency of mechanism-based inactivation
(MBI) against CYP3A4. The DDI which resulted from MBI is
known to occur after the dosing period of the inhibitory
agent. When bupropion (CYP2B6 substrate), dextromethor-
phan (CYP2D6), or lovastatin (CYP3A4) were administered
with multiple doses of brexpiprazole in a clinical DDI study
(Otsuka America Pharmaceutical, Inc 2015), the AUC values
of these three agents were not changed, when compared to
each alone.
In the in vitro CYP induction studies using human hepato-
cytes, brexpiprazole did not exhibit strong induction potency
for CYP1A2, CYP2B6, CYP2C9, or CYP3A4 (Table 2). For
in vitro evaluation of drug–drug interaction for an NME, CYP
enzymes CYP3A4, CYP2B6 and CYP1A2 should always be
included as markers of induction in the basic methodology
of CYP induction as described by the EMA (2012) guideline.
This approach is based on the molecular mechanisms of CYP
induction via nuclear receptors of pregnane X receptor (PXR,
for CYP3A4 and CYP2C9), constitutive androstane receptor
(CAR, for CYP2B6) and the aryl hydrocarbon receptor (AhR,
for CYP1A2). Recently, CYP2D6 has been reported to be indu-
cible in one paper (Farooq et al. 2016). However, this induc-
tion is currently limited to endogenous and exogenous
corticosteroids and rifampin, and its exact inductive molecu-
Table 6. Inhibitory effects of brexpiprazole and its metabolite, DM-3411, on
BSEP-mediated vesicular uptake of [3H]TCA.
Concentration
Uptake volume into BSEP-expressing vesicles
(lmol/L)
(lL/mg protein/2 min)
0 (Control)
341.5 65.5
% of control
brexpiprazole
340.2 102.0
Inhibitor
DM-3411
0.3
1
3
10
99.1
134.4
119.7
103.9
66.4¶
0.0#
83.7
108.8
102.5
81.8
30
54.0¶¶
9.1#
30 (Cyclosporin A)
The BSEP-mediated uptake volume of [3H]TCA is the value in the BSEP-
expressing vesicles minus that in the non-expressing control vesicles.
The % of control represents the ratio of the uptake volume in the presence of
inhibitor to that in the absence of inhibitor (concentration 0 lmol/L).
The statistical significance of differences for the test compound and positive
control compound for inhibition studies was determined by the Williams test
(lower-tailed) and unpaired t-test (Student’s or Welch), respectively, using
#
Statistical SAS software (Release 9.4). ¶p < 0.05, ¶¶P < 0.01, p < 0.05.
The uptake volume and the % of control are expressed as mean SD or
mean of triplicate determinations.
itraconazole (CYP3A4) or quinidine (CYP2D6), both of AUC
values increased to approximately two fold (Garnock-Jones
2016), respectively. Additionally, when brexpiprazole was also
administered to the poor metaboliser of CYP2D6, the AUC
value was approximately twice of that in the extensive
metaboliser (Ishigooka et al. 2018). Therefore, CYP3A4 and
CYP2D6 are thought to contribute equally to the metabolic
elimination profile of brexpiprazole. In the labelling sheet of
brexpiprazole, the results were summarised as ‘the dosage
adjustments of brexpiprazole are recommended when brex-
piprazole is administered with strong CYP2D6 inhibitors (e.g.
paroxetine, fluoxetine, and quinidine) or to CYP2D6 poor lar mechanisms and direct implications for CYP2D6 induction
metaboliser’. Conversely as demonstrated in this study, in a clinical setting are still not clear. Though brexpiprazole

XENOBIOTICA
533
Table 7. Summary of the inhibitory effects of brexpiprazole and its metabolite, DM-3411, on ABC transporters and SLC transporters.
Brexpiprazole
DM-3411
Brexpiprazole
DM-3411
ꢁ
ꢁ
ꢁ
ꢁ
max,u/IC50
Transporters
IC50 [SE] (mmol/L)
Dose/250 mL/IC50
Cinlet,max,u/IC50
Cmax,u/IC50
C
MDR1
BCRP
OATP1B1
OATP1B3
OAT1
OAT3
OCT1
6.31 [2.38]
1.16 [0.32]
8.39 [5.57]
>30
>30
7.84 [5.04]
3.04 [1.56]
9.13 [2.49]
>30
>30
>30
11.4 [2900]
6.50 [0.75]
1.83 [1.07]
0.154 [0.014]
>30
5.8
32
–
0.0015
0.0084
0.0012
0.00060
0.0033
0.00045
–
0.00066
0.0017
0.00057
ꢁꢁ
ꢁꢁꢁ
–
–
–
–
–
–
–
–
–
–
–
–
–
>30
¶
13.0 [9.3]
4.27 [1.82]
2.79 [1.65]
1.57 [0.77]
>30
0.00075
0.00029
0.00089
0.0014
0.0024
–
0.00046
0.00080
0.0028
0.034
–
OCT2
–
MATE1
MATE2-K
BSEP
0.0035
–
–
The value and inequality sign indicate the maximum concentration of the test compound in the inhibition study when 50% inhibition was not
observed even at the maximum concentration. The IC50 and the estimated SE were obtained by the nonlinear regression of the mean remaining
activity (% of control, triplicate determinations) from the control (0 lmol/L) to the Emax model equation, using WinNonlin (Ver. 6.1).
ꢁ
The parameters when brexpiprazole was administered to Japanese patients at 4 mg for 14 days were used to calculate the ratios for drug–drug
interaction (Ishigooka et al. 2018). The Cmax,u/IC50 value was calculated using free fraction (fu: 0.01 for brexpiprazole, 0.035 for DM-3411) and
plasma Cmax (0.38 mmol/L for brexpiprazole and 0.15 mmol/L for DM-3411) . Cinlet,max,u was calculated using the equation fu ꢃ [Cmax þ (ka ꢃ dose/
Qh)], where the absorption rate constant (ka: 0.1 minꢀ1, regulatory maximum) was obtained from plasma concentration profiles and hepatic blood
flow (Qh: 23.4 mL/min/kg).
ꢁꢁ
–: not applicable.
ꢁꢁꢁ
>30: Inhibitory effects less than 50% at 30 lmol/L of the test compound.
¶The estimated SE is large compared to the IC50 value. The two point values at the lower concentration of the test compound are poorly fitted
because they show the maximum variation that could be tolerated, resulting in a large computer estimate of the SE value. As there was sufficient
inhibition at 30 lmol/L which is a higher concentration than the estimated IC50, we concluded that was confirmed that the estimated IC50 value
could be adopted.
was mainly metabolised by CYP2D6 and CYP3A4, given this primary metabolie, DM-3411, will have low to negligible DDI
background, we performed the CYP induction study with risk for CYP enzymes in the clinical setting.
respect to CYP1A2, CYP2B6 and CYP3A4, based on the
Brexpiprazole was transported neither by P-gp nor by
molecular mechanisms of CYP induction, not self-induction. BCRP (Figure 2), which limit the absorption of oral hydropho-
For CYP1A2, CYP2C9 and CYP3A4, both mRNA level and bic compounds in the apical membrane of the intestine.
activity were measured and compared to levels in untreated These in vitro results are consistent with the report that the
control hepatocytes. The results indicated very similar con- bioavailability of brexpiprazole in human is nearly 100%, and
clusions that brexpiprazole has no induction potency. For the absorptive fraction is thought to complete when brexpi-
CYP2B6, changes in the mRNA level were hardly observed on prazole is orally administered (Garnock-Jones 2016).
treatment with brexpiprazol. Considering that mRNA is a Contrarily, the inhibition of brexpiprazole for the transport
much more sensitive method that provides greater dynamic functions of P-gp and BCRP was observed in in vitro trans-
range, while showing a good relationship with enzyme activ- port studies across the expressing cell monolayers. When
ity (Fahmi and Ripp 2010), the enzyme activity of CYP2B6 is their IC50 values were compared with the expected max-
also expected not to be changed by drug treatment.
imum concentration in the gut lumen after oral dosing of
The more hepatocyte sample are used in the testing, the 4 mg (4 mg ꢅ 250 mL ¼ 37 mmol/L), the ratios were greater
better the system will be. For in vitro evaluation of drug–- than 10, which is the cut-off position for three regulatory
drug interactions for a New Molecular Entity (NME), the FDA, agencies for conduction of clinical study to evaluate of DDI
EMA, and PMDA specify use of hepatocytes derived from risk (Table 7). It may follow that brexpiprazole has a potential
three or more donors, since cultured human hepatocytes for DDI risk by P-gp and BCRP. However, the AUC values of
have great inter-individual variability or lot-to-lot variation fexofenadine and rosuvastatin, which are substrates for P-gp
(EMA 2012, PMDA 2019, U.S. Department of Health and and BCRP, respectively, were not changed when both were
Human Services 2020). With this background, we used hepa- co-administered with brexpiprazole in a clinical DDI study
tocytes from three donors under established and validated (Otsuka America Pharmaceutical, Inc 2015).
experimental procedures (Supplementary Tables 3 and 4),
When inhibition or transport studies of intestinal trans-
with which the increase seen with control inducers could be porters such as P-gp and BCRP, are conducted in vitro, these
observed at a level that is comparable to reported results, in evaluations are required at a broad range of inhibitor and
this study and also in previous in-house studies. Therefore, substrate’s concentrations, considering the physiologically
we believe that the results presented here could verify the relevant concentrations described above. However, the inhib-
inductive potency of test compounds. In addition, many ition of brexpiprazole was evaluated in the experiment of
results using hepatocytes from three individuals have been the inhibitor concentration until 30 mmol/L, which is limited
reported from pharmaceutical company researchers and aca- due to the solubility in the transport buffer. But, the IC50 val-
demic researchers, just as in our experiment, and the results ues of brexpiprazole could be obtained accurately.
of in vitro studies are conclusive. From the results obtained Meanwhile, the transport experiment of brexpiprazole was
in these studies, one can conclude that brexpiprazole and its performed in the cell monolayers only at low donor

534
H. SASABE ET AL.
concentrations of 1 mmol/L. In the cell monolayers, the mem- DM-3411, the ratio (0.034) of the maximum plasma unbound
brane permeability generally constitutes passive diffusion concentration and IC50 values are above the regulators’ cut-
and transporter-mediated efflux. Thereby, the function of the off (0.02). The accurate prediction of interaction for MATEs is
transporter at high concentration may be diminished by sat- challenging, because MATEs and OCT2 are thought to often
uration. For example, the apical to basal permeability of the share the same substrate, and the inhibition of MATEs may
cause only an increase in intracellular concentration of sub-
strates, not reflecting the systemic exposure (Yoshida et al.
2017). Although the systemic unbound concentration
(5.3 nmol/L) is sufficiently much lower than the IC50 value, a
close observation on the renal function may be needed
when a MATE substrate, like the antidiabetic agent, metfor-
min is concomitantly administered in the clinic.
BSEP is expressed on the bile canalicular membrane of
hepatocytes and functions as an exporter of bile acids to
bile. Its drug-mediated functional dysfunction leads to the
intrahepatic accumulation of bile acids, which results in
severe liver damage, a form of drug-induced liver injury
(DILI) (Funk et al. 2001, Yoshikado et al. 2013). Brexpiprazole
did not inhibit BSEP and therefore, may have a low clinical
risk for DILI due to the function of BSEP.
In summary, in the in vitro assessments using the human
hepatic microsomes, brexpiprazole and its major metabolite,
DM-3411 exhibited weak inhibitory effects on the activities
of several CYPs (CYP2B6, CYP2D6, CYP2C9, CYP2C19, and
CYP3A4). In the comparisons with the IC50 (or Ki value) and
the systemic concentration (Cmax,u or Cinlet,max,u), the ratio
was low and therefore, not believed to induce any drug
interaction when a potential CYP substrate is concomitantly
administered with brexpiprazole. In studies using human
transporter-expressing-cells, brexpiprazole exhibited weak or
negligible inhibitory effects at the hepatic and renal SLC
transporters (OATPs, OCTs, OATs, and MATEs) and efflux
transporters (MDR1, BCRP, and BSEP) investigated. When a
comparison of IC50 to systemic Cmax,u or Cinlet,max,u is per-
formed, the risk for DDI is considered to be minimal, this is
also true for CYPs, as well. Overall, brexpiprazole did not
exhibit relevant changes in the clinical DDI studies per-
formed together with their CYP or transporter substrates.
These in vitro data suggest that brexpiprazole is unlikely to
cause clinically relevant drug interactions resulting from the
effects on CYPs or transporters mediating the absorption,
metabolism, and/or disposition of co-administered drugs.
These findings are particularly significant for patients with
mental disorders who are treated with brexpiprazole in add-
ition to other medications to manage their psychi-
atric symptoms.
substrates for P-gp (quinidine, verapamil, and vinblastine)
increased when their apical side concentration was increased
and reached a maximum value at the higher concentration
range, whereas the basal to apical permeability was
decreased (Shirasaka et al. 2008). These results demonstrated
that saturation of the efflux transporters would decrease the
permeability ratio, suggesting that the assay for a substrate
of efflux transporters would be less sensitive at higher con-
centrations. It was confirmed that brexpiprazole is not a sub-
strate of P-gp and BCRP at a sufficiently low concentration
(1 lmol/L); thereby, it is considered that brexpiprazole would
not be a substrate of these transporters even when testing
at assumed higher concentrations in the gut lumen.
Next, when the interaction risks of DM-3411 for P-gp and
BCRP are considered by comparison of the compound con-
centration with IC50, the systemic unbound concentration
should be used rather than the concentration in the gut
lumen, because, the metabolite is produced in the systemic
compartment after absorption. The ratios of systemic Cmax,u
to the IC50 values are lower than 0.02, which is 3 regulator’s
criteria (Table 7), and are sufficiently low, thus not affecting
the transport function of P-gp and BCRP. The P-gp- and
BCRP-mediated interactions of brexpiprazole and the main
metabolite, DM-3411, are considered to be not induced in
the clinical setting.
Brexpiprazole is mainly eliminated through metabolism, as
the parent compound was not detected in human urine
probably due to its high lipophilic property and high protein
binding (Garnock-Jones 2016). Brexpiprazole has undergone
several oxidative reactions, for example, hydroxylation,
dehydrogenation, and N-dealkylation. These reactions usually
occurred in the liver after transferring to the inside of hepa-
tocytes. SLC transporters, OATPs and OCT1 recognise anionic
organic compounds and cationic organic compounds,
respectively, on the plasma membrane of hepatocytes. These
transporters take in such substrates into the inside of hepa-
tocytes from circulating blood. As a result, they accelerate
the subsequent hepatic metabolism (Izumi et al. 2017).
Brexpiprazole is not a substrate for any uptake transporters
(Figures 2 and 3). Thus, it is considered that brexpiprazole
transfers through the plasma membrane only by passive dif-
fusion. This evidence suggests that brexpiprazole has hardly
undergone the uptake transporter-mediated pharmacokinetic
changing by any concomitant agents. On the contrary, brex-
piprazole and DM-3411 exhibit inhibitory effects on the
transport activities of OATP1B1, OCT1, OCT2, MATE1, and
MATE2-K in the in vitro studies on transporter-expressing
cells (Table 7). Except MATE2-K, the ratio values of systemic
concentration and IC50 values were much lower than 0.1
(FDA, PMDA guideline’s cut-off) and 0.02, (EMA’s cut-off)
(Table 7). Thereby, DDI through these uptake transporters is
not considered to be induced in the clinical concomitant
Disclosure statement
The authors report no declarations of interest.
Acknowledgments
We are grateful to Kenji Maeda Ph.D. and Mikio Suzuki Ph.D. and the
many members at REXULTI Project of Otsuka Pharmaceutical Co., Ltd. for
their advices with the scientific discussion. The authors thank Takuya
Kuno Ph.D. in our division for his help in statistical analyses. And, we are
grateful to the many members at the ADME & Toxicology Research
dosing with brexpiprazole. For the MATE2-K inhibition of Institute of Sekisui Medical Co., Ltd. and XenoTech, LLC, and at the

XENOBIOTICA
535
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