Enantioselective in vitro metabolism and in vitro-in vivo correlation of the herbicide ethofumesate in a human model

Article abstract of DOI:10.1016/j.jpba.2020.113349

Ethofumesate (ETO) is a chiral herbicide that is marketed as a racemic mixture in the European Union and the United States. The growing consumption of pesticides in the world, along with their presence in water and food, has increased human exposure to these chemicals. Another issue concerning these compounds is that each enantiomer of a chiral pesticide may interact with biomolecules differently. For this reason, this study aimed to investigate the in vitro metabolism of ethofumesate (the racemic mixture as well as the isolated enantiomers) by human liver microsomes (HLM) and to explore the in vitro-in vivo correlation. Before the kinetics was determined, the method was fully validated by evaluating its selectivity, linearity, precision, accuracy, carryover, and stability. All the evaluated parameters agreed with the European Medicines Agency guideline. The enzyme kinetic parameters and the in vitro-in vivo correlation demonstrated that there was no enantioselective difference for the metabolism and bioavailable fraction of each enantiomer. The enzyme kinetics was biphasic; the K_M1 values were 15, 5.8, and 5.6 for rac-ETO, (+)-ETO, and (–)-ETO, respectively. The total in vitro intrinsic clearance was 0.10 mg mL min^?1 mg^?1 for rac-ETO and its enantiomers. The enantiomer (–)-ETO was only metabolized by CYP2C19, while (+)-ETO was metabolized by both CYP2C19 and CYP3A4. CYP2C19 polymorphism and/or inhibition may represent a risk for humans exposed to this pesticide.

Full text of DOI:10.1016/j.jpba.2020.113349

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Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
journal homepage: www.elsevier.com/locate/jpba
Enantioselective in vitro metabolism and in vitro-in vivo correlation of
the herbicide ethofumesate in a human model
a
a
a
Icaro Salgado Perovani , Daniel Blascke Carrão , Nayara Cristina Perez de Albuquerque ,
a,b,∗
Anderson Rodrigo Moraes de Oliveira
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil
a
b
National Institute for Alternative Technologies of Detection, Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT–DATREM),
Unesp, Institute of Chemistry, P.O. Box 355, 14800-900, Araraquara, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethofumesate (ETO) is a chiral herbicide that is marketed as a racemic mixture in the European Union
and the United States. The growing consumption of pesticides in the world, along with their presence
in water and food, has increased human exposure to these chemicals. Another issue concerning these
compounds is that each enantiomer of a chiral pesticide may interact with biomolecules differently. For
this reason, this study aimed to investigate the in vitro metabolism of ethofumesate (the racemic mixture
as well as the isolated enantiomers) by human liver microsomes (HLM) and to explore the in vitro-in
vivo correlation. Before the kinetics was determined, the method was fully validated by evaluating its
selectivity, linearity, precision, accuracy, carryover, and stability. All the evaluated parameters agreed
with the European Medicines Agency guideline. The enzyme kinetic parameters and the in vitro-in vivo
correlation demonstrated that there was no enantioselective difference for the metabolism and bioavail-
able fraction of each enantiomer. The enzyme kinetics was biphasic; the KM1 values were 15, 5.8, and
5.6 for rac-ETO, (+)-ETO, and (–)-ETO, respectively. The total in vitro intrinsic clearance was 0.10 mg mL
Received 9 January 2020
Received in revised form 27 April 2020
Accepted 28 April 2020
Available online xxx
Keywords:
Herbicide
ethofumesate
human liver microsomes
enantioselective in vitro metabolism
in vitro-in vivo correlation
GC-MS
−1
−1
min mg for rac-ETO and its enantiomers. The enantiomer (–)-ETO was only metabolized by CYP2C19,
while (+)-ETO was metabolized by both CYP2C19 and CYP3A4. CYP2C19 polymorphism and/or inhibition
may represent a risk for humans exposed to this pesticide.
©
2020 Elsevier B.V. All rights reserved.
1
. INTRODUCTION
electivity bioactivity, and toxicity. Its most bioactive enantiomer
(2R,4S)-difenoconazole) is the least toxic against non-target organ-
(
In the modern world, expansion of the world population has
isms, thus marketing of the single enantiomer rather than the
racemic mixture has been recommended [3].
made the use of pesticides crucial for food production [1]. Accord-
ing to the Food and Agriculture Organization (FAO), pesticide use
in the world increased from 2.2 million tons in 1990 to 4.1 mil-
lion tons in 2017 [2]. This increase suggests that human exposure
to these chemicals has become more frequent. About 30% of the
currently marketed pesticides are chiral, and only 7% of them are
marketed as a single enantiomer [1]. In a chiral environment, such
as biological organisms, each enantiomer of a chiral pesticide may
engage in different interactions. The enantioselective behavior of
pesticides in biological organisms has been extensively explored
recently. The triazole fungicide difenoconazole displays stereos-
This enantioselective behavior may also affect enzyme-
mediated processes including absorption, disposition, metabolism,
and excretion. The enantioselective metabolism could lead to pref-
erential clearance of one enantiomer and bioaccumulation of the
other [1]. This behavior has been demonstrated for the chiral tri-
azole fungicide myclobutanil in an in vitro metabolism study that
employed human liver microsomes (HLMs). HLMs did not metab-
olize the enantiomer R-(–)-myclobutanil, while cytochrome P450
(CYP450) mediated the metabolism of the other enantiomer [4].
This has raised concern as to whether pesticides, which are still
marketed as racemates, are safe regarding human exposure.
Ethofumesate (ETO) is a chiral herbicide (Fig. 1). It acts by
inhibiting mitosis and reducing photosynthesis and plant respi-
ration [5]. It is currently marketed as racemate in the European
Union [6], the US [7], and other countries. ETO has been detected
in surface water in Hungary [8] and Spain [9], in air in France
∗ Corresponding author at: Departamento de Química, Faculdade de Filosofia,
Ciências e Letras de Ribeirão Preto – USP, Av. dos Bandeirantes, 3900, Ribeirão Preto,
São Paulo, 14040-901, Brasil.
E-mail address: deoliveira@usp.br (A.R.M. de Oliveira).
https://doi.org/10.1016/j.jpba.2020.113349
0
731-7085/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

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I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (xxxx) xxx
Fig. 1. Chemical structure of the herbicide ethofumesate and its metabolite etofumesate-2-hydroxy. *chiral center.
[
10], and in human blood serum in China [11]. These findings
Human liver microsomes (HLMs, from a pool of 150 donors) and
recombinant CYP450 isoforms (rCYP450 s) were purchased from
Corning Life Science (Phoenix, AZ, USA) and stored at −80 C.
suggest that humans are exposed to ETO, and that human pop-
ulations have already been contaminated by the pesticide. The
metabolism and cytotoxicity of ETO enantiomers are enantiose-
lective in rat hepatocytes [12]. In rat and rabbit liver microsomes,
ETO metabolism displays an enantioselective behavior [13]. In
fact, in rabbit liver microsomes, formation of the metabolite
ethofumesate-2-hydroxide (ETO-2-OH, Fig. 1) maintains the stere-
oselectivity that had been previously observed for ETO depletion
in rabbit liver microsomes [13]. Additionally, interspecies differ-
ences have been observed for ETO [12,13], which may impact risk
evaluation of this pesticide in humans based on results obtained in
other species. From this perspective, this work aims to determine
whether the cytochrome P450 enzymes present in HLMs metabo-
lize ETO enantioselectively and to find out which enzyme(s) is(are)
involved in this process.
◦
All the solvents were HPLC grade. Methanol and acetonitrile
were obtained from Panreac (Barcelona, Spain); ethyl acetate,
hexane, and isopropanol were acquired from Honeywell Riedel-
de Häenm (Seelze, Germany). The other reagents were analytical
grade. Tris(hydroxymethyl)aminomethane was acquired from J.T.
Baker (Phillipsburg, NJ, USA); potassium chloride was obtained
from Mallinckrodt (Xalostoc, MX, Mexico); and sodium phosphate
monobasic, sodium phosphate dibasic, and hydrochloric acid were
purchased from Synth (Diadema, SP, Brazil). Ultrapure water was
obtained from a Direct-Q3 system acquired from Millipore (Bil-
lerica, MA, USA). The chiral column that was used to isolate the
enantiomers was Chiralcel OD-H (150 × 4.6 mm, 5 ␮m), acquired
from Daicel Chemical Industries (Tokyo, Japan).
The in vitro metabolism studies were conducted with the
racemic mixture and with the isolated enantiomers, and the results
were compared. In addition, an in vitro-in vivo correlation was per-
formed, and the hepatic clearance, the hepatic extraction ratio,
and the bioavailable fraction were predicted. The analysis was
performed by gas chromatography tandem mass spectrometry
2
.2. ETO isolation and determination of its optical rotation sign
The enantiomers were isolated in a Shimadzu HPLC sys-
tem (Kyoto, Japan) consisting of two LC-6AD solvent pumps, an
SCL-10AVP system controller, and a UV/VIS SPD-10AV detector
operating at 230 nm. The chiral separation was carried out at room
(
GC-MS) and the bioanalytical method was validated to ensure data
reliability.
◦
temperature (22 ± 2 C) on a Chiralcel OD-H column (150 x 4.6 mm,
5
␮m) and hexane:isopropanol (98:2, v/v) was employed as mobile
−
1
phase at a flow rate of 1.0 mL min . Sequential manual injec-
2
. MATERIALS AND METHODS
−
1
tions of 100 ␮L of rac-ETO (10 mg mL ) solutions prepared in
hexane:isopropanol (90:10, v/v) were conducted. After that, each
enantiomer was collected separately at the column end, dried
under vacuum in a sample concentrator model Concentrator Plus
acquired from Eppendorf (Hamburg, Germany), and solubilized in
methanol. The enantiomer fraction of each enantiomer after isola-
tion was greater than 98%. To determine the optical rotation sign of
the enantiomers, the electronic circular dichroism (ECD) spectrum
of each enantiomer solubilized in the mobile phase was recorded
with Jasco J-810 (Tokyo, Japan). The spectrum was registered from
190 to 350 nm after correction with a blank solution. A cell with
2.1. Chemicals, reagents, and chiral columns
Racemic ethofumesate (rac-ETO) (≥99%) was purchased from
Sigma-Aldrich (St. Louis, MO, USA), and racemic ethofumesate-
2
-hydroxy (rac-ETO-2-OH) (≥99%) was obtained from Dr. Ehren-
storfer (Augsburg, Germany). Phenacetin (≥98%), used as internal
standard (IS), was acquired from Sigma-Aldrich (St. Louis, MO,
−
1
USA). Standard of rac-ETO (10000 ␮mol L ) and rac-ETO-2-OH
−
1
(
975 ␮mol L ) stock solutions were prepared in methanol. A
−
1
standard IS stock solution (6.70 ␮mol L ) was prepared in ace-
tonitrile. The following selective CYP450 chemical inhibitors were
employed in the CYP450 phenotyping study: ␣-naphthoflavone
◦
path length of 1 mm was employed at 25 C; four scans were accu-
mulated.
(
≥98%), ticlopidine (≥99%), montelukast (≥98%), sulfaphenazole
(
≥98%), ketoconazole (≥98%), and 4-methylpirazole (≥95%), which
2.3. Solubility in the incubation medium
were supplied by Sigma-Aldrich (St. Louis, MO, USA). The stan-
−
1
−1
dard ␣-naphthoflavone (80 ␮mol L ), ticlopidine (240 ␮mol L
and 800 ␮mol L ), montelukast (80 ␮mol L ), sulfaphenazole
(
L
ETO solubility in the incubation medium was evaluated to
ensure that all the ETO in the incubation medium would be soluble
and, therefore, available for metabolism. For this assay, an aliquot
−1
−1
− −1
1
800 ␮mol L ), quinidine (80 ␮mol L ), ketoconazole (40 ␮mol
−
1
−1
−1
), and 4-methylpirazole (4000 ␮mol L ) stock solutions were
of standard rac-ETO stock solution (10000 ␮mol L ) was pipet-
−
1
prepared in acetonitrile. The NADPH regeneration system com-
ted into a microtube, to yield 200, 250, or 300 ␮mol L in 200 ␮L
of incubation medium. The solvent was evaporated in a speed
vacuum system model Concentrator Plus. The incubation medium
comprised 4 ␮L of methanol, 146 ␮L of phosphate buffer solution
ponents ␤-nicotinamide adenine dinucleotide phosphate hydrate
+
(
NADP ), glucose-6-phosphate sodium salt, and the enzyme
glucose-6-phosphate dehydrogenase were purchased from Sigma-
Aldrich (St. Louis, MO, USA). The ␤-nicotinamide adenine
−
1
−1
(100 mmol L , pH 7.4), and 50 ␮L of tris-KCl buffer (50 mmol L
+
−1
−1
dinucleotide phosphate hydrate (NADP ) (2.5 mmol L ), glucose-
tris(hydroxymethyl)aminomethane and 150 mmol L KCl pH 7.4).
Control samples for each concentration were prepared in 200 ␮L
of methanol, which represented the condition in which rac-ETO
was completely soluble. The incubation medium samples were cen-
−
1
6
-phosphate sodium salt (50 mmol L ), and glucose-6-phosphate
dehydrogenase (8.0 U mL ) stock solutions were prepared in a tris-
tris(hydroxymethyl)aminomethane and
50 mmol L KCl pH 7.4). All the solutions were stored at −20 C.
−
1
−1
KCl buffer (50 mmol L
1
−
1
◦
◦
trifuged at 1600 × g and 4 C for 10 min in a Hitachi HIMAC CF 15D2
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

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3
centrifuge (Tokyo, Japan). Next, 50 ␮L of the incubation medium
samples were collected, and 50 ␮L of methanol was added. For the
control samples, 50 ␮L was collected, and 50 ␮L of phosphate buffer
ratio and the analyte concentration in the incubation medium was
evaluated by using 1/x as the weighting factor. The simplest math-
2
ematical model that best fitted the data and presented random
residual distribution was employed. The ANOVA lack of fit test
with a 95% confidence level and n–1 degrees of freedom was used
to ensure that the mathematical model fit the data well. The sta-
tistical calculation was performed with the Minitab 18 Statistical
software (State College, PA, USA). Accuracy and precision were
expressed as the relative error (RE) and the coefficient of varia-
tion (CV), respectively. Accuracy and precision were evaluated for
four concentration levels: (i) LLOQ, (ii) low-quality control (LQC),
(iii) medium-quality control (MQC), and (iv) high-quality control
(HQC). Accuracy and precision were evaluated within-day (n = 5)
and between-days (n = 3). The RE and CV acceptance criteria were
± 20% and 20% for the LLOQ, respectively, and ± 15% and 15% for
the other quality controls (LQC, MQC, and HQC), respectively. Car-
ryover was assessed by analyzing a blank sample before and after
analysis of the upper limit of quantification. If the signal for each
analyte and the IS was lower than 20% of the LLOQ and 5% of the
IS signal, respectively, the carryover effect was considered absent.
rac-ETO and rac-ETO-2-OH stability was evaluated in three differ-
ent conditions: (i) autosampler for the total analysis time (27 h),
−1
solution (100 mmol L , pH 7.4) was added. The incubation medium
and the control samples were analyzed by HPLC. The HPLC system
used was acquired from Shimadzu (Kyoto, Japan). It was equipped
with an LC-20AT and an LC-20AD solvent pumps, a CTO-20A column
oven, an SPD-M20A operating at 230 nm, and a CBM-20A com-
munication module. The analyses were carried out on an Ascentis
®
Express Fused Core C18 (150 × 4.6 mm, 5 ␮m) analytical column
◦
at 30 C and methanol:water (80:20, v/v) was used as mobile phase
−1
at a flow rate of 0.8 mL min . The solubilities of the samples and the
controls were compared and evaluated by Student t-test with 95%
confidence interval using the software GraphPad Prism 6 software
(
San Diego, CA, USA).
2.4. In vitro incubation medium and sample preparation
The incubation medium consisted of 4 ␮L of rac-ETO, (+)-ETO,
−
1
or (–)-ETO; 40 ␮L of HLMs (1.0 mg mL microsomal protein), and
1
−1
06 ␮L of phosphate buffer (100 mmol L , pH 7.4) in a total
incubation volume of 200 ␮L. Preincubation was conducted in a
◦
◦
thermostatic water bath at 37 C for 5 min. The reaction was started
(ii) storage at −20 C for 27 h, and (iii) in the incubation medium
◦
by adding 50 ␮L of the NADPH cofactor solution. The reaction was
stopped by adding ethyl acetate (800 ␮L) and beginning the sample
preparation procedure. The samples were extracted at 1000 rpm in
at 37 C for 30 min. The stability study was carried out at two con-
centration levels (LQC and HQC). The concentration determined for
each stability condition was compared to the nominal concentra-
tion, and samples were considered stable if degradation was lower
than 15%.
®
a Vibrax VXR sample shaker (IKA, Staufen, Germany) for 5 min.
◦
After extraction, the samples were centrifuged at 1600 × g and 4 C
for 10 min in a Hitachi HIMAC CF 15D2 centrifuge. Next, 640 ␮L
of the organic phase was collected and evaporated in a sample
concentrator model Concentrator Plus. Finally, the samples were
reconstituted in 100 ␮L of acetonitrile and analyzed by GC-MS.
2.7. ETO racemization
ETO enantiomer racemization was evaluated in the incuba-
◦
tion medium at 37 C for 30 min to ensure that no racemization
2
.5. GC-MS analysis
occurred during the enzyme kinetic study. For this evaluation, the
samples were spiked with the isolated ETO enantiomers and sub-
jected to the same incubation conditions used in the metabolism
studies. The samples were analyzed by using the enantioselec-
tive chromatographic conditions described in section 2.2 with
slight modifications. The chiral separation was performed on the
rac-ETO, (+)-ETO, (–)-ETO, and rac-ETO-2-OH were analyzed
by GC-MS in a Shimadzu apparatus (Kyoto, Japan). The GC-MS
equipment consisted of a GC-2010 column oven, an AOC-20i auto-
matic injector, and a GC-MS QP2010 Plus mass spectrometer. The
data were acquired with the GCMS solution software version 2.72,
also from Shimadzu. Helium (≥99.999%), purchased from White
Martins (Rio de Janeiro, RJ, Brazil), was used as carrier gas. The
chromatographic column was a Zebron ZB-1MS (30 m x 0.25 mm,
◦
Chiralcel OD-H (150 × 4.6 mm, 5 ␮m) column at 30 C and hex-
ane:isopropanol (90:10, v/v) was used as mobile phase. The flow
−
1
rate was 0.8 mL min , and detection was set at 280 nm.
0
.25 ␮m) column acquired from Phenomenex (Torrance, CA, USA).
2.8. Enzyme kinetics
−
1
The carrier gas total flow was 11.5 mL min , the purge flow was
3
was 0.5 ␮L. The injection, ion source, and interface temperatures
were 250 C, and electron ionization (EI) was used as the ionization
method. The temperature program started by heating the column
from 120 to 240 C (15 C min ). After that, the column was heated
from 240 to 280 C (40 C min ). The monitored ions were m/z 286
for ETO and m/z 179 for IS and ETO-2-OH.
−1
.0 mL min , the split ratio was 1:5, and the injection volume
Enantioselective metabolism was assessed by incubating rac-
ETO, (+)-ETO, or (–)-ETO with HLMs. The initial reaction rate (V0)
of rac-ETO-2-OH formation was monitored. To this end, an achiral
GC-MS method was employed to quantify the metabolite rac-ETO-
2-OH in the incubation medium. Initially, the V0 conditions were
◦
◦
◦
◦
◦
−1
−1
−1
determined according to protein concentration (0.2 mg mL ) and
incubation time (10, 20, 30, or 40 min); three different rac-ETO con-
−
1
centrations (1.50, 100, or 200 ␮mol L ) were employed. Linearity
in the formation of the metabolite rac-ETO-2-OH was investigated
for the four incubation times. The enzyme kinetic parameters after
rac-ETO, (+)-ETO, or (–)-ETO metabolism by HLM was determined
by using substrate concentrations ranging from 0.6 to 180 ␮mol
2.6. Bioanalytical Method Validation
The selectivity, linearity, lower limit of quantification (LLOQ),
accuracy and precision, carryover, and stability of the analytes were
evaluated during the bioanalytical method validation. The accep-
tance criteria for each assay was based on the European Medicines
Agency (EMA) bioanalytical method validation guidelines [14].
Selectivity was evaluated by analyzing blank samples of the
incubation medium in the absence of rac-ETO, rac-ETO-2-OH, and
the IS. The result was compared to the analysis of the incubation
medium with HLMs in the presence of ETO and ETO-2-OH. Lin-
−
1
L
(n = 5). The results were plotted as V0 versus the substrate con-
centration. The Eadie-Hofstee plot was employed to determine the
enzyme kinetic model. Finally, the first component of the enzyme
kinetics was fitted with the Michaelis Menten model, and the sec-
ond component was fitted by linear regression to determine the
slope of the enzyme kinetic curve in this phase [15]. The kinetic
parameters (Michaelis Menten constant (KM) and maximum rate
(VMAX)) were obtained by nonlinear regression analysis with the
GraphPad Prism 6 software (San Diego, CA, USA), and the intrinsic
−
1
earity was assessed for ETO (0.0999–200 ␮mol L ) and ETO-2-OH
−1
(
0.0506–19.5 ␮mol L ). The correlation between the normalized
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

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ARTICLE IN PRESS
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I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (xxxx) xxx
clearance of the first component (CLINT1) was calculated by using
Eq. 1 [15].
the bioavailable fraction after hepatic metabolism (f ) is related to
EH.
h
KM1
CLINT1 = V
(1)
2.10. CYP450 reaction phenotyping
MAX1
Where CLINT1 is the intrinsic clearance, KM1 is the Michaelis-
Menten constant of the first component, and VMAX1 is the maximum
rate of the first component. The intrinsic clearance of the second
component (CLINT2) was obtained by the slope of the kinetic curve
in the second phase of the enzyme kinetics. The second component
was distinguished from the first component by using the Eadie-
Hofstee graph. The inflection point indicated the boundary between
both components.
The CYP450 reaction phenotyping study was carried out by
using two different methods: (i) selective chemical inhibitors and
(ii) rCYP450 s. The reaction phenotyping assay was evaluated at two
−
1
concentrations: the KM of the first component, and 180 ␮mol L
to determine the CYP450 isoforms involved in the first and second
components of the biphasic kinetics. In the first method, the fol-
lowing selective chemical inhibitors were used: ␣-naphthoflavone
−
1
−1
(1.0 ␮mol L ) for CYP1A2, ticlopidine (3.0 and 10.0 ␮mol L ) for
−
1
The total in vitro intrinsic clearance (CLINT) was calculated by
using Eq. 2 [16].
CYP2B6 and CYP2C19, montelukast (1.5 ␮mol L ) for CYP2C8, sul-
−
1
faphenazole (10.0 ␮mol L ) for CYP2C9, ketoconazole (0.5 ␮mol
−
1) for CYP3A, and 4-methylpirazole (50.0 ␮mol L−1) for CYP2E1.
L
CLINT = CLINT1 + CLINT2
.9. In vitro-in vivo correlation
The in vitro-in vivo correlation was carried out to predict the
(2)
Ticlopidine is a time-dependent selective inhibitor for CYP2B6,
thus it requires 10-min preincubation with HLMs and NADPH with
further ten- times dilution in the assay incubation medium. The
incubation medium consisted of the selective chemical inhibitor,
2
−
1
−1
rac-ETO (15 and 180 ␮mol L ); (+)-ETO (5 and 180 ␮mol L ), or
−
1
−1
toxicokinetic parameters: hepatic clearance (CLH), hepatic extrac-
tion ratio (EH), and bioavailable fraction (f). For this purpose, the
enantioselective unbound fractions between ETO and plasma (fu,p)
and microsomal (fu,mic) proteins were determined by using the
ultracentrifugation method (n = 3). The rac-ETO, (+)-ETO, and (–)-
ETO concentrations corresponded to their KM values for the first
component of the biphasic kinetics. The plasma samples con-
(–)-ETO (5 and 180 ␮mol L ); HLM (0.2 mg mL ); NADPH; and
phosphate buffer (100 mmol L−1, pH 7.4). The metabolite rac-ETO-
2-OH was quantified in the presence and absence of the chemical
selective inhibitor, and the percentage of inhibition (%I) was deter-
mined as described in Eq. 6 [21]. Participation of the CYP450 isoform
in ETO metabolism was considered significant if the percentage of
inhibition (%I) was greater than 40% [22]. The results were analyzed
with the GraphPad Prism 6 software.
−
1
sisted of human plasma (42 mg mL protein concentration [17]);
−1
−1
rac-ETO (15 ␮mol L ), (+)-ETO (5 ␮mol L ), or (–)-ETO (5 ␮mol
v − v
0
i
−
1); and phosphate buffer (100 mmol L−1, pH 7.4). The micro-
L
%I =
(6)
v
0
somal incubation samples consisted of tris-KCl buffer (50 mmol
−
1
−1
L
tris(hydroxymethyl)aminomethane and 150 mmol L KCl pH
Where %I is the percentage of inhibition, v0 is the reaction rate in
the absence of the chemical selective inhibitor, and vi is the reaction
rate in the presence of the selective chemical inhibitor.
−1
−1
7
.4); phosphate buffer (100 mmol L , pH 7.4); HLMs (0.2 mg mL
−
1
microsomal proteins); and rac-ETO (15 ␮mol L ), (+)-ETO (5 ␮mol
−
1
−1
L
), or (–)-ETO (5 ␮mol L ). The control samples consisted of
For the second method, incubations with rCYP450 s were carried
out. The incubation medium consisted of rac-ETO (15 and 180 ␮mol
−1
phosphate buffer (100 mmol L , pH 7.4); tris-KCl buffer (50 mmol
−
1
−1
−1
−1
−1
L
tris(hydroxymethyl)aminomethane and 150 mmol L KCl pH
L
L
); (+)-ETO (5 and 180 ␮mol L ) or (–)-ETO (5 and 180 ␮mol
−
1
−1
7
.4); and rac-ETO (15 ␮mol L ), (+)-ETO (5 ␮mol L ), or (–)-ETO
); rCYP450 (rCYP1A2, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C19,
−
1
(
5 ␮mol L ). The plasma and the microsomal samples were prein-
rCYP3A4, or rCYP3A5); NADPH; and phosphate buffer (100 mmol
◦
◦
−1
cubated at 37 C for 10 min and centrifuged at 150060 × g and 4 C
for 3 h in a Beckman Optima XL-100 K (Brea, CA, USA). After cen-
trifugation, 200 ␮L of the control, plasma, and microsomal samples
were subjected to the sample preparation procedure described in
section 2.4. To determine the unbound fractions, the microsomal
and the plasma samples were compared to the control samples
representing completely unbound rac-ETO, (+)-ETO, or (–)-ETO. The
L
, pH 7.4). After the incubations, the metabolite rac-ETO-2-OH
was quantified. The metabolite formation rate was multiplied by
the abundance of each CYP450 in the native HLM, which represents
a normalized rate (NR). The NRs for each CYP were added, to provide
a total normalized rate (TNR). The NR of each CYP was expressed
as a percentage of the total normalized rate (%TNR) according to
Eq. 7 [21]. The results were analyzed with the GraphPad Prism 6
software.
CLINT was scaled to in vivo intrinsic clearance (CL
) by using
INT,in vivo
−1
−1
4
0 mg microsomal protein g liver and 21.4 g kg body weight as
NR x 100
TNR
%
TNR =
(7)
scaling factor [18]. Eqs. 3–5 describe the calculation involved in the
prediction of the toxicokinetic parameters CLH, EH, and fh [19,20].
Where %TNR is the total normalized ratio percentage, NR is the
normalized ratio, and TNR is the total normalized ratio.
fu,p
fu,m
Q x
x CL
INT, in vivo
CLH =
(3)
fu,p
fu,m
Q + (
x CLINT, in vivo)
3. RESULTS AND DISCUSSION
fu,p
fu,
x CLINT,in vivo
3.1. ETO and ETO-2-OH enantioseparation
EH =
x 100%
(4)
(5)
fu,p
fu,m
Q + (
x CLINT,in vivo)
Initially, to perform the in vitro enantioselective metabolism
study, the possibility of ETO and ETO-2-OH enantioseparation in
the same run was investigated. However, the metabolite ETO-2-
OH is stereolabile [23]. Therefore, evaluating enantioselective ETO
metabolism by monitoring enantioselective ETO-2-OH formation
would not be feasible. Thus, enantioselective metabolism was eval-
uated by incubating each ETO enantiomer separately (rac-ETO,
(+)-ETO, (−)-ETO) and monitoring rac-ETO-2-OH formation. The
fh = 1 − EH
Where CLH is the hepatic clearance, Q is the hepatic blood flow
− −1
1
(
20 mL min kg [19,20]), fu,p is the fraction of the substrate that
is not bound to the plasmatic proteins, fu,m is the fraction of the
substrate that is not bound to the microsomal proteins, CLINT,in vivo
is the in vivo intrinsic clearance, EH is the hepatic extraction rate and
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

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5
Table 1
Non-linear calibration curve employed to quantify ETO and its metabolite ETO-2-OH.
Lack of fit
Fb
Analyte
Model
ra
pc
rac-ETO
rac-ETO-2-OH
y = −1.32 10− x3 - 4.46 10 x2 + 3.77 10 x + 9.22 10
7
−4
−1
−3
−3
0.999
0.999
1.69
0.64
0.215
0.601
y = −6.48 10−
4
x
3
+ 1.81 10
−2
x
2
+ 4.06 10 x + 2.84 10
−1
a
Correlation Coefficient.
Fcritical95% = 2.85.
pcritical 95% = 0.05.
b
c
Table 2
Within-run accuracy and precision for ETO and its metabolite ETO-2-OH, (n = 5).
Nominal Concentration (␮mol L 1
−
)
Obtained concentration (␮mol L
−1
)
CV (%)a
E (%)b
Analytes
rac-ETO
rac-ETO-2-OH
0.0506 / 0.152 / 8.86 / 14.7
0.0999 / 0.300 / 99.9 / 160
0.0524 / 0.148 / 8.84 / 14.7
0.102 / 0.313 / 98.6 / 160
10 / 7 / 1 / 1
7 / 6 / 1 / 2
4 / −3 / −0.2 / 0
2 / 4 / −1 / 0
a
Precision expressed as coefficient of variation.
Accuracy expressed as relative standard error.
b
method that was developed to quantify rac-ETO-2-OH was based
on GC-MS.
Figure S3 shows the results of the solubility assay. ETO was soluble
−
1
in the incubation medium up to 200 ␮mol L
.
3.2. Method validation
3.5. Enzyme kinetics
Method selectivity was demonstrated by analyzing blank sam-
ples consisting of incubation medium in the presence of HLMs and
NADPH. As seen in Figure S1, the method was selective for rac-ETO
and rac-ETO-2-OH analysis. Method linearity for rac-ETO was eval-
Initially, rac-ETO metabolism was evaluated in the presence
and absence of the NADPH cofactor. The metabolite rac-ETO-2-
OH was the only metabolite that was formed in the presence of
NADPH, indicating that CYP450 mediated the metabolism. The
incubation conditions (protein concentration and incubation time)
yielding V0 for rac-ETO-2-OH formation were determined. Forma-
tion of the metabolite rac-ETO-2-OH was linear within 30 min and
–1
uated from 0.0999 to 200 ␮mol L (0.0999, 0.300, 4.00, 30.0, 99.9,
−
1
1
60, 200 ␮mol L ), and method linearity for rac-ETO-2-OH was
−
1
assessed from 0.0506 to 19.5 ␮mol L (0.0506, 0.152, 1.52, 4.81,
.86, 14.7, 19.5 ␮mol L ). For both rac-ETO and rac-ETO-2-OH, the
−1
8
−
1
GC-MS response was non-linear. For this reason, on the basis of
visual fitting and the lack of fit test, the non-linear mathematical
model that fitted the data the best was employed. The cubic model
for 0.2 mg mL microsomal protein (Figure S4). The enantioselec-
tive metabolism was assessed by incubating rac-ETO, (+)-ETO, and
(–)-ETO, separately, and monitoring rac-ETO-2-OH formation. Rac-
ETO, (+)-ETO, and (–)-ETO metabolism kinetic profile was biphasic
(Fig. 2). The Eadie-Hofstee plots revealed a biphasic kinetic profile
for all the substrates (Fig. 2, minor graphs).
(
1
Eq. 8) was the best model for both rac-ETO and rac-ETO-2-OH with
/x as the weighting method.
2
3
2
y = ax + bx + cx + d
(8)
The data obtained for rac-ETO, (+)-ETO, and (–)-ETO were ana-
lyzed by fitting the Michaelis-Menten model to the first component
to determine the Michaelis-Menten constant (KM1) and its maxi-
mum rate (VMAX1). After that, CLINT1 was calculated. Table 6 lists the
kinetic parameters for rac-ETO, (+)-ETO, and (–)-ETO metabolism.
Comparison between CLINT1 and CLINT evidenced that the first com-
ponent was mainly responsible for CLINT and ETO metabolism.
There was no significant difference for rac-ETO CLINT and its enan-
tiomers even though the KM of the first component was lower for
each enantiomer than for the racemic mixture. On the other hand,
CLINT2 was significantly different for both enantiomers, indicat-
ing enantioselective in vitro metabolism. However, participation
of the second component in CLINT was minimal, thus enantios-
elective in vitro metabolism was very unlikely. Table 6 presents
the CLINT for rac-ETO, (+)-ETO, and (–)-ETO. Comparison of these
results with reported literature values for in vitro intrinsic clearance
in rat and rabbit liver microsomes revealed a significant differ-
ence. The in vitro clearance in the rac-ETO, (+)-ETO, and (–)-ETO
Table 1 describes the calibration curves for both rac-ETO and rac-
ETO-2-OH used in this study as well as the lack of fit test results.
–
1
The LLOQ for rac-ETO and for rac-ETO-2-OH was 0.0999 ␮mol L
–
1
and 0.0506 ␮mol L , respectively, with RE and CV below ± 20 and
0%, respectively. The chromatographic method demonstrated pre-
cision and accuracy in both within-day (Table 2) and between-day
Table 3) assays. rac-ETO and rac-ETO-2-OH stability was evaluated
2
(
in the sample injector for 27 h (maximum batch analysis time), in
◦
the incubation medium at 37 C for 30 min, and for 27 h storage at
◦
−
20 C. Tables 4 and 5 summarize the results for analyte stability.
Both rac-ETO and rac-ETO-2-OH were stable in the analysis condi-
tions. The carryover evaluation demonstrated that carryover was
not significant (data not shown).
3
.3. ETO racemization
Evaluation of the racemization extent during incubation indi-
−1 −1
cated that no ETO enantiomer racemization should take place
during the metabolism kinetics. Figure S2 shows the analysis of
each enantiomer before and after being subjected to the incubation
conditions.
human model was much lower (0.10 mL min mg ) as compared
−
1
−1
to the rat (18.56 and 15.79 mL min mg ) and rabbit (23.64 and
−
1
−1
21.14 mL min mg ) models [13]. This difference was associated
with interspecies differences resulting from distinct amino acid
compositions across different species, leading to different enzyme
activity [24]. This result is particularly interesting because animal
models are still being used in toxicokinetic studies aiming at human
risk assessment, and these interspecies differences may clearly con-
duct to unreliable data extrapolation [25]. Unfortunately, direct
comparison of the results in HLMs, rat liver microsomes, and rab-
3
.4. ETO solubility in the incubation medium
ETO solubility was evaluated in the incubation medium at three
−
1
different concentrations (200, 250, and 300 ␮mol L ) to ensure
that all the analyte would be soluble in the incubation medium.
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
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Table 3
Between-run accuracy and precision for ETO and its metabolite ETO-2-OH, (n = 3).
Nominal Concentration (␮mol L 1
−
)
Obtained concentration (␮mol L
−1
)
CV (%)a
E (%)b
Analytes
rac-ETO
rac-ETO-2-OH
0.0506 / 0.152 / 8.86 / 14.7
0.0999 / 0.300 / 99.9 / 160
0.0504 / 0.147 / 8.75 / 14.7
0.103 / 0.298 / 96.7 / 159
9 / 7 / 2 / 4
6 / 6 / 2 / 3
−0.4 / −3 / −1 / 0
3 / −0.7 / −3 / −0.6
a
Precision expressed as coefficient of variation.
Accuracy expressed as relative standard error.
b
Table 4
◦
Stability for rac-ETO in the incubation medium, in the autosampler for 27 h, and for storage at −20 C for 27 h.
Nominal concentration (␮mol L−1)
Obtained concentration (␮mol L
−1
)
E (%)a
Condition
0
1
0
1
0
1
.300
60
.300
60
.300
60
0.297
159
0.338
170
0.332
161
−1
−0.6
12
6
11
◦
Incubation (37 C for 30 min)
Autosampler
Storage at −20 ◦
C
0.6
a
Relative standard error.
Table 5
Stability for rac-ETO-2-OH in the incubation medium, in the autosampler for 27 h, and for storage at −20 C for 27 h.
◦
Nominal concentration (␮mol L−1)
Obtained concentration (␮mol L
−1
)
E (%)a
Condition
0
1
0
1
0
1
.158
4.7
.158
4.7
.158
4.7
0.167
15.7
0.152
15.7
0.178
15.7
6
7
◦
Incubation (37 C for 30 min)
−4
Autosampler
7
12
7
Storage at −20 ◦
C
a
Relative standard error.
Fig. 2. Biphasic enzyme kinetic profile for rac-ETO (A), (+)-ETO (B), and (–)-ETO (C). The biphasic profile is denoted by the characteristic Eadie-Hofstee plot (smaller graphs).
Table 6
Kinetic parameters obtained for rac-ETO and its enantiomers. KM1 represents the Michaelis Menten constant for the first phase of the enzyme kinetics, and VMAX1 is the
maximum rate for the first phase of the enzyme kinetics. CLINT1 and CLINT2 represent the in vitro intrinsic clearance of the first and second phase, respectively. CLINT represents
the total in vitro intrinsic clearance.
KM1 (␮mol L 1
−
)
VMAX1 (nmol mg min
−1
)
CLINT1 (mL min mg
−1
−1
)
CLINT2 (mL min mg
−1
−1
)
CLINTa (mL min mg
−1
−1
)
Analyte
rac-ETO
15 ± 1
1.59 ± 0.04
0.54 ± 0.02
0.53 ± 0.01
0.102 ± 0.007
0.10 ± 0.01
0.0052 ± 0.0007
0.0017 ± 0.0004
0.005 ± 0.001
0.107 ± 0.007
0.10 ± 0.01
0.10 ± 0.01
(
+)-ETO
5.8 ± 0.6
5.6 ± 0.5
(−)-ETO
0.094 ± 0.008
a
CLINT = CL1 + CL2.
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

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7
Fig. 3. Reaction phenotyping results for rac-ETO, (+)-ETO, and (–)-ETO obtained by using both recombinant CYP450 isoforms and selective chemical inhibitors.
Table 7
fering from liver diseases or other conditions that can impact the
parameters described before, which means that these people may
be more susceptible to the ETO toxic effects on the organism as
compared to healthy people.
In vitro-in vivo correlation results for ETO metabolism by HLMs.
CLH (mL min 1 kg
−
−1
)
EH
fh
Analyte
fuPlasma
fuMicrosome
rac-ETO
0.21
0.20
0.26
0.92
0.84
1.00
10
9.8
10
0.52
0.49
0.52
0.48
0.51
0.48
(
(
+)-ETO
−)-ETO
3.7. CYP450 reaction phenotyping
fuPlasma: plasma unbound fraction.
fuMicrosome: microsome unbound fraction.
CLH: Hepatic clearance.
EH: Hepatic extraction rate.
fh: bioavailable fraction.
The reaction phenotyping study allowed identification of the
main CYP450 isoforms involved in ETO metabolism. Because no
method alone is suitable for a reliable conclusion, CYP450 reaction
phenotyping should be accomplished by two different methods
to evaluate whether the results are similar [28]. Additionally, this
study was performed by using two concentrations: the KM1 of
rac-ETO, (+)-ETO, or (–)-ETO and the highest concentration eval-
bit liver microsomes for each enantiomer is not possible. The ECD
signal depends on the solvent in which the enantiomers are dis-
solved [26]. Since the results are based on the attribution of the
optical activity of each enantiomer and because the authors did
not report the mobile phase in which the circular dichroism analy-
sis was performed, a direct comparison for each enantiomer is not
possible.
−1
uated in the enzyme kinetic study (180 ␮mol L ), to assess the
main isoforms involved in both phases of the biphasic kinetics
[
21]. Fig. 3 illustrates the results. The reaction phenotyping results
with rCYP450 isoforms indicated that CYPs 1A2, 2B6, 2C19, and
A4 were the main isoforms involved in the metabolism of rac-ETO
3
and its enantiomers at the two evaluated concentrations. On the
other hand, the results using selective chemical inhibitors showed
that only ticlopidine significantly inhibited ETO metabolism at its
KM1. This suggested that CYP2C19 was the only isoform that was
responsible for the first component of rac-ETO, (+)-ETO, and (–)-ETO
enzyme kinetics. At the second component, ticlopidine (CYP2C19)
and ketoconazole (CYP3A) inhibited ETO metabolism significantly.
Thus, CYP2C19 and CYP3A were involved in the second compo-
nent of rac-ETO metabolism. Additionally, CYP3A and CYP2C19
mediated (+)-ETO and (–)-ETO metabolism, respectively. Integrat-
ing the results from reaction phenotyping with rCYPs and selective
chemical inhibitors, both CYP2C19 and CYP3A4 were the major
isoforms responsible for ETO metabolism. Reaction phenotyping
using rCYPs also indicated that CYP1A2 and CYP2B6 metabolized
ETO. However, the study with chemical selective inhibitors did not
suggest significant CYP1A2 and CYP2B6 inhibition by the selec-
tive chemical inhibitors, indicating that these two CYPs might play
a minor role in ETO metabolism. Considering each enantiomer,
reaction phenotyping also suggested that CYP2C19 was the only
responsible for (–)-ETO metabolism in both kinetic components.
Human CYP2C19 may yield a biphasic enzyme kinetic profile. For
(+)-ETO, CYP2C19 was also the major responsible for the first com-
3
.6. In Vitro-In Vivo Correlation
Determination of the ETO free fraction in plasma did not indi-
cate enantioselective binding to plasma proteins (Table 7). Neither
the metabolism nor the protein binding was enantioselective, thus
CLH was not expected to be enantioselective. Table 7 also shows
no enantioselectivity for CLH of both enantiomers and the racemic
mixture, which evidenced that there was no enantioselective accu-
mulation of one enantiomer in the organism. EH estimation for
both enantiomers and the racemic mixture also described a non-
enantioselective behavior (Table 7). EH prediction indicated that
half of the rac-ETO, (+)-ETO, or (–)-ETO amount reaching the liver
was transformed into the metabolite ETO-2-OH, so half of the
amount would be bioavailable after hepatic first-pass metabolism.
Such an EH may indicate that rac-ETO and its enantiomers are mod-
erately metabolized in the liver (0.3 ≤ EH ≤ 0.7) [27]. For such
xenobiotics, changes in the Q, fu,P, and CLINT,
may significantly
invivo
impair CLH [27]. For instance, liver diseases may impact Q, protein
concentration in plasma, and hepatocyte capacity to metabolize
xenobiotics, thus increasing the ETO bioavailable fraction in the
organism [27]. Thus, ETO may be more bioavailable in people suf-
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

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I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (xxxx) xxx
ponent, while CYP3A4 was responsible for the second component.
The results obtained with reaction phenotyping agreed with the
enzyme kinetics results. No difference was observed for each of the
enantiomers in the first component of the kinetics in terms of KM
and in vitro intrinsic clearance, which was consistent with the same
isoform (CYP2C19) metabolizing both (+)-ETO and (–)-ETO. On the
other hand, the difference was significant for the second phase
of the kinetics for both enantiomers in terms of in vitro intrinsic
clearance, which agreed with two different isoforms metabolizing
review & editing, Validation. Daniel Blascke Carrão: Writing
- review & editing, Methodology. Nayara Cristina Perez de
Albuquerque: Writing - review & editing, Methodology. Ander-
son Rodrigo Moraes de Oliveira: Conceptualization, Supervision,
Project administration, Funding acquisition.
ACKNOWLEDGMENTS
The authors are grateful to the São Paulo Research Founda-
tion (FAPESP, Grant numbers: 2014/50945-4 and 2018/07534-4],
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(
+)-ETO (CYP3A4) and (–)-ETO (CYP2C19).
In terms of pesticide-drug interactions, some drugs that are
commonly used by the population such as fluoxetine, clopidogrel,
and omeprazole inhibit CYP2C19 [29]. Since the metabolism data
suggested that CYP2C19 was the main isoform responsible for ETO
metabolism in the liver, the presence of such drugs in the organism
could inhibit ETO metabolism, thus increasing its bioavailable frac-
tion in the organism. From this point of view, subjects taking these
drugs might be more susceptible to the ETO toxic effects. Addition-
ally, commonly used drugs including clarithromycin, nicardipine,
and ketoconazole inhibit CYP3A4, a very important isoform in drug
metabolism [29]. Although this isoform metabolizes ETO to a lesser
extent, the pesticide-drug interactions indicated that patients tak-
ing these drugs could also be more susceptible to the ETO toxic
effects.
(
CNPq – INCT-DATREM) [Grant n. 465571/2014-0], Coordena c¸ ão
de Aperfei c¸ oamento de Pessoal de Nível Superior – Brasil (CAPES)
Finance Code 001 and to the Electrocatalysis and Environmental
–
Electrochemistry Laboratory for the GC-MS use.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.jpba.2020.
1
13349.
Keeping that in mind, if CYP2C19 is inhibited, (–)-ETO may accu-
mulate in this organism because it is solely cleared by this isoform,
whereas (+)-ETO is also cleared by CYP3A4. Since the CYP2C19 iso-
form is polymorphic, the possibility of differences in the extent
of ETO metabolism across human ethnic groups should not be
overlooked [30]. Moreover, the frequency of individuals with poor
metabolizer phenotype for CYP2C19 among Caucasians and black
Africans ranges from 1 to 8%, whereas it may reach 23% of the indi-
viduals in Asian populations [30]. This suggests that Asians might
be more susceptible to the toxic effects of ETO on the organism as
compared to other ethnic groups because its bioavailable fraction is
expected to be higher in this ethnical group. Furthermore, recently,
the presence of ETO in human blood serum has been reported in
China [11]. This result corroborates with the in vitro metabolism
results in HLMs and may represent a concerning result for this
population.
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ETO metabolism by monitoring the metabolite rac-ETO-2-OH is
feasible even though this metabolite is stereolabile. The enzyme
kinetic parameters and the in vitro-in vivo correlation indicated
no difference in terms of metabolism and clearance for rac-
ETO, (+)-ETO, and (–)-ETO. From this perspective, enantioselective
enrichment in the organism is not expected. However, the pheno-
typing results indicated a stereoselective difference. Only CYP2C19
metabolized the enantiomer (–)-ETO, whereas both CYP2C19 and
CYP3A4 metabolized (+)-ETO. This indicated that pesticide-drug
interactions for (–)-ETO may lead to enantioselective enrichment
of this enantiomer in the organism. Additionally, it is expected
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CRediT authorship contribution statement
[
Icaro Salgado Perovani: Investigation, Conceptualization, Data
curation, Formal analysis, Writing - original draft, Writing -
Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349

G Model
PBA-113349; No. of Pages9
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Please cite this article as: I.S. Perovani, D.B. Carrão, N.C.P. de Albuquerque et al., Enantioselective in vitro metabolism and in vitro-in vivo
correlation of the herbicide ethofumesate in a human model, J. Pharm. Biomed. Anal., https://doi.org/10.1016/j.jpba.2020.113349