Accepted Manuscript

Title: Apigenin, a novel candidate involving herb-drug

interaction (HDI), interacts with organic anion transporter 1
(OAT1)

Authors: Ting Wu, Haixin Li, Jiasheng Chen, Ying Cao,

Weimin Fu, Pingzheng Zhou, Jianxin Pang

PII: S1734-1140(17)30027-0
DOI: http://dx.doi.org/doi:10.1016/j.pharep.2017.06.012
Reference: PHAREP 751

To appear in:

Received date: 16-1-2017

Revised date: 23-4-2017
Accepted date: 22-6-2017

Please cite this article as: Ting Wu, Haixin Li, Jiasheng Chen, Ying Cao, Weimin
Fu, Pingzheng Zhou, Jianxin Pang, Apigenin, a novel candidate involving herb-
drug interaction (HDI), interacts with organic anion transporter 1 (OAT1) (2010),
http://dx.doi.org/10.1016/j.pharep.2017.06.012

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Title Page

Title: Apigenin, a novel candidate involving herb-drug interaction (HDI), interacts

with organic anion transporter 1 (OAT1).

Ting Wu, Haixin Li, Jiasheng Chen ,Ying Cao, Weimin Fu, Pingzheng Zhou, Jianxin
Pang *

Corresponding Author: Jianxin Pang, School of Pharmaceutical Sciences, Southern

Medical University, 1838 North Guangzhou Ave, Guangzhou, China 510515. Tel.:
+86(20)61648671. Fax: +86(20)61648671. Email: pjx@smu.edu.cn
Conflict of interest statement
We wish to confirm that there are no known conflicts of interest associated with this
publication titled “Apigenin, a novel candidate involving herb-drug interaction (HDI),
interacts with organic anion transporter 1 (OAT1)” and there has been no significant
financial support for this work that could have influenced its outcome.

Apigenin, a novel candidate involving herb-drug interaction (HDI), interacts with

organic anion transporter 1 (OAT1).

1
Abstract
Background: Apigenin is a flavonoid compound, widely distributed in natural plants.
Various studies have suggested that apigenin has inhibitory effects towards several drug
transporters, such as the organic anion transporting (OAT) polypeptides, 1B1 and 1B3
(OATP1B1 and OATP1B3). However, the mechanism by which apigenin interacts with
OAT1 has not been well studied.
Methods: MDCK cells stably-expressing OAT1 were used to examine the inhibitory
effects of apigenin on OAT1. UPLC-MS/MS was used to evaluate the in vitro and in
vivo effects of apigenin on the uptake of acyclovir by OAT1. Cytotoxicity was
determined by the cell viability, MTT assays.
Results: Apigenin effectively inhibited the activity of OAT1 in a dose-dependent
manner with an IC50 value of 0.737 μM. Pre-incubation of cells with apigenin caused
a time-dependent inhibition (TDI) of OAT1. Additionally, we examined the interactions
between apigenin and acyclovir or adefovir. Data showed that apigenin (1 μM)
significantly blocked the uptake of acyclovir by OAT1 in vitro with an inhibition rate
of 55%. In vivo, apigenin could increase the concentration of acyclovir in plasma when
co-administered with acyclovir. Importantly, the MTT assays showed that, at a dose of
50 μM, apigenin significantly reduced the cytotoxicity of adefovir and substantially
increased cell viability from 50.6% to 112.62%.
Conclusion: Our results demonstrate that apigenin regulates OAT1, and can cause TDI
or herb-drug interaction (HDI) when used in combination with acyclovir or adefovir.
Therefore, apigenin could be used as a nephroprotective agent when used in
combination with the substrates of OAT1.

Abbreviations: OAT1, organic anion transporter 1; TDI, time-dependent inhibition;

HDI, herb-drug interaction.

Keywords: Apigenin; ; ; ; ; , MDCK-OAT1 cells, TDI, acyclovir, adefovir, HDI.

2
Introduction
Pharmaceutical agents are commonly used worldwide for diagnostic and treatment
purposes. However, some of the drugs can cause side effects such as nephrotoxicity.
Induction of kidney injury is mediated by renal uptake transporters, which are
responsible for the clearance of drugs from the blood circulation. Several members of
the organic anion transporter (OAT) family mediate the transport of circulating
substrates. The OAT family belongs to the SLC22 (solute carrier 22) subfamily of the
major facilitator superfamily (MFS) including over 10 transmembrane proteins (OAT1
- OAT10)， which are composed of about 540–560 amino acids comprising 12
transmembrane domains [1]. Among OATs, OAT1 is predominantly expressed at the
basolateral membrane of kidney proximal tubular epithelial cells and is responsible for
the transport of anionic substrates from the systemic blood circulation into the cells,
where cell-mediated substrate clearance occurs [2-4]. It is well known that, OAT1 plays
an important role in the elimination of a variety of compounds, such as antivirals,
nonsteroidal anti-inflammatory drugs, β-lactam antibiotics and diuretics [5, 6], but it
also might be implicated in nephrotoxicity arising from the accumulation of some drugs
(especially some antivirals as adefovir and cidofovir) in renal tubules.
Interestingly, co-administration of drugs in some cases can improve the half-life of the
drugs and reduce renal clearance and nephrotoxicity [7], and this can be mediated by
OATs. One classic example is the co-administration of probenecid, a known OAT1
inhibitor, which has shown to diminish the renal clearance of adefovir and
benzylpenicillin, resulting in an increased concentration of circulating adefovir and
benzylpenicillin in the body [8]. Similarly, nonsteroidal anti-inflammatory drugs,
including ketoprofen, ibuprofen, and naproxen, have been proven to exhibit protective
effect against the OAT1-mediated cytotoxicity of adefovir [9]. Therefore, the drug-drug
interaction (DDI) or the transporter-drug interaction (TDI) might affect the inhibition
of OAT1 activity. This can result in reversed pharmacokinetics or unexpected severe
adverse effects in combination therapy, particularly for those drugs that are transported
via the OATs process [7, 10-12].
In the recent years, with the increased interest in using herbal supplements, it has
become obvious that interactions can also occur between herbs and drugs. Numerous
studies have been conducted on the interaction of herbs with drug transporters,
including P-gp, MRP1, MRP2, ABCG2，OATP1B1 and so on, or with detoxifying

3
enzymes, such as CYPs and UGTs. However, only few studies have been conducted to
elucidate the interaction of herbs with OATs in the kidney [13-16].
Some findings indicate that edible herbal medicine might cause herb-drug interaction
(HDI), when co-administered in vivo with substrates of OAT1. For example, rhein, a
component in rhubarb, has been recently reported to be an inhibitor of OATs and could
lead to HDI when co-administered with furosemide [17]. Flavonoids, such as luteolin,
morin, dietary phenolic acids and so on, have also demonstrated potent inhibitory effect
on OAT1, and some of them showed strong inhibitory activities towards OAT1 in vitro,
with IC50 even lower than 0.5 μM [4]. Recently, one of flavonoid conjugates, quercetin-
3’-O-sulfate was shown to attenuate OAT1-mediated cytotoxicity of adefovir in a dose-
dependent manner [18]. Apigenin, known as 4′, 5, 7, -trihydroxyflavone, is abundant in
vegetables and fruits, especially in celery. Compared with other flavonoids, apigenin
has gained its superiority due to its low intrinsic toxicity. In addition, recent findings
revealed that apigenin can inhibit enterovirus 71 replication, through suppression of the
viral IRES activity and modulation of the JNK pathway [19]. These results demonstrate
that apigenin has antiviral activity and suggest that it has the potency to be used in the
combined treatment of viral infections. In addition, several reports have proved that
apigenin could show inhibitory activity towards some hepatic drug transporters
(OATP1B1 and OATP1B3). Thus, exploring the potential HDI between apigenin and
OAT1-transported drugs is of great value.
Considering the important role of OAT1 in the drug-induced nephrotoxicity, co-
administration of nephrotoxic drugs with OAT1 inhibitors may provide the key in
reducing OAT1-mediated drug nephrotoxicity. Research on identifying potent OAT1
inhibitors and understanding their mechanism(s) of action is of great importance for
drug nephrotoxicity prevention. Therefore, in this study, we aimed to explore whether
apigenin interacts with OAT1 in vitro. A previous study conducted by Ma et al. showed
that some anthraquinones presented TDI toward OAT1 [20]. For this reason, our
experimental approach was focused on investigating the potential inhibitory role of
apigenin towards OAT1, in a time-dependent manner. In addition, two antiviral drugs,
acyclovir and adefovir, were used to explore whether apigenin can infer any potential
herb-drug interaction. Acyclovir is in a class of antiviral medications called synthetic
nucleoside analogues, whose main elimination route is through the OAT1 process.
Adefovir is an antiviral drug that can cause nephrotoxicity, as a side effect, due to

4
intracellular drug accumulation, via the OAT1-mediated transport. To assess the in vitro
effect of apigenin on the uptake of acyclovir and the protective effect of apigenin on the
cytotoxicity caused by adefovir mediated by OAT1, we used cells stably expressing
OAT1 (MDCK-OAT1 cells). Finally, assays were performed in rats to evaluate the in
vivo effect of apigenin on the pharmacokinetics of acyclovir in rats.
Materials and Methods
Chemicals and reagents
Apigenin, baicalein, wogonin and emodin (purity >99% by HPLC), were purchased
from Chengdu Must Bio-Technology Co, Ltd. Probenecid was obtained from Sigma-
Aldrich Chem Co. (St. Louis, MO, USA). Acyclovir and adefovir were from Aladdin
Reagent Company (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM),
fetal bovine serum (FBS) and trypsin were bought from Gibco (Gaithersburg, MD,
USA). Geneticin (G418) was purchased from Calbiochem (San Diego, CA). BCA
protein assay kit was bought from Pierce Chemical, Rockford, IL. Formic acid and
methanol (purity > 99% by HPLC), were analytical grade and purchased from J&K
Chemical (Beijing, China).
Cell culture
MDCK-OAT1 cells used were kept in our lab, and grown in DMEM supplemented
with 10% fetal bovine serum, with 100 units/ml penicillin and 100 mg/ml streptomycin
(all from Sigma-Aldrich) and 350 μg/mL geneticin. Cells were kept at 37 °C in an
incubator with 5% CO2. Geneticin was not added in each experiment. All experiments
were performed with MDCK cells between passages 1 and 20.
6-CFL cellular uptake and inhibition assays
6-carboxyfluorescein (6-CFL), a fluorescent dye, was used to perform uptake assays
since it has been established as an effective OAT1 substrate. MDCK (Madin-Darby
canine kidney) cells and their derivative cell line MDCK-OAT1, which stable
expresses OAT1, were used to assess their 6-CFL uptake capacity. The uptake assay
was adapted from previously published papers with few modifications [17, 21].
MDCK-OAT1 and MDCK-mock cells were seeded into 24-well plates at a density of
2×105 cells/well. After incubating for 48 h, the cells were washed twice with preheated
Hank's Balanced Salt Solution (HBSS), and then pre-incubated with HBSS, for 10 min
at 37 °C. Then, 6-CFL alone or 6-CFL with tested compounds were incubated for the
designated time. The uptake process was terminated by washing quickly three times

5
with phosphate-buffered saline (PBS). The cells in each well were lysed with 300 μL
of 0.1 M sodium hydroxide. An aliquot of 200 μL lysate was used to measure
fluorescence by using the TECAN Microplate Reader. Cellular protein content in each
well was determined by the bicinchoninic acid (BCA) protein assay. The ratio of 6-
CFL uptake to total protein content was used to evaluate the inhibition activity. The
compounds were dissolved in dimethyl sulfoxide and diluted with HBSS buffer before
each assay. The final concentration of dimethyl sulfoxide in the assay was less than
0.1%.

Concentration-dependent inhibition assays were conducted to test the inhibitory effects

of the tested compounds towards OAT1. Increasing concentrations of apigenin (0.25-
8 μM), baicalein (0.5-16 μM), wogonin (0.5-16 μM) and probenecid (25-500 μM) were
added to the uptake buffer to determine the inhibitory effects. 6-CFL (40 μM) was used
as substrate and incubated for 4 min with different drugs as mentioned above. IC50
values were estimated by non-linear regression analysis from three separate
experiments using Graphpad Prism 5 and the results were expressed as mean ± SD.

For the time-dependent inhibition assays, the MDCK-OAT1 and mock cells were
prepared as described above and were pre-incubated with HBSS buffer or buffer
containing 100 μM probenecid, 0.8 μM apigenin, 20 μM baicalein and 8 μM wogonin
or various concentrations of apigenin for 30 min at 37 oC. After 30 min, the pre-
incubation medium was replaced with HBSS buffer containing the same concentration
of apigenin and 6-CFL to initiate the uptake procedure. Increasing concentrations of
apigenin (0.25-8 μM) were added to investigate whether pre-incubation could enhance
its inhibitory effects towards OAT1. To verify the TDI, 0.8 μM apigenin was used, and
the termination time was set at 0-60 min. Following, the samples were processed as
described above to measure fluorescence; the values were normalized to the total
protein content.

Apigenin cellular uptake assays

For the apigenin accumulation assay, MDCK cells were seeded in 6-well plates at a
density of 5×105 cells/well. Cells were grown for 48 h at 37 oC before the experiments.
The uptake process was conducted as described above, with the exception that HBSS
containing apigenin (20 μM) was used as the incubation media, and the incubation time

6
was set for 20 min. The uptake medium containing 20 μM apigenin, with or without
probenecid (500 μM), was then added to each well. The final DMSO concentration in
the transport buffer was no more than 0.2%. Uptake procedure was also terminated as
described above. An aliquot of 200 μL of 0.1% Triton X-100 was added into each well
to lyse the cells. The samples were analyzed by UPLC-MS/MS to measure the apigenin
concentrations in the cell lysates, while the cellular protein content in each well was
measured by the BCA kit.

Apigenin-drug interaction assays

To determine whether apigenin could influence the uptake of acyclovir by MDCK-
OAT1 cells, the cells were seeded in 24-well plates with a density of 1×105 cells/well.
The experiments were conducted after 48-h incubation. The uptake process was
conducted as described above, with the exception that HBSS containing apigenin (1
μM), probenecid (500 μM), and emodin (1 μM) were used as the inhibitors, and the
incubation time was set at 60 min. The uptake medium containing 400 μM acyclovir,
with or without the inhibitors above, was then added to each well. Uptake procedure
was also terminated as described above. The concentration of acyclovir in the cell
lysates were measured by UPLC-MS/MS, meanwhile the cellular protein content in
each well was determined by the BCA kit.

Plasma apigenin measurements

Male Sprague-Dawley rats, weighting 200 ± 20 g, were obtained from the
Experimental Animal Center of the Southern Medical University. Rats were kept at
room temperature (22 - 24 oC), with ～50 - 70% relative humidity. The animals were
normally fed ad libitum with rodent food and water before the experiments. The rats
were deprived of food 12 h before the experiments. All procedures were strictly done
in accordance to the local animal handling guidelines.
Rats were divided randomly into two groups: control group (acyclovir, 35 mg/kg) and
experimental group (acyclovir, 35 mg/kg + apigenin, 70 mg/kg). Each group included
5 rats. Apigenin was dissolved in saline and was given 2 h before administration of 35
mg/kg acyclovir [suspended in 0.5% carboxymethyl cellulose sodium solution (CMC-
Na)] by intravenous injection in the tail vein. Serial blood samples were collected via
the orbital venous sinus into heparinized tubes at the designated time points (10, 20,
30, 60, 120, 240, 360, 480 min). The blood samples were prepared as follows.
7
Sample preparation for UPLC-MS/MS analysis
For the cell lysates preparation, a 100 μL aliquot of cell lysates was deproteinized by
adding an equal volume of methanol. Following, the mixture was vortexed for 3 min
and then centrifuged at 13,200 rpm for 30 minutes. Afterwards, 100 μL of the
supernatant was used for UPLC-MS/MS analysis. External standard method was
applied in the analysis of apigenin, and internal standard method was chosen for
acyclovir.
The analysis of acyclovir in the plasma was performed with the same method with few
modifications [22]. Briefly, 300 μL of methanol was added to 100 μL of plasma sample
[containing 200 ng/mL of nolatrexed dihydrochloride (AG337) as internal standard
(IS)]. The mixture was vortexed for at least 5 min and then centrifuged at 13, 200 rpm
for 15 min. The supernatant was transferred to another clean tube and evaporated to
dryness at 37 oC under a gentle stream of nitrogen. The residue was then re-suspended
in 100 µL of methanol and mixed by vortexing for 3 min. Subsequently, the mixture
was centrifuged for further 15 min, at 13,200 rpm, and 100 µL of the supernatant was
injected into the UPLC-MS/MS system.

UPLC-MS/MS analysis
Chromatographic analysis was conducted by using an ACQUITY UPLC/HSS T3 C18
column (1.8 μm, 2.1×50 mm, Waters, USA). The mobile phase of apigenin consisted
of 0.1% formic acid (A) and acetonitrile (B). Besides, methanol (A) and 0.2 % formic
acid (B) were chosen for acyclovir. The flow rates were set at 0.25 and 0.3 mL/min
and the column temperature was set at 35 oC and 30 oC, for apigenin and acyclovir,
respectively. The gradient elution steps were as follows: For apigenin, 0-4.5 min: 85%
B, 4.5-5 min: 85%-10% B, 5-5.5 min: 10%-85% B; For acyclovir, 0-1 min: 95% B, 1-
2.5 min: 95%-40% B, 2.5-3.0 min: 40% B, 3-3.5 min: 40%-95% B.
Apigenin and acyclovir were ionized by the ESI source in a negative and positive ion
mode, respectively [22, 23]. Additional details about other important multiple reaction
monitoring parameters in this assay are summarized in Table 1. The results of the
method validation of apigenin and acyclovir are presented in Figure 1S and Table 2S-
5S.

MTT assays

8
MDCK-OAT1 and MDCK-mock cells were plated onto 96-well plates with a density
of 5×103 cells/well. After incubating for 24 h, the medium was replaced with medium
containing various concentrations of adefovir and/or apigenin (three replicates for
each sample), and the cells were further incubated for 24 h. At the designated time,
cell viability was tested through MTT assays. In brief, the culture medium was
removed and replaced with 100 μL of complete media containing 0.5 mg/mL MTT.
After a 4 h incubation at 37 oC, 150 μL of DMSO was added to each well and the
samples were mixed by gentle shaking for 10 min; the absorbance was measured at
570 nm on the TECAN Microplate Reader.

Data Analysis
All results were expressed as mean ± SD. IC50 values were calculated using GraphPad
Prism 5. Student's t-test was used to evaluate the different groups. Differences between
groups were assumed significant for p values ≤0.05.

Results

Inhibition of OAT1-mediated uptake of 6-CFL by apigenin

Besides probenecid (a typical inhibitor of OAT1), we also used baicalein and wogonin
as positive controls, since they have been previously reported to inhibit OAT1 [24]. As
shown in (Fig. 1B), we confirmed that the tested compounds presented inhibitory
activities on the OAT1-mediated uptake of 6-CFL, in a concentration-dependent
manner. The estimated IC50 values of MDCK-OAT1 cells were 0.737, 7.691, 16.72
and 56.45 μM, after apigenin, wogonin, baicalein and probenecid treatment,
respectively. The IC50 values for baicalein, wogonin were very close to the previously
published values of 18.0 μM and 8.2 μM [24], respectively, which confirmed the
validity of our assay.

TDI-mediated inhibition of OAT1 by apigenin

As shown in Fig. 2, pre-incubation of MDCK-OAT1 cells with apigenin for 30 min

increased its inhibitory potency towards OAT1, while treatment with wogonin,
baicalein and probenecid had no effect on the inhibition of OAT1 (Fig. 2A). The IC50
value of apigenin was 0.295 μM after the treatment of pre-incubation, which is
significantly lower (55% lower) than the value observed without pre-incubation (0.737
μM) (Fig. 2B). These results demonstrate that pre-incubation with apigenin can
9
substantially enhance its inhibition potency towards OAT1 and suggest that the
inhibitory effect of apigenin should not be underestimated. As shown in Fig. 2C, as the
pretreating time increased from 0 to 60 min, apigenin displayed an analogous increase
in inhibitory activity, in a time-dependent manner.

Intracellular uptake of apigenin and the effect of apigenin on the OAT1-mediated

uptake of acyclovir in vitro

Our results confirmed that apigenin is an inhibitor of OAT1. However, the mechanism
by which apigenin exerts its inhibitory effect on OAT1 remained unknown. In order to
investigate if apigenin acts as a substrate of OAT, we further studied the interaction of
apigenin and OAT1 by investigating the uptake of apigenin by MDCK-OAT1 and
MDCK-mock cells, in the absence or presence of probenecid (500 μM). Probenecid is
an established inhibitor and substrate of OAT1 which should compete with other
substrates for binding to OAT1. However, in both cell lines, the presence of probenecid
did not alter the intracellular uptake of apigenin (Fig. 3A), indicating no binding
competition, which shows that apigenin is a not a substrate of OAT1.

As shown in Fig. 3B, apigenin, probenecid and emodin, as inhibitorts of OAT1, all
reduce the transport of acyclovir. Apigenin (1 μM) reduced by half the uptake of
acyclovir in MDCK-OAT1 cells. In addition to probenecid, we have also tested emodin
since it has been reported to be a strong inhibitor [17], we firstly wanted to explore the
interactions between emodin and OAT1 in this experiment and to compare the
inhibitory effects of emodin and apigenin on the OAT1-mediated uptake of acyclovir.
Initially, we explored the interactions between emodin and OAT1 and then compared
the inhibitory effects of emodin and apigenin on the OAT1-mediated uptake of
acyclovir. Consequently, a decrease of OAT1-mediated uptake of acyclovir was
observed in the presence of apigenin

Apigenin interacts with acyclovir in rats.

Apigenin recently has been reported to have antiviral activity, thus we intended to
investigate whether apigenin could influence the concentration of acyclovir when
used in combination with acyclovir in vivo. The time-depended mean plasma
concentration profiles of acyclovir in the presence and absence of apigenin in rats is
shown in Fig. 4. The data revealed that the concentration of acyclovir in the plasma

10
of rats treated with both compounds was significantly higher in comparison to the
plasma after treatment with acyclovir alone, especially during the first 60 min.

Apigenin attenuates the OAT-mediated cytotoxicity of adefovir

As was clearly shown, the cytotoxicity of adefovir in MDCK-OAT1 was largely

enhanced when compared to MDCK-mock cells. In OAT1-expressing cells, the IC50
value of adefovir was 139.04 μM, while the IC50 value in the control cells was
substantially higher (Fig. 5B). To investigate whether apigenin could reduce the
cytotoxicity of adefovir in MDCK-OAT1 cells, we co-incubated MDCK-OAT1 and
MDCK-mock cells with 200 μM adefovir containing various concentrations of
apigenin. The results showed that apigenin (1.56-50 μM) can indeed reduce the
cytotoxicity caused by adefovir. Importantly, apigenin alone showed no effect on cell
viability in both MDCK-OAT1 and MDCK-mock cells (Fig. 5A). In addition, apigenin
(1.56-50 μM) gradually attenuated the cytotoxicity of adefovir in MDCK-OAT1 cells
in a dose-dependent manner (Fig. 5C). In the presence of 50 μM of apigenin, the
viability of cells treated with 200 μM adefovir considerably increased from 50.6% to
112.62%.

11
Discussion
It is generally known that OAT1 plays a critical role in the elimination of numerous
organic anions in the kidney [1, 25]. The transport process mediated by OAT1 is
beneficial for the pharmacological, pharmacokinetic, and toxicological behavior of
many drugs commonly used in clinical practice. Drug-drug interactions (DDI) that arise
during combinational therapy can influence the treatment of a disease; therefore this
area of research has attracted the interests of many scientists. The interaction between
drugs and substrates or inhibitors of transporters can have unexpected side effects, as it
has been demonstrated for example in the DDI cases of benzylpenicillin and acyclovir,
JBP485 and acyclovir, and bezafibrate and mizoribine [6, 26, 27]. Therefore, advanced
prediction and evaluation of DDI potential in combinational therapy becomes of great
importance for the clinical prognosis of a patient’s treatment. In recent years, the use of
herbal medication has increased; however, there are limited studies on herb-drug
interactions (HDI), especially on those that specifically target transporters, which
deserves further investigation.

Our study is the first to explore the inhibitory effect of the plant compound, apigenin,
on OAT1, and to demonstrate that HDI occurs between apigenin and acyclovir, both in
vitro and in vivo, as well as with adefovir, in vitro. Apigenin is a widely used traditional
flavone compound, which has gathered a lot of attention recently, owing to its
antioxidant, antibacterial, antiviral and anti-inflammatory properties and its ability to
prevent hypertension [28]. Our results revealed that apigenin could obviously inhibit
OAT1, and the IC50 value was determined to be 0.737 μM. Therefore, we speculate
that apigenin has the potential to be used as a promising nephroprotectant, as it can
suppress the cytotoxicity of drugs, which are also substrates of OAT1.

Considering the structures of the studied monomers (Shown in Table 1S), our results
show that the presence of a hydroxyl group on the C2 position of the benzene ring may
be responsible for the stronger inhibitory effect of apigenin on OAT1 in comparison to
baicalein and wogonin. Previous studies on morin and leteolin, have also shown strong
inhibitory effects on OAT1, with IC50 values of < 0.3 µM and 0.47 µM [4],
respectively. Collectively, the reported results and our findings in this study indicate
that the structure of the flavonoids with stronger inhibitory activity against OAT1, need
to follow certain requirements: The C2=C3 bond should be retained, and no large
12
groups should be attached to the C3. Besides, certain hydroxyl groups on the benzene
ring are necessary; the presence of 2 ', 4’-OH correlates with the strongest inhibitory
effects; the 2', 3'-OH moderately reduces the effect, and the 2', 3’, 4'-OH significantly
reduces the inhibitory activity, such as in the case of myricetin.

TDI in CYP450 enzyme-mediated metabolism has gained broad interest, which might
be caused by the inhibition of in vitro incubation [29]. However, drug-transporter-based
TDI has not been widely investigated. In this study, we accidentally found that pre-
incubating MDCK-OAT1 with apigenin for 30 min could increase its inhibitory effect
on OAT1. At the same time, pre-incubation of the cells with probenecid, baicalein and
wogonin had no influence on their inhibitory effects toward OAT1 (Fig. 2A). Pre-
incubation of cells with apigenin for variable time intervals revealed a correlation
between the pre-incubation time and the inhibitory potency of apigenin towards OAT1.
Full inhibition of OAT1 was not achieved after 60 min of pre-incubation (Fig. 2C). The
IC50 values in the pre-incubation and not-pre-incubation groups were 0.295 μM and
0.737 μM, respectively, which suggests that the inhibitory effect of apigenin towards
OAT1 cannot be underestimated (Fig. 2B).

However, the mechanism deserved further exploration. We first guessed that pre-
incubation with apigenin increased its intracellular concentration, which in turn
enhanced its inhibitory effect on OAT1. However, our apigenin uptake study showed
that apigenin is not a substrate of OAT1, so this hypothesis could not explain the TDI
mediated by apigenin. An alternative explanation is that apigenin inhibits the
glycosylation of OAT1 or affects the substrate recognition regions of OAT1. It has been
reported that the inhibition of the glycosylation modification processes of some OAT
transporters could down-regulate their transporting activities [30, 31]. However, the
exact mechanism(s) of TDI caused by apigenin deserves further investigation in the
future.

Previous work demonstrated that OAT1-expressing cells showed higher affinity

towards some commonly used antiviral drugs, such as acyclovir, cidofovir, adefovir
and tenofivir, compared to control cells [10]. Caffeic acid had been reported to be a
novel candidate for OAT1 inhibitor, and thus contributed to food-drug interaction
between methotrexate, aminopterin, and acyclovir in vitro [32]. This triggered our

13
interest to investigate if a similar interaction can be observed between apigenin and
acyclovir. Co-incubation of MDCK-OAT1 and mock cells with acyclovir and apigenin
revealed that apigenin did influence the uptake of acyclovir in the OAT1 expressing
cells. Moreover, a similar effect was observed with probenecid.

The OAT1-mediated HDI of apigenin and acyclovir was further investigated in vivo by
performing assays on rats. Apigenin, at a dose of 70 mg/kg, increased the concentration
of acyclovir in the plasma (Fig. 4), which indicated that apigenin might interact with
acyclovir when co-administered, and thus might strengthen and lengthen the curative
effects of acyclovir. However, care should be taken to adjust the dosage of acyclovir
when the two compounds are prescribed together to avoid possible drug toxicity.

Being potent inhibitor of OAT1, quercetin-3-O-sulfate, which is one of the main

metabolites of the natural flavonoid quercetin in humans, was shown to effectually
reduce the cytotoxicity of adefovir [18]. This provides a possibility that flavonoids
might limit the incidence rate of drug-induced renal injury through the inhibition of
OAT1. Morin, a common flavonoid, has been demonstrated to be protective against the
nephrotoxicity induced by imipenem, which was mediated by OAT3 and led to
accumulation of this antibiotic in the renal tubular epithelial cells [33]. In this study,
apigenin was found, to reduce to some extent the cytotoxicity of adefovir in MDCK-
OAT1 cells (Fig. 5C), which might play a crucial role in suppressing the nephrotoxicity
associated with antiviral agents.

Conclusions
In conclusion, our study has shown for the first time, to the best of our knowledge, that
apigenin acts as an inhibitor of OAT1 but is not a substrate of it. TDI was observed
between apigenin and OAT1, however the exact mechanism deserves further study. In
addition, apigenin could strongly reduce the OAT1-mediated uptake of acyclovir in
vitro and lead to pharmacokinetic interaction when used in combination with acyclovir
in vivo. Besides, apigenin was shown to effectively reduce the cytotoxicity of adefovir
in MDCK-OAT1 cells. Although probenecid could be used as a nephroprotective agent,
it always requires a relatively high dose which might cause higher risk of side effects.
Therefore, identification of OAT1 inhibitors with great security is more beneficial to
reduce the OAT1-mediated nephrotoxicity of certain drugs. Altogether, our results

14
indicate that apigenin might have the potential to decrease the incidence rate of renal
injury caused via the inhibition of OAT1. Since up to now there are no reported safety
issues concerning apigenin, we suggest that apigenin might be safely used to reduce the
OAT1-mediated nephrotoxicity. Considering that apigenin exists in our diet, and that
in recent years there is a great interest in flavonoids as health supplements, the clinical
importance of identifying food and drug interaction deserves further investigation.

Acknowledgements

Our work was supported by the Foundation of science and technology of Guangdong.
Thanks are due to the teacher Shanhe Wan for his assistance in UPLC-MS/MS tests of
apigenin and acyclovir. This work was supported by the Foundation of science and
technology of Guangdong (2014A020210013).

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Figure Legends
Fig. 1 The effects of the tested compounds on the OAT1-mediated uptake of 6-
CFL.
(A) 6-CFL uptake by MDCK-OAT1 and MDCK-mock cells for the quantification of
the inhibitory effects of apigenin (10 μM), baicalein (10 μM), wogonin (10 μM), and
probenecid (500 μM). (B) Concentration-dependent 6-CFL uptake assays for the
quantification of the inhibitory effect of apigenin (B-1), baicalein (B-2), wogonin (B-
3), and probenecid (B-4) on MDCK-OAT1. The values shown are the mean ± SD
(n=3). * p<0.05; ** p<0.01; *** p<0.001.
Fig. 2 Time-dependent inhibition of apigenin on OAT1-mediated 6-CFL
transport. (A) 6-CFL uptake results, showing the inhibition of OAT1-mediated 6-
CFL transport after 30 min pre-incubation with (Gray Bars) or without (Black Bars)
100 μM probenecid, 0.8 μM apigenin, 20 μM baicalein and 8 μM wogonin, in MDCK-
OAT1 cells. (B) 6-CFL uptake results after pre-incubating MDCK-OAT1 cells with
various concentrations of apigenin. (C) Time-dependent inhibitory effect of 0.8 μM
apigenin on the OAT1-mediated 6-CFL uptake. The values shown are the mean ± SD
(n=3). * p<0.05; ** p<0.01.
Fig. 3 Intracellular uptake of apigenin by OAT1 and the effect of apigenin on the
OAT1-mediated transport of acyclovir. (A) UPLC/MS/MS analysis for the cellular
uptake of apigenin (20 μM) in the presence or absence of 500 μM probenecid, after
20 min of co-incubation; (B) 400 μM acyclovir in the presence or absence of apigenin
(1 μM), probenecid (500 μM), and emodin (1 μM), after 1 h of co-incubation. MDCK-
OAT1 and MDCK-mock cells were used to perform the uptake assays. The values
shown are the mean ± SD (n=3). * p<0.05; ** p<0.01; *** p<0.001.
Fig. 4 Apigenin increases the plasma concentration of acyclovir.
Time course analysis of the acyclovir plasma concentration in Male SD rats following
intravenous administration of acyclovir (35 mg/kg) in the presence or absence of
apigenin (70 mg/kg) (n=5).
Fig. 5 Apigenin attenuates the cytotoxicity of adefovir induced by OAT1. (A)
Viability of MDCK-OAT1 and MDCK-mock cells after 24 h incubation with (A)
apigenin (1.56-50 μM); (B) adefovir (12.5-800 μM); (C) variable concentrations of

18
apigenin (1.56-50 μM) in the presence of 200 μM adefovir. MTT assays were utilized
to perform the cell viability assays. * p<0.05; ** p<0.01; *** p<0.001.

19
Figr-1

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Figr-2 21
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Figr-3

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Figr-4

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Figr-5

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 Table 1 MRM parameters of apigenin and acyclovir.

cone collision capillary Source desolvation dwell

Compound Q1 (m/z) Q3(m/z)
（V） （V） (kv) (℃) (℃) (s)
Apigenin 269.04 117 40 34 3.00 108 350 0.100
Acyclovir 226.1 151.95 20 15 5.00 105 350 0.100
AG337 285.2 268.1 30 25 5.00 105 350 0.100

26