Journal of Functional Foods 40 (2018) 292–298

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Journal of Functional Foods

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

Food–drug interactions involving multiple mechanisms: A case study with MARK

effect of Capsaicin on the pharmacokinetics of Irinotecan and its main
metabolites in rat
⁎ ⁎
Nanxi Wang, Chaoran Zhu, Xinlin Zhang, Xuejia Zhai , Yongning Lu
Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

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

Keywords: Capsaicin (CAP), the main ingredient of chili peppers, displays potent anti-tumor activity in several human
Capsaicin cancers. Irinotecan, an anticancer agent with narrow therapeutic index, is subjected to disposition by several
Irinotecan enzymes or transporters modulated by CAP. This study systematically investigated the CAP–drug interactions in
Metabolite vitro, in situ and in animals. Results indicated that after 7 days of 3 mg/kg CAP treatment, the AUC0-∞ of SN-38
Mechanism
was significantly increased to 1.48-fold compared to the vehicle treated rats. Meanwhile, it caused a significant
Food–drug interactions
plasma protein binding displacement of SN-38, lowered the liver to plasma concentration ratio and slight re-
duction of biliary excretion after CAP treatment. These results demonstrated that ingestion of CAP would in-
crease the systemic exposure and toxicity of SN-38 to a significant extent in rats by affecting multiple enzymes
and transporters at various stages.

1. Introduction
Hot pepper fruits, as traditional food or additives, are among the
most heavily consumed spices throughout the world (Hernandez-Ortega
et al., 2012). Capsaicin (CAP, trans-8-methyl-N-vanillyl-6-nonenamide)
derived from the natural food, is the most abundant pungent compo-
nent in chili pepper (Bode & Dong, 2011). It is a valuable pharmaco-
logical agent that has been used for chronic pain treatment, gastro
protection and pruritus in various clinical conditions (Gooding, Canter,
Coelho, Boddy, & Ernst, 2010). Moreover, emerging studies indicated
that CAP displayed potent anti-tumor activity in various types of cancer
and could act as a cancer preventive agent, which has motivated re-
generated attraction for human health and disease (Chapa-Oliver &
Mejia-Teniente, 2016; Reyes-Escogido, Gonzalez-Mondragon, &
Vazquez-Tzompantzi, 2011).
In daily life, the food or spice supplements such as CAP are generally
considered safe. However, several studies demonstrated that CAP might
widely influence the drug metabolizing enzymes (DMEs) and drug
transporters (DTs), which may cause complex drug-drug interactions
(DDIs) (Babbar, Chanda, & Bley, 2010; Takanohashi, Isaka, Ubukata,
Mihara, & Bernard, 2010). Our previous investigations demonstrated
that CAP could significantly decrease the bioavailability of digoxin and
simvastatin, which were mainly metabolized by CYP3A (Kato et al.,
2012; Zhai, Chen, Liu, Shi, & Lu, 2013). However, some researchers
found CAP could increase the systemic exposure of the substrate of the
CYP3A enzyme and P-glycoprotein (P–gp) transporter, and showed a
dual inhibition of CYP3A and P–gp (Zhai, Shi, Chen, & Lu, 2013).
Meanwhile, other reports indicated that CAP exhibited inhibition on
OATP1B1 and BCRP transporter (Chen, Zhai, Zhu, & Lu, 2015; Duan
et al., 2013), which also mediated the pharmacokinetic behaviors of
abovementioned drugs. Hence, these paradoxical results indicated that
other intrinsic underlying mechanisms such as OATP1B1 and BCRP
transporter inhibition may play an important role in these complex
DDIs. These significant CAP–drug interactions have been recognized for
many years, but the biochemical mechanisms have not been fully ex-
plained.
Irinotecan (CPT-11) plays an important role in several cancer regi-
mens mainly in advanced colorectal cancer (Vanhoefer et al., 2001). Its
pharmacokinetics and pharmacodynamics were extremely complex,
involving multiple DMEs and DTs (Newton, Newman, & Hill, 2012). In
short, it was subjected to extensive metabolism by various enzymes
including CES2 to form its active metabolite SN-38, then UGT1A1 to

Abbreviations: APC, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecin; AUC, Area under the plasma concentration–time curve; CAP, Capsaicin; CES,
Carboxylesterase; CPT, Camptothecin; CPT-11, Irinotecan; CYP, cytochrome P450; DMEs, Drug-metabolizing enzymes; DTs, Drug transporters; LC–MS/MS, Liquid chromatography-
tandem mass spectrometry; RLM, Rat liver microsomes; SD rats, Sprague-Dawley rats; SN-38, 7-ethyl-10-hydroxycamptothecin; SN-38G, SN-38 glucuronide; UGT, Uridine diphosphate
glucuronosyl transferase esterase
⁎
Corresponding authors at: No. 1277, Jiefang Avenue, Wuhan, Hubei Province, China.
E-mail addresses: zhaixuejia@163.com (X. Zhai), luyn_union@163.com (Y. Lu).

https://doi.org/10.1016/j.jff.2017.11.014
Received 23 July 2017; Received in revised form 8 November 2017; Accepted 11 November 2017
1756-4646/ © 2017 Elsevier Ltd. All rights reserved.
N. Wang et al.
form SN-38G and CYP3A4 to form APC. Meanwhile, several membrane
transporters, notably BCRP and P–gp, play important roles in their bile
elimination, which greatly affected their pharmacokinetics in vivo
(Smith, Figg, & Sparreboom, 2006). For SN-38, the hepatic uptake
process was mainly dependent on OATP1B1 transporter (Iusuf et al.,
2014). Based on the aforementioned modulations on DMEs and DTs,
CAP was highly suspected to cause the complex interaction with CPT-11
and its metabolites. In addition, SN-38 and CAP both were strongly
bound to albumin in plasma (Fujita et al., 2016), then whether CAP
would increase the free fraction of SN-38 at the target site to cause
adverse effects that was still unknown.
This study performed the in vivo pharmacokinetics study of CPT-11
and its main metabolites with or without CAP pretreatment, and me-
chanistically investigated the behavioral change in the distribution,
metabolism and excretion phases to elucidate the underlying mechan-
isms. It would provide some suggestions for the rational use of CPT-11
and foster the hope of innovative applications of CAP in human disease.
2. Materials and methods
2.1. Chemicals and reagents
CPT-11, SN-38, CPT (internal standard, IS) and CAP were purchased
from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). SN-
38G and APC were purchased from TRC (Toronto, Canada).
HPLC–grade methanol and acetonitrile were obtained from Merck
(Darmstadt, Germany). Formic acid was purchased from Sigma (St.
Louis, MO, USA). The water used for HPLC was purified by use of a
Milli–Q system (Millipore, Milford, MA, USA). All other chemicals were
analytical grade and were commercially available.
2.2. Pharmacokinetic studies in rat
Male Sprague–Dawley rats (200 ± 20 g) were obtained from the
Laboratory Animal Center of the Tongji Medical College of Huazhong
University of Science and Technology (Hubei, China quality certifica-
tion number: SCXK (E) 2016-0009). The rats were allowed to standard
rat chow and water and maintained under controlled temperature and
humidity (22 ± 2 °C and 45 ± 5%). All animal experimentation pro-
cedures were carried out according to the ‘‘Guiding Principles in the
Use of Animals in Toxicology’’ adopted by the Society of Toxicology
(USA) in July 1989 and revised in March 1999 and were approved by
the Ethics Committee of Union Hospital of Huazhong University of
Science and Technology (Permit Number: 2016-S216, Wuhan, China).
The rats were randomly divided into three groups (n = 6, each):
control group (pretreated with 0.5% CMC-Na); positive control group
(pretreated with repeated doses 10 mg/kg of CsA in 0.5% CMC-Na);
CAP pretreated group (pretreated with repeated doses 3 mg/kg of CAP
in 0.5% CMC-Na). All the rats were given vehicle or drug daily by oral
gavage for 7 days. In oral gavage operation, wiped the gavage needle
clean after drawing CAP solution, then injected the CAP directly into
stomach to avoid leaving CAP on the mouth and skin, thereby stimu-
lating animals showed spicy and hot reaction. Twelve hours before
intravenous administration of CPT-11, access to diet was removed and
only water was provided. On the 7th day, 30 min after pretreatment
with CAP, all rats were intravenously injected with CPT-11(HENGRUI
MEDICINE, Jiangsu, China) at the dose of 20 mg/kg. Blood samples
were collected after injection of CPT-11 at the times of 10, 20, 30 min
and 1, 1.5, 2, 3, 4, 6, 8, 10, 24 h. Immediately after sampling, sample
tubes were briefly immersed into ice-water, then immediately cen-
trifuged to obtain plasma, which were stored at −80 °C until analysis.
2.3. Analytical methods
In present paper, a rapid, robust and sensitive LC–MS/MS method
was developed and validated to detect CPT-11 and its main metabolites
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Journal of Functional Foods 40 (2018) 292–298
in biological samples. Briefly, an aliquot of 15 μL of CPT (IS, 1 μg/mL)
was added to 150 μL of rat plasma/tissue homogenate in an Eppendorf
tube. After vortex-mixing for 30 s, the mixture was added with 300 μL
extraction solvent (methanol containing 0.1% formic acid) to pre-
cipitate protein, and then the sample was vortex mixed for 5 min and
centrifuged at 10,000g for 10 min at 4 °C. The clear supernatant fluid
was injected into the LC–MS/MS system for concentration analysis.
The chromatographic separation work was carried on an Agilent
1200 HPLC system with Agilent ZORBAX Eclipse XDB–C18 column
(2.1 × 50 mm, 3.5 μm). The mobile phase was composed of acetonitrile
– water containing 0.1% formic acid (27:73, v/v) at flow rate of 0.3 mL/
min, under 35 °C. The mass spectrometer was operated using an AB
Sciex Qtrap® 4000 tandem mass spectrometer equipped with an elec-
trospray (ESI) source in positive mode. The optimized condition was
consisted of a collision-activated dissociation (CAD) gas of 8 psi, a
curtain gas of 25 psi, a nebulizer gas (GS1) of 40 psi, a Turbo Ion–Spray
gas (GS2) of 40 psi, an ion spray voltage of 5500 V, a source tem-
perature of 400 °C. The monitored ion transitions and collision energy
(CE) were as follows: m/z 587.0 → 124.2, 54 V for CPT-11, m/z
393.2 → 349.1, 36 V for SN-38, m/z 569.4 → 393.2, 39 V for SN-38G,
m/z 619.3 → 393.2, 42 V for APC and m/z 349.1 → 305.1, 34 V for CPT.
Calibration curves were linear over the concentration range of
16.9–6760 ng/mL for CPT-11, 0.58–920 ng/mL for SN-38,
2.5–1000 ng/mL for SN-38G and 1.25–250 ng/mL for APC. The intra–
and inter– batch precision and accuracy of the validated method were
within the acceptable limits of less than 15%. The assay was sufficiently
robust; there was no significant matrix effect from different bio-ma-
trices or tissues. Stability of the samples under the assay conditions was
secured. Data acquisition and integration were carried out by the
Analyst 1.6.1 software (AB SCIEX).
2.4. Estimation of plasma protein binding (PPB) by ultrafiltration
The plasma protein binding rates of SN-38 in the presence or ab-
sence of CAP (0.5 μg/mL) were determined by ultrafiltration method.
Briefly, the fresh rat plasma was prepared from untreated rats and then
SN-38 was added to the blank plasma in vitro to prepare SN-38 plasma
samples at three concentration levels (0.2, 0.5, 1 μg/mL). The series
concentrations of SN-38 were enough to cover the effective con-
centration of clinical dosing range (Marangon, Posocco, Mazzega, &
Toffoli, 2015) and CAP were incubated at plasma concentration ob-
tained from published literature (Suresh & Srinivasan, 2010). The
prepared samples were placed in a water bath incubator set at 37 °C. At
the end of the incubation time, 400 μL plasma was put in the ultra-
filtration apparatus (Molecular weight cutoff: 30 KDa, Millipore), cen-
trifuged to obtain ultrafiltrate. The concentrations in the plasma and
filtrate measured at each point were used to calculate the unbound
fraction through dividing the unbound concentration by the total
plasma concentration. For sample analysis, 100 μL of filtrate was mixed
with 10 μL of the internal standard solution, then added with 100 μL of
methanol, vortex-mixed for 30 s and centrifuged at 10,000g for 5 min at
4 °C, and take the supernatant fluid for concentration analysis. For re-
covery test of ultrafiltration, SN-38 PBS buffer samples at high, medium
and low (375, 40, 5 ng/mL) concentrations (Cpre) were respectively
placed in blank ultrafiltration tube, and centrifuged to obtain ultra-
filtrate (Cpost) at the same ultrafiltration condition. The recovery rates
of the ultrafiltration apparatus were calculated as: Recovery
rate = Cpost/Cpre × 100%. Followed by initial experiment to de-
termine the incubation time and ultrafiltration condition of SN-38, each
experiment was conducted in 5 replicates.
2.5. Distribution of CPT-11 and its active metabolite in rat liver
In this liver distribution study, rats were randomized and orally
administered by 0.5% CMC-Na or 3 mg/kg CAP in 0.5% CMC-Na for
7 days. On the 7th day, 30 min after pretreatment with CAP, all rats
N. Wang et al.
Fig. 1. Plasma concentrations of CPT-11 and its main metabolites after intravenous administration of CPT-11 (20 mg/kg) to rats given oral 0.5% CMC-Na (control group), CsA (10 mg/kg,
positive control group) and CAP (3 mg/kg) for seven consecutive days (n = 6). Data are expressed as mean ± S.D.
were intravenously given by CPT-11 at the dose of 20 mg/kg. Then, the
blood and liver samples were immediately collected at 1, 2, 4 h (n = 5,
each). The liver tissue was washed with cold physiological saline,
blotted on filter paper and weighed, then cut into pieces. Each gram of
liver tissue was ground into homogenate with 3 mL ice-cold physiolo-
gical saline. The samples were processed by above described method for
analysis.
2.6. Metabolism studies
To examine the inhibitory potential of CAP against the key enzymes
mediated CPT-11 metabolism, we investigated the CAP effect on the
activity of Ces2 and Ugt1a1 enzymes in short- and long-term treatment.
For short-term treatment, normal rat liver microsomes (RLM) were
incubated CPT-11 or SN-38 with increasing concentrations of CAP
(1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM and 100 μM) to de-
termine IC50. The rats were given oral 0.5% CMC-Na (control group)
and CAP (3 mg/kg) for seven consecutive days and sacrificed on the
seventh day to get the long-term treated RLM. Then, we also detected
the enzyme activity by only incubation with CPT-11 or SN-38.
According to the above process, incubated metabolites with CPT-11 and
SN-38 were performed at the final concentration corresponding to the
relative activity and the IC50 value for Ces2 and Ugt1a1 enzymes, re-
spectively.
The RLM was prepared by the classic differential centrifugation
method, then the protein concentration of microsome was detected by
BCA Protein kit (Bradford, 1976). We had optimized the incubation
system conditions, such as the concentration of microsome protein and
the incubation time by univariate analysis. Then, we chose the best
condition for the incubation system. The microsome protein and the
substrates were pre-incubated separately for 5 min at 37 ± 1 °C. After
pre–incubation, 30 min incubation for samples were carried out in a
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shaking water bath. The Ces2 enzyme incubation mixture with a total
volume of 100 μL contained 40 μL phosphate buffer (pH = 7.4), 10 μL
CPT-11 solution (10 μM at final concentration) and 50 μL of microsome
protein (0.3 mg/mL at final concentration). The cofactors and substrate
of Ugt1a1 enzyme incubation system were performed separately in a
37 °C water bath for 5 min. Then, the reaction was started by adding
10 μL UDPGA–generating system, 12.5 μL MgCl2, 5 μL SN-38 (5 μM at
final concentration) and 22.5 μL phosphate buffer to 50 μL of micro-
some protein (0.3 mg/mL protein). All above system were terminated
by 200 μL of cold methanol contained 0.1% formic and 1000 ng/mL of
IS, and placed on ice for 3 min. Then, the samples were vortexed for
30 s, centrifuged at 10,000g for 10 min at 4 °C, and the supernatant
injected for further analysis.
2.7. Drug interaction in biliary excretion
In the biliary excretion experiments, male SD rats were randomly
divided into control group and CAP pretreated group as described
above, to give vehicle or CAP daily by oral gavage for 7 days. On the
7 th day, the abdominal cavity was opened after induction of anesthesia
with 10% chloral hydrate and the common bile duct was ligated and a
catheter was inserted into the gallbladder. 30 min after pretreatment
with CAP, all rats were intravenously given with CPT-11. The bile
samples were collected and weighted during 0–0.5, 0.5–1, 1–2, 2–3,
3–4 h after which the rats were sacrificed. Bile samples were analyzed
as described below. An aliquot of 10 μL of bile samples was added with
500 μL of extraction solvent (0.1% formic acid methanol), vortex mixed
and centrifuged, and then the clear supernatant fluid was injected into
the LC–MS/MS system.
N. Wang et al. Journal of Functional Foods 40 (2018) 292–298

2.8. Statistical analysis

The pharmacokinetic parameters (AUC, CL, and T1/2) were calcu-

lated by the Drug and Statistics (DAS) 2.0.1 pharmacokinetics fitting
software. The inhibitory IC50 values were calculated and obtained by
fitting the data with nonlinear regression analysis using Prism 5.0
(Graph Pad Software Inc., San Diego, CA, USA). The results of control
and test groups were compared by one-way ANOVA followed by
Fisher’s Protected Least Significance Difference (PLSD) test. All ex-
perimental data were expressed by Mean ± SD. All statistical tests
were performed with SPSS Statistics version 17 (IBM Inc., Armonk,
USA).

3. Results

Fig. 2. PPB displacement of SN-38 in the presence of CAP. Data represent the mean and
3.1. The effect of CAP on the pharmacokinetics of CPT-11 and its main
standard error of mean of five independent experiments. **P < .01, compared with
metabolites
control group.

There were no significant differences in mean body weight between

the control and CAP pretreatment groups after 7 days of treatment.
After intravenous injection at the dose of 20 mg/kg, the plasma con-
centration–time curves of CPT-11 and its main metabolites with dif-
ferent pretreatments were shown in Fig. 1.
Upon seven consecutive days of pretreatment with CAP at the daily
dosages of 3 mg/kg, the blood concentration of SN-38 had significantly
increased (P < .05). Compared to the control group, the AUC0-∞ were
significantly increased to 1.48-fold in the CAP pretreated group, as
shown in Table 1. The difference in T1/2 did not quite reach statistical
significance, but the CL was significantly decreased by 29.92%
(P < .05). At the same time, it was interesting to note that the plasma
concentration of SN-38 was higher than the group pretreated with ve-
hicle during 1 h to 4 h, and the difference was statistically significant
(P < .05). However, the main pharmacokinetic parameters of CPT-11,
SN-38G and APC didn’t hint statistically significant difference between
the group pretreated with vehicle and CAP (P > .05), as presented in
Supplementary Table 1.
3.2. PPB displacement
The time to reach PPB equilibrium of SN-38 with an oscillation
frequency of 100 r/min was 60 min. Considering no leakage of protein
and proper ultrafiltration ratio, the sample was centrifuged at 7000g for
15 min to obtain filtrate. The recovery rates at three concentrations
(375, 40, 5 ng/mL) were (97.5 ± 1.70)%, (97.1 ± 0.61)% and
(97.3 ± 1.23)%, respectively, which indicating that SN-38 had no
significant adsorption to the ultrafiltration apparatus in the experi-
mental concentration range. PPB displacement studies clarified that SN-
38 in the presence of CAP resulted in significant decrease of the bound
ratios of SN-38 in rat plasma. The binding rates of SN-38 (0.2, 0.5, 1 μg/
mL) were (95.7 ± 0.72)%, (94.1 ± 0.97)%, (94.4 ± 0.49)% in rat
plasma, respectively. In the presence of CAP (0.5 μg/mL), the plasma
protein binding rates of SN-38 were significantly decreased to
(90.7 ± 1.83)%, (88.5 ± 1.91)%, (86.2 ± 2.89)%. Accordingly, the
unbound SN-38 concentrations were significantly increased to 2.15,
1.95 and 2.48-fold (P < .01), shown in Fig. 2.
3.3. CPT-11 and SN-38 concentrations in the rat liver
To estimate distribution ratio between liver and plasma tissue (L/P
ratio) and gain insight into the liver distribution, the concentration of
CPT-11 and SN-38 were quantified in rat liver and plasma samples. The
liver tissue concentrations of SN-38 in the CAP pretreated group were a
little lower at the 1, 2 and 4 h, which were not significantly different
between the different treatments. The further statistical study indicated
that the L/P ratios of SN-38 in the group pretreated with CAP were
lower than the control group. Moreover, the difference reach statistical
significance at 2 h point (P < .05). For the CPT-11, there were not
significant difference in liver distribution between CAP treatment group
and control group (P > .05), as shown in Fig. 3.
3.4. Effect of CAP on the key enzyme mediated CPT-11 metabolism in RLM
CPT-11 and SN-38 as the probe substrates, were mainly metabolized
by Ces2 enzyme and by Ugt1a1 enzymes, respectively (Zhang, Liu, &
Jeong, 2015). Therefore, the effect of CAP on Ces2 and Ugt1a1 activity
were further examined using CPT-11 and SN-38, respectively. The
conditions of incubation system in vitro have been optimized, and IC50
values were estimated in RLM incubation system. It was found that the
IC50 of CAP for Ces2 and Ugt1a1 enzyme respectively were 60.74 μM
and 408.2 μM respectively (Fig. 4). It demonstrated that CAP exhibited
weak inhibition on Ces2 enzyme and nearly no inhibition on Ugt1a1
enzyme. However, the Ces2 enzyme activity in RLM was not sig-
nificantly different after CAP long-term pretreatment. Meanwhile, the
amount of SN-38G was decreased by 8.41%, which indicated the
Ugt1a1 enzyme activity was decreased by 8.41%, but the difference still
didn’t reach significant level (P > .05), as shown in Fig. 4.
3.5. The effect of CAP on bile excretion of the CPT-11 and its main
metabolites in rat
The cumulative bile excretion of CPT-11, SN-38 and SN-38G in the
positive control group pretreated with CsA, were much lower than the
control group due to the inhibition of the BCRP and P-gp transporters

Table 1
The main pharmacokinetic parameters of SN-38 in rat plasma (Mean ± SD, n = 6).

Analyte Parameter 0.5% CMC-Na CsA 3 mg/kg CAP

* ^3 ^3**
SN-38 AUC(0-∞) (μg h/L) 0.91 ± 0.17 × 10 1.55 ± 0.31 × 10 1.35 ± 0.35 × 10^3*
t1/2 (h) 2.72 ± 1.41 3.11 ± 0.35 3.21 ± 1.42

*Indicates P < .05 and ** Indicates P < .01 compared to the 0.5% CMC-Na (control group).

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N. Wang et al.
(Li et al., 2014). Those target analytes were significantly decreased to
53.4%, 53.5%, 62.9%, respectively (P < .05). Meanwhile, the cumu-
lative bile excretion of CPT-11, SN-38 and SN-38G were slightly re-
duced in CAP pretreated group (Fig. 5), and the detail cumulative bile
excretions of CPT-11, SN-38 and SN-38G data were shown in Table 2.
4. Discussion
Currently, CAP has potential antitumor effects and induces apop-
tosis in many types of malignant cell lines, including colon adeno-
carcinoma, hepatocellular carcinoma, breast cancer and many others,
suggesting that CAP would be a promising therapeutic agent for cancer
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Journal of Functional Foods 40 (2018) 292–298
Fig. 3. The distribution concentrations and L/P
ratio of CPT-11 and SN-38 after intravenous ad-
ministration of CPT-11 (20 mg/kg) to rats given
oral 0.5% CMC-Na (control group) and CAP (3
mg/kg) for seven consecutive days (n = 5).
*
P < .05, compared with control group.
(Lin et al., 2017). In the present study, the potential CAP–CPT-11
pharmacokinetic interactions were evaluated, which would foster the
hope of innovative applications of CAP in human cancer. This study
investigated the effects of CAP on the pharmacokinetics of CPT-11 and
its main metabolites in rats and preliminary examine the multiple
mechanisms underlying the drug interactions, including PPB displace-
ment and liver distribution of CPT-11 and SN-38, role of metabolism
and biliary excretion in vitro and in situ.
In the pharmacokinetic study, the rats were pretreated with CAP at
3 mg/kg CAP for 7 day based on the average daily intake of CAP (Zhai
et al., 2013) and the pretreatment period reported in several published
literatures (Chen et al., 2015; Duan et al., 2013). The pharmacokinetic
Fig. 4. Inhibitory effect of CAP on Ces2 and
Ugt1a1 enzyme. For short-term, reaction velo-
cities are expressed as a ratio (in percentage) re-
lative to the velocity without CAP in RLM. For
long-term, reaction velocities are expressed as
generation amount of SN-38 and SN-38G.
N. Wang et al.
Fig. 5. Biliary excretion of CPT-11, SN-38 and SN-38G over 4 h after CPT-11 intravenous injection to rats given oral 0.5% CMC-Na (control group), CsA (10 mg/kg, positive control group)
and CAP (3 mg/kg) for seven consecutive days (n = 6). Data are expressed as mean ± S.D.
Table 2
Effect of CAP on the biliary excretion cumulative of CPT-11, SN-38 and SN-38G
(Mean ± SD, n = 6).
Experimental groups Cumulative excretion of bile (μg/kg/4h)
CPT-11 SN-38 SN-38G
^3
Control group 1.36 ± 0.36 × 10 69.3 ± 14.1 76.5 ± 17.3
CsA pretreated group 0.73 ± 0.14 × 10^3* 37.1 ± 4.09* 48.1 ± 7.88*
CAP pretreated group 1.02 ± 0.32 × 10^3 60.8 ± 19.9 70.8 ± 15.4
* Significantly different with P < .05 versus 0.5% CMC-Na (control group).
results illustrated that co-administration of 3 mg/kg CAP increased the
plasma concentration and the systemic exposure of SN-38. Meanwhile,
the AUC0-∞ were significantly increased to 1.48-fold (P < .05), after
the rats had ingested a CAP-supplemented diet for 7 days. As we all
know, SN-38 is the active metabolite of CPT-11 and exert the phar-
macological effect. The increased bioavailability of SN-38 was pro-
mising to achieve a higher anticancer efficacy in colon carcinoma
treatment (Chu et al., 2014). However, SN-38 has a narrow therapeutic
index, and its cytotoxicity is much greater than the parent drug. Higher
SN-38 accumulation level may lead to acute diarrhea and other choli-
nergic syndrome and fatal hematological toxicity. Therefore, CAP pre-
administered in a relevant supplementary concentration would enhance
the pharmacological efficacy and increase the supra-therapeutic level
risk of SN-38 by heightening plasma concentration levels after intake.
Then, if a patient on CPT-11 treatment intakes excessive CAP-con-
taining food/drug, it’s better to reduce the dose of CPT-11 accordingly.
It was generally accepted that an increase in free fraction of the drug
would have effect on its disposition process in vivo, and significantly
alter AUC of the drug. For further research, we conducted the PPB
displacement experiment to investigate the unbound plasma con-
centration changes of SN-38 in rat plasma. Our findings suggested that
CAP significantly enhanced the unbound fractions of SN-38 and in-
creased its unbound concentrations in vitro (P < .01). However, other
published literatures (Benet & Hoener, 2002; Shaik, Bohnert, Williams,
Gan, & LeDuc, 2016) indicated that the changes in plasma protein
binding in vitro rarely leaded to significant pharmacokinetic interac-
tion, as increased free drug concentration would lead to rapid elim-
ination of the drug until steady state is obtained. Besides, some in-
vestigators (Fujita et al., 2016) have shown that the significant PPB
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Journal of Functional Foods 40 (2018) 292–298
displacement of SN-38 ex vivo was not the main reason for the in-
creased unbound concentration of SN-38 in vivo, which wouldn't result
in clinically significant interactions. The findings from this study need
to be understood in conjunction with DMEs inhibition, as well as dis-
position mediated by DTs.
In the positive control group, CsA as the reported inhibitor of P–gp
and BCRP transporter, had increased systemic availability of CPT-11,
SN-38 and SN-38G in rat. Our previous studies also found CAP could
increase systemic exposure of other substrate drugs of P–gp and BCRP
transporters that mediated the biliary excretion of CPT-11, SN-38 and
SN-38G. However, through observing the serum profiles, it was inter-
esting to note that there were difference of effect period on pharma-
cokinetic behavior of SN-38 between CAP and CsA treatments. The
former affected pharmacokinetics mainly during 1–4 h (Fig. 1b) and the
latter increased concentration of SN-38 during 4–8 h. Moreover, the
plasma concentrations of SN-38 were obviously higher than the group
pretreated with vehicle during 1–4 h, and the difference were still sta-
tistically significant (P < .05). It was illustrated that the biochemical
mechanism may be different in some aspects between the effects of CsA
and CAP on the pharmacokinetic of SN-38. To test this hypothesis, we
investigated the biliary excretion outputs of CPT-11 and it metabolites.
Compared to the control group, the positive control group pretreated
with CsA, significantly decreased the biliary excretion, which was
consistent with the pharmacokinetic results. The accumulation bile
excretion of SN-38 had also decreased in the group pretreated with
CAP, but it didn’t reach the statistical significance (p > .05). The re-
sults explained satisfactorily the above different pharmacokinetic be-
havior, and indicated that CAP had a slight regulation in bile elimina-
tion of SN-38.
According to the aforementioned knowledge, there were still several
DMEs and DTs in the distribution and metabolism system that would
influence the disposition behavior of SN-38, which were also key
pathways for SN-38 disposition. Hence, the findings from this study
need to be understood in conjunction with tissue distribution, as well as
the role of several DMEs.
Distribution refers to the drug within the blood circulation to reach
the body's tissues and organs process, which has close relationship with
the tissue storage, efficacy and adverse effects. Many studies have
shown Oatp1b2 transporter in rat (OATP1B1 in human) may mediate
hepatic uptake of SN-38 from plasma in rat. Based on the pharmaco-
kinetic results, we investigated the effect of CAP on the distribution
N. Wang et al.
from blood to liver during 1–4 h. Interestingly, CAP could reduce the
distribution of SN-38 in liver, and the L/P ratio had a significantly
difference at 2 h (P < .05). Meanwhile, other published literature
(Suresh & Srinivasan, 2010) showed that the concentration of CAP in
liver was highest at 3 h after oral CAP, and then the concentrations of
CAP in liver were decreased rapidly, which suggested the CAP in-
hibitory concentration may be effective only during 1–4 h, thereby
leading to transient inhibition of Oatp1b2 transporter. Besides, the
tissue distribution results were consistent with our previous study
finding that CAP could increase the bioavailability of pitavastatin, the
substrate of Oatp1b2 transporter in rat (Chen et al., 2015). The results
indicated that CAP may inhibit the Oatp1b2 mediated SN-38 uptake to
some extent, which would mainly contribute in this DDIs.
Metabolism is structural change process for drugs in the body,
which impact on the pharmacokinetics of the drug greatly. Liver mi-
crosomal incubation is the commonly used method for the drug inter-
action study in vitro, because it’s easy to operate and has good re-
producibility. We evaluated the activity of the key enzymes including
Ces2 and Ugt1a1 by RLM incubation system. Our findings indicated
that CAP had a weak inhibitory effect on Ces2 (IC50 = 60.74 μM) in
RLM. The results in vitro appeared to be inconsistent with the phar-
macokinetic result in vivo, as SN-38 exposure amount was increased in
the CAP pretreated group. However, other study indicated that systemic
exposure to CAP which follows from either the ingestion of CAP-con-
taining foods or topical administration of a high-concentration cuta-
neous patch is highly unlikely to inhibit or induce the activities of
metabolic enzymes (Babbar et al., 2010). Besides, upon seven con-
secutive days of pretreatment with CAP, the Ces2 enzyme activity in the
CAP pretreated group was not significantly different compared to the
control group. Therefore, the inhibition enzyme activity of CAP on Ces2
is negligible in vivo; it might be masked or reversed by other me-
chanism interventions.
Based on our findings, we could draw a conclusion that CAP cer-
tainly alters the pharmacokinetic of SN-38 and increased the bioavail-
ability of SN-38 to a significant extent in the rat. The complex DDIs
mechanisms were originated from the net–effect, which integrated in
significant plasma protein binding displacement of SN-38, lower L/P
ratio in distribution phase and slight reduction of biliary excretion in
elimination phase after CAP treatment. The net–effects have been de-
scribed as a “see–saw” phenomenon (Wu, 2012), and the importance of
this effect has been elucidated via in vitro, in situ and in animal studies,
which would be helpful for the rational prediction of possible interac-
tion. Meanwhile, for many drugs, such as CPT-11 that modulate by a
variety of enzymes and transporters in vivo, it’s not suitable to study
and evaluate this series of complex food–drug interactions associated
with multiple mechanisms by only considering single DME and DT.
Otherwise, it could lead to ignorance or wrongly prediction of severe
adverse reactions and/or pharmacological effects reduction.
In summary, since the SN-38 disposition was altered when CAP co-
administered to rats, we suggest that care should be taken of the pos-
sible CAP–drug interactions when patient’s concurrent intake of CAP.
With regard to potential pharmacokinetic drug–CAP interactions, it is
necessary to confirm and clarify these effects of CAP to gain insight into
any possible interactions between CAP and CPT-11 or other drugs in
further clinical use.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (Grants 81473287, 81673715 and 81403012).
298
Journal of Functional Foods 40 (2018) 292–298
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.jff.2017.11.014.
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