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Biomedicine & Pharmacotherapy 108 (2018) 208–215

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Biomedicine & Pharmacotherapy


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

Metabolism of liver CYP450 and ultrastructural changes after long-term T


administration of aspirin and ibuprofen
Congcong Wena,1, Zaishou Zhuangb,1, Huanchun Songc, Shuhua Tongc, Xianchuan Wanga,
⁎ ⁎
Yijing Lina, Haichao Zhanc, Zhibin Chend, , Lufeng Hue,
a
Laboratory Animal Center, Wenzhou Medical University, Wenzhou 325035, China
b
Department of Intensive Care Unit, Cangnan People’s Hospital, Wenzhou 325000, China
c
Department of Pharmacy, Jinhua Municipal Central Hospital, Jinhua, China
d
Department of Nephrology, The Affiliated Yueqing Hospital of Wenzhou Medical University, Yueqing, China
e
Department of Pharmacy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China

A R T I C LE I N FO A B S T R A C T

Keywords: Worldwide, aspirin and ibuprofen are the most commonly used non-steroidal anti-inflammatory drugs (NSAIDs).
Liver Some adverse reactions, including gastrointestinal reactions, have been concerned extensively. Nevertheless, the
Aspirin mechanism of liver injury remains unclear. In the present study, we focused on the metabolism of liver cyto-
Ibuprofen chrome P450 (CYP450) and ultrastructural morphology of liver cells. A total of thirty rats were divided into
CYP450
three groups of 10. Rats in the aspirin and ibuprofen groups were given enteric-coated aspirin (15 mg/kg) and
Rat
ibuprofen (15 mg/kg), respectively by gavage for four weeks. The body weights were recorded every two days.
Liver function and metabolic capacity of CYP450 were studied on days 14 and 28. We then conducted ultra-
structural examinations. Body weights in the Ibuprofen group were lower than those of the Control group, and
ALT and AST levels were significantly higher (P < 0.05). There were no significant differences in terms of body
weight, ALT or AST between the Aspirin and Control groups. The metabolic capacity of CYP450 was evaluated
using five probe drugs, phenacetin, tolbutamide, metoprolol, midazolam, and bupropion. We found that ibu-
profen and aspirin induced metabolism of the probe drugs. Moreover, according to the pharmacokinetic data, the
Control, Aspirin and Ibuprofen groups could be discriminated accurately. Ultrastructural examination showed
that the number of mitochondria was increased in both the Ibuprofen and Aspirin groups. Long-term adminis-
tration of enteric-coated aspirin and ibuprofen induced the metabolic activity of the CYP450 enzyme. Aspirin
had better tolerability than did ibuprofen, as reflected by pharmacokinetic data of probe drug metabolism.

1. Introduction of fever, pain, and inflammation, including post immunization fever,


painful menstruation, osteoarthritis and headaches [10]. Ibuprofen can
Aspirin and ibuprofen are among the most widely used non-ster- also be used to close patent ductus arteriosus [11] and to enhance
oidal anti- inflammatory drugs (NSAIDs) in the world. Aspirin, also pyrazinamide treatment of murine tuberculosis [12].
called acetylsalicylic acid, is used to treat certain types of pain, fever Because aspirin and ibuprofen are NSAIDs, both cause a variety of
and inflammation, including neuropathic pain [1], migraine and epi- adverse effects, including stomach ulcers, stomach bleeding, coagula-
sodic tension-type headache [2] and rheumatoid arthritis [3,4]. Long- tion disorders, anaphylaxis and cerebral microbleeds [13]. Moreover,
term aspirin use prevents heart attacks and strokes [5]. For example, both cause liver damage [10]. Laster [14] reported a patient with
administration of 75–1500 mg/d of aspirin has substantial benefits for pericarditis treated with aspirin who developed subsequent acute liver
the prevention of myocardial infarction [6]. Furthermore, aspirin re- injury. Liver injuries are more dangerous than gastrointestinal injuries.
duces the risk of colorectal cancer [7], breast cancer [8] and en- Nevertheless, there have been few studies focusing on liver injury and
dometrial cancer [9]. Ibuprofen [2-(4-isobutylphenyl)- propionic acid], the mechanism of that injury.
had effects similar to those of aspirin, but had better effect in treatment In a previous study [15], we showed that intragastric administration


Corresponding authors.
E-mail addresses: czb-888@163.com (Z. Chen), hulufeng79@sina.com (L. Hu).
1
These authors contributed equal to this work.

https://doi.org/10.1016/j.biopha.2018.08.162
Received 14 June 2018; Received in revised form 20 August 2018; Accepted 31 August 2018
0753-3322/ © 2018 Published by Elsevier Masson SAS.
C. Wen et al. Biomedicine & Pharmacotherapy 108 (2018) 208–215

Table 1
UPLC-MS/MS conditions of five probe drugs and their metabolites.
Compound Parent ion Daughter ion Dwell Cone (V) Collision
(m/z) (m/z) (s)

Bupropion 240.1 184.1 0.009 32 15


Metoprolol 268.1 115.8 0.009 32 10
Midazolam 326.0 291.0 0.009 20 12
Phenacetin 180.1 109.9 0.009 32 10
Tolbutamide 271.2 155.1 0.009 30 16
Hydroxybupropion 256.1 238.1 0.009 30 12
Hydroxymetoprolol 284.1 116.0 0.009 32 10
Hydroxylmidazolam 342.2 203.1 0.009 30 16
Acetaminophen 152.0 110.0 0.009 30 12
Hydroxytolbutamide 287.0 107.0 0.009 30 12
Internal standard 285.1 193.1 0.009 30 12

Fig. 1. Monitoring of body weight of rats in Control, Aspirin, and Ibuprofen


groups, the number of rats n = 10 in every groups. 2.3. Determination of probe drugs

of aspirin (15 mg/kg) or ibuprofen (15 mg/kg) for 3 weeks caused 2.3.1. Analytical conditions
metabolomic changes in blood and increased the level of alanine Chromatographic separation of the five probe drugs, metabolites,
transaminase (ALT) and aspartate aminotransferase (AST), indicating tolbutamide, bupropion, phenacetin, metoprolol and testosterone and
there was liver injury with long-term administration of aspirin or ibu- (IS) was carried out on an ACQUITY UPLC and a XevoXevo TQ-S Micro-
profen. In the present study, we focused on the metabolic changes of triple quadrupole mass spectrometer (Waters Corporation, USA).
liver CYP450 enzymes and ultrastructural morphological characteristics An BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used at 40 °C.
of liver cells. We developed a cocktail method and used it to evaluate The mobile phase consisted of 0.1% formic acid in water (A) and
the metabolic capacity of liver CYP450 isoenzymes. We also evaluated acetonitrile (B) in a gradient elution as follows (Tmin / acetonitrile):
the values of liver CYP450 isoenzyme measurement for early diagnosis 0.0–0.5/45%, 0.5–3.0/90% and 3.0–4.0/45%. The flow rate was set at
of liver injury. The whole schematic picture of study design was showed 0.4 mL/min. The injection volume was 2 μL. The probe drugs and me-
in supplemental Fig. 1. tabolites were measured in multiple reaction monitoring (MRM) mode,
as shown in Table 1.
2. Materials and methods
2.3.2. Sample preparation
2.1. Reagents Aliquots of 100 μL plasma were added to 1.5 mL centrifuge tubes
after the frozen plasma samples thawed at room temperature.
Aspirin enteric tablet, 0.1 g/tablet, batch number: J20130078, was Subsequently, 0.3 mL of acetonitrile containing 1.0 μg/mL of IS was
obtained from Bayer China Co., LTD (Beijing, China). Ibuprofen, 0.3 g/ added and vortexed for 0.5 min. The mixture was centrifuged at
capsule, batch number: H109000089, was provided by the Sino- 15,000 rpm for 10 min, and 2 μL supernatants were injected into the
American Tianjin Pharmaceutical Co., LTD (Tianjin, China). The five LC–MS system for analysis.
probe drugs: phenacetin, tolbutamide, metoprolol, midazolam, bupro-
pion and their metabolites hydroxybupropion, hydroxymetoprolol, 2.3.3. Method validation
hydroxymidazolam, paracetamol, hydroxytolbutamide (all purity > The calibration curves of all probe drugs and metabolites were
98%), were purchased from Sigma-Aldrich (St. Louis, MO, USA). The constructed over the range of 1–2000 ng/mL (1, 5, 10, 20, 50, 100, 200,
internal standard was diazepam, purchased from Sigma-Aldrich. HPLC 500, 1000 and 2000 ng/mL). Quality-control (QC) samples were pre-
grade acetonitrile and methanol were from Merck Company pared at 20, 400 and 1800 ng/mL. The samples for calibration curves
(Darmstadt, Germany). Ultra-pure water (resistance > 18 mΩ) was and QC were precipitated with acetonitrile, as with the sample pre-
prepared on a Millipore Milli-Q purification system (Bedford, USA). All paration.
other chemicals were of analytical grade. The intra-day and inter-day precision of the method was evaluated
at three QC levels (20, 400, and 1800 ng/mL) with five replicates. The
2.2. Animals and experimental design extraction recovery of probe drugs and IS investigated in rat plasma was
determined by comparing the peak area of probe drugs to pure standard
The 30 male Sprague Dawley (SD) rats weighing 200–250 g were solution at the same concentration. The matrix effect was investigated
purchased from Wenzhou Medical College Laboratory Animal Center by comparing the peak area of probe drugs to extracted sample added
(Wenzhou, China). They were randomly divided into 3 groups: Aspirin probe drugs with the same concentration (20, 400 and 1800 ng/mL).
group (n = 10), Ibuprofen group (n = 10), and Control group (n = 10). The stability of the probe drugs was investigated by analyzing QC
Rats in the Aspirin-group were given intragastric administration with samples at room temperature for 4.0 h and stored at -80 °C. For more
aspirin (15 mg/kg) or ibuprofen (15 mg/kg) for four weeks, while the detailed information, please refer to our prior studies [16,17].
rats in the control group were given saline solution. Every two days, the
weight of all rats was recorded and the administration doses were ad- 2.4. Pharmacokinetic studies of probe drugs
justed.
The metabolic capacity of CYP450 enzymes and liver function were On the morning of days 14 and 28, the five probe drugs (tolbuta-
studied on days 14 and 28, followed by ultrastructural examinations of mide, bupropion, phenacetin, metoprolol and testosterone) were si-
livers. Liver function was tested on a fully-automatic biochemical in- multaneously administered to rats by gavage at 1, 10, 10, 10 and
strument (AU 5800, Beckman). The metabolic capacity of CYP450 en- 10 mg/kg. Then, the blood samples (0.3 mL) were collected from the
zymes was evaluated by a cocktail method, with co-administration of caudal vein into heparinized 1.5 mL polythene tubes at 0, 0.25, 0.5, 1,
the five probe drugs. The UPLC-MS/MS determination method of five 2, 3, 4, 6, 8, 12 and 24 h. The samples were immediately centrifuged at
probe drugs was developed as described below. 8000 rpm for 5 min. The plasma was separated and stored in -80oC until

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LC–MS analysis. 3.2. Blood biochemical examination


Based on the development of the LC–MS method, the plasma con-
centrations of five probe drugs were determined and analyzed. The We determined a total of 13 blood biochemical indexes: alanine
pharmacokinetic parameters of the five probe drugs were calculated in transaminase (ALT), aspartate aminotransferase (AST), alkaline phos-
a two-compartment model with 1/cc weight coefficients using Drug and phatase (AKP), total bilirubin (TBIL), direct bilirubin (DBIL), albumin
Statistics 2.0 software (DAS 2.0). (ALB), total protein (TP), uric acid (UA), creatinine (CR), total choles-
terol (CHO), total triglyceride (TG), low density lipoprotein (LDL) and
high-density lipoprotein (HDL). On day 14, there was no significant
2.5. Ultrastructure of liver tissue difference in terms of ALT and AST between the Aspirin and Ibuprofen
groups. On day 28, the levels of AST and ALT were higher in the
On day 28, after the pharmacokinetic studies of probe drugs, rats in Ibuprofen and Aspirin groups, with a significant difference between the
all three groups were anesthetized by intraperitoneal injection of Control and Ibuprofen groups (P < 0.05), suggesting that ibuprofen
20 mg/kg chloral hydrate (10% aqueous solutions). The liver was ra- caused more serious liver damage than did aspirin. The results of blood
pidly isolated and immersed in fixative (2.5% glutaraldehyde, 0.1 M biochemical examinations are shown in Table 2.
cacodylate buffer, pH 7.2). After the tissue was washed in 0.1 M caco-
dylate buffer five times, it was post-fixed in 1% osmium tetroxide for
24 h. Then it was washed in 0.1 M cacodylate buffer again and em- 3.3. Pharmacokinetics of probe drugs
bedded in Epon. An ultramicrotome (Leica Ultracut-UCT) was used to
obtain the ultrathin sections that were counterstained with uranyl Typical UPLC-MS/MS chromatograms (Fig. 2) of the five probe
acetate and lead citrate. The stained sections were examined by a drugs and metabolites were smooth and clean without interference
Hitachi H-7500 transmission electron microscope. from endogenous compounds. The study of method validation showed
that UPLC-MS/MS method had good linearity, accuracy and precision
2.6. Statistical analysis in the range of 1–2000 ng/mL. The UPLC-MS/MS linear regression is
listed in supplemental Table 3. The concentrations of the five probe
The statistical differences of the pharmacokinetic parameters of the drugs and their metabolites in rat plasma were measured by UPLC-MS/
five probe drugs, their metabolites and indices of liver function among MS and analyzed by DAS. The pharmacokinetic profile was calculated
the groups were analyzed by ANOVA. The diagnostic values of phar- in two compartment models.
macokinetic parameters of probe drugs, metabolites and liver indices The main pharmacokinetic parameters and concentration-time
were analyzed by the Fisher discriminant, performed using the stepwise curve of probe drugs, determined on days 14 and 28 in the three groups
method with Wilks' lambda option. The ANOVA and Fisher dis- are displayed in Tables 3 and 4 and Figs. 3 and 4. The main pharma-
criminant were performed using SPSS16.0 software. cokinetic parameters and concentration-time curves of metabolites of
probe drugs determined on day 14 and 28 are listed in supplemental
Tables 4 and 5 and supplemental Figs. 2 and 3.
3. Results

3.1. Monitoring of body weight 3.4. Fisher discriminant

The average body weights in the three groups were shown in Fig. 1. All pharmacokinetic data of the five probe drugs (bupropion, me-
Body weights in the control group increased rapidly, followed by the toprolol, midazolam, phenacetin, and tolbutamide) and their metabo-
Aspirin and Ibuprofen groups. There was a significant difference lites were used to discriminate the three groups by a stepwise method.
(P < 0.05) between the Control group and the Ibuprofen group on On day 14, original grouped cases and cross-validated grouped cases
days 3–28, however, there was no significant difference between the were correctly classified 100.0% and 93.3% for probe drugs, respec-
Control and Aspirin groups even at day 28 (supplemental Tables 1 and tively, and 83.3% and 80.0% for their metabolites, respectively. On day
2). On days 15–19, body weights in all three groups were decreased. 28, the original grouped cases and cross-validated grouped cases cor-
This was because there were pharmacokinetic studies of probe drugs on rectly classified were 96.7% and 96.7% for probe drugs, respectively,
day 14 that required that the animals fast for 24 h prior to collection of and 89.7% and 82.8% for their metabolites, respectively. The scatter
blood samples. plot of pharmacokinetic data of the five probe drugs is shown in Fig. 5.

Table 2
Blood biochemical indices in three groups and ANOVA analysis (Mean ± SD).
Index 14 day 28 day

Control Aspirin Ibuprofen Control Aspirin Ibuprofen

ALT 55.60 ± 8.88 65.40 ± 12.42 47.40 ± 8.26 54.60 ± 9.10 70.20 ± 20.66 141.20 ± 59.81*
AST 229.20 ± 34.04 201.00 ± 50.85 206.20 ± 37.85 240.80 ± 42.73 264.80 ± 36.28 510.60 ± 17.29*
AKP 151.40 ± 10.81 178.40 ± 25.75 155.00 ± 31.25 160.00 ± 21.86 153.80 ± 37.43 122.80 ± 18.75
TP 68.54 ± 6.84 83.73 ± 10.62 77.28 ± 4.28 68.54 ± 6.84 69.28 ± 4.34 75.08 ± 5.56
ALB 25.20 ± 5.38 32.08 ± 4.71 28.96 ± 3.03 25.20 ± 5.38 21.48 ± 4.40 28.34 ± 1.76
TC 1.54 ± 0.07 2.15 ± 0.43* 1.73 ± 0.10 1.54 ± 0.07 1.35 ± 0.17 1.49 ± 0.18
TG 1.25 ± 0.30 1.00 ± 0.22 0.85 ± 0.12* 1.25 ± 0.30 1.64 ± 0.54 1.08 ± 0.10
DBIL 1.94 ± 1.68 8.30 ± 8.90 2.02 ± 1.00 1.94 ± 1.68 1.34 ± 1.51 6.52 ± 3.97
TBIL 0.75 ± 0.34 0.15 ± 0.07 0.57 ± 0.64 2.28 ± 3.43 0.34 ± 0.50 2.64 ± 0.78*
HDL 0.83 ± 0.05 0.98 ± 0.25 1.02 ± 0.09 0.83 ± 0.05 0.67 ± 0.12 0.56 ± 0.11
LDL 0.31 ± 0.06 0.38 ± 0.05 0.29 ± 0.05 0.31 ± 0.06 0.33 ± 0.03 0.30 ± 0.04
Crea 35.40 ± 17.23 22.00 ± 2.65 21.40 ± 4.72 35.40 ± 17.23 19.80 ± 4.09* 16.00 ± 6.96*
UA 90.20 ± 27.17 79.60 ± 41.96 69.40 ± 31.64 90.20 ± 27.17 62.00 ± 23.61 88.20 ± 8.53

Note: the number of rats n = 10 in every groups, *Compared with control group, P < 0.05.

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Fig. 2. The UPLC-MS/MS chromatograms of five probe drugs and metabolites.

3.5. Ultrastructure of liver cell are shown in Fig. 6. In the Control group, the ultrastructure of orga-
nelles, including nucleoli, endoplasmic reticula, Golgi complexes and
Transmission electron micrographs of liver cells in the three groups mitochondria were clearly observed. The membrane structures of

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Table 3
The pharmacokinetic parameters of five CYP probe drugs in three groups at day14.
Drug Group AUC(0-t) AUC(0-∞) t1/2z Tmax CLz/F Cmax
(mg/L*h) (mg/L*h) (h) (h) (mL/h/kg) (mg/L)

Tolbutamide Control 22,694.0 ± 6012.7 33802.1 ± 21,834.6 12.0 ± 8.7 4.0 ± 2.2 0.2 ± 0.1 1897.9 ± 497.9
Aspirin 11475.0 ± 2510.1*# 14,535.1 ± 9487.7*# 8.9 ± 9.9 3.1 ± 1.5 0.3 ± 0.2 1100.4 ± 294.8
Ibuprofen 9590.1 ± 1592.1* 10,283.0 ± 2052.1* 5.6 ± 1.6* 2.9 ± 1.2 0.3 ± 0.1 1071.8 ± 294.0*
Metoprolol Control 4559.9 ± 1441.9 5198.2 ± 1570.9 8.4 ± 10.0 1.0 ± 0.6 2.0 ± 0.6 1409.6 ± 739.7
Aspirin 5091.8 ± 1670.6# 5393.2 ± 1934.3# 2.6 ± 2.6 0.7 ± 0.2 2.2 ± 0.8# 2075.2 ± 871.0*#
Ibuprofen 6647.4 ± 1967.1* 7012.1 ± 2187.5* 3.0 ± 2.8 0.9 ± 0.5 1.5 ± 0.7 2661.8 ± 1328.8
Bupropion Control 4367.6 ± 944.9 4536.4 ± 868.7 5.1 ± 2.6 0.7 ± 0.4 2.4 ± 0.5 855.62 ± 203.6
Aspirin 3562.9 ± 1301.0# 3767.2 ± 1261.8# 2.8 ± 1.0* 1.0 ± 0.8 3.1 ± 1.3*# 1088.1 ± 509.4
Ibuprofen 5036.6 ± 2014.7 5522.5 ± 2292.6 3.3 ± 1.3 0.4 ± 0.3 2.0 ± 0.8 1605.9 ± 813.9*
Phenacetin Control 3915.9 ± 1405.4 4191.7 ± 1318.6 4.5 ± 4.6 0.5 ± 0.1 2.6 ± 1.0 2854.5 ± 1236.4
Aspirin 2307.2 ± 760.4*# 2588.1 ± 1074.0* 3.7 ± 8.2 0.5 ± 0.1 4.4 ± 1.6* 1842.5 ± 690.9*#
Ibuprofen 2585.3 ± 1018.5* 2730.5 ± 1057.2* 4.3 ± 3.9 0.5 ± 0.4 4.2 ± 1.6* 1728.9 ± 472.9*
Midazolam Control 1495.7 ± 646.6 1621.2 ± 626.6 3.4 ± 1.0 1.6 ± 1.5 3.4 ± 1.0 447.4 ± 278.5
Aspirin 1293.7 ± 601.1 1763.2 ± 1673.6 4.3 ± 2.6 1.0 ± 1.1 8.1 ± 4.2* 350.5 ± 144.1#
Ibuprofen 976.22 ± 342.7* 1055.5 ± 361.4* 4.0 ± 4.3 0.6 ± 0.3 10.4 ± 3.1* 427.8 ± 188.6

Note: the number of rats n = 10 in every groups, *Compared with control group, P < 0.05, # Compared with Ibuprofen group, P < 0.05.

hepatic nucleoli were round, intact and clear. The mitochondria were mucous layer of stomach became thin in aspirin group. Nevertheless, in
oval, with a clear ridge and rich medium. In the Aspirin and Ibuprofen ibuprofen group, there was ulcerous formation in the lesser and greater
groups, except for the nucleoli, the stainings of endoplasmic reticula, curvature of the stomach (supplemental Fig. 4).
Golgi complexes and mitochondria were weak. The numbers of mi- In the present study, we measured liver biochemical indexes, liver
tochondria were significantly greater, some mitochondria were en- CYP450 enzymes and ultrastructure of liver cell to determine the in-
larged, and mitochondrial cristae disintegrated and disappeared. In the fluence of aspirin and ibuprofen on the liver. The metabolic ability of
Ibuprofen group, the liver nuclei were irregular and inclined to py- liver CYP450 enzymes was investigated by a cocktail method, con-
knosis. sisting of co-administration of several probe drugs simultaneously
[21,22]. Various probe drugs are specifically metabolized by various
4. Discussion CYP450 isozymes, therefore, the metabolic ability of CYP450 isozymes
can be evaluated according to the pharmacokinetics of probe drugs
It is widely acknowledged that ibuprofen is better tolerated than is [23].
aspirin. A large-scale, randomized, investigator-blinded study showed On day 14, the AUC(0–t), AUC(0–∞), Cmax and t1/2z of tolbuta-
that overall tolerability of ibuprofen was better than that of aspirin mide were lower in the Aspirin and Ibuprofen groups than in the
[18], especially in terms of gastrointestinal tolerability [19]. Con- Control group, while the AUC(0–t), AUC(0–∞) and Cmax of hydro-
sidering the risks of gastrointestinal reaction, aspirin is commonly used xytolbutamide were higher. Although the AUC(0–t), AUC(0–∞) and
in enteric-coated dosage form. Nevertheless, whether enteric-coated Cmax of metoprolol were higher in the Ibuprofen group, AUC(0–t),
aspirin is better than ibuprofen in terms of gastrointestinal tolerability AUC(0–∞) and Cmax of hydroxybupropion did not decrease accord-
remains unclear. Therefore, enteric-coated aspirin was selected in this ingly. Therefore, the increased AUC(0–t), AUC(0–∞) and Cmax of
study. According to the monitoring of body weight, rats in the Ibu- metoprolol may be caused by increasing absorption of metoprolol. As
profen group were lighter than those in the Aspirin group, suggesting for bupropion, AUC(0–t), AUC(0–∞) and T1/2 were lower in the
that ibuprofen caused more gastrointestinal damage than did aspirin. Aspirin group, and AUC(0–t), AUC(0–∞), Cmax and CL of hydro-
Although many studies have reported that ibuprofen had better gas- xybupropion were higher, suggesting that the metabolism of bupropion
trointestinal tolerability than did aspirin [10,19,20], our study showed was accelerated. In the Ibuprofen group, the metabolism of bupropion
that enteric-coated aspirin had better gastrointestinal tolerability than was same as that of the Aspirin group, in that the AUC(0–t), AUC(0–∞),
did ibuprofen. Further morphological examinations showed that the Cmax and CL of hydroxybupropion were also higher, suggesting that

Table 4
The pharmacokinetic parameters of five probe drug in three groups at day 28.
Drug Group AUC(0-t) AUC(0-∞) t1/2z Tmax CLz/F Cmax
(mg/L*h) (mg/L*h) (h) (h) (mL/h/kg) (mg/L)

Tolbutamide Control 14,456.0 ± 5975.4 15269 ± 5810.0 5.9 ± 1.7 1.3 ± 1.7 0.3 ± 0.2 1790.9 ± 889.1
Aspirin 20585.0 ± 8530.6# 22,787 ± 10,024.5# 6.5 ± 2.8# 1.8 ± 1.5 0.3 ± 0.1 3000.1 ± 2462.0
Ibuprofen 16078.0 ± 5945.7 16927 ± 6232.8 5.3 ± 1.1 0.9 ± 0.9 0.4 ± 0.3 2203.0 ± 1095.9
Metoprolol Control 2905.2 ± 2049.9 2932.7 ± 2049.7 2.0 ± 0.6 0.7 ± 0.5 5.3 ± 3.4 1392.0 ± 900.8
Aspirin 1776.9 ± 1123.8 1833 ± 1128.6 2.2 ± 1.1 1.0 ± 0.8 8.9 ± 7.4 848.3 ± 680.0
Ibuprofen 1770.5 ± 1183.9 1849.8 ± 1209.1 2.7 ± 1.9 0.6 ± 0.2 7.1 ± 3.3 846.6 ± 482.5
Bupropion Control 1570.7 ± 732.3 1615.5 ± 738.5 2.7 ± 1.0 0.4 ± 0.5 7.2 ± 2.7 877.0 ± 358.9
Aspirin 4473.3 ± 5196.2* 4564 ± 5256.9* 2.4 ± 0.9 0.7 ± 0.8 6.0 ± 6.2# 1509.6 ± 1351.1
Ibuprofen 3977.4 ± 2115.5* 4222.6 ± 2113.0* 3.2 ± 1.5 0.4 ± 0.7 3.0 ± 1.5* 1743.2 ± 1028.2*
Phenacetin Control 3588.8 ± 2231.3 3622 ± 2241.3 3.5 ± 1.6 0.3 ± 0.1 3.5 ± 1.7 3187.3 ± 1381.0
Aspirin 1433.7 ± 932.7* 1457.2 ± 934.3* 3.5 ± 3.7 0.3 ± 0.1 16.1 ± 21.5* 1116.7 ± 791.5*
Ibuprofen 1439.0 ± 615.1* 1472.3 ± 613.1* 5.3 ± 4.4 0.3 ± 0.1 8.6 ± 5.22* 1240.1 ± 454.4*
Midazolam Control 1069.1 ± 1148.5 1083.9 ± 1149.7 2.5 ± 0.9 0.8 ± 1.2 15.9 ± 9.5 552.0 ± 513.3
Aspirin 465.6 ± 493.2 485.6 ± 491.0 3.3 ± 2.1# 0.6 ± 0.4 46.1 ± 45.8*# 237.4 ± 268.8*#
Ibuprofen 650.8 ± 275.3 655.2 ± 276.2 2.0 ± 0.7 0.3 ± 0.1 21.0 ± 18.31 422.6 ± 223.1

Note: the number of rats n = 10 in every groups, *Compared with control group, P < 0.05, # Compared with Ibuprofen group, P < 0.05.

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Fig. 3. Concentration-time curve of five probe drugs in Control, Aspirin, and Ibuprofen groups at day 14, the number of rats n = 10 in every groups.

the metabolisms of tolbutamide and bupropion were accelerated in the Cmax of acetaminophen and 1-hydroxy-midazolam were both higher.
Aspirin and Ibuprofen groups. The AUC(0–t), AUC(0–∞), and Cmax of Therefore, except for metoprolol, the metabolism of tolbutamide, bu-
phenacetin and midazolam had similar changes: all were lower in the propion, phenacetin and midazolam were all accelerated after admin-
Aspirin and Ibuprofen groups. Moreover, the AUC(0–t), AUC(0–∞) and istration of aspirin and ibuprofen.

Fig. 4. Concentration-time curve of five probe drugs in Control, Aspirin, and Ibuprofen groups at day 28, the number of rats n = 10 in every groups.

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Fig. 5. The canonical discriminant functions of three groups in day 14 (A) and 28 (B) analyzed by using pharmacokinetic data (1 ○ Control-group, 2 ◇ Aspirin
group, 3 Ibuprofen group).

Fig. 6. Electron ultrastructure of liver cell in Control group (A), Aspirin group (B), and Ibuprofen group (C).

Tolbutamide, bupropion, phenacetin and midazolam are used as were not accordingly higher as on day 14. As a group, the metabolisms
probe drugs for CYP2C9, CYP2B6, CYP1A2 and CYP3A4. These of tolbutamide, bupropion, phenacetin and midazolam on day 28 were
CYP450 s are controlled by transcription factors in the nucleus, called not accelerated further with continuous administration of aspirin and
nuclear receptors (NR), including thyroid and vitamin D receptors, ibuprofen. This situation may be correlated with damage of liver cells.
constitutive androstane receptor (CAR) and the pregnane X receptor The AST and ALT were higher in the Aspirin and Ibuprofen groups on
(PXR) [24,25]. The accelerated metabolism of these probe drugs sug- day 28, in accordance with results from our previous work [27].
gested that the catalytic abilities of CYP2C9, CYP2B6, CYP1A2, and Moreover, the ultrastructural examination showed that morphological
CYP3A4 were induced. One of the possible mechanisms is that the NR characteristics of liver nuclei were altered to irregular, suggesting that
was activated by aspirin and ibuprofen, resulting in expression of these there was damage to liver cells.
CYPs. Another mechanism may be related to the increased numbers of Regarding differences between the Aspirin and Ibuprofen groups on
mitochondria according to ultrastructure examination. Ibuprofen pro- day 14, compared with the Aspirin group, the t1/2 of metabolites of all
moted the opening of inner mitochondrial membrane pores by acti- five probe drugs were lower in the Ibuprofen, and there was statistical
vating Ca2+ and phosphate [26]. significance for hydroxyltolbutamide (P < 0.05). On day 28, tolbuta-
On day 28, there was no AUC(0–t), AUC(0–∞), Cmax decreased for mide, hydroxytolbutamide and midazolam were all significantly lower
tolbutamide and no AUC(0–t), AUC(0–∞), Cmax of increase for hy- in the Ibuprofen group (P < 0.05). Therefore, the metabolic ability of
droxytolbutamide in the Aspirin and Ibuprofen groups. Although CYP450 in the Ibuprofen group was stronger than that of the Aspirin
AUC(0–t), AUC(0–∞), Cmax of metoprolol decreased in the Aspirin and group.
Ibuprofen groups, the t1/2 did not decrease and AUC(0–t), AUC(0–∞), Some studies have been conducted on the activities of CYP450
Cmax for hydroxymetoprolol were also decreased, suggesting that the [28–31]. For example, Chen et al. reported that low-dose aspirin in-
absorption of metoprolol was reduced. Although the AUC(0–t), duced the in vivo activity of CYP2C19 in healthy subjects [30], Krasniqi
AUC(0–∞) and Cmax of bupropion were increased in the Aspirin and et al. reported that CYP2C8*3 and CYP2C9*2*3 variants correlated
Ibuprofen groups, there were no changes in AUC(0–t), AUC(0–∞) and with ibuprofen-induced hepatotoxicity and gastrointestinal bleeding
Cmax for hydroxybupropion. Moreover, the AUC(0–t) and AUC(0–∞) [31]. Nevertheless, the activity of other CYP450 isozymes and phar-
for bupropion were 1570.7 ± 732.3 and 1615.5 ± 738.5 at day 28, macokinetics changes remain unclear, especially with long-term ad-
respectively, lower than those of day 14. Therefore, the difference of ministration. In thepresent study, the metabolic activity and pharma-
bupropion was most likely caused by experimental error. As for phe- cokinetics of five liver CYP450 enzymes (CYP1A2, CYP2C9, CYP2B6,
nacetin and midazolam, the AUC(0–t), AUC(0–∞), and Cmax were all CYP2D6 and CYP3A4) were simultaneously studied using a cocktail
decreased in the Aspirin and Ibuprofen groups. However, the AUC(0–t), approach. The diagnostic value of those five CYP450 enzymes was
AUC(0–∞), and Cmax of acetaminophen and 1-hydroxy-midazolam evaluated by Fisher discriminant by using pharmacokinetic data.

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C. Wen et al. Biomedicine & Pharmacotherapy 108 (2018) 208–215

Moreover, the ultrastructural alterations of liver cells were investigated, a systematic review and meta-analysis of observational studies, Int. J. Gynecol.
providing a different perspective on aspirin and ibuprofen. Cancer 26 (6) (2016) 1111–1120.
[10] K.D. Rainsford, Ibuprofen: pharmacology, efficacy and safety,
Inflammopharmacology 17 (6) (2009) 275–342.
5. Conclusions [11] M. Akar, T.G. Yildirim, G. Sandal, S. Bozdag, O. Erdeve, N. Altug, N. Uras,
S.S. Oguz, U. Dilmen, Does ibuprofen treatment in patent ductus arteriosus alter
oxygen free radicals in premature infants? Cardiol. Young 27 (3) (2017) 507–511.
Enteric-coated aspirin had better gastrointestinal tolerability than [12] S.T. Byrne, S.M. Denkin, Y. Zhang, Aspirin and ibuprofen enhance pyrazinamide
did ibuprofen. Long-term administration of aspirin and ibuprofen in- treatment of murine tuberculosis, J. Antimicrob. Chemother. 59 (2) (2007)
creased the number of mitochondria and the levels of ALT and AST. 313–316.
[13] L. Ge, G. Niu, X. Han, Y. Gao, Q. Wu, H. Wu, Y. Zhang, D. Guo, Aspirin treatment
Aspirin and ibuprofen induced the metabolic activity of liver CYP450 increases the risk of cerebral microbleeds, the Canadian journal of neurological
enzymes, and ibuprofen was stronger in this respect. The pharmacoki- sciences, Le journal canadien des sciences neurologiques 38 (6) (2011) 863–868.
netic data from probe drugs with respect to liver CYP450 discriminated [14] J. Laster, R. Satoskar, Aspirin-induced acute liver injury, ACG Case Rep. J. 2 (1)
(2014) 48–49.
among the Control, Aspirin and Ibuprofen groups.
[15] J. Zhang, H. Song, S. Jiang, Z. Chen, S. Tong, F. Lin, C. Wen, X. Zhang, L. Hu, Fisher
discrimination of metabolic changes in rats treated with aspirin and ibuprofen,
Conflicts of interest Pharmacology 100 (3-4) (2017) 194–200.
[16] Y.N. Sun, F.Y. Lin, D. Wang, J.S. Ma, X.C. Wang, C.C. Wen, H.C. Cao, L.F. Hu, Early
changes in blood biochemistry and CYP450 metabolism in a rat model of alcoholic
The authors declare no conflict of interest. fatty liver disease, Int. J. Clin. Exp. Med. 11 (3) (2018) 2232–2239.
[17] L.F. Hu, X.Z. Yang, X.Q. Wang, J.Y. Zhu, S.H. Tong, G.Z. Cao, Rapid LC-APCI-MS-MS
Acknowlegements method for simultaneous determination of Phenacetin and its metabolite para-
cetamol in rabbit plasma, Chromatographia 70 (3-4) (2009) 585–590.
[18] N. Luyk, Review of pain study of aspirin, ibuprofen and paracetamol, N. Z. Dent. J.
This work was supported by fund of Natural Science Foundation of 96 (424) (2000) 66.
Zhejiang province (LY16H300005), the Health Department of Zhejiang [19] P. Rampal, N. Moore, E. Van Ganse, J.M. Le Parc, R. Wall, H. Schneid, F. Verriere,
Gastrointestinal tolerability of ibuprofen compared with paracetamol and aspirin at
province (2015KYA154); Public Project of Wenzhou Science and over-the-counter doses, J. Int. Med. Res. 30 (3) (2002) 301–308.
Technology Bureau (Y20160304) the social development of Jinhua [20] R.E. Harris, J. Beebe-Donk, H. Doss, D. Burr Doss, Aspirin, ibuprofen, and other
Technology Bureau (key projects: 2016-3-013, public service project: non-steroidal anti-inflammatory drugs in cancer prevention: a critical review of
non-selective COX-2 blockade (review), Oncol. Rep. 13 (4) (2005) 559–583.
2016-4-005). [21] Z.B. Chen, A.Y. Zhi, F.Y. Lin, D. Li, X.G. Yu, W.H. Chen, L.F. Hu, Pharmacokinetic of
four probe drugs in adriamycin-induced nephropathy rat, Eur. Rev. Med.
Appendix A. Supplementary data Pharmacol. Sci. 18 (10) (2014) 1439–1447.
[22] Q. Wu, Q. Zhang, C. Wen, L. Hu, X. Wang, G. Lin, The effect of MS-275 on CYP450
isoforms activity in rats by cocktail method, Int. J. Clin. Exp. Pathol. 8 (8) (2015)
Supplementary material related to this article can be found, in the 9360–9367.
online version, at doi:https://doi.org/10.1016/j.biopha.2018.08.162. [23] L.F. Hu, Z. Wang, R.N. Xu, J.S. Ma, X.Q. Wang, X.H. Zhang, Determination of bu-
propion and its main metabolite in rat plasma by LC-MS and its application to
pharmacokinetics, Pharmazie 66 (12) (2011) 924–928.
References [24] R. Skowronek, P. Czekaj, A. Suszka-Switek, E. Czech, A. Wiaderkiewicz, D. Plewka,
A. Bryzek, Expression of cytochrome P450 2c and 3a in female rat liver after long-
[1] Z.F. Wang, Q. Li, S.B. Liu, W.L. Mi, S. Hu, J. Zhao, Y. Tian, Q.L. Mao-Ying, term administration of gonadoliberin analogs, Int. J. Occup. Med. Environ. Health
J.W. Jiang, H.J. Ma, Y.Q. Wang, G.C. Wu, Aspirin-triggered Lipoxin A4 attenuates 29 (2) (2016) 293–314.
mechanical allodynia in association with inhibiting spinal JAK2/STAT3 signaling in [25] C. Duret, M. Daujat-Chavanieu, J.M. Pascussi, L. Pichard-Garcia, P. Balaguer,
neuropathic pain in rats, Neuroscience 273 (2014) 65–78. J.M. Fabre, M.J. Vilarem, P. Maurel, S. Gerbal-Chaloin, Ketoconazole and mico-
[2] C. Lampl, M. Voelker, T.J. Steiner, Aspirin is first-line treatment for migraine and nazole are antagonists of the human glucocorticoid receptor: Consequences on the
episodic tension-type headache regardless of headache intensity, Headache 52 (1) expression and function of the constitutive androstane receptor and the pregnane X
(2012) 48–56. receptor, Mol. Pharmacol. 70 (1) (2006) 329–339.
[3] M.E. Csuka, D.J. McCarty, Aspirin and the treatment of rheumatoid arthritis, [26] I.A. Al-Nasser, Ibuprofen-induced liver mitochondrial permeability transition,
Rheum. Dis. Clin. North Am. 15 (3) (1989) 439–454. Toxicol. Lett. 111 (3) (2000) 213–218.
[4] T. Kurth, C.H. Hennekens, J.E. Buring, J.M. Gaziano, Aspirin, NSAIDs, and COX-2 [27] J. Zhang, H.C. Song, S.Y. Jiang, Z.B. Chen, S.H. Tong, F.Y. Lin, C.C. Wen,
inhibitors in cardiovascular disease: possible interactions and implications for X.H. Zhang, L.F. Hu, Fisher discrimination of metabolic changes in rats treated with
treatment of rheumatoid arthritis, Curr. Rheumatol. Rep. 6 (5) (2004) 351–356. aspirin and ibuprofen, Pharmacology 100 (3-4) (2017) 194–200.
[5] J.C. Sun, R. Whitlock, J. Cheng, J.W. Eikelboom, L. Thabane, M.A. Crowther, [28] M. Bojic, C.A. Sedgeman, L.D. Nagy, F.P. Guengerich, Aromatic hydroxylation of
K.H. Teoh, The effect of pre-operative aspirin on bleeding, transfusion, myocardial salicylic acid and aspirin by human cytochromes P450, Eur. J. Pharm. Sci. 73
infarction, and mortality in coronary artery bypass surgery: a systematic review of (2015) 49–56.
randomized and observational studies, Eur. Heart J. 29 (8) (2008) 1057–1071. [29] G. Di Nardo, V. Dell’Angelo, G. Catucci, S.J. Sadeghi, G. Gilardi, Subtle structural
[6] T.N. Patel, K.C. Goldberg, Use of aspirin and ibuprofen compared with aspirin alone changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new ac-
and the risk of myocardial infarction, Arch. Intern. Med. 164 (8) (2004) 852–856. tivity toward diclofenac, tolbutamide and ibuprofen, Arch. Biochem. Biophys. 602
[7] F. Andreotti, R. De Caterina, F. Crea, Aspirin and the prevention of a common (2016) 106–115.
disease: colorectal cancer, Int. J. Cardiol. 248 (2017) 394–395. [30] X.P. Chen, Z.R. Tan, S.L. Huang, Z. Huang, D.S. Ou-Yang, H.H. Zhou, Isozyme-
[8] G. Curigliano, A. Balduzzi, A. Cardillo, R. Ghisini, G. Peruzzotti, L. Orlando, specific induction of low-dose aspirin on cytochrome P450 in healthy subjects, Clin.
R. Torrisi, S. Dellapasqua, L. Lunghi, A. Goldhirsch, M. Colleoni, Low-dose aspirin Pharmacol. Ther. 73 (3) (2003) 264–271.
for the prevention of venous thromboembolism in breast cancer patients treated [31] V. Krasniqi, A. Dimovski, I.K. Domjanovic, I. Bilic, N. Bozina, How polymorphisms
with infusional chemotherapy after insertion of central vein catheter, Support. Care of the cytochrome P450 genes affect ibuprofen and diclofenac metabolism and
Cancer 15 (10) (2007) 1213–1217. toxicity, Arh. Hig. Rada Toksikol. 67 (1) (2016) 1–8.
[9] D. Zhang, B. Bai, Y. Xi, Y. Zhao, Can aspirin reduce the risk of endometrial Cancer?:

215