In Vivo Cardioprotective Effects and Pharmacokinetic Profile of N-Propyl Caffeamide Against Ischemia Reperfusion Injury

Article abstract of DOI:10.1007/s00005-016-0413-y

Caffeic acid derivatives constitute a class of potent anti-inflammatory and cardioprotective drug candidates. We recently synthesized a new caffeic acid derivative N-propyl caffeamide (PCA). Our pilot experiments demonstrated that PCA enhanced the survival of rat cardiomyocyte H9c2 cells against oxygen glucose deprivation and reoxygenation challenge in a concentration-dependent manner. Interestingly, PCA exhibited better cardioprotective potential than caffeic acid phenethyl ester and propyl caffeate. Thus, we hypothesized that PCA could protect heart against ischemia reperfusion (I/R) injury in mice. We first determined the stability and pharmacokinetic profile of PCA in male Sprague–Dawley rats by ultra-performance liquid chromatography coupled with UV and MS/MS detections. The stability of PCA in rat plasma was defined by the half-life of 31.39, 7.19 and 1.37?h in rat plasma at 25, 37 and 60?°C, respectively. To study the pharmacokinetic profiles, PCA was injected into male SD rats at the dose of 15?mg/kg via intravenous bolus administration. PCA showed the elimination half-life of approximate 235?min in rats. We subsequently evaluated the cardioprotective potential of PCA in mice model of myocardial infarction. Our results demonstrated that PCA effectively reduced infarct size and release of myocardial enzymes (e.g., CK, CK-MB and LDH). Biochemical analyses suggested that PCA increased the activities of antioxidant enzymes (e.g., CAT and SOD) while attenuated lipid peroxidation. Moreover, PCA profoundly reduced the number of apoptotic cells in infarcted myocardium. Consistently, PCA increased the expression level of anti-apoptotic protein Bcl2 whereas suppressed the expression of pro-apoptotic protein Bax in cardiac tissues. Collectively, PCA appears to be a novel bioavailable and stable pharmacological treatment for myocardial infarction.

Full text of DOI:10.1007/s00005-016-0413-y

Arch. Immunol. Ther. Exp.
DOI 10.1007/s00005-016-0413-y
ORIGINAL ARTICLE
In Vivo Cardioprotective Effects and Pharmacokinetic Profile
of N-Propyl Caffeamide Against Ischemia Reperfusion Injury
Yuan-Yuan Cheng1 Dan Luo1 Zhengyuan Xia2 Hung-Fat Tse3
•
•
•
•
Xuechen Li4 Jianhui Rong1
•
Received: 28 February 2016 / Accepted: 9 May 2016
Ó L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland 2016
Abstract Caffeic acid derivatives constitute a class of
potent anti-inflammatory and cardioprotective drug can-
didates. We recently synthesized a new caffeic acid
derivative N-propyl caffeamide (PCA). Our pilot experi-
ments demonstrated that PCA enhanced the survival of rat
cardiomyocyte H9c2 cells against oxygen glucose depri-
vation and reoxygenation challenge in a concentration-
dependent manner. Interestingly, PCA exhibited better
cardioprotective potential than caffeic acid phenethyl
ester and propyl caffeate. Thus, we hypothesized that
PCA could protect heart against ischemia reperfusion (I/
R) injury in mice. We first determined the stability and
pharmacokinetic profile of PCA in male Sprague–Dawley
rats by ultra-performance liquid chromatography coupled
with UV and MS/MS detections. The stability of PCA in
rat plasma was defined by the half-life of 31.39, 7.19 and
1.37 h in rat plasma at 25, 37 and 60 °C, respectively. To
study the pharmacokinetic profiles, PCA was injected into
male SD rats at the dose of 15 mg/kg via intravenous
bolus administration. PCA showed the elimination half-
life of approximate 235 min in rats. We subsequently
evaluated the cardioprotective potential of PCA in mice
model of myocardial infarction. Our results demonstrated
that PCA effectively reduced infarct size and release of
myocardial enzymes (e.g., CK, CK-MB and LDH). Bio-
chemical analyses suggested that PCA increased the
activities of antioxidant enzymes (e.g., CAT and SOD)
while attenuated lipid peroxidation. Moreover, PCA pro-
foundly reduced the number of apoptotic cells in infarcted
myocardium. Consistently, PCA increased the expression
level of anti-apoptotic protein Bcl2 whereas suppressed
the expression of pro-apoptotic protein Bax in cardiac
tissues. Collectively, PCA appears to be a novel
bioavailable and stable pharmacological treatment for
myocardial infarction.
Keywords N-Propyl caffeamide Á Bioavailability Á
Pharmacokinetics Á Cardioprotection Á
Ischemia reperfusion injury
Abbreviations
AAR
BOP
Area at risk
Benzo-triazol-1-yloxy-
tris(dimethylamino)phosphonium
hexafluorophosphate
BSA
Bovine serum albumin
Caffeic acid phenethyl ester
Catalase
CAPE
CAT
CK
Creatine kinase
& Jianhui Rong
jrong@hkucc.hku.hk
CK-MB
DMEM
DMSO
ECL
Myocardial muscle creatine kinase
Dulbecco’s modified Eagle’s medium
Dimethyl sulfoxide
1
School of Chinese Medicine, University of Hong Kong,
10 Sassoon Road, Pokfulam, Hong Kong, China
Enhanced chemiluminescence
Slectrospray ionization-mass spectrometry
Glyceraldehydes-3-phosphate
dehydrogenase
2
Department of Anesthesiology, University of Hong Kong,
Hong Kong, China
ESI-MS
GAPDH
3
Department of Medicine, Li Ka Shing Faculty of Medicine,
University of Hong Kong, Hong Kong, China
HRP
IgG
Horseradish peroxidase
Immunoglobulin G
4
Department of Chemistry, University of Hong Kong,
Hong Kong, China
123

Arch. Immunol. Ther. Exp.
evaluated for cardioprotective potential in the treatment of
myocardial infarction (Ho et al. 2014; Lee et al. 2015;
Rajan et al. 2001). Nevertheless, the pharmacological and
toxicological profiles of these caffeic acid derivatives have
not been fully characterized.
LC/MS/MS Liquid chromatography/mass
spectrometry/mass spectrometry
LAD
LDH
MPO
OGD
PCA
ROS
RNS
SOD
TTC
Left anterior descending coronary artery
Lactate dehydrogenase
Myeloperoxidase
In this study, we developed a chemical procedure to
synthesize N-propyl caffeate amide (PCA) as a potential
structurally simple and pharmacologically stable caffeic
acid derivative as described in Fig. 1a. We investigated the
cardioprotective effect of PCA in cardiomyocyte H9c2
cells against oxygen glucose deprivation (OGD) and
reoxygenation challenge, and also in a rat model of acute
myocardial infarction by the ligation of left coronary
artery. We also developed a rapid ultra-performance liquid
chromatography (UPLC) method and liquid chromatogra-
phy/mass spectrometry/mass spectrometry (LC/MS/MS)
method to determine the stability and pharmacokinetic
profile of PCA.
Oxygen glucose deprivation
N-Propyl caffeate amide
Reactive oxygen species
Reactive nitrogen species
Superoxide dismutase
Triphenyltetrazolium chloride
Introduction
Myocardial infarction is a leading cause of morbidity and
mortality worldwide (Bonow et al. 2002; Yellon and
Hausenloy 2007). In the patients with myocardial infarc-
tion, ischemia and reperfusion trigger a sequence of
inflammatory reactions involving the infiltration, activa-
tion, apoptosis and clearance of inflammatory leukocytes
(e.g., neutrophils and macrophages) (Jordan et al. 1999).
Neutrophils and macrophages not only cause tissue damage
but also contribute to wound healing, cardiac remodeling
and scar formation (Jordan et al. 1999; Nahrendorf et al.
2010). Upon reperfusion, neutrophils are rapidly recruited
into infarcted hearts and activated to release reactive
oxygen species (ROS), reactive nitrogen species (RNS),
proteases, and specific chemoattractant mediators (Jordan
et al. 1999). Different forms of ROS cause oxidative
damage of proteins and lipids, leading to apoptotic cell
death in myocardium and dysfunctions of heart. Previous
studies suggested that neutrophil depletion dramatically
reduced infarct size in animal models of myocardial
infarction (Jolly et al. 1986; Litt et al. 1989).
Materials and Methods
Biochemical Reagents
Antibodies against glyceraldehyde-3-phosphate dehydro-
genase (GAPDH), caspase-3, Bax and Bcl2 were obtained
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA,
USA). The anti-rabbit horseradish peroxidase (HRP)-con-
jugated IgG secondary antibody was purchased from
Sigma-Aldrich (St. Louis, MO, USA). Protein Assay Dye
Reagent Concentrate was purchased from Bio-Rad (Her-
cules, CA, USA). Select chemiluminescence (ECL)
detection kit was purchased from GE Healthcare (Uppsala,
Sweden). PCE was a synthetic product from Hangzhou
Yuhao Chemical Technology Co., Ltd (Zhejiang, China).
CAPE was obtained from Santa Cruz Biotechnology, Inc.
(Dallas, TX, USA). Caffeic acid and other biochemical
reagents were purchased from Sigma-Aldrich (St. Louis,
MO, USA) unless otherwise indicated.
Caffeic acid and related derivatives are well-known to
exhibit anti-oxidant, anti-inflammatory, chemopreventive,
anticancer and antibacterial properties in a structure-de-
pendent and cell-type-specific manner (da Cunha et al.
2004; Fiuza et al. 2004; Rajan et al. 2001). As an example,
caffeic acid phenethyl ester (CAPE) was initially isolated
from propolis, a honeybee hive product as a specific NF-jB
inhibitor (Natarajan et al. 1996). Several recent studies
demonstrated that CAPE could effectively attenuate
ischemia reperfusion (I/R) injury in different in vitro and
in vivo models (Kart et al. 2009; Koltuksuz et al. 1999;
Wei et al. 2004). However, caffeic acid esters are not
stable enough and have short life-time in male Sprague–
Dawley (SD) rats (Wang et al. 2007). For this reason,
several different caffeic acid amide derivatives including
N-methyl, N-propargyl, N-anilide, N-phenethyl and pyrro-
lidinyl caffeamide have been developed and partly
Animal Husbandry
Male C57BL/6N mice (6–8 weeks, 20 5 g) and male SD
rats (7–8 weeks, 250 20 g) were obtained from Labo-
ratory Animal Unit, University of Hong Kong (China). All
experimental procedures were in compliance with the
guidelines of the Committee on the Use of Live Animals in
Teaching and Research of the University of Hong Kong.
Animals were housed in a temperature- and humidity-
controlled environment on a 12 h light–dark cycle, and
allowed free access to standard laboratory mice chow and
drinking water. Animals were acclimated in the laboratory
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Arch. Immunol. Ther. Exp.
Fig. 1 Chemical synthesis and in vitro cytoprotective effect of PCA.
a Synthesis of PCA via direct amidation of caffeic acid with
propylamine. BOP benzotriazol-1-yloxy-tris(dimethylamino) phos-
MTT assay. The results were expressed as mean SD from three
independent experiments. *p \ 0.05 and **p \ 0.01 (PCA ? OGD/
R vs OGD/R). c Different cytoprotective effect of PCA, CAPE and
PCE. H9c2 cells were exposed to OGD condition and subsequent
reoxygenation under normal growth condition in the absence or
presence of caffeic acid derivatives at the concentration of 20 lM.
The results were expressed as mean SD of three independent
experiments. *p \ 0.05 and **p \ 0.01 (Drug ? OGD/R vs OGD/R)
phonium
hexafluorophosphate,
CH2Cl2
dichlormethane,
Et3N trimethylamine. b Effect of PCA on the survival of H9c2 cells
against OGD and reoxygenation challenge. H9c2 cells were exposed
to OGD condition and subsequent reoxygenation under normal
growth condition in the absence or presence of PCA with varying
concentrations. The cell viability was determined by colorimetric
for 7 days prior to the experiment and fasted for 12 h but
allowed water before experiments.
characterized by mass spectroscopy (MS) and nuclear
magnetic resonance (NMR) spectroscopy. MS analysis was
performed on an ABI/Sciex triple quadrupole 3200
QTRAP mass spectrometer (Framingham, MA, USA)
equipped with an TurboV Source operating in positive
ionization mode under the control of Analyst v1.4.2 data
system (Applied Biosystems/MDS Sciex, Concord, ON,
Canada). ESI–MS (m/z): 222.1 [M?H]?. HRMS for
C12H15NO3: calculated, 222.1125 [M?H]?; experimental,
222.1127 [M?H]?. NMR spectra were recorded in DMSO-
d6 on a Varian Unity plus NMR 600 MHz spectrometer
Heparinized male SD rat plasma was obtained from
Laboratorial Animal Unit, The University of Hong Kong.
Water used in the experiments was passed through a Mil-
lipore Milli-Q water purification system.
Synthesis and Chemical Characterization of PCA
Mixtures of 100 mg caffeic acid, 1.2 eq propylamine and
0.08 mL Et3N were dissolved in 1 mL dichloromethane.
While stirring on ice, a solution of benzotriazol-1-yloxytris
(dimethylamino)phosphonium hexafluorophosphate (BOP)
(1.2 equiv) in 5 mL dichloromethane was subsequently
added into caffeic acid solution. The reaction mixture was
stirred at 0 °C for 30 min and at room temperature over-
night. At the end of reaction, the solvent was removed by
rotary evaporator under vacuum. The residues were
extracted five times with ethyl acetate (EtOAc). The
organic phase was sequentially washed with 3 M HCl
aqueous solution, and 10 % NaHCO3 in water. The product
PCA was purified by column chromatography on silica gel
with chloroform and methanol (PE:EtOAc = 1:1.5) to
yield a yellow powder (86 mg, 70 %). PCA was further
1
(Varian Inc., Palo Alto, CA, USA). H NMR (DMSO-d6,
600 MHz): 9.22 (s, 2-OH), 7.95 (t, J = 5.6 Hz, 1-NH),
7.22 (d, J = 15.7 Hz, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.83
(dd, J = 8.2, 1.9 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.33
(d, J = 15.7 Hz, 1H), 3.11 (m, 2H), 1.46 (m, 2H), 0.87 (t,
J = 7.4, 3H); and 13C NMR (DMSO-d6, 150 MHz): 165.8,
147.7, 146.0, 139.3, 126.9, 120.7, 119.2, 116.2, 114.3,
40.9, 22.9, 11.9.
Measurement of Cell Viability
Rat cardiomyocyte H9c2 cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
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Arch. Immunol. Ther. Exp.
Pharmacokinetics of PCA
2 mM glutamine, antibiotics (100 U/mL of penicillin A
and 100 U/mL of streptomycin), and 10 % heat-inacti-
vated fetal bovine serum (Gibco/BRL, Gaithersburg,
MD, USA) and maintained in a 37 °C humidified
incubator containing 5 % CO2. For drug treatment,
H9c2 cells were seeded at the density of 1.0 9 105 in a
96-well plate. After overnight incubation, the cells were
incubated in degassed and glucose-free DMEM, and
subsequently exposed to OGD condition in an anaerobic
chamber saturated with 5 % CO2 and 95 % N2 (v/v) at
37 °C for 10 h. The cells were subsequently treated
with PCA, CAPE and PCE in complete growth medium
under normoxia condition for another 14 h. The cell
viability was evaluated by a colorimetric assay of MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] reduction. Briefly, at the end of drug treat-
ment, MTT (final concentration of 0.5 mg/mL) was
added to cell culture in a 96-well plate. After 4 h
incubation at 37 °C, the supernatant was removed. The
formazan products in viable cells were solubilized in
150 lL of DMSO and quantified by measuring the
absorbance at 570 nm on a Bio-Rad microplate reader
(Hercules, CA, USA).
The pharmacokinetic profile of PCA was studied in male
SD rats. A solution of PCA (0.8 mL, 15 mg/kg) in 80 %
sterile saline and 20 % ethanol was administrated intra-
venously over a period of 20 s via femoral vein. Blood
samples (0.3 mL) were collected into 1.5 mL tubes before
injection, at 5, 15, 30 min, 1, 2, 4, 6 and 8 h after injection.
After immediate centrifugation at 5700 rpm for 10 min, the
plasma samples were recovered and stored at -80 °C until
analysis.
For LC/MS/MS analysis, plasma sample (100 lL) was
mixed with 200 ng of resveratrol as internal standard (IS)
in 200 lL acetonitrile. The samples were vortexed for
5 min and then centrifuged at 13,000 rpm for 10 min. The
supernatants were evaporated and reconstitution with
100 lL of MeOH. The methanolic solution (20 lL) was
subsequently analyzed on a Synergi hydro-RP C18 column
(150 9 4.6 mm, 4 lm) at the flow rate of 0.8 mL/min. The
separation was operated under the control of an Agilent
HPLC system equipped with a 1525 Separations Module
and a 2998 DAD Unit. Mobile phase compositions were
(A) water with 0.4 % (v/v) formic acid and (B) acetonitrile.
Gradient was set as follows: 0–2 min, 80 % A; 2–5 min,
80–50 % A; 5–7 min, 50–80 % A; 7–8 min, 80 % A. The
elution was analyzed on an ABI/Sciex triple quadrupole
mass spectrometer 3200 QTRAP system (Framingham,
MA, USA). The MS system was equipped with an ESI-
Turbo V source operating in positive ionization mode
under the control of Analyst v1.4.2 data system (Applied
Biosystems/MDS Sciex, Concord, ON, Canada). Mass
spectrometry was operated under the conditions as follows:
drying gas N2, 10 L/min; capillary voltage, 20 V; pressure
of nebulizer, 30 psi; ion spray voltage, 5.5 kV; and capil-
lary temperature, 325 °C. Multiple reaction monitoring
(MRM) parameters were optimized for analysis of PCA
and IS, respectively. Analyst 1.5.1 software (AB Sciex,
Foster, CA, USA) was used for the control of equipment,
data acquisition and analysis. Plasma PCA concentration vs
time data for each rat was analyzed by Kinetica 2000
software (Version 3.0).
In Vitro Stability of PCA
The in vitro stability of PCA was studied by incubating
the drug with rat plasma as previously described (Yang
et al. 2012). Plasma was collected from male SD rats
(body weight 250 20 g). PCA (10 lL, 2 mg/mL) was
incubated with rat plasma (180 lL) at 25, 37 and 60 °C
for different times. After adding 10 lL of the internal
control resveratrol solution (400 lg/mL), the samples
were immediately extracted with 600 lL of ethyl acet-
ate. The solvent was removed by passing a stream of
nitrogen at 25 °C. The samples were reconstituted and
mixed in 200 lL of MeOH. After brief centrifugation,
the supernatants were transferred to glass tubes with
caps (Agilent Inc, USA). The methanolic solution (5 lL)
was analyzed through a C18 reverse phase UPLC column
(5 l, 4.6 9 150 mm) under the control of a Thermo
(Thermo Scientific, USA) DIONEX series LC system.
The UPLC system was equipped with a ultimate 3000
RS pump, 3000 RS auto-sampler, 3000 RS column
compartment along with an 3000 RS diode array DAD
detector. The flow rate was set at 0.4 mL/min. Detection
wavelength was set to UV 320 nm. The concentration of
intact PCA was determined at minimal five time points
for each temperature condition. Three replicates were
analyzed at each time point. The stability of PCA was
correlated with the half-life of PCA at each respective
temperature and the energy of activation was also
calculated.
Evaluation of Infarct Size and Cardiac Damage
after Myocardial Infarction
Myocardial infarction was induced by the ligation of the
left anterior descending coronary artery (LAD) in male
C57BL/6N mice as previously described (Xu et al. 2014).
Mice were randomly divided into the following four groups
(n = 13): (1) Sham, receiving sham surgery and
intraperitoneal (i.p.) injection of vehicle (a mixture of 50 %
saline and 50 %1,2-propanodiol); (2) I/R, receiving 30 min
ligation of coronary artery, 24 h reperfusion and i.p.
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Arch. Immunol. Ther. Exp.
injection of vehicle (i.p.); (3) I/R ? PCA5, receiving
30 min ligation of coronary artery, 24 h reperfusion and
i.p. injection of 5 mg/kg PCA at a time point of 10 min
after ischemia; (4) I/R ? PCA20, receiving 30 min liga-
tion of coronary artery, 24 h reperfusion and i.p. injection
of 20 mg/kg PCA at a time point of 10 min after ischemia.
To induce myocardial infarction, mice were anesthetized
by i.p. injection of a mixture of midazolam (5 mg/kg),
fentanyl (0.5 mg/kg) and medetomidine (0.05 mg/kg) and
ventilated under a mouse volume-control ventilator. Fol-
lowing thoracotomy between the third and fourth
intercostal space, a surgery was performed to expose the
heart. The left main coronary artery was ligated with a 6–0
silk suture for 30 min. At 10 min after ligation, PCA was
administered via i.p. injection, whereas equal volume of
vehicle was injected into the animals in Sham and I/R
groups. After 30 min ischemia, the suture was loosened to
allow reperfusion in the mice for the recovery from anes-
thetization for 24 h. At the end of I/R operation, the LAD
was relegated and Evans blue dye was injected to visualize
the area at risk (AAR) of infarction showing no infiltration
of Evans blue dye. Five slices produced by cutting the
myocardium were staining in 0.5 % triphenyltetrazolium
chloride (TTC) for 15 min, and infarct area (IA), which
was not stained with TTC, was assessed. The areas of AAR
and IA were quantified by computerized planimetry of
digital images using a free downloadable NIH Image J
software.
buffer. After centrifugation, the supernatants were col-
lected for the measurement of SOD, CAT, MDA and MPO
with specific commercial assay kit from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China) according to the
manufacturer’s instructions. The activities of SOD and
CAT were expressed as U/mg proteins. The MDA level
was represented in nmol/mg proteins. MPO activity was
expressed as U/mg wet tissues.
TUNEL Staining of Myocardial Apoptosis
Formalin-fixed heart tissues were embedded in paraffin and
sectioned into 4 lm thick slices (n = 3/group). The sec-
tions were deparaffinized, rehydrated and stained with
TUNEL kit (Roche, Mannheim, Germany) according to the
manufacturer’s instructions. The sections were imaged on a
Leica DME Basic Microscope (Hicksville, NY, USA) with
Photometrics CoolSNAP Camera (Tucson, AZ, USA). The
apoptotic cells and the overall cell nuclei in the heart tissue
were counted. The positive-cells were calculated from five
random field of view on the images with 4009 magnifi-
cation. All these images were collected and counted in a
blinded fashion.
Western Blot Analysis of Apoptosis Biomarkers
Myocardial tissues were homogenized in ice-cold RIPA
buffer (Sigma St. Louis, MO, USA) containing proteinase
inhibitor for protein extraction. For the detection of protein
expression, 70 lg of the cardiac proteins were resolved in
12 % SDS-PAGE, and subsequently transferred onto
polyvinylidene difluoride membrane (EMD Millipore,
Billerica, MA, USA). Membranes were incubated in tris-
buffered saline buffer (pH 7.6) containing 0.1 % Tween-20
and 5 % BSA for 2 h at room temperature. After several
washes, membranes were probed with specific primary
antibodies against Bcl-2, Bax and GAPDH (1:500 dilution)
overnight at 4 °C. Following the removal of excessive non-
bound antibodies, membranes were detected with HRP-
conjugated secondary antibody (1:1000 dilution) for
another 3 h at 4 °C. The bound antibodies were visualized
with enhanced chemiluminescence reagents (GE Health-
care, Uppsala, Sweden).
For hematoxylin and eosin (H&E) staining, the fresh
heart tissues in each group (n = 3) were fixed in 4 %
formalin and embedded in paraffin. The embedded tissues
were sectioned at the thickness of 4 lm and stained with
H&E stain for detailed observation of the potential patho-
logical changes.
Determination of the Cardiac Enzymes
and Oxidative Stress Biomarkers
To determine the release of myocardial muscle creatine
kinase (CK-MB), creatine kinase (CK) and lactate dehy-
drogenase (LDH), at the end of reperfusion, the blood
samples (n = 3) were collected in heparinized tubes and
centrifuged at 5700 rpm for 10 min. The supernatants were
recovered for the measurement of CK-MB with a com-
mercial ELISA kit from USCN Life Science Inc (Wuhan,
China), CK and LDH using corresponding detection kit
from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China) according to the manufacturer’s instructions. To
determine the levels of superoxide dismutase (SOD),
catalase (CAT), malondialdehyde (MDA) and myeloper-
oxidase (MPO), on the other hand, the heart tissues (n = 3)
were harvested and homogenized in 0.9 % NaCl saline
Statistical Analysis
The results were presented as mean SD from the indi-
cated experiments. The data were compared by one-way
analysis of variance (ANOVA), followed by least signifi-
cant difference post hoc test using IBM software SPSS
version 20.0 (Amonk, NY, USA). Differences with
p \ 0.05 were considered as statistically significant.
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Arch. Immunol. Ther. Exp.
were below 9.98 % for PCA in terms of relative standard
deviation (RSD; shown in Table 1). By plotting the natural
logarithm (Ln) of the percentage remaining PCA (Ln %
Remaining PCA) against time, we obtained the stability
profiles of PCA as shown in Fig. 2a. Based on linear
regression on these data points, we calculated the first-
order rate constant of decomposition (k, h-1) as 0.0302,
0.1181, 0.4879 for 25, 37 and 60 °C, respectively. Simi-
larly, we obtained the half-life (t1/2, h) of PCA in Table 2
as 31.39, 7.19, 1.37 for 25, 37 and 60 °C, respectively. By
applying the Arrhenius equation, we calculated the energy
of activation of 16.7 kcal/mol for PCA (R2 = 0.981). In
addition, the extraction of 100 lg/mL PCA in plasma
allowed the recovery of drug at 97.9 % (1.11 % RSD)
following three freeze–thaw cycles compared with the
analysis of samples prior to freezing. In the study of long
term stability, PCA samples were recovered at 96.4 %
(3.72 % RSD).
Results
PCA Enhanced the Survival of Cardiomyocytes
Under OGD and Reoxygenation Condition
To develop new structurally simple and pharmacologically
stable caffeic acid derivatives, we synthesized PCA in
70 % yield by direct amidation of caffeic acid with
propylamine as outlined in Fig. 1a. The chemical structure
of PCA was verified by NMR and MS techniques. We
explored the effect of PCA on the survival of cardiomy-
ocyte H9c2 cells under OGD and reoxygenation condition
as an in vitro model of I/R injury. In practical, H9c2 cells
were exposed to OGD condition for 10 h, and subsequent
reoxygenation condition with/without drugs in normal cell
culture medium for 14 h. The cell viability of H9c2 was
determined by a colorimetric MTT assay as described
(Cheng et al. 2016). As shown in Fig. 1b, PCA enhanced
the survival of H9c2 cells against OGD and reoxygenation
challenge in a concentration-dependent manner. Interest-
ingly, by examining the cytoprotective effects of PCA with
CAPE and PCE at the same concentration (20 lM), PCA
showed better cytoprotective effect than CAPE and PCE
(Fig. 1c).
Determination of Pharmacokinetic Profile
by a LC–MS/MS Method
To determine the pharmacokinetic profile of PCA, we
developed a LC–MS/MS method for the detection of PCA
(m/z, 222 to [163) and IS (m/z, 229 to [135) in MRM
mode (shown in Table 3). We first confirmed that the
detection was highly selective for PCA and IS at the
retention time of 5.93 and 6.45 min, respectively, while no
interference was detectable from the endogenous sub-
stances. We subsequently established the calibration curve
with good linearity (y = 31.926x, R2 = 0.9948) over the
concentration range of 2–3000 ng/mL in rat plasma. Based
on the assessment of the sensitivity, precision and accuracy
in Table 4, we concluded that such a LC–MS/MS assay
was accurate and reproducible for the determination of
PCA in rat plasma. Finally, we successfully applied the
LC–MS/MS method to study the pharmacokinetics of PCA
in SD rats following i.v. administration of 15 mg/kg PCA.
By plotting the PCA concentrations against the detection
time as shown in Fig. 2b, we obtained the pharmacokinetic
parameters for PCA in Table 5 as follows: elimination
half-life (t1/2), 235.02 56.67 min; AUC0–?, 1225.14
Characterization of Pharmacological Stability
by an Ultra-Fast UPLC-UV Method
To characterize the stability of PCA in plasma, we devel-
oped an ultra-fast UPLC-UV method to determine the
intact PCA after the incubation with fresh rat plasma for
different time points at 25, 37 and 60 °C. We excluded any
endogenous interference that could be co-eluted with PCA
and IS under the experimental chromatographic conditions.
Prior to the actual chromatographic determination, we
established
a
calibration curve (slope = 0.4608,
R = 0.9995) by five replicated determinations of PCA at
six concentrations of 2.5, 5, 10, 50, 100 and 250 lg/mL.
We satisfied with the overall reproducibility of the method
for the deviation within 13.01 %. By calculating the ratio
of analyte response from plasma extracted samples to un-
extracted samples, the absolute recovery of PCA was ran-
ged 91.8–98.9 %. The intra-day and inter-day precisions
162.39 ng h/mL; AUC0–t
,
1346.06 161.99 ng h/mL;
Table 1 Method validation for HPLC–DAD detection of PCA
Nominal
concentration
(lg/mL)
Observed concentration
(lg/mL; average SD)
Intra-day precision
(% RSD)
Inter-day precision
(% RSD)
Accuracy
(% deviation)
Absolute recovery
(% RSD)
2.5
50
2.19 0.127
45.65 0.95
252.47 1.78
2.00–9.60
0.98–2.01
1.5–2.09
9.98
3.63
4.36
13.01
4.21
1.44
91.8 (4.7)
95.1 (5.7)
98.9 (0.5)
250
HPLC–DAD high performance liquid chromatography–diode array detector
123

Arch. Immunol. Ther. Exp.
Fig. 2 Stability and pharmacokinetic profiles of PCA. a Stability of
PCA in rat serum. PCA was incubated in rat serum at different
temperature [i.e., 25 °C (solid triangles), 37 °C (solid squares), and
60 °C (solid rhombus)] for different time points. The remaining PCA
was quantified by HPLC–UV method. b Pharmacokinetic profile of
PCA in male Sprague–Dawley rats. PCA (15 mg/kg) was adminis-
trated to male Sprague–Dawley rats via intravenous injection. Plasma
samples were collected at different time points and determined by
HPLC–MS method. Data are presented as mean SD (n = 3)
AAR. PCA treatment attenuated myocardial infarction in a
dose-dependent manner (Fig. 3c). PCA at the dose of
20 mg/kg effectively reduced myocardial infarction to
*16 % relative to the AAR. Consistently, H&E staining in
Fig. 3d showed that I/R challenge disrupted the organiza-
tion of myocardial fibers compared with sham surgery. I/R
challenge appeared to induce the infiltration of polymor-
phonuclear leukocytes into the myocardial interstitial space
compared with sham surgery. Interestingly, PCA not only
preserved the structure of myocardial fibers but also
appeared to limit the infiltration of polymorphonuclear
leukocytes compared to I/R challenge.
Table 2 Rate constant, half-life, and activation energy of PCA
Temperature (°C)
k (h-1
)
t1/2 (h)
Ea (kcal/mol)
25
37
60
0.0302
0.1181
0.4879
31.39
7.19
1.37
16.7
Ea energy of activation
Table 3 Parameters for MRM-based analysis
Components
RT
MRM
CE (eV)
DP (eV)
CPA
RE
5.93
6.45
222–163
229–135
19
29
51
PCA Diminished the Release of Cardiac Enzymes
and Oxidative Stress
236
To verify the effect of PCA on I/R-induced cardiac dam-
age, we determined the release of cardiac enzymes
including CK-MB, CK and LDH into the circulation. As
shown in Fig. 4a–c, I/R challenge indeed markedly
increased the serum levels of CK-MB, CK and LDH
compared with sham surgery. However, administration of
PCA significantly suppressed the level of LDH, CK and
CK-MB (I/R-PCA vs I/R).
total plasma clearance (CLt), 53.06 7.79 mL/min; mean
volume of distribution (Vd), 17.99 6.12 L; MRT0–t
,
2.73 0.64 h.
PCA Reduced Infarct Size and Cardiac Damage
in Mouse Model of Myocardial Infarction
The in vivo cardioprotective potential of PCA was evalu-
ated in a mouse model of myocardial infarction as
illustrated in Fig. 3a. PCA was administrated by i.p.
injection 10 min after ischemia was induced by the ligation
of left coronary artery. Twenty minutes later, animals were
subjected to reperfusion for 24 h. Based on TTC staining of
viable myocardium in Fig. 3b, ischemia and reperfusion (I/
R) induced *42 % of myocardial infarction relative to the
To examine the antioxidant effects of PCA against
myocardial injury, we harvested the hearts from each
experimental group, and analyzed for the levels of CAT,
SOD and MDA. As shown in Fig. 4d–f, I/R challenge
markedly decreased the enzymatic activities of CAT and
SOD while increased the production of MDA in heart tis-
sues. PCA pre-treatment could prevent the loss of CAT and
SOD activities, and diminish the production of MDA.
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Arch. Immunol. Ther. Exp.
Table 4 Method validation for LC/MS/MS detection of PCA
Nominal
concentration
(ng/mL)
Observed concentration
(ng/mL; average SD)
Intra-day precision
(% RSD)
Inter-day
precision
(% RSD)
Accuracy
(% deviation)
20
20.93 0.92
520.1 9.21
2959 8.21
4.44–7.12
1.58–6.73
0.75–1.53
4.35
2.66
1.20
11.6
9.40
1.42
500
3000
LC/MS/MS liquid chromatography/mass spectrometry/mass spectrometry
Table 5 Pharmacokinetic parameters of PCA
tissues. PCA pre-treatment ameliorated the infiltration of
polymorphonuclear leukocytes in response to I/R
challenge.
t1/2 (min)
235.02 56.67
1225.14 162.39
1346.06 161.99
53.06 7.79
AUC0–? (ng h/mL)
AUC0–t (ng h/mL)
CLT (mL/min)
Vd (L)
PCA Reduced Myocardial Apoptosis via Regulating
Bcl2 and Bax Expression
17.99 6.12
MRT0–t (h)
2.73 0.64
To understand the potential cardioprotective mechanism, we
examined the effects of PCA on I/R-induced myocardial
apoptosis by TUNEL assay. As shown in Fig. 5a, I/R chal-
lenge triggered apoptosis in cardiomyocytes. PCA
pretreatment effectively protected cardiomyocytes against
I/R-induced injury in a dose-dependent manner. Based on
TUNEL staining, PCA profoundly reduces the ratio of
To further clarify the effects of PCA on the infiltration
of polymorphonuclear leukocytes, we harvested the hearts
from each experimental group, and analyzed for the levels
of MPO activity. As shown in Fig. 4g, I/R challenge
markedly increased the enzymatic activity of MPO in heart
Fig. 3 Effect of PCA on infarct size and myocardial histology in
mouse model of myocardial infarction. a Experimental design. The
animals were randomly divided into four groups: Sham, I/R,
I/R ? PCA5 and I/R ? PCA5. Acute myocardial infarction was
induced by the ligation of the LAD artery as outlined. After the
animals were euthanized, the hearts and blood samples were
recovered for histological and biochemical assessments. b Detection
of viable myocardium by TTC staining. c Quantification of I/R injury
in hearts. Values represent mean SD (n 4). *p \ 0.05; **p \ 0.01
(PCA ? I/R vs I/R). d Histological evaluation of I/R injury in hearts
by H&E staining. Scale bar 25 lm
123

Arch. Immunol. Ther. Exp.
Fig. 4 Effects of PCA on the biomarkers of cardiac damage,
oxidative stress and leukocyte infiltration. After 30 min ischemia
and 24 h reperfusion, blood samples and heart tissues were collected.
Serum levels of CK-MB (a), CK (b) and LDH (c) were determined by
commercial assay kits as described in ‘‘Materials and methods’’. On
the other hand, cardiac tissues were homogenized and determined for
the levels of CAT (d), SOD (e), MDA (f) and MPO (g) by commercial
assay kits as described in ‘‘Materials and methods’’. (n = 3)
##p \ 0.01 and ###p \ 0.01 (I/R vs Sham); *p \ 0.05; **p \ 0.01
and ***p \ 0.001 (PCA ? I/R vs I/R)
apoptotic nuclei in overall cardiomyocytes from 33.7 % for
I/R group to 26.8 % for I/R ? PCA5 group and 14.7 % for
I/R ? PCA20 group. To further explore the apoptotic
mechanisms, we analyzed the protein levels of Bcl2 and Bax
by Western blotting using specific antibodies. As shown in
Fig. 4b, I/R challenge down-regulated the expression of anti-
apoptotic protein Bcl2 while up-regulated the expression of
pro-apoptotic protein Bax, causing a significant decrease in
the ratio of Bcl2/Bax. PCA pre-treatment markedly up-reg-
ulated the expression of Bcl-2 while suppressed the
expression of Bax, conferring a significant increase in the
ratio of Bcl2/Bax for I/R ? PCA20 group.
stable in various biological systems. In this study, we
synthesized a simple caffeic acid amide derivative PCA,
and subsequently discovered that PCA exhibited better
protection of cardiomyocyte H9c2 cells against OGD and
reoxygenation challenge, compared with CAPE and PCE.
These results stimulated us to evaluate the in vivo cardio-
protective effects and pharmacokinetics of PCA. We
further discovered that PCA effectively reduced cardiac
damages in a mouse model of myocardial infarction pos-
sibly via suppressing oxidative stress and infiltration of
polymorphonuclear leukocytes.
Ischemia induces oxidative stress in myocardial cells by
stimulating ROS generation, but reperfusion recruits
enormous polymorphonuclear leukocytes and further
amplifies the production of toxic ROS (Chen and Zweier
2014). ROS is known to directly damage proteins and
membrane lipids in cardiac tissues (Hori and Nishida
2009). Thus, the cardiac damage is often assessed by
assaying different biomarkers. First, CK-MB, CK and LDH
Discussion
The biological activities of caffeic acid derivatives are
highly dependent on the chemical structures. For example,
caffeic acid amide derivatives were found to be more
123

Arch. Immunol. Ther. Exp.
Fig. 5 Effects of PCA on the apoptosis of myocardial cells and the
expression of Bcl-2 and Bax. a Representative images of TUNEL-
stained myocardial tissues in different groups. Scale bar 50 lm. The
images were quantified for TUNEL-positive cells (n = 5).
***p \ 0.001 (treatment vs Sham group); ##p \ 0.01 and
###p \ 0.01 (treatment vs I/R group). b Western blot analyses for
Bcl-2 and Bax expression. The results were expressed as the ratio of
Bcl-2/Bax (n = 3). **p \ 0.01 and ***p \ 0.001 (treatment vs Sham
group); #p \ 0.05, ##p \ 0.01 and ###p \ 0.01 (treatment vs I/R
group). c Proposed mechanisms underlying the cardioprotective
effects of PCA. Red arrow activation, blue line inhibition
in serum have widely used as the biomarkers for the
assessment of myocardial damage. In this study, I/R chal-
lenge elevated the serum levels of CK-MB, CK and LDH.
PCA prevented the release of cardiac enzymes into the
blood. Consistently, PCA effectively reduced infarct size
and preserved the structure of myocardial fibers. Second,
the oxidative damage of cell membrane is mainly mediated
by the peroxidation of lipids into MDA. For this reason,
MDA is another important biomarker to evaluate the
oxidative damage of myocardial cells (Del Rio et al. 2005).
Our results showed that I/R challenge significantly
increased the formation of MDA. PCA almost abolished
the production of MDA. Third, ROS overproduction
overwhelms the antioxidant capacity in the heart. CAT and
SOD are two key antioxidant enzymes for scavenging free
radicals, serving as the first line defenders against oxidative
tissue damage (Rashid et al. 2013). I/R challenge pro-
foundly down-regulated the expression of CAT and SOD.
PCA could rescue the loss of CAT and SOD activities to
certain extent, helping the scavenger of free radicals.
Polymorphonuclear leukocytes are progressively
recruited and persistently activated within infarcted myo-
cardium. Neutrophils not only produce proteases and
different forms of toxic ROS and RNS to destroy the sur-
rounding tissues, but also secret various mediators to
recruit more pro-inflammatory cells (Schofield et al. 2013).
Neutrophils infiltration is often represented by assaying
MPO activity in injured cardiac tissues (Goldmann et al.
2009). In this study, we observed that I/R challenge
induced while PCA markedly attenuated the deposition of
polymorphonuclear leukocytes in myocardium by H&E
staining. We detected that PCA effectively suppressed I/R-
induced increase in cardiac MPO activity. However, little
is known about the mechanisms by which PCA regulates
the infiltration of polymorphonuclear leukocytes against
I/R challenge. Current anti-inflammatory drugs mainly
target the production and the signaling pathways of various
proinflammatory mediators (Hakonarson et al. 2005; Werz
and Steinhilber 2006). Leukotriene B4 (LTB4) is one of the
key pro-inflammatory mediators for regulating the
123

Arch. Immunol. Ther. Exp.
infiltration and survival of neutrophils in myocardial injury
(Borgeat and Naccache 1990; Helgadottir et al. 2006;
Yokomizo et al. 2001). We recently discovered that plant
natural products (e.g., gallic acid and calycosin) could
induce LTB4DH to convert LTB4 into less bioactive
12-oxo-LTB4. We demonstrated that LTB4DH induction
might attenuate neutrophil-mediated myocardial injury via
inactivating LTB4. Interestingly, natural product curcumin
as a structural analogue of PCA could also induce
LTB4DH expression (Yu et al. 2012). Thus, we speculated
that PCA might limit neutrophils infiltration partly via the
induction of LTB4DH expression.
results suggest that PCA may be a potent drug candidate
for the treatment of I/R injury.
Acknowledgments The authors are grateful to Dr. Chi-Wai Cheung
for offering the access to small animal ventilator and Mr. Hao-Bo Li
for his excellent technical assistance on mouse model of myocardial
infarction. This work was supported by General Research Fund (i.e.,
HKU 775812M and 17120915) from the Research Grants Council of
Hong Kong and the Seed Fund for Basic Research Programme, the
University of Hong Kong (to J.R.).
Compliance with ethical standards
Conflict of interest The authors declare that there is no conflict of
interest about the article.
I/R injury is well-known to damage myocardial tissues
via activating apoptosis cascade in hearts (Buja et al.
2007). The anti-apoptotic proteins are commonly sup-
pressed whereas pro-apoptotic protein Bax is activated in
myocardial mitochondria during I/R treatment (Kumar and
Jugdutt 2003). Dysregulated Bcl-2/Bax ratio induces the
activation of caspase-3 and caspase-9 leading to apoptosis
(Dong et al. 2003). On the other hand, Bcl-2 could inhibit
myocardial apoptosis through suppressing the opening of
mitochondrial permeability transition pore, blocking the
release of cytochrome c and decreasing the activities of
various caspases (Cook et al. 1999). In our study, we used
TUNEL assay to detect myocardial apoptosis. As shown in
Fig. 5a, PCA treatment could significantly reduce
myocardial apoptosis. We further determined the protein
levels of Bcl-2 and Bax in the infracted hearts compared
with that in untreated hearts. As shown in Fig. 5b, PCA
treatment markedly up-regulated the ratio of Bcl-2/Bax
whereas I/R operation negatively regulated Bcl-2/Bax ratio
in myocardial tissues. Collectively, the pharmacological
actions of PCA could be summarized in Fig. 5c. In acute
myocardial I/R model, PCA exhibited cardioprotective
effects via promoting the expression of antioxidant
enzymes (1.e., CAT, SOD) and anti-apoptotic Bcl-2, and
suppressing the expression of pro-apoptotic Bax, lipid
peroxidation, and neutrophil infiltration. Ultimately, PCA
could effectively reduce cell apoptosis.
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