Carvacryl acetate, a novel semisynthetic monoterpene ester, binds to the TRPA1 receptor and is effective in attenuating irinotecan-induced intestinal mucositis in mice

Article abstract of DOI:10.1111/jphp.12818

Objectives: We aimed to determine whether carvacryl acetate acts as a TRPA1 receptor agonist and its effects against irinotecan (CPT-11) induced intestinal mucositis in mice. Methods: TRPA1 structure was obtained from a protein databank, and the 3D structure of carvacryl acetate was determined. Appropriate binding conformations were discovered via automatic docking simulations. To determine the effect of carvacryl acetate in vivo, mice were treated with either DMSO 2%, CPT-11, carvacryl acetate followed by CPT-11, or HC-030031, a TRPA1 antagonist, followed by carvacryl acetate. Jejunum samples were taken and structural, inflammatory and antioxidant parameters were studied. Key findings: Eight amino acids residues in TRPA1 established stable interactions with carvacryl acetate, which led to pharmacological efficacy against CPT-11-induced intestinal mucositis via reduction of both neutropenia and bacteremia, increase in villi height and crypt depth, decrease in pro-inflammatory cytokines (interleukin-1β, keratinocyte chemoattractant and tumour necrosis factor-α) and decrease in malondialdehyde and nitric oxide metabolite levels in the jejunum. Conclusions: Carvacryl acetate is a promising anti-inflammatory and antioxidant agent, a fact confirmed through observations of its interactions with TRPA1 in CPT-11-induced intestinal mucositis in mice.

Full text of DOI:10.1111/jphp.12818

Research Paper
Carvacryl acetate, a novel semisynthetic monoterpene
ester, binds to the TRPA1 receptor and is effective in
attenuating irinotecan-induced intestinal mucositis in mice
a
a
a
a
b
Elenice M. Alvarenga , Nayara A. Sousa , Simone de Ara uꢀ jo , Jos eꢀ L. P. J uꢀ nior , Alyne R. Ara uꢀ jo ,
a
c
c
c
d
Bruno Iles , Dvison M. Pac ꢀı fico , Gerly Anne C. Brito , Emmanuel P. Souza , Dami a~ o P. Sousa
Jand Venes R. Medeiros
and
a
a
Laboratory of Pharmacology of Inflammation and Gastrointestinal Disorders (Lafidg), Federal University of Piau ꢀı , Parna ꢀı ba, PI, Brazil,
b
c
Biotechnology and Biodiversity Center Research, BIOTEC, Federal University of Piau ꢀı , Parna ꢀı ba, PI, Brazil, Department of Morphology, Faculty of
d
Medicine, Postgraduate Program in Morphofunctional Sciences, Federal University Cear ꢀa , Fortaleza, CE, Brazil and Department of Pharmaceutical
Sciences, Federal University of Para ꢀı ba, Jo ~a o Pessoa, Para ꢀı ba, Brazil
Keywords
Abstract
carvacryl acetate; CPT-11; cytokines;
molecular docking; oxidative stress
Objectives We aimed to determine whether carvacryl acetate acts as a TRPA1
receptor agonist and its effects against irinotecan (CPT-11) induced intestinal
mucositis in mice.
Correspondence
Jand Venes R. Medeiros, BIOTEC/LAFFEX/
UFPI, Av. S ~a o Sebasti ~a o, no 2819, CEP
Methods TRPA1 structure was obtained from a protein databank, and the 3D
structure of carvacryl acetate was determined. Appropriate binding conforma-
tions were discovered via automatic docking simulations. To determine the effect
of carvacryl acetate in vivo, mice were treated with either DMSO 2%, CPT-11,
carvacryl acetate followed by CPT-11, or HC-030031, a TRPA1 antagonist, fol-
lowed by carvacryl acetate. Jejunum samples were taken and structural, inflam-
matory and antioxidant parameters were studied.
6
4202-020, Parna ꢀı ba, PI, Brazil.
E-mail: jandvenes@ufpi.edu.br
Received July 5, 2017
Accepted August 26, 2017
doi: 10.1111/jphp.12818
Key findings Eight amino acids residues in TRPA1 established stable interactions
with carvacryl acetate, which led to pharmacological efficacy against CPT-11-
induced intestinal mucositis via reduction of both neutropenia and bacteremia,
increase in villi height and crypt depth, decrease in pro-inflammatory cytokines
(
interleukin-1b, keratinocyte chemoattractant and tumour necrosis factor-a) and
decrease in malondialdehyde and nitric oxide metabolite levels in the jejunum.
Conclusions Carvacryl acetate is a promising anti-inflammatory and antioxidant
agent, a fact confirmed through observations of its interactions with TRPA1 in
CPT-11-induced intestinal mucositis in mice.
between carvacrol and carvacryl acetate is the replacement
of the hydroxyl group by an ester group in the latter, which
Introduction
Carvacryl acetate (5-isopropyl-2-methylphenyl acetate,
C H O ), a semisynthetic monoterpene ester, has shown
anti-inflammatory and antinociceptive effects in experi-
is attributable to carvacryl’s lower toxicity and higher sta-
bility. Carvacryl acetate inhibits inflammatory mediators
12
16 2
[
1]
and neutrophil migration. However, information about
the effect of carvacryl acetate on specific inflammatory dis-
eases is scarce, particularly on disorders of the gastrointesti-
nal tract.
[1]
mental models of peritonitis and paw oedema. The main
properties of carvacryl acetate include: molecular weight –
1
7
92.26; refractive index – 1.497; boiling point – 94.56 °C at
60 mmHg; enthalpy of vapourization – 48.414 kJ/mol;
Widely distributed in the gastrointestinal tract, the
ankyrin-repeat transient receptor potential (TRPA1) is a
cation channel and sensor for cell damage signals, including
3
[2]
density – 0.994 g/cm . Due to the relatively high toxicity
of its precursor carvacrol compared to that of other phenols
and esters, carvacryl acetate was thought to be a less toxic
semisynthetic derivative of carvacrol with the same or
improved pharmacological properties. The difference
[
3]
reactive oxygen species and inflammatory mediators.
[
4]
Some compounds, such as carvacrol and HC-030031, act
as a TRPA1 agonist and antagonist, respectively.
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
1

Carvacryl acetate attenuates mucositis
Elenice M. Alvarenga et al.
Intestinal mucositis, an inflammatory process that results
in morphological and physiological changes in the small
intestine mucosa, is generally caused by the use of antineo-
plastic drugs, such as irinotecan hydrochloride (CPT-11).
CPT-11 is a semisynthetic analogue of camptothecin and a
experimental procedures were performed in accordance
with the Guide for Care and Use of Laboratory Animals
(National Institute of Health, Bethesda, MD, USA) and
were approved by the local ethics committee (protocol no.
080/15). The animals were divided into experimental
groups of 5–7 animals each.
[
5,6]
selective inhibitor of the enzyme topoisomerase I.
It is
one of the main neoplastic drugs in use today. As intestinal
mucositis is severe, studies on how to minimize its negative
effects, or that clarify how intestinal mucositis develops are
essential, because they can help discover potential thera-
peutic targets. Due to its wide distribution in the intestinal
mucosa, TRPA1 is a molecule of interest in studies that
attempt to uncover the mechanisms triggering mucositis.
The aim of this study was to investigate whether car-
vacryl acetate is a TRPA1 receptor agonist. We also aimed
to verify whether this interaction between carvacryl acetate
and TRPA1 could reduce inflammation and increase muco-
sal protection in the gut of mice with CPT-11-induced
intestinal mucositis.
Effect of carvacryl acetate on intestinal
mucositis: experimental protocol
The experimental protocol to induce intestinal mucositis
was based on a previously described protocol with modifi-
[
10,11]
cations.
the experimental groups received only carvacryl acetate (25,
In this study, which lasted 8 days, the mice in
[
1,12]
75, or 150 mg/kg, i.p.) on the first day.
Between the
second and fifth days the mice received CPT-11 (75 mg/kg,
i.p.), followed by carvacryl acetate 30 min later. From days
6–8, the mice received either carvacryl acetate or DMSO
(2%, i.p.). To verify the putative involvement of TRPA1
receptors in the carvacryl acetate effect on mucositis, a dif-
ferent group of mice was administered HC-030031
Materials and Methods
(
0.025 mg/kg, i.p.) (4), a TRPA1 antagonist, 30 min before
carvacryl acetate from day 2 onwards. Negative and positive
controls received only DMSO (2%, i.p.) or only CPT-11
(75 mg/kg, i.p.), respectively.
Molecular docking
Structures of the agonist carvacryl acetate and TRPA1
(
PDB ID: 3J9P), determined using cryo-electronic micro-
In the experimental period, the mass and survival of the
mice were measured daily. The mice were then killed with
an overdose of ketamine/xylazine (>100/10 mg/kg, s.c.).
Blood samples were obtained by cardiac puncture to per-
form leucogram and bacterial counts, and segments of the
jejunum (80–100 mg) were removed for further analysis
described below.
ꢁ
scopy at a resolution of 4.24 A, were obtained from the
Research Collaboratory for Structural Bioinformatics pro-
tein databank. A docking simulation was used to study
the binding of TRPA1 and carvacryl acetate. As previ-
[7]
ously mentioned, the area of the binding site was con-
sidered the most desirable region for fitting the agonist.
The binding ability of carvacryl acetate and its corre-
sponding binding affinity scores were used to determine
better molecular interactions. The results were visualized
and analysed using ADT, Maestro, PyMOL and Discovery
Studio.
Blood leucocyte and bacteria counts
Blood samples obtained by cardiac puncture were trans-
ferred to test tubes, which were then heparinized and
diluted with Turk’s solution (380 ll of blood and 20 ll of
diluent solution). The leucocytes were counted in a Neu-
bauer Chamber with a light microscope.
Preparation of carvacryl acetate
Carvacryl acetate (98% purity; Figure 1a) was obtained via
acetylation of carvacrol, using acetic anhydride for the
acetylation and pyridine as a catalyst. Carvacryl acetate was
To count the bacteria, blood samples obtained by cardiac
puncture were quantified according to a previously devel-
[
13]
oped methodology
with modifications.
[
8,9]
obtained (4.779 g; 0.025 mol) at a 76% yield.
The
structural identification of carvacryl acetate was performed
by spectroscopic techniques (1H and 13C NMR, IR) and by
Histological and morphometric evaluation
[1]
[12]
Segments of jejunum were prepared according to. The
severity of intestinal mucositis was determined by
comparison with the literature data.
[
11,14]
grades.
The length of the intestinal villi and depth of
Animals
the crypts were measured (ImageJ Software, version 1.4,
NIH, USA). Between 5 and 10 villi and crypts were mea-
sured per slice, and a range of 5–8 slices were analysed per
Female Swiss mice (25–30 g) were randomly maintained in
appropriate cages at 25 Æ 2 °C under a 12-h light/dark
cycle with food and water provided ad libitum. All
[
15]
group.
2
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Elenice M. Alvarenga et al.
Carvacryl acetate attenuates mucositis
Figure 1 Chemical structure of carvacryl acetate (5-isopropyl-2-methylphenyl acetate, C12
H
16
O
2
). Models of molecular docking of carvacryl acet-
ate onto TRPA1 (a); Details of interactions between carvacryl acetate and the activation site of TRPA1 (b); 3D stereo view of carvacryl acetate
docking in the activation site, showing interactions with neighbouring residues through hydrophobic interactions (c); 2-D illustration of the types
of interactions between carvacryl acetate and TRPA1, containing a legend with the various interaction types (d).
Glutathione (GSH) and superoxide dismutase (SOD)
levels were determined according to the method described
+
+
Determination of Na /K -ATPase activity
+
+
[19]
[20]
Na /K -ATPase activity of enterocytes was measured in
segments of mouse jejunum according to a previously
by
and,
respectively. The malondialdehyde (MDA)
was used as an indicator of lipid peroxidation. The MDA
concentration for jejunum tissue was measured using the
[16]
described methodology with modifications.
expressed as a relation between the inorganic phosphate
Results are
[
21]
method described previously by.
[22]
[17]
and protein concentration (lmol P /mg protein/h).
The Griess reaction was used to quantification of the
À
nitric oxide (NO) metabolites, nitrate (NO3 ) and nitrite
i
À
(
according to.
NO2 ) (collectively NOx), in mouse small intestine
[
Effect of carvacryl acetate on inflammatory
and oxidative stress markers in intestinal
mucositis
22]
Immunohistochemical assay for NF-jB and
COX-2
[18]
Myeloperoxidase activity was assessed according to.
Cyto-
kine levels (TNF-a, IL-1b and KC) were measured using an
enzyme-linked immunosorbent assay (ELISA) according
Segments of mouse jejunum were incubated with goat anti-
COX-2 (1:200, SC-1747; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) or rabbit anti-NF-kB p50 NLS antibodies
[15]
to and the manufacturer’s instructions (DuoSet ELISA
Development kit R&D Systems, Minneapolis, MN, USA).
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
3

Carvacryl acetate attenuates mucositis
Elenice M. Alvarenga et al.
(
1:200, SC-114; Santa Cruz Biotechnology). The quantita-
tive estimation of DAB products from immunostaining was
performed using digital images of at least ten different areas
[
23]
of each section.
Statistical analysis
Results are expressed as mean Æ SEM of 5–8 animals per
group, and statistical analysis was performed using one-
way analysis of variance (ANOVA) followed by the New-
man–Keuls post hoc test, when appropriate. Statistical
threshold was set at P < 0.05.
Results
Molecular docking
The chemical structure of carvacryl acetate is shown in Fig-
ure 1a. The protein–ligand complexes of carvacryl acetate
were inserted at the TRPA1 cavity through Autodock 4.2
software, as shown in Figure 1c. Interactions between car-
vacryl acetate and the TRPA1 receptor occurred at eight
amino acids residues at the active site of TRPA1, (ILE803,
LEU807, LEU848, LEU863, GLU864, LEU867, LYS868 and
PHE947) (Figure 1b). A hydrogen bond formed from the
interaction between the acetylate ring and LYS868. GLU864
interacted with carvacryl acetate through a pi-anionic inter-
action. The other amino acid residues in TRPA1 interacted
with carvacryl acetate via alkyl interactions. The binding
energy that resulted in the carvacryl acetate-TRPA1 interac-
tion was negative (À6.1 kcal/mol), in a single conforma-
tion with an inhibition constant of 36.8 lM. These results
show that carvacryl acetate binds tightly to the active site of
TRPA1. All molecular docking models of interactions
between carvacryl acetate and the TRPA1 receptor are illus-
trated in Figures 1b–d.
Figure 2 Carvacryl acetate effect on survival and change in body
mass in mice. Carvacryl acetate treatment (75 mg/kg) in mice
increased the survival (a) and decreased body mass (b) of mice with
induced mucositis compared to CPT-11 (75 mg/kg) treatment. Rows
represent the mean Æ standard error of the mean expressed as
change in body mass and were analysed using one-way analysis of
variance (ANOVA) followed by a Newman–Keuls test. *P < 0.05 vs
#
control group; P < 0.05 vs CPT-11 (75 mg/kg) group.
compared to that observed for the CPT-11 group
(
4.6 Æ 1.7 g, 4.4 Æ 1.7 g and 3.4 Æ 1.3 g, respectively).
Therefore, as carvacryl acetate (75 mg/kg) caused no deaths
and caused a small change in body mass, this dose was cho-
sen for all further experimental procedures. Moreover, this
dose and its route of administration have been previously
reported as effective in other mice models of inflammation
by our research group (1).
Effect of carvacryl acetate on survival and
body mass variation
The mouse survival rate and changes in body mass were
observed throughout the intestinal mucositis experiment
(
Figure 2). Mice treated with CPT-11 (75 mg/kg, i.p.) had
the lowest survival rate (62.5%) compared to that of mice
treated with DMSO 2% (negative control, 100% survival)
or those treated with carvacryl acetate at any concentration
Effect of carvacryl acetate on leucogram
and bacterial counts
After intestinal mucositis induction, leucograms were
determined. CPT-11 treatment caused a decrease in total
(
25, 75, or 150 mg/kg, i.p., 87.5%, 100% and 87.5% sur-
vival, respectively). The only dose at which there were no
deaths was 75 mg/kg of carvacryl acetate (100% survival).
Body mass increased only in mice treated with DMSO
3
leucocyte number (3382 Æ 613.7 leucocytes/mm ) relative
to that observed for DMSO 2% treatment (4967 Æ 1145
3
leucocytes/mm ). Conversely, the density of leucocytes in
2
% (2 Æ 0.9 g at 7 days), while those treated with CPT-11
mice treated with carvacryl acetate was higher
3
lost body mass (5.4 Æ 2.1 g at 7 days). Mice treated with
(
5710 Æ 660.9 leucocytes/mm ) than that in the CPT-11
carvacryl acetate (25, 75 and 150 mg/kg, i.p.) lost less mass
4
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Elenice M. Alvarenga et al.
Carvacryl acetate attenuates mucositis
and DMSO groups. Mice treated with HC-030031
acetate on intestinal mucosa, resulting in mucositis of
degrees 2–4; this shows that carvacryl acetate likely acts
through TRPA1 receptors.
(
(
0.025 mg/kg) had the lowest density of leucocytes
2570 Æ 1142), as shown in Figure 3a.
Carvacryl acetate effectively reduced bacterial growth
Figure 3b) relative to that reported with CPT-11. The
Morphometric analysis also showed that treatment with
carvacryl acetate restored villi length (185 Æ 37.2 lm) and
enlarged crypt depths (86.5 Æ 17.34 lm), as shown in Fig-
ure 5a,b. CPT-11 caused a significant shortening of the villi
height (104.5 Æ 12.63 lm) and a reduction in crypt depth
(33 Æ 12 lm), while DMSO left the intestinal architecture
intact (villi height, 199 Æ 13.4 lm; crypt depth,
(
levels of bacterial growth in mice treated with carvacryl
acetate were similar to those in mice treated with DMSO.
Pretreatment with HC-030031 increased the bacterial count
to the level observed after CPT-11 treatment.
8
9.6 Æ 14.9 lm). Pretreatment with HC-030031 reduced
villi height (77.26 Æ 8 lm) and crypt depth
40.79 Æ 5.9 lm) to levels similar to those observed after
CPT-11 treatment.
Effect of carvacryl acetate on morphological
and morphometric parameters
(
As determined by degrees of mucositis, mice treated with
CPT-11 showed extensive loss of crypt architecture, includ-
ing necrotic, shortened and flattened villi (Figure 4b).
Mucositis of degree 3–4, necrotic cells and infiltration of
inflammatory cells such as neutrophils into more than 20%
of the lamina propria were observed. The injury scores in
mice treated with carvacryl acetate (Figure 4c) were signifi-
cantly lower (0–2) than those for the CPT-11 group, sug-
gesting that carvacryl acetate significantly restored the
histopathological alterations caused by intestinal mucositis.
This result is corroborated by the observation of a reduc-
tion in infiltration of inflammatory cells and the general
alteration of architecture, largely restoring normal tissue
structure of villi and crypts. Figure 4a shows that there was
no injury to mouse jejunum in samples treated with DMSO
+
+
Na /K -ATPase activity
+
+
Treatment with CPT-11 reduced the activity of Na /K -
ATPase enzyme (1027 Æ 299 lmol/mg/h) compared to
that reported with DMSO treatment (1894 Æ 514.6 lmol/
mg/h). However, after treatment with carvacryl acetate,
+
+
Na /K -ATPase activity returned to baseline values
(2156 Æ 884.3 lmol/mg/h), as shown in Figure 6.
Effect of carvacryl acetate on inflammatory
and oxidative stress parameters
Results related to the effects of carvacryl acetate (75 mg/kg)
on MPO, GSH, MDA, NOx and cytokines (TNF-a, IL-1b
and KC) are shown in Table 1. Higher levels of MPO were
2% (0–1). Moreover, Figure 4d shows that pretreatment
with HC-030031 nullified the beneficial action of carvacryl
Figure 3 Carvacryl acetate effect on leucocyte levels and bacterial load in blood of mice. CPT-11 (75 mg/kg) treatment decreased the total num-
ber of leucocytes in mice with induced intestinal mucositis, and treatment with carvacryl acetate (75 mg/kg) restored leucocyte levels to normal
(
(
a). Bacterial load, expressed as a logarithm of the number of bacterial colonies, was also measured in blood samples. In mice treated with CPT-11
75 mg/kg), an increase in bacteremia was observed, which was reduced by treatment with carvacryl acetate (75 mg/kg) (b). In both cases, pre-
treatment with HC-030031 (0.025 mg/kg) prevented the action of carvacryl acetate. Bars and dots represent the mean Æ standard error of the
#
mean and were analysed using one-way analysis of variance (ANOVA) followed by a Newman–Keuls test. *P < 0.05 vs control group; P < 0.05
vs CPT-11 (75 mg/kg) group.
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
5

Carvacryl acetate attenuates mucositis
Elenice M. Alvarenga et al.
(
a)
(c)
(
b)
(d)
(
e)
Figure 4 Photomicrographs from mouse jejunum mucosa stained with haematoxylin and eosin. (Panel a) DMSO 2% group (negative control)
exhibits normal histological features, with intact tissue architecture (preservation of villi and crypts) and no evidence of mucositis. (Panel b) CPT-11
(
75 mg/kg) group shows pronounced mucositis with shortened villi caused by vacuolated cells, loss of crypt architecture, and infiltration of inflam-
matory cells into the lamina propria. (Panel c) Carvacryl acetate (75 mg/kg) partly restored the tissue architecture after induction of mucositis with
some preservation of villi height and crypt depth. (Panel d) HC-030031 (0.025 mg/kg) pretreatment group shows prevention of the carvacryl acet-
ate action, with villi shortening and loss of crypt architecture. Scale bars: 50 mm. (Panel e) Histological grading of mucositis for mouse jejunum
treated with carvacryl acetate (75 mg/kg), which significantly reduced the grade of CPT-11-induced intestinal mucositis. Data represent median
#
values of scores and were analysed using a Kruskal–Wallis test. *P < 0.05 vs control group; P < 0.05 vs CPT-11 (75 mg/kg) group.
6
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Elenice M. Alvarenga et al.
Carvacryl acetate attenuates mucositis
Figure 5 Villi height (a) and crypt depth (b) of mouse jejunum mucosa showing villi shortening and decrease of crypt depth in mice subjected to
induced intestinal mucositis. Carvacryl acetate (75 mg/kg) reverted the effects of CPT-11 (75 mg/kg) and pretreatment with HC-030031
(
0.025 mg/kg) showed similar effects as CPT-11. Bars represent the mean Æ standard error of the mean and were analysed using one-way analy-
#
sis of variance (ANOVA) followed by a Newman–Keuls test. *P < 0.05 vs control group; P < 0.05 vs CPT-11 (75 mg/kg) group.
Treatment with CPT-11 significantly increased the levels
of the cytokines IL-1b, KC and TNF-a (447.9 Æ 53.8 pg/
ml, 1024 Æ 147.9 pg/ml and 144.5 Æ 51 pg/ml, respec-
tively) compared to that reported for the DMSO group
(
12.57 Æ 6.2 pg/ml, 8 Æ 9.98 pg/ml and 9.83 Æ 8.42 pg/
ml, for IL-1b, KC and TNF-a, respectively). Carvacryl acet-
ate reduced the CPT-11-induced increase in cytokine levels
(
148.4 Æ 51.12 pg/ml,
497.9 Æ 151.4 pg/ml
and
4
6.24 Æ 42.38 pg/ml for IL-1b, KC and TNF-a, respec-
tively). Pretreatment with HC-030031 increased the cytoki-
nes levels (426 Æ 54.9 pg/ml, 810.9 Æ 133.3 pg/ml,
1
39.1 Æ 40.91 pg/ml, for IL-1b, KC and TNF-a, respec-
tively), close to those observed after CPT-11 treatment,
clearly preventing carvacryl acetate action.
The presence of GSH, a cellular defence agent, is inferred
by hepatic non-protein sulfhydryl (NPSH) levels. Thus,
after intestinal mucositis induction with CPT-11, a reduc-
tion of NPSH levels (165 Æ 90.91 lg/ml) was seen com-
pared to that with DMSO treatment (385.8 Æ 77.59 lg/
ml). Treatment with carvacryl acetate increased NPSH
levels (610.5 Æ 95.09 lg/ml), and pretreatment with HC-
+
+
Figure 6 The Na /K -ATPase activity in the mouse jejunum was
measured by determining the release of inorganic phosphate from the
hydrolysis of ATP. Pretreatment with carvacryl acetate (75 mg/kg)
+
+
increased the activity of Na /K -ATPase significantly as compared to
the CPT-11 group. Bars represent the mean Æ standard error of the
mean and were analysed using one-way analysis of variance (ANOVA)
0
30031 prevented this increase (216 Æ 92.96 lg/ml).
followed by
#
a Newman–Keuls test. *P < 0.05 vs control group;
P < 0.05 vs CPT-11 (75 mg/kg) group.
The levels of SOD, another cellular defence agent, after
intestinal mucositis induction with CPT-11 was reduced
(
2.72 Æ 0.49 U SOD/lg) if compared to that observed
observed in mice that received CPT-11 (4.08 Æ 2.1 U
after DMSO treatment (3.92 Æ 0.18 U SOD/lg). Treat-
ment with carvacryl acetate increased SOD levels
(5.04 Æ 0.31 U SOD/lg), and pretreatment with HC-
030031 prevented this increase (3.83 Æ 0.20 U SOD/lg).
Another commonly used marker of oxidative stress is
MDA, a product of lipidic peroxidation. Mice treated with
CPT-11 had higher MDA levels (280.1 Æ 119.7 nmol/ml
MPO/mg of tissue) than in mice treated with DMSO
(0.95 Æ 1.4 U MPO/mg of tissue). Treatment with car-
vacryl acetate decreased the levels of MPO in the jejunum
(1.16 Æ 1.12 U MPO/mg of tissue) while pretreatment
with HC-030031 induced a significant increase in MPO
levels (4.01 Æ 1.6 U MPO/mg of tissue).
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
7

Carvacryl acetate attenuates mucositis
Elenice M. Alvarenga et al.
than mice that received DMSO did (161.4 Æ 83.02 nmol/
ml). Treatment with carvacryl acetate reduced MDA levels
(
111.8 Æ 81.85 nmol/ml) to those observed in the DMSO
group, which supports the protective action of carvacryl
acetate. Mice pretreated with HC-030031 showed slightly
elevated MDA levels (194.1 Æ 38.72).
NOx levels represent the amount of oxidative damage in
inflamed tissues. Thus, mice that received CPT-11 had
higher levels of NOx (0.47 Æ 0.16 lM) than mice that
received DMSO treatment did (0.24 Æ 0.03 lM). Carvacryl
acetate lowered NOx levels (0.24 Æ 0.15 lM), to those
observed in the DMSO group, confirming its protective
action on intestinal mucosa. Pretreatment with HC-030031
increased NOx levels (0.48 Æ 0.21 lM), to those observed
in the CPT-11 group. This result confirms that carvacryl
acetate exerts its protective action through interactions
with the TRPA1 receptor.
Immunochemical detection of NF-jB and
COX-2
Jejunum sections submitted to immunochemical detection
of NF-jB and COX-2 showed intensely stained areas in
CPT-11-treated samples (33.89 Æ 12.66 cells immunos-
tained for NF-jB detection and 47.6 Æ 11.14 cells
immunostained for COX-2 detection) compared to the
results of the DMSO group (NF-jB, 7.26 Æ 3.63 cells
immunostained; COX-2, 19.5 Æ 6.9 cells immunostained).
Treatment with carvacryl acetate significantly reduced these
immunostained areas (NF-jB, 23.58 Æ 12.62 cells
immunostained; COX-2, 12.14 Æ 3.6 cells immunos-
tained); in the case of COX-2, the staining intensity
decreased to levels close to those of the DMSO group. In
the DMSO and carvacryl acetate groups, there were few
immunostained cells in the epithelium. However, in the
CPT-11 group, the epithelium and muscle layers showed
intense immunostaining, as shown in Figures 7 and 8,
respectively.
Discussion
Results showed that carvacryl acetate stably binds to
TRPA1, both because of the number and the type of inter-
actions between them. Carvacryl acetate interacted with
TRPA1 and activates, acting as an agonist, through eight
amino acids residues, which contributed to the high stabil-
ity in this interaction. It bound to the TRPA1 receptor via
hydrogen bonds and pi-anionic interactions, both of which
are among the strongest kind of chemical interactions,
which also contributed to the strength of the interaction.
Hydrogen bonds are very strong due to the presence of high
hydrogen electropositivity and high oxygen, nitrogen and
[
24]
fluorine electronegativity.
8
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Elenice M. Alvarenga et al.
Carvacryl acetate attenuates mucositis
(
c)
(
a)
(
b)
(d)
Figure 7 Photomicrographs from immunohistochemical staining for NF-jB in jejunum of mouse with induced intestinal mucositis. Moderate NF-
jB staining was seen in normal jejunum (a). A jejunum from a mouse treated with CPT-11 showed intense immunostaining for NF-jB in the
epithelium and muscular layer (b). Treatment with carvacryl acetate considerably reduced the immunostaining in the surface epithelium (c), which
was demonstrated by a reduction in the number of immunostained cells following carvacryl acetate (75 mg/kg) treatment (d). Bars represent the
mean Æ standard error of the mean and were analysed using one-way analysis of variance (ANOVA) followed by a Newman–Keuls test. *P < 0.05
#
vs control group; P < 0.05 vs CPT-11 (75 mg/kg) group.
Comparison of the molecular docking models shows that
the interactions between carvacryl acetate and TRPA1 are
more stable and lasting than those between carvacrol and
constant than that reported for the carvacrol-TRPA1 inter-
[
action.
12]
To demonstrate that the interaction between carvacryl
acetate and TRPA1 could culminate in a relevant biological
effect against intestinal mucositis, we observed some clini-
cal parameters. Carvacryl acetate did not affect the mice’s
survival and induced a loss in body mass.
[
12]
TRPA1. This is because carvacrol and TRPA1 interacted
through only six amino acids residues using bonds weaker
[
12]
than hydrogen bonds.
Some molecules can show a large
conformational variation, with even a few rotatable bonds
[25]
creating many different conformations.
vacrol can bind to TRPA1 through seven different confor-
In this way, car-
Some of the main side effects of CPT-11 are neutropenia
[
26]
due to myelosuppression,
the blood due to tissue damage.
and the increase in bacteria in
[13,27]
[
12]
mations.
Carvacryl acetate, however, only showed a
Results demonstrated
single conformation in the TRPA1 receptor-binding site.
This stable and lasting interaction between carvacryl
acetate and TRPA1 is also corroborated by observation of
the inhibition constant, which is a parameter associated
with the dissociation constant. Interactions between car-
vacryl acetate and TRPA1 showed a smaller inhibition
that carvacryl acetate restored leucocyte levels in the blood
and minimized bacteremia, which were again increased
through HC-030031 pretreatment. Bacteremia may have
been triggered by the disruption of the mucus barrier in the
small intestine. Normally, bacteria are located in the loose
[
27,28]
mucus and cannot penetrate the inner mucus layer.
©
2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
9

Carvacryl acetate attenuates mucositis
Elenice M. Alvarenga et al.
(
a)
(
c)
d)
(
(
b)
Figure 8 Photomicrographs from immunohistochemical staining for COX-2 in jejunum mucosa in a mouse with induced intestinal mucositis trea-
ted with DMSO 2% (a). The jejunum of a mouse that received CPT-11 showed intense immunostaining for COX-2 in the epithelium and muscular
layer (b). Treatment with carvacryl acetate considerably reduced the immunostaining present in the surface epithelium (arrow, c). This was demon-
strated by a reduction in the number of immunostained cells following carvacryl acetate (75 mg/kg) treatment (d). Bars represent the mean Æ s-
tandard error of the mean and were analysed using one-way analysis of variance (ANOVA) followed by a Newman–Keuls test. *P < 0.05 vs
#
control group; P < 0.05 vs CPT-11 (75 mg/kg) group.
However, in intestinal mucositis, because of the tissue
destruction caused by CPT-11 treatment, there was damage
to the mucus layer, which may have allowed bacteria to
reach the bloodstream through contact with injured epithe-
lial cells.
its reversion after carvacryl acetate treatment. Moreover, as
[
30]
proposed in other studies,
the neutrophil migration to
the injured site itself may help explain the decrease in leu-
cocyte number in the blood.
To confirm that neutrophils trigger the release of inflam-
[
31]
Furthermore, in intestinal mucositis, we observed high
levels of MPO, indicating a large amount of neutrophil
migration to the injured tissue. Treatment with carvacryl
acetate reduced MPO levels in the jejunum tissue, although
there was an increase in the systemic levels of leucocytes.
This is because CPT-11 causes systemic neutropenia, along
with an increase in neutrophil migration to the injured tis-
sue to defend against inflammation. Oxidative stress, which
is common in inflamed tissues, can induce p53 activation
resulting in senescence, arrest cycle and cell death in the
matory mediators,
we observed the levels of some pro-
inflammatory cytokines (IL-1b, KC – an ortholog of
human IL-8 in mouse and TNF-a), which decreased after
treatment with carvacryl acetate. It is well established that
these cytokines have important functions in the develop-
ment of intestinal mucositis by regulating and amplifying
[11,32]
the immune response, contributing to tissue damage.
These actions are related to the stimulation of cytokine
release by IL-1b; induction of metabolite generation from
arachidonic acid by IL-1b, which can act as a trigger for
inflammation; and activation by TNF-a of some proteases
released by epithelial cells, fibroblasts and neutrophils,
[29,30]
hematopoietic compartment. This could explain the
low leucocyte systemic levels after CPT-11 treatment and
1
0
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**

Elenice M. Alvarenga et al.
Carvacryl acetate attenuates mucositis
[
11,31]
+
Na /K -ATPase activity in intestinal mucositis is scarce.
+
related to tissue injury.
vant chemokine recruiting neutrophils to the inflammation
Moreover, KC acts as a rele-
+
+
This study showed that Na /K -ATPase activity was dimin-
ished with CPT-11 treatment and returned after carvacryl
acetate treatment. In this manner, carvacryl acetate may
also help prevent diarrhoea found in intestinal mucositis,
encouraging survival.
[
11,33]
site and, thus, acting as a feedback agent in this pro-
cess, as more neutrophils causes more cytokine activation.
TNF-a, through interactions with TNF-R1, is also associ-
ated with activation of the transcription factor NF-jB.
[34]
Additionally, the presence of bacteria in injured mucosa, as
observed in intestinal mucositis, can lead to the activation of
Based on these results, it is possible to infer that TRPA1
act as a sensitive channel for cell damage. Other authors
showed this and the fact of its sensitivity are increased
under inflammatory conditions in mouse sensory neurons,
[35]
NF-jB.
an increase in pro-inflammatory cytokine levels, such as IL-
b and TNF-a, and can induces apoptosis, contributing to
The transcription factor NF-jB is associated with
[
3,39]
1
as demonstrated by other authors.
Through interac-
the pathology of intestinal mucositis. Furthermore, NF-jB
activates the expression of cell surface receptors to molecules
related to the inflammation, cell adhesion molecules and
tions with carvacryl acetate, which act as an agonist for this
receptor, TRPA1 could act in the modulation of the cells
responses to the damage made by ROS and stimulated for
other inflammation related factors. When it is used an
antagonist of TRPA1, it is possible to notice that this results
are reverted and there is no response to the inflammation
and cell damage, which is in concordance with the action
of carvacryl acetate on TRPA1.
[34]
molecules related to the response to oxidative stress.
Another pro-inflammatory mediator shown to be highly
expressed in CPT-11-induced intestinal mucositis was
COX-2, which was also decreased by carvacryl acetate treat-
ment. COX-2 is involved in the synthesis of prostaglandins,
which are responsible for the development of inflamma-
[
34,36]
tion.
As in other studies, we noted that COX-2 and
Conclusion
NF-jB levels seemed to follow a similar pattern of expres-
sion; both increased after CPT-11 treatment and decreased
after carvacryl acetate treatment. This agrees with the
understanding that NF-jB upregulates COX-2 expression,
Results show that carvacryl acetate presented a strong and
stable interaction with the TRPA1 receptor. This may be
responsible for the anti-inflammatory and antioxidant
action of carvacryl acetate in intestinal mucositis. There-
fore, our results suggest that carvacryl acetate may be an
effective drug to treat intestinal mucositis, especially
because of its route of action through TRPA1.
[37]
which can also be increased by IL-1b.
Results also showed a concomitant reduction in oxida-
tive stress parameters (MDA and NOx) and increase in cel-
lular defence agents (GSH and SOD) after treatment with
carvacryl acetate. This is consistent with the fact that
increasing levels of reactive oxygen and nitrogen species
can activate many pro-inflammatory pathways and upregu-
Declarations
[29,36]
late pro-inflammatory cytokines.
The mechanisms by
which carvacryl acetate reduced MDA levels and increased
GSH and SOD levels may have been related to the decrease
in neutrophil migration to the small intestine mucosa, or
by the reduction in the direct toxic action of CPT-11 on
the epithelial cells. The increase in GSH levels may also be
secondary to the consumption of other reactive oxygen spe-
Conflict of interest
The Authors confirm that there is no conflict of interests.
Acknowledgements
[
36]
This work was supported by the National Counsel of Tech-
nological and Scientific Development (CNPq, grant no.
cies.
+
+
The enzyme Na /K -ATPase is responsible for the active
transport of many electrolytes in the small intestine. Its
303032/2013-8). Dr. Brito, Dr. Sousa and Dr. Medeiros are
+
+
recipients of CNPq fellowships. We thank Rivelilson Men-
des de Freitas (in memoriam) for his assistance with the
obtention of chemical compounds used in this work. The
authors declare that no competing interests exist.
inhibition prevents the absorption of Na and K in the
small intestine and, thus, induces intestinal fluid accumula-
[
17,38]
tion, which contributes to diarrhoea,
side effects of CPT-11 therapy.
one of the
Information about
[11]
response in mice. Life Sci 2014; 94:
8–66.
esquistossomose. BR
1
O
2012
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