ENP11, a potential CB1R antagonist, induces anorexia in rats

Article abstract of DOI:10.1016/j.pbb.2015.06.007

Over the past decade, pharmacological manipulation of cannabinoid 1 receptor (CB1R) has become an interesting approach for the management of food ingestion disorders, among other physiological functions. Searching for new substances with similar desirable effects, but fewer side-effects we have synthesized a SR141716A (a cannabinoid receptor inverse agonist also called Rimonabant) analog, 1-(2,4-Difluorophenyl)-4-methyl-N-(1-piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxamide, ENP11, that so far, as we have previously shown, has induced changes in glucose availability, i.e. hypoglycemia, in rats. In this study we tested the effects, if any, of ENP11 (0.5, 1.0, and 3.0 mg/kg) in food ingestion, core temperature, pain perception and motor control in adult Wistar rats. Results showed that ENP11 reduced food ingestion during the first hour immediately after administration. Likewise, ENP11 (1.0 mg/kg) blocked anandamide (AEA)-induced hyperphagia during the first 4 h of the dark phase of the light-dark cycle, and it also blocked AEA-induced hypothermia. However, none of the ENP11 doses used affected pain perception or motor control. We believe that ENP11 is a potential useful CB1R antagonist that reduces food ingestion and regulates core temperature.

Full text of DOI:10.1016/j.pbb.2015.06.007

Pharmacology, Biochemistry and Behavior 135 (2015) 177–181
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior
journal homepage: www.elsevier.com/locate/pharmbiochembeh
ENP11, a potential CB1R antagonist, induces anorexia in rats
Mónica Méndez-Díaz a, Octavio Amancio-Belmont a, Eduardo Hernández-Vázquez b,
Alejandra E. Ruiz-Contreras c, Francisco Hernández-Luis b, Oscar Prospéro-García a,
⁎
a
Grupo de Neurociencias de la UNAM, Laboratorio de Canabinoides, Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México, D. F. 04510 Mexico
Grupo de Neurociencias de la UNAM, Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, México, D. F. 04510 Mexico
Grupo de Neurociencias de la UNAM, Laboratorio de Neurogenómica Cognitiva, Coordinación de Psicofisiología, Facultad de Psicología, Universidad Nacional Autónoma de México,
b
c
México, D. F. 04510 Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Over the past decade, pharmacological manipulation of cannabinoid 1 receptor (CB1R) has become an interesting
approach for the management of food ingestion disorders, among other physiological functions. Searching for
new substances with similar desirable effects, but fewer side-effects we have synthesized a SR141716A (a canna-
binoid receptor inverse agonist also called Rimonabant) analog, 1-(2,4-Difluorophenyl)-4-methyl-N-(1-
piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxamide, ENP11, that so far, as we have previ-
ously shown, has induced changes in glucose availability, i.e. hypoglycemia, in rats. In this study we tested the ef-
fects, if any, of ENP11 (0.5, 1.0, and 3.0 mg/kg) in food ingestion, core temperature, pain perception and motor
control in adult Wistar rats.
Results showed that ENP11 reduced food ingestion during the first hour immediately after administration. Like-
wise, ENP11 (1.0 mg/kg) blocked anandamide (AEA)-induced hyperphagia during the first 4 h of the dark phase
of the light–dark cycle, and it also blocked AEA-induced hypothermia. However, none of the ENP11 doses used
affected pain perception or motor control.
Received 14 January 2015
Received in revised form 12 May 2015
Accepted 10 June 2015
Available online 11 June 2015
Keywords:
ENP11
Food ingestion
Core temperature
Endocannabinoids
CB1R
We believe that ENP11 is a potential useful CB1R antagonist that reduces food ingestion and regulates core
temperature.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
CB1R is widely expressed throughout the central nervous system in
presynaptic GABAergic and glutamatergic terminals (Huang et al., 2001;
The incidence of obesity and associated metabolic disorders has
become a health problem worldwide (World Health Organization,
WHO 2015). Due to the fact that extensive scientific literature has
supported the endocannabinoid system (eCBs) as a food ingestion
modulator, the phyto and synthetic cannabinoids seem to be useful to
affect such a function (Koch, 2001; Martínez-González et al., 2004;
Méndez-Díaz et al., 2012; Merroun et al., 2009; Pérez-Morales et al.,
2012; Soria-Gómez et al., 2010). In this context, it is now known that
CB1R antagonists reduce food intake thereby promoting weight loss.
Moreover, CB1R antagonists improve the metabolic profile in dogs, ro-
dents and humans (Bennetzen et al., 2008; Bergholm et al., 2013; Cota
et al., 2009; Van Gaal et al., 2008, Mølhøj et al., 2010; Randall et al.,
2010; Richey et al., 2009; Van Gaal et al., 2005; Verty et al., 2009).
Katona et al., 1999; Marsicano and Lutz, 1999). It is a protein Gi coupled
receptor that promotes potassium channel opening while blocking N/P/
Q-type calcium channels, thus reducing neuronal excitability and neu-
rotransmitter release (Twitchell et al., 1997). Although the mechanism
by which CB1R activation induces food intake is not quite clear, we
believe it is part of a negative feedback loop that regulates neurotrans-
mitter release, thereby modulating various CNS circuits, including
those involved in food ingestion and energy expenditure such as the hy-
pothalamic and reward systems. We recently synthesized a CB1R high
affinity, SR141716A analog, to wit: 1-(2,4-Difluorophenyl)-4-methyl-
N-(1-piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-
carboxamide (ENP11). We have shown that ENP11 causes hypoglyce-
mic effects in experimentally induced diabetic rats. ENP11-induced
food ingestion reduction was an expected effect based on the estimation
made by the Prediction of Activity Spectra for Substances (PASS®) pro-
gram (Hernández-Vázquez et al., 2013).
Abbreviations: ENP11, 1-(2,4-Difluorophenyl)-4-methyl-N-(1-piperidinyl)-5-[4-
(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxamide; CB1R, cannabinoid 1 receptor;
AEA, anandamide; eCBs, endocannabinoid system; DMSO, dimethyl sulfoxide.
1.1. Present study
⁎
Corresponding author at: Laboratorio de Canabinoides, Grupo de Neurociencias,
Depto. de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México,
México, D. F. 04510 Mexico.
In this study we have tested the effects of ENP11 on food ingestion,
pain perception, core temperature, and motor control, seeking to
E-mail address: opg@unam.mx (O. Prospéro-García).
http://dx.doi.org/10.1016/j.pbb.2015.06.007
0091-3057/© 2015 Elsevier Inc. All rights reserved.

178
M. Méndez-Díaz et al. / Pharmacology, Biochemistry and Behavior 135 (2015) 177–181
describe a potential influence of ENP11 on these variables. To further
support its properties to antagonize endocannabinoid activity, we test-
ed its efficacy to inhibit anandamide (AEA)-induced effects on these
variables and compare it to AM251 and Rimonabant efficacy.
AM251 (1.2 mg/kg). The pain perception test was performed using a
Hot Plate device (Socrel, model DS37). The stainless steel plate
(25 × 25 cm) was maintained at 55 °C 1 °C. Rats were placed on the
plate and maintained within the plate area by means of a Plexiglas cyl-
inder (20 cm diameter and 20 cm height). The time elapsed from the
moment the rat was placed on the plate to the time the rat showed
signs of discomfort such as licking its hind paw or jumping to the
highest border of the cylinder was recorded (s) and was considered
the reaction time (latency). Rats received their treatment 30 min before
the test. One additional assessment was performed 60 min after injec-
tion. The hot plate was cleaned thoroughly with a 5% chlorine solution
after each trial. Data are expressed as mean SEM of latency (s) and
were analyzed using a one-way ANOVA test followed by post-hoc
Tukey test (p b 0.05).
2. Material and methods
2.1. Animals
Adult male Wistar rats (250 g) were used. Rats remained housed
in standard Plexiglas cages (42 × 25.5 × 20 cm) with sawdust bed-
ding and maintained on a controlled 12/12 h light/dark cycle (lights
on 20:00 h), in climate controlled rooms (21 1 °C and 52% humid-
ity). Water and food (Rat Chow, Purina) were provided ad libitum.
The rats were allowed one week of habituation prior to experimental
manipulation. All rats were used in one experiment only. Every effort
was made to minimize the number of animals used and their poten-
tial suffering. All experiments were carried out during the dark phase
of the photoperiod. The management and animal care adhered strict-
ly to the provisions of the Official Mexican Regulation on “Technical
specifications for the production, care and use of laboratory animals”
(NOM-062-ZOO-1999). Additionally, our protocol was approved by
the Research and Ethics Committee of the Facultad de Medicina,
Universidad Nacional Autónoma de México (UNAM).
2.5. Motor control
Rats were randomly assigned to one of the following treatment
groups (n = 5–9): DMSO, AEA (1.0 mg/kg), ENP11 (0.5, 1.0, and
3.0 mg/kg), SR 141716A (1.0 mg/kg), AM251 (1.2 mg/kg), to evalu-
ate motor control. We used a Rota-Rod (Ugo Basile Rota-Rod for
rats 7750 Ugo Basile North America, PA, CA), the apparatus consists
of a set of four drums, 50 cm high, on which 4 subjects are positioned
simultaneously. These drums are separated by opaque disks to pre-
vent the animals from being distracted. The speed of the drum's ro-
tation increases steadily. Each drum has its own digital timer and
display. The time elapsed from the time the rat is placed on the
drum to the time the rat falls on a switch that shuts down a timer,
is quantified.
The assay consists of 2 phases: the first phase is training, and it
consists of placing the animals on the drum and turning on rotation
5 times, 1 min each time. Once the training phase is completed the
animals are returned to their home cages allowing them to rest for
30 min. After this rest period the test begins, for which the animals
are again placed on the Rota-Rod 5 more times, 1 min each time,
while the drum's rotation increases steadily. In all trials, we quanti-
fied the time it took for the animals to fall from the drum (latency
to fall). The Rota-Rod apparatus was cleaned thoroughly with a 5%
chlorine solution after each trial. Data are expressed as mean
SEM of time (s), and were analyzed using a one-way ANOVA test
followed by post-hoc Tukey test (p b 0.05).
2.2. Food ingestion
The election of dose used to evaluate the effect of ENP11 on food
ingestion is based on the fact that 1 mg/kg SR141716A reduces food
ingestion (Gómez et al., 2002; Dore et al., 2014), hence, we decided
to use an equimolar dose of ENP11 that is 1 mg/kg, as well as a
lower dose, 0.5 mg/kg and a higher dose, 3 mg/kg.
Rats were randomly assigned to each one of the following groups
(n = 8–11): DMSO (300 μl), ENP11 (0.5, 1.0 or 3.0 mg/kg), SR141716A
(1 mg/kg), AM251 (1.2 mg/kg), AEA (1 mg/kg), AEA (1 mg/kg)/ENP11
(1 mg/kg); AEA (1 mg/kg)/AM251 (1.2 mg/kg), AEA (1 mg/kg)/
SR141716A (1 mg/kg). Rat body weight was measured immediately be-
fore treatment. Once rats received the treatment for the group they
belonged to, the amount of food consumption was quantified 1 h later,
and every hour, during the first 4 h of the dark phase of the cycle, and
24 h later. The amount of food consumed is reported as an index of the
amount of food eaten (g) divided by the rat's body weight (g) × 100.
Data are expressed as mean
SEM. Data analyses were carried out
2.6. Drugs
every hour during the first 4 h, for the total 4 h, and for the total of
24 h, by means of a repeated-measures ANOVA test (p b 0.05).
AEA, SR141716A and DMSO were purchased from Sigma Aldrich
Inc., MO, USA. AM251 was obtained from Tocris Bioscience MO, USA.
AEA, Rimonabant, and AM251 were prepared in 100% DMSO. Drugs
were injected intraperitoneally. Regular food used was Purina Lab
Chow. ENP11 synthesis is described below.
2.3. Core temperature
Rats were randomly assigned to one of the following treatment
groups (n = 5–8): DMSO (300 μl), AEA (1 mg/kg), ENP11 (1 mg/kg),
SR141716A (1 mg/kg), AM251 (1.2 mg/kg), AEA (1 mg/kg)/ENP11
(1 mg/kg), AEA (1 mg/kg)/SR141716A (1 mg/kg), AEA (1 mg/kg)/
AM251 (1.2 mg/kg). Core temperature was measured using a rectal
thermometer with a digital display. Once rats were weighed, they re-
ceived the corresponding treatment and returned to their home cage.
Core temperature was measured 30, 60 and 240 min after administra-
2.7. ENP synthesis
The synthesis of ENP11 was published elsewhere (Hernández-
Vázquez et al., 2013). Briefly, the enolate of substituted 4-
trifluoromethyl propiophenone was obtained in methylcyclohexane
by treatment with Lithium bis(trimethylsilyl)amide and was then
treated with diethyl oxalate to obtain the tricarbonylic lithium salt
in 70% yield. The next step was the cyclocondensation of 2,4-
difluorophenylhydrazine and the previously obtained tricarbonylic
compound in a sulfuric acid–ethanol solution, which afforded the
ethyl pyrazole-5-carboxylate. The ester was converted to the corre-
sponding carboxylic acid by treatment with potassium hydroxide at
50 °C, resulting in a yield of 59% for two steps. Finally, the target product
was achieved with 81% yield by formation of the acyl chloride derivative
from the pyrazole 5-carboxylic acid and the subsequent reaction with
1-aminopiperidine and N,N-Diisopropylethylamine in chloroform.
tion. Data are expressed as mean
SEM and were analyzed using a
one-way repeated-measures ANOVA test followed by post-hoc Tukey
test (p b 0.05).
2.4. Pain perception
Rats were randomly assigned to one of the following treatment
groups (n = 7 each group): DMSO, AEA (1 mg/kg), ENP11 (1 mg/kg),
SR 141716A (1 mg/kg), AM251 (1.2 mg/kg), AEA (1 mg/kg)/ENP11
(1 mg/kg), AEA (1 mg/kg)/SR141716A (1 mg/kg), AEA (1 mg/kg)/

M. Méndez-Díaz et al. / Pharmacology, Biochemistry and Behavior 135 (2015) 177–181
179
3. Results
The analysis per hour of ENP11 (0.5, 1.0 and 3.0 mg/kg) is depicted
ANOVA was used to analyze the 24 h effect of ENP11 [F(3,37) =
4.680, p = 0.0072]. Tukey's analysis shows that the higher ENP11
dose (3.0 mg/kg) tested decreased the total amount of food for the fol-
lowing 24 h (p = 0.0038) (insert 1a).
in Fig. 1a [F(3,37) = 8.155; p = 0.0003]. Tukey's analysis shows
that the ENP11 at doses tested does not have significant effect on
the amount of food ingested 1 h after its systemic administration
(p N 0.4 vs. DMSO). During the 2nd h, ENP11 (1.0 and 3.0 mg/kg) de-
creases the amount of food ingested significantly compared to DMSO
(p = 0.049 and p = 0.048 respectively). All ENP11 doses tested reduced
the quantity of food ingested compared to DSMO 3 h after administra-
tion (ENP11 0.5 mg/kg p = 0.006; ENP11 1.0 mg/kg p = 0.0001 and
ENP11 3.0 mg/kg p b 0.0001). ENP11 (1.0 and 3.0 mg/kg), significantly
reduced food ingestion as compared to DMSO, 4 h after administration
(ENP11 1.0 mg/kg p = 0.028 and ENP11 3.0 mg/kg p b 0.0001). 1 way
Regarding the effect of all CB1R antagonists Fig. 1b depicts the ef-
fect of ENP11 (1.0 mg/kg); SR141716A (1.0 mg/kg) and AM251
(1.2 mg/kg) on food ingestion [F(3,35) = 13.40; p b 0.0001]. Tukey's
analysis revealed that none of the treatments affected significantly
the amount of food ingested during the 1st h after administration
(p N 0.4). However, during the 2nd h both AM251 (p = 0.0038)
and ENP11 (p = 0.0442) reduced the amount of food ingested as
compared to DMSO. The total amount of food ingested 3 h after
ENP11 or AM251 administration was reduced as compared to
DMSO (p b 0.0001 for both treatments). SR141716A does not affect
food ingestion compared to DMSO group and therefore exhibits sig-
nificant differences from AM251 effects (p = 0.0041). At the end of
the 4th h post administration, all treatments reduced significantly
the total amount of food ingested as compared to DMSO (ENP11
p = 0.0252; AM251 p b 0.0001 and SR141716A p = 0.0001). Both
ENP11 and SR141716A treatments are significantly different from
AM251 treatment (p b 0.0001 and p = 0.0003 respectively). The
inset in Fig. 1b shows the 1 way ANOVA by 24 h [F(3,34) = 9.677;
p b 0.0001]. Tukey's analysis shows that AM251 continues decreas-
ing the amount of food ingested 24 h after administration (p =
0.0003 vs. DMSO) (inset 1b).
Two way ANOVA by h [F(4,44) = 9.677; p b 0.0001] reveals dif-
ferences by AEA treatment. After Tukey's analysis, Fig. 1c shows the
significant AEA-induced increase in food ingestion 1 h after its
administration compared to DMSO (p b 0.0384), this effect was
prevented by AM251 (p = 0.0121 vs. AEA). During the 2nd h all an-
tagonists prevented AEA-induced increase in food ingestion (ENP11
p = 0.046; SR141716A p = 0.0051 or AM251 p b 0.0001 vs. AEA).
During the 3rd h ENP11 (p = 0.0100) and AM251 (p b 0.0001)
prevented AEA-induced increase in food ingestion. The analysis of the
4th h shows that ENP11 (p b 0.0001 vs. AEA), SR141716A (p = 0.0010
vs. AEA) or AM251 (p b 0.0001 vs. AEA) continues antagonizing the
AEA effect. Tukey's analysis shows that AM251 remains antagonizing
AEA-induced increase in food ingestion (p = 0.0179 vs. AEA) (inset 1c).
The effect of ENP11 (1.0 mg/kg), SR141716A (1.0 mg/kg) and
AM251 (1.2 mg/kg) on core temperature 30, 60 or 240 min after ad-
ministration was analyzed using 2 way RM ANOVA [F(3,24) = 1.566,
p = 0.2235]. After Tukey's analysis we observed that no antagonist
significantly affected the core temperature (p N 0.05 vs. DMSO)
(Fig. 2a). Two way RM ANOVA was used to analyze AEA and
AEA + antagonist treatments on core temperature 30, 60 or
240 min after administration [F(4,38) = 3.090; p = 0.0090]. Fig. 2b
depicts the decrease in core temperature induced by AEA on core
temperature 30 (p = 0.0215 vs. DMSO) and 60 (p = 0.0018 vs.
DMSO) min after administration. This effect was blocked by ENP11
(1 mg/kg), SR141716A (1 mg/kg) and AM251 (1.2 mg/kg) at both
times evaluated: 30 and 60 min (p N 0.05 vs. DMSO).
The effect of ENP11 (1.0 mg/kg), SR141716A (1.0 mg/kg) or AM251
(1.2 mg/kg) on pain perception was analyzed by two way ANOVA
[F(4,30) = 0.6348, p = 0.6416] (Fig. 3a). Tukey's analysis shows that
AEA (1.0 mg/kg) or any antagonist combination treatment affects pain
perception (p N 0.05) (Fig. 3b).
Fig. 1. Food intake. Graphs show the accumulative amount of food intake (g/body weight
(g) × 100) during the first 4 h after the treatment and the inset shows the total amount of
food intake (g/body weight (g) × 100) 24 h after the administration. a) Illustrates the ef-
fect of 0.5, 1.0 and 3.0 mg/kg of ENP 11. Two way ANOVA followed by Tukey, *p b 0.05
DMSO vs. all doses of ENP 11; #p b 0.05 DMSO vs. 1.0 and 3.0 mg/kg of ENP 11;
+p b 0.05 DMSO vs. 3.0 mg/kg of ENP 11. b) Shows the effect of ENP 11 (1.0 mg/kg),
SR141716A (1.0 mg/kg) and AM251 (1.2 mg/kg). Two way ANOVA followed by Tukey,
*p b 0.05 DMSO vs. ENP11, SR1417161A and AM251; #p b 0.05 DMSO vs. ENP11 and
AM251; &p b 0.05 DMSO vs. AM251; +p b 0.05 AM251 vs. SR141716A; $p b 0.05 AM vs.
ENP and SR141716A. And c) shows the orexigenic effect of AEA (1 mg/kg), as well as an-
tagonism of ENP11 (1 mg/kg), SR141716A (1 mg/kg), and AM251 (1.2 mg/kg). Two way
ANOVA followed by Tukey, *p b 0.05 AEA vs. all treatments; #p b 0.05 DMSO vs. AEA:
+p b 0.05 AEA vs. AM; &p b 0.05 AEA vs. ENP.
Fig. 4 shows that none of the treatments used affected motor control
tested in the Rota-Rod [F(6,43) = 0.4618, p = 0.8326].
4. Discussion
In the present study, we have described the pharmacological effect
on food ingestion, core temperature, pain perception and motor control,
of a recently synthesized CB1R antagonist, ENP11, a SR141716A related
derivative, in rats. As expected ENP11 reduced food ingestion. ENP11
(1 mg/kg) effects on food ingestion were comparable to those induced

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M. Méndez-Díaz et al. / Pharmacology, Biochemistry and Behavior 135 (2015) 177–181
Fig. 4. Motor control. None of the treatments used affected motor control compared to
DMSO.
that this drug also binds to CB1R. As we have previously demonstrated
that ENP11 causes hypoglycemia in rats, it is very likely that the food in-
gestion reduction contributes to induce such hypoglycemia.
Fig. 2. Effect of ENP 11 on core temperature. Graph a) shows changes in core temperature
(°C) 30, 60 and 240 min after administration of vehicle and antagonists ENP 11 (1 mg/kg),
SR141716A (1 mg/kg), and AM251 (1.2 mg/kg); b) shows the hypothermic effect of AEA
(1 mg/kg), as well as the antagonistic effect of ENP 11, SR141716A, and AM251 at the same
doses used. *p b 0.05 AEA vs. all treatments.
None of the antagonists used, at equimolar doses, affected the core
temperature by themselves. But all of them, ENP11 included, prevented
AEA's hypothermic effect (Fig. 2b). Moreover, none of these antagonists
modified pain threshold (Fig. 3) or motor control (Fig. 4). Hence, we
have gathered some evidence suggesting that ENP11 reduces food
intake (even for 24 h at the higher dose used in this study), without in-
ducing side effects on pain perception or motor control, suggesting it as
a therapeutic option to decrease food ingestion. However, further ex-
periments assessing its potential to induce depression-like symptoms
will complement the present findings.
by SR141716A or AM251 at equimolar dose used. ENP11 was unable to
modify pain perception and motor control. Likewise, ENP11 prevented
anandamide's effects on food ingestion and core temperature.
Although none of the antagonists administered had an effect on food
ingestion 1 h post administration, in the 2nd h the expected effect of
ENP11 appeared, that is similar to the well-characterized AM251 ano-
rectic effect (Méndez-Díaz et al., 2012; Tallett et al., 2009). This finding
reveals that both antagonists have the same latency to affect food inges-
tion. Apparently, SR141716 is less potent than the other antagonists at
this equimolar dose, because the latency to significantly decrease food
ingestion is about 4 h. The higher ENP11 (3 mg/kg) dose used to de-
creased food ingestion induced this effect for the following 24 h after
administration, as AM251 (1.2 mg/kg) did. In short, ENP11 exhibited,
at equimolar dose, a shorter latency than SR141716A and shorter half-
life than AM251 to decrease food ingestion. This ENP11 short half-life
seems to be convenient due to the fact that in some therapeutic circum-
stances a limited effect is desirable.
5. Conclusion
This study sheds some evidence supporting ENP11's potential an-
orexigenic properties. Moreover, ENP11's capacity to block some
AEA effects supports its interaction with the CB1R.
Acknowledgment
This study was supported by Grant IN224314 from DGAPA to OPG.
We would like to thank to Edith Monroy for her careful assistance
with English language review of this manuscript.
In addition, since ENP11 (1 mg/kg) antagonized AEA's orexigenic
effect just as SR141716A and AM251 did (Colombo et al., 1998;
Martínez-González et al., 2004; Méndez-Díaz et al., 2012), we believe
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