Toxicology Letters 382 (2023) 41–46

Contents lists available at ScienceDirect

Toxicology Letters
journal homepage: www.journals.elsevier.com/toxicology-letters

A pharmacologically pre-contracted smooth muscle bowel model for the

study of highly-potent opioid receptor agonists and antagonists
Niko Amend *, Horst Thiermann, Franz Worek, Timo Wille
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany

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

Editor : Dr. Angela Mally Isolated organ models are a versatile tool for pharmacological and toxicological research. Small bowel has been
used to assess the inhibition of smooth muscle contraction by opioids. In the present study, we set out to establish
Keywords: a pharmacologically stimulated rat bowel model. The effects of carfentanil, remifentanil and the new synthetic
Opioids opioid U-48800 and their respective antagonists naloxone, nalmefene and naltrexone were studied in a small
Isolated organ
bowel model in rats. The IC50 values of the tested opioids were as follows: carfentanil (IC50 = 0.02 µmol/L, CI
Dose response relationship
0.02–0.03 µmol/L) ≫ remifentanil (IC50 = 0.51 µmol/L, CI 0.40–0.66 µmol/L) ≫ U-48800 (IC50 = 1.36 µmol/L,
CI 1.20–1.54 µmol/L). The administration of the opioid receptor antagonists naloxone, naltrexone and nalmefene
led to progressive, parallel rightward shifts of the dose-response curves. Naltrexone was most potent in antag­
onizing the effects of U-48800, whereas naltrexone and nalmefene were most effective in antagonizing the effects
of carfentanil. In summary, the current model seems to be a robust tool to study opioid effects in a small bowel
model without the necessity of using electrical stimulation.

1. Introduction opioid overdose is naloxone as a μ-, δ- and κ-competitive opioid receptor

Highly-potent synthetic opioids show an increasing prevalence in the
illicit drug market, challenging health care providers worldwide due to
frequent overdosing (Misailidi et al., 2018). In fact, fentanyl and its
synthetic analogs have contributed to a significant number of deaths in
North America. Pain relievers like oxycodone, available legally by pre­
scription are oftentimes, if abused, the first step towards addiction (Lee
et al., 2016; Shanks and Behonick, 2017; Allibe et al., 2019; Krausz et al.,
2021). The current COVID-19 pandemic has further deteriorated the
opioid crisis in North America (Volkow, 2020). The selective µ-receptor
agonists carfentanil and remifentanil deserve thorough consideration
due to their extremely high potency and their use in veterinary and
human medicine, respectively (Scott and Perry, 2005; George et al.,
2010). These two compounds were allegedly used in the 2002 Dubrovka
theatre siege in Moskow, causing the death of 125 hostages (Riches
et al., 2012). The exact lethal dose of carfentanil in humans is unclear,
but it was shown in animal experiments that its potency was approxi­
mately 100-fold higher than the respective potency of fentanyl (Misailidi
et al., 2018). Hence, carfentanil might even exceed the toxicity of the
nerve agent VX, having an estimated LD50 (VX) of 5 mg/human (Her­
rera, 1991; Amend et al., 2020). The ”gold standard” for the treatment of
antagonist (Jordan, 2000; Skolnick, 2018, 2021). However, there is
evidence that naloxone alone might not be a sufficient treatment in the
era of highly-potent synthetic opioids (Krieter et al., 2019; Skolnick,
2021). Naltrexone or nalmefene are additional treatment options, worth
further investigation (Misailidi et al., 2018; Krieter et al., 2019).
Ex-vivo models to study concentration-response relationships that
rely on the inhibitory effects of opioids on gastrointestinal muscle ac­
tivity are isolated small bowel models (Gray et al., 2005; Bian et al.,
2015; Hussain et al., 2016). They can be a helpful tool in basic research
on opioids (Luca et al., 1994; Yuan et al., 1995; Hustveit, 1996) and
might help to reduce animal trials in the sense of the 3R principle
(Russell and Burch, 1959). These tissue bath models use electrically or
pharmacologically pre-contracted small bowel segments which are
exposed to opioid receptor agonists and antagonists (Hustveit, 1996;
Gray et al., 2005; Hussain et al., 2016). After exposure,
concentration-dependent changes to isometric contraction can be
measured (Jespersen et al., 2015). Previous models to investigate opioid
receptors relied for the most part on transmural (coaxial) stimulation
(Luca and Coupar, 1996). There is evidence that the experimental results
are highly dependent on the species and the type of electrical stimula­
tion (Luca and Coupar, 1996). A study by Luca et al. (1994) showed that

* Corresponding author.
E-mail address: nikoamend@bundeswehr.org (N. Amend).

https://doi.org/10.1016/j.toxlet.2023.05.010
Received 18 July 2022; Received in revised form 24 February 2023; Accepted 22 May 2023
Available online 26 May 2023
0378-4274/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
N. Amend et al.
high concentrations of morphine which caused a maximum inhibition of
contractions of the transmurally stimulated guinea pig ileum at 0.1 Hz
had no effect at pulse frequencies of 1 Hz or above. Hence, a model that
offers a more robust experimental setting and does not depend on
electric stimulation might be of interest. The current study set out to
establish a pharmacologically pre-contracted smooth muscle bowel
model. There is evidence that acetylcholine, as excitatory transmitter in
the enteric nervous system might be suitable for this purpose (Erhirhie
et al., 2018; Marquart et al., 2019). Since guinea pig bowel that has been
used in various studies, was not suitable for the current study due to a
lack of response upon cholinergic stimulation, the selection of a different
species was necessary. Previous results showed that acetylcholine,
respectively its analog carbamoylcholine (carbachol) induced a stable
contraction in rat ileum by cholinergic stimulation (Neumaier et al.,
2016; Marquart et al., 2019). In fact, after cholinergic stimulation,
smooth muscle tissue shows an initial fast rising component (phasic
contraction), due to intracellular calcium mobilization. This is followed
by a stable tonic (sustained) contraction which is caused by extracellular
calcium influx into the smooth muscle (Sabeur, 1996). Opioids inhibit
acetylcholine release from enteric interneurons and motor neurons,
alleviating the respective tonic contraction and allowing the study of
dose-response relationships in the current tissue bath study (Sobczak
et al., 2014; Galligan and Sternini, 2017). Even though in a clinical
setting, the cholinergic and opioid toxidrome are difficult to differen­
tiate due to similar signs and symptoms (Ciottone, 2018), the current
small bowel model is suitable to assess agonist and antagonist effects due
to the abovementioned different pathways leading to contraction or
inhibition of the smooth muscle.
2. Material and methods
2.1. Chemicals
Carbamoylcholine chloride (carbachol) (≥ 98 %), naloxone hydro­
chloride (≥ 98 %), naltrexone hydrochloride (≥ 98 %) and nalmefene
hydrochloride (≥ 98 %) were supplied by Sigma-Aldrich (Taufkirchen,
Germany). NaCl, NaHCO3, glucose, KCl, CaCl2, MgCl2, NaH2PO4 were
purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Ke­
tamine was purchased from Zoetis Deutschland GmbH (Berlin, Ger­
many) and xylazine from Selectavet Dr. Otto Fischer GmbH (Weyarn-
Holzolling, Germany). Remifentanil (pharmaceutical-grade) was ob­
tained from Fresenius Kabi AG (Bad Homburg vor der Höhe, Germany).
Carfentanil (≥ 98 %) and U-48800 (≥ 98 %) were provided by LGC
(Wesel, Germany).
2.2. Animals
In accordance with previous studies (Königer et al., 2013; Neumaier
et al., 2016; Marquart et al., 2018), male Wistar rats (Charles River,
Sulzfeld, Germany), weighing 250–300 g, were held in small groups
with a 12/12 h light-dark cycle. The standard laboratory diet and water
were provided. Male Dunkin-Hartley guinea pigs (350–400 g) were
supplied by Charles River and also kept under the abovementioned
standard conditions. A sex-bias, using male animals only might not be
ruled out, but studies using electrical stimulation did not indicate major
differences between male and female animals (in an electrically stimu­
lated model) (Gray et al., 2005). All experiments were in accordance
with the German Animal Welfare Act of 18th May 2006 (BGB1, I S. 1206,
1313) and the European Parliament and Council Directive of 22nd
September 2010 (2010/63/EU).
2.3. Experimental protocol
Male Wistar rats: the experiments were performed as described pre­
viously (Neumaier et al., 2016). In brief, 11 cm of the aboral part of the
jejunum were removed and cut into 10 pieces of about 1 cm each and the
42
Toxicology Letters 382 (2023) 41–46
first centimeter was discharged. The small bowel specimens were placed
into organ baths (Glasatelier Schimmer, Waging am See, Germany)
containing 15 ml gassed (95 % O2 and 5 % CO2) Tyrode buffer (NaCl
137.0 mmol/L, NaHCO3, 22.0 mmol/L, glucose 5.5 mmol/L, KCl 2.68
mmol/L, CaCl2 1.8 mmol/L, MgCl2 1.05 mmol/L, NaH2PO4 0.42
mmol/L; pH= 7.4; T = 37.0 ◦ C). The tissue stripes were mounted be­
tween a force transducer, connected to an amplifier (HSE PLUGSYS ®
TAMA, type 705/1, Hugo Sachs, March-Hugstetten, Germany). After an
initial equilibration phase of 30 min with a basic tension of 1 g and two
buffer changes, the baseline (basic contraction) was evaluated for 2 min
and X‾baseline (equals the activity in the 2 min interval on the x-axis) was
calculated. Then, the tissue was exposed to 2 µmol/L carbachol as stable
acetylcholine analogue. Following an additional equilibration phase of
7 min, the contraction induced by carbachol was calculated (AUCcarba­
chol). Afterwards, the test compounds were added in a cumulative way in
the presence of carbachol. To study the effects of the opioid receptor
antagonists naloxone, naltrexone and nalmefene, a 10 min
pre-incubation period was added before the administration of opioids.
This pre-exposure method was chosen due to the setup of the current
model. After a maximum relaxation of the smooth muscle by opioids, it
was technically difficult to regain a stable contraction. The respective
dose of the antagonists was 1 µmol/L, which was chosen deliberately
high for model establishment and to address the issue that very high
doses of antagonists might be necessary to counteract highly potent
opioids (Moe et al., 2020).
The relaxing effects on the smooth muscle stripes were recorded for
5 min, including the last 2 min from this interval according to previous
protocols from Marquart et al. (Marquart et al., 2018) to calculate
AUCsubstance.
The effects of the compounds were calculated as follows:
∑
AUCcarbachol = (y - baseline)
∑
AUCsubstance = (y - baseline)
y equals the recorded value and the isometric force was calculated as
% of maximal carbachol stimulation by the equation:
% isometric force = (AUCsubstance/AUCcarbachol) × 100
The IC50 values were calculated by non-linear normalized regression
analysis from semi-logarithmic plots of the test compound concentration
versus % of the isometric force. The constrains were 0 (bottom) and 100
(top).
The data are shown as means plus the corresponding confidence
intervals and standard error of the mean (SEM). The experiments were
conducted with n ≥ 5 segments from the small bowel of at least three
animals per concentration of one compound. A one-way analysis of
variance (ANOVA) with Bonferroni post-test was employed to test for
significant differences (p < 0.05). For data recording and analysis, the
following software was used: HSE Acad W (Hugo Sachs Electronics),
GraphPad Prism 9.4.0 (San Diego, CA, USA) and Microsoft-Excel (Red­
mond, WA, USA).
Guinea pigs: In contrast to rat small bowel, the cholinomimetic drug
carbachol induced only a brief and unstable contraction in both small
and large guinea pig bowel. With the exception of the anatomical dif­
ferences, the experimental protocol for guinea pigs was identical to the
protocol used in rats as described above.
3. Results
3.1. Method adaptation
The administration of 2–8 µmol/L carbachol in guinea pig small and
large bowel did not result in a stable tonic contraction (Fig. 2C). How­
ever, 2 µmol/L carbachol induced a stable contraction in rat ileum.
Therefore, subsequent experiments were performed with this species
N. Amend et al.
and 2 µmol/L carbachol.
3.2. Comparison of the smooth muscle relaxing effects of the tested opioid
receptor agonists and antagonists
The smooth muscle relaxing effects after stimulation with 2 µmol/L
carbachol were evaluated and allowed the determination of dose-
response curves (Fig. 1). The opioid receptor antagonists naloxone,
naltrexone and nalmefene induced progressive, parallel rightward shifts
of the respective U-48800, remifentanil and carfentanil dose-response
curves.
The IC50 values (concentration which showed 50 % of the maximal
relaxing effect) and the corresponding confidence intervals were
calculated for each opioid and for the respective antagonists (Table 1).
To highlight the effects of the respective antidotes, the IC50 ratio (IC50
agonist combined with antagonist/IC50 agonist) was calculated
(Table 2).
Fig. 1. Decrease of muscle force by the highly-potent opioids U-48800 (A),
remifentanil (B) and carfentanil (C) and the respective effects of the opioid
receptor antagonists naloxone, naltrexone and nalmefene. Data are shown as
muscle contraction (% of control) (mean ± SEM) after pre-treatment with
2 µmol/L carbachol (n ≥ 5 segments of rat small intestine (ileum) of at least
three animals per concentration).
43
Toxicology Letters 382 (2023) 41–46
4. Discussion
Isolated small bowel models that rely on pre-contracted smooth
muscle are considered as a versatile tool to study the effects of opioid
receptor agonists and their respective antagonists (Hustveit, 1996;
Mundey et al., 2000; Gray et al., 2005; Erhirhie et al., 2018). Due to
plethora of opioid receptors in the myenteric plexus,
concentration-dependent changes to isometric muscle contraction can
be measured after the administration of opioids and their respective
antagonists (Jespersen et al., 2015; Hussain et al., 2016). Cholinergic
stimulation induces calcium mobilization. After an initial fast rising
component (phasic contraction), a stable contraction (sustained) can be
measured (Sabeur, 1996). Opioids inhibit the respective acetylcholine
release by various mechanisms. Among them is the inhibition of Ca2+
channel function (Galligan and Akbarali, 2014; Sobczak et al., 2014;
Galligan and Sternini, 2017). The electrical field stimulation of isolated
ileum is one of the established models (Luca and Coupar, 1996). How­
ever, as shown in the studies of Luca et al. and Gray et al., these models
might lack interinstitutional comparability due to different stimulation
protocols and varying hardware manufacturers of the respective electric
field stimulation equipment (Luca et al., 1994; Gray et al., 2005). Hence,
the current study set out to establish a pharmacologically pre-contracted
small bowel model for the study of opioid receptor agonists and antag­
onists. Fig. 2 illustrates the respective effects of carbachol, highly potent
opioids and the respective opioid receptor antagonists. Besides, the lack
of effect of carbachol on guinea pig bowl is highlighted.
The most frequently used species for studies using transmural stim­
ulation are guinea pigs (Hustveit, 1996; Gray et al., 2005). However, due
to species differences and in contrast to rats, Blackwood et al. indicated
that guinea pigs might not be suitable for a pharmacological stimulation
with carbachol and that even high carbachol concentrations could not
induce a stable contraction (Blackwood and Bolton, 1993). This is in line
with the current study as doses of 8 µmol/L carbachol were not able to
generate the required stable tonic contraction in guinea pigs (Fig. 2C). In
contrast, it was possible to show that 2 µmol/L carbachol induced a
stable tonic contraction in rat ileum. In accordance with previous
studies, only male animals were used (Königer et al., 2013; Neumaier
et al., 2016; Marquart et al., 2018). Even though a sex-bias cannot be
ruled out, previous data did not indicate sex differences (Gray et al.,
2005). The exposure to the respective antagonist was prior to opioid
exposure. This approach does not mimic the clinical exposure, which is
of course after the intoxication. It was chosen due to model specifica­
tions in this area of research. Most studies used this approach, since a
binding of the respective agonist would no longer allow restoration of
contractile tension (Luca et al., 1994; Yuan et al., 1995; Hustveit, 1996;
Gray et al., 2005). The opioids carfentanil and remifentanil were
examined in the current study due to their high potency as underlined by
the Dubrovka theatre siege in Moskow resulting in 125 casualties
(Riches et al., 2012). In Fig. 1, dose-response curves are shown for the
opioids and their respective antagonists naloxone, naltrexone and nal­
mefene. There is evidence that naloxone might lack efficiency in car­
fentanil poisoning (Tuet et al., 2019; Moe et al., 2020). In the current
study, the combination of carfentanil and naloxone was not significantly
different from carfentanil alone, whereas naltrexone and nalmefene
were able to significantly reduce carfentanil effects. The comparability
of the determined IC50 values with existing data is difficult due to the
abovementioned challenges regarding species, model and the general
lack of data for highly potent opioids in smooth muscle models. To the
best of our knowledge, there is no data available on carfentanil, remi­
fentanil and U48800 that would allow a comparison of IC50 values.
The analgetic profile of opioids is mainly determined by affinity to
and efficacy at the receptor (Drewes et al., 2013). The affinity of car­
fentanil to the μ-opioid receptor in radioligand competitive binding as­
says is estimated at 0.19 ± 0.01 nmol/L and the affinity of remifentanil
is estimated at 0.60 ± 0.08 nmol/L (Lipiński et al., 2019). Hence, car­
fentanil and remifentanil effects are largely determined by effects at the
N. Amend et al. Toxicology Letters 382 (2023) 41–46

Table 1
Smooth muscle relaxing effects of the tested highly-potent opioids and the effects of the respective opioid receptor antagonists. The IC50 is given as mean and the
corresponding confidence interval (CI) is depicted (µmol/L; n ≥ 5 segments of at least three animals per concentration; * indicates significance vs. the respective
agonist; * p ≤ 0.03; ** p ≤ 0.002; *** p ≤ 0.0002; **** p ≤ 0.0001).
Structure IC50 CI Naloxone Naltrexone Nalmefene
(µmol/L) IC50 (µmol/L) IC50 (µmol/L) IC50 (µmol/L)
CI (µmol/L) CI (µmol/L) CI (µmol/L)

U-48800 1.36 1.20–1.54 4.76* 10.06* 2.72

3.05–7.42 4.76–23.40 1.96–3.72

Remifentanil 0.51 0.40–0.66 1.33 12.55**** 54.06****

0.82–2.15 10.01–15.63 19.72–148.20

Carfentanil 0.02 0.02–0.03 0.04 0.28*** 0.90***

0.03–0.06 0.08–0.99 0.10–8.43

1000 fold, depending on the design of the experiment and the species
Table 2
(Gray et al., 2005). In future studies, it might be helpful to characterize
The IC50 Ratio (IC50 of the antagonist and the respective antagonist / IC50
the respective effects of fentanyl, since the abovementioned studies use
Agonist) of the tested opioids and the respective antidotes (n ≥ 5 segments of at
this compound to compare potencies. Due to these diverse studies and
least three animals per concentration).
the respective heterogeneous results, it was tempting to use a pharma­
Naloxone Naltrexone Nalmefene
cologically stimulated model which might help standardizing the
IC50-Ratio IC50-Ratio IC50-Ratio
experimental setup. The opioid receptor agonist U-48800 is a
U-48800 3.50 7.40 2.00 non-fentanyl derived new synthetic opioid (Solimini et al., 2018). This
Remifentanil 2.61 24.61 106.00
Carfentanil 1.67 11.67 37.50
class of substances comprises compounds that were designed for recre­
ational use, to circumvent law enforcement, but have no therapeutic use
(Solimini et al., 2018). There have been only few studies regarding the
receptor and not by their affinity (Lipiński et al., 2019). It is generally pharmacological and toxicological properties of U-48800 (Gampfer
estimated that carfentanil shows 30–100 higher potency than fentanyl et al., 2019; Fogarty et al., 2022). The current study indicates that this
(in rats) (Wilde et al., 2019), and remifentanil is approximately equi­ substance is less potent than remifentanil. Besides, the opioid receptor
potent to fentanyl (in humans) (Scott and Perry, 2005). In the current rat antagonists naltrexone and nalmefene were effective in antagonizing the
model, carfentanil is 24-fold more potent than remifentanil. In fact, the opioid effects of U-48800 in the present model (Table 1). The antago­
inhibitory potencies of opioids in a small bowel model might vary up to nists caused progressive, parallel rightward shifts of the U-48800

Fig. 2. Original representative registration of the effects of carbachol on rat (A; B) and guinea pig bowel (C) in the current model. The inhibitory effects of remi­
fentanil (A) and the ability of naloxone to antagonize these effects (2 B) are shown. Besides, the lack of effect of carbachol on guinea pig bowel (C) is highlighted. The
x-axis shows the tension in Newton and the y-axis the time in minutes.

44
N. Amend et al.
dose-response curve. This is crucial for the treatment of overdoses, since
an effective antidote dose depends on the opioid to be displaced from the
µ-receptor (Boyer, 2012). The highly potent opioid remifentanil was
more potent than U-48800. There is abundant clinical data concerning
the suitability of remifentanil for general anesthesia and the reversibility
of its respiratory depressive effects by naloxone (Amin et al., 1995;
Battershill and Keating, 2006). In the current study, naloxone had no
significant effects against remifentanil which might have been due to the
above-mentioned differences in species. Naltrexone was most efficient in
antagonizing the effects of U-48800, whereas naltrexone and nalmefene
were both efficient in antagonizing the respective effects of carfentanil.
As mentioned in the introduction, the dose of the antagonists was
1 µmol/L, which was chosen deliberately high for model establishment.
Besides, very high doses might be necessary in a clinical context to
counteract highly potent opioids (Moe et al., 2020).
To highlight the effects of the respective antidotes, the IC50 ratios
were obtained. A higher ratio indicates a higher potency of the respec­
tive antagonist in the current model (Table 2). Nalmefene with a ratio of
106 was the most effective antidote for remifentanil and showed the
highest ratio of all antagonists.
In conclusion, the current model appears to be a suitable method to
study opioid agonist/antagonist relationships in rat small bowel. One
major benefit of this pharmacologically stimulated model might be the
generic approach concerning stimulation, allowing a possible stan­
dardization and interinstitutional comparability.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Prof. Dr. Franz Worek is a member of the editorial board of Toxicology
Letters.
Data Availability
Data will be made available on request.
Acknowledgements
The authors are grateful to Katharina Kettner, Julia Schürger and
Jasmin Rauch for expert technical assistance.
Conflict of interest statement
The authors declare that there are no conflicts of interest. The study
was funded by the German Ministry of Defence. However, the design,
performance, data interpretation and manuscript writing were under the
control of the authors and has not been influenced by the German
Ministry of Defence.
References
Allibe, N., Fouilhe Sam-Lai, N., Willeman, T., Jourdil, J.-F., Bartoli, M., Mallaret, M.,
Nemoz, B., Stanke-Labesque, F., Eysseric-Guerin, H., 2019. Norcarfentanil:
carfentanil misuse or remifentanil treatment? Forensic Toxicol. 37 (2), 488–495,
10.1007/s11419-019-00481-2.
Amend, N., Niessen, K.V., Seeger, T., Wille, T., Worek, F., Thiermann, H., 2020.
Diagnostics and treatment of nerve agent poisoning-current status and future
developments. Ann. N. Y. Acad. Sci. 1479 (1), 13–28. https://doi.org/10.1111/
nyas.14336.
Amin, H.M., Sopchak, A.M., Esposito, B.F., Henson, L.G., Batenhorst, R.L., Fox, A.W.,
Camporesi, E.M., 1995. Naloxone-induced and spontaneous reversal of depressed
ventilatory responses to hypoxia during and after continuous infusion of remifentanil
or alfentanil. J. Pharmacol. Exp. Ther. 274 (1), 34–39.
Battershill, A.J., Keating, G.M., 2006. Remifentanil: a review of its analgesic and sedative
use in the intensive care unit. Drugs 66 (3), 365–385. https://doi.org/10.2165/
00003495-200666030-00013.
Bian, X., Zhou, R., Yang, Y., Li, P., Hang, Y., Hu, Y., Yang, L., Wen, D., 2015. Divergent
effect of dezocine, morphine and sufentanil on intestinal motor function in rats. Int.
J. Med. Sci. 12 (11), 848–852. https://doi.org/10.7150/ijms.12616.
45
Toxicology Letters 382 (2023) 41–46
Blackwood, A.M., Bolton, T.B., 1993. Mechanism of carbachol-evoked contractions of
guinea-pig ileal smooth muscle close to freezing point. Br. J. Pharmacol. 109 (4),
1029–1037. https://doi.org/10.1111/j.1476-5381.1993.tb13725.x.
Boyer, E.W., 2012. Management of opioid analgesic overdose. N. Engl. J. Med. 367 (2),
146–155. https://doi.org/10.1056/NEJMra1202561.
Ciottone, G.R., 2018. Toxidrome recognition in chemical-weapons attacks. N. Engl. J.
Med. 378 (17), 1611–1620. https://doi.org/10.1056/NEJMra1705224.
Drewes, A.M., Jensen, R.D., Nielsen, L.M., Droney, J., Christrup, L.L., Arendt-Nielsen, L.,
Riley, J., Dahan, A., 2013. Differences between opioids: pharmacological,
experimental, clinical and economical perspectives. Br. J. Clin. Pharmacol. 75 (1),
60–78. https://doi.org/10.1111/j.1365-2125.2012.04317.x.
Erhirhie, E., Emudainohwo, T., Igboeme, S., Ajaghaku, D., Ujam, T., Okezie, U.,
Ilodigwe, E., 2018. Preclinical screening techniques for antidiarrheal drugs: a
comprehensive review. A J. Physiol. Biochem. Pharmacol. 7 (2), 61. https://doi.org/
10.5455/ajpbp.20180329014330.
Fogarty, M.F., Mohr, A.L.A., Papsun, D.M., Logan, B.K., 2022. Analysis of the illicit
opioid U-48800 and related compounds by LC-MS-MS and case series of fatalities
involving U-48800. J. Anal. Toxicol. 46 (1), 17–24. https://doi.org/10.1093/jat/
bkaa180.
Galligan, J.J., Akbarali, H.I., 2014. Molecular physiology of enteric opioid receptors. Am.
J. Gastroenterol. Suppl. (Print.) 2 (1), 17–21. https://doi.org/10.1038/
ajgsup.2014.5.
Galligan, J.J., Sternini, C., 2017. Insights into the role of opioid receptors in the GI tract:
experimental evidence and therapeutic relevance. Handb. Exp. Pharmacol. 239,
363–378. https://doi.org/10.1007/164_2016_116.
Gampfer, T.M., Richter, L.H.J., Schäper, J., Wagmann, L., Meyer, M.R., 2019.
Toxicokinetics and analytical toxicology of the abused opioid U-48800 - in vitro
metabolism, metabolic stability, isozyme mapping, and plasma protein binding.
Drug Test. Anal. 11 (10), 1572–1580. https://doi.org/10.1002/dta.2683.
George, A.V., Lu, J.J., Pisano, M.V., Metz, J., Erickson, T.B., 2010. Carfentanil–an ultra
potent opioid. Am. J. Emerg. Med. 28 (4), 530–532. https://doi.org/10.1016/j.
ajem.2010.03.003.
Gray, A.C., White, P.J., Coupar, I.M., 2005. Characterisation of opioid receptors involved
in modulating circular and longitudinal muscle contraction in the rat ileum. Br. J.
Pharmacol. 144 (5), 687–694. https://doi.org/10.1038/sj.bjp.0706107.
Herrera, J., 1991. The reproductive biology of a riparian Mediterranean shrub, Nerium
oleander L. (Apocynaceae). Bot. J. Linn. Soc. 106 (2), 147–172. https://doi.org/
10.1111/j.1095-8339.1991.tb02289.x.
Hussain, Z., Rhee, K.W., Lee, Y.J., Park, H., 2016. The effect of DA-9701 in opioid-
induced bowel dysfunction of guinea pig. J. Neurogastroenterol. Motil. 22 (3),
529–538. https://doi.org/10.5056/jnm15194.
Hustveit, O., 1996. Interaction between opioid and muscarinic receptors in the guinea-
pig ileum preparation // Interaction between opioid and muscarinic receptors in the
guinea-pig ileum preparation: a mathematical model: a mathematical model.
Pharmacol. Toxicol. 78 (3), 167–173. https://doi.org/10.1111/j.1600-0773.1996.
tb00199.x.
Jespersen, B., Tykocki, N.R., Watts, S.W., Cobbett, P.J., 2015. Measurement of smooth
muscle function in the isolated tissue bath-applications to pharmacology research.
J. Vis. Exp.: JoVE 95, 52324. https://doi.org/10.3791/52324.
Jordan, B., 2000. Opioids and their complicated receptor complexes.
Neuropsychopharmacology 23 (4), S5–S18. https://doi.org/10.1016/S0893-133X
(00)00143-3.
Königer, C., Worek, F., Thiermann, H., Wille, T., 2013. Effect of MB327 and oximes on
rat intestinal smooth muscle function. Chem. -Biol. Interact. 204 (1), 1–5. https://
doi.org/10.1016/j.cbi.2013.04.004.
Krausz, R.M., Westenberg, J.N., Ziafat, K., 2021. The opioid overdose crisis as a global
health challenge. Curr. Opin. Psychiatry 34 (4), 405–412. https://doi.org/10.1097/
YCO.0000000000000712.
Krieter, P., Gyaw, S., Crystal, R., Skolnick, P., 2019. Fighting fire with fire: development
of intranasal nalmefene to treat synthetic opioid overdose. J. Pharmacol. Exp. Ther.
371 (2), 409–415. https://doi.org/10.1124/jpet.118.256115.
Lee, D., Chronister, C.W., Broussard, W.A., Utley-Bobak, S.R., Schultz, D.L., Vega, R.S.,
Goldberger, B.A., 2016. Illicit fentanyl-related fatalities in florida: toxicological
findings. J. Anal. Toxicol. 40 (8), 588–594. https://doi.org/10.1093/jat/bkw087.
Lipiński, P.F.J., Kosson, P., Matalińska, J., Roszkowski, P., Czarnocki, Z., Jarończyk, M.,
Misicka, A., Dobrowolski, J.C., Sadlej, J., 2019. Fentanyl family at the Mu-opioid
receptor: uniform assessment of binding and computational analysis. Molecules 24
(4). https://doi.org/10.3390/molecules24040740.
Luca, A. de, Coupar, I.M., 1996. Insights into opioid action in the intestinal tract.
Pharmacol. Ther. 69 (2), 103–115. https://doi.org/10.1016/0163-7258(95)02053-
5.
Luca, A., de, Korszniak, N.V., Coupar, I.M., 1994. Opiate agonist and antagonist action on
guinea-pig isolated ileum. Gen. Pharmacol. 25 (1), 79–83. https://doi.org/10.1016/
0306-3623(94)90013-2.
Marquart, K., Prokopchuk, O., Wilhelm, D., Worek, F., Thiermann, H., Martignoni, M.E.,
Wille, T., 2019. Human small bowel as model for poisoning with organophosphorus
compounds. Toxicol. Vitr.: Int. J. Publ. Assoc. BIBRA 57, 76–80. https://doi.org/
10.1016/j.tiv.2019.02.010.
Marquart, K., Prokopchuk, O., Worek, F., Thiermann, H., Martignoni, M.E., Wille, T.,
2018. Human small bowel as a useful tool to investigate smooth muscle effects of
potential therapeutics in organophosphate poisoning. Toxicol. Lett. 293, 235–240.
https://doi.org/10.1016/j.toxlet.2017.11.012.
Misailidi, N., Papoutsis, I., Nikolaou, P., Dona, A., Spiliopoulou, C., Athanaselis, S., 2018.
Fentanyls continue to replace heroin in the drug arena: the cases of ocfentanil and
carfentanil. Forensic Toxicol. 36 (1), 12–32. https://doi.org/10.1007/s11419-017-
0379-4.
N. Amend et al.
Moe, J., Godwin, J., Purssell, R., O’Sullivan, F., Hau, J.P., Purssell, E., Curran, J., Doyle-
Waters, M.M., Brasher, P.M.A., Buxton, J.A., Hohl, C.M., 2020. Naloxone dosing in
the era of ultra-potent opioid overdoses: a systematic review. CJEM 1–9. https://doi.
org/10.1017/cem.2019.471.
Mundey, M.K., Ali, A., Mason, R., Wilson, V.G., 2000. Pharmacological examination of
contractile responses of the guinea-pig isolated ileum produced by mu-opioid
receptor antagonists in the presence of, and following exposure to, morphine. Br. J.
Pharmacol. 131 (5), 893–902. https://doi.org/10.1038/sj.bjp.0703659.
Neumaier, K., Worek, F., Thiermann, H., Wille, T., 2016. Bispyridinium non-oximes: an
evaluation of cardiac effects in isolated hearts and smooth muscle relaxing effects in
jejunum. Toxicol. Vitr.: Int. J. Publ. Assoc. BIBRA 35, 11–16. https://doi.org/
10.1016/j.tiv.2016.05.005.
Riches, J.R., Read, R.W., Black, R.M., Cooper, N.J., Timperley, C.M., 2012. Analysis of
clothing and urine from Moscow theatre siege casualties reveals carfentanil and
remifentanil use. J. Anal. Toxicol. 36 (9), 647–656. https://doi.org/10.1093/jat/
bks078.
Russell, W., Burch, 1959. The principles of humane experimental technique.
Sabeur, G., 1996. Effect of temperature on the contractile response of isolated rat small
intestine to acetylcholine and KCl: calcium dependence. Arch. Physiol. Biochem. 104
(2), 220–228. https://doi.org/10.1076/apab.104.2.220.12891.
Scott, L.J., Perry, C.M., 2005. Remifentanil: a review of its use during the induction and
maintenance of general anaesthesia. Drugs 65 (13), 1793–1823. https://doi.org/
10.2165/00003495-200565130-00007.
Shanks, K.G., Behonick, G.S., 2017. Detection of carfentanil by LC-MS-MS and reports of
associated fatalities in the USA. J. Anal. Toxicol. 41 (6), 466–472. https://doi.org/
10.1093/jat/bkx042.
46
Toxicology Letters 382 (2023) 41–46
Skolnick, P., 2018. On the front lines of the opioid epidemic: rescue by naloxone. Eur. J.
Pharmacol. 835, 147–153. https://doi.org/10.1016/j.ejphar.2018.08.004.
Skolnick, P., 2021. Treatment of overdose in the synthetic opioid era. Pharmacol. Ther.,
108019 https://doi.org/10.1016/j.pharmthera.2021.108019.
Sobczak, M., Sałaga, M., Storr, M.A., Fichna, J., 2014. Physiology, signaling, and
pharmacology of opioid receptors and their ligands in the gastrointestinal tract:
current concepts and future perspectives. J. Gastroenterol. 49 (1), 24–45, 10.1007/
s00535-013-0753-x.
Solimini, R., Pichini, S., Pacifici, R., Busardò, F.P., Giorgetti, R., 2018.
Pharmacotoxicology of non-fentanyl derived new synthetic opioids. Front.
Pharmacol. 9, 654. https://doi.org/10.3389/fphar.2018.00654.
Tuet, W.Y., Pierce, S.A., Racine, M.C., Tressler, J., McCranor, B.J., Sciuto, A.M.,
Wong, B., 2019. Changes in murine respiratory dynamics induced by aerosolized
carfentanil inhalation: efficacy of naloxone and naltrexone. Toxicol. Lett. 316,
127–135. https://doi.org/10.1016/j.toxlet.2019.09.012.
Volkow, N.D., 2020. Collision of the COVID-19 and addiction epidemics. Ann. Intern.
Med. 173 (1), 61–62. https://doi.org/10.7326/M20-1212.
Wilde, M., Pichini, S., Pacifici, R., Tagliabracci, A., Busardò, F.P., Auwärter, V.,
Solimini, R., 2019. Metabolic pathways and potencies of new fentanyl analogs.
Front. Pharmacol. 10, 238. https://doi.org/10.3389/fphar.2019.00238.
Yuan, C.S., Foss, J.F., Moss, J., 1995. Effects of methylnaltrexone on morphine-induced
inhibition of contraction in isolated guinea-pig ileum and human intestine. Eur. J.
Pharmacol. 276 (1–2), 107–111. https://doi.org/10.1016/0014-2999(95)00018-g.