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Article history:
Received 15 May 2014
Received in revised form
21 May 2014
Accepted 23 May 2014
Available online 2 June 2014
Keywords:
Nalmefene
Carfentanil
Loss of righting reex
Respiratory depression
Chemical compounds studied in this article:
Nalmefene (PubChem CID: 5284594)
Carfentanil (PubChem CID: 62156)
1. Introduction
Carfentanil is an analog of the synthetic opioid analgesic fentanyl.
It is one of the most potent opioids, with a quantitative potency
10,000-times that of morphine and 100-times that of fentanyl (Lust
et al., 2011). Initially, carfentanil was intended for use only as a
tranquilizing agent to rapidly incapacitate large animals for examinations and procedures by veterinarians (Ramsay et al., 1995; Shaw
et al., 1995). Nowadays, carfentanil is also used for in vivo positron
emission tomography studies of mu-opioid receptors in the laboratory and clinic (Hagelberg et al., 2012; Ly et al., 2013).
In recent years, there have been reports of humans developing
symptoms of drug toxicity induced by carfentanil (George et al.,
2010). As with other opioid analgesics, carfentanil can produce loss
of righting reex (LORR) and even induce respiratory depression
(recognized as one of the most serious adverse events) (Moresco
http://dx.doi.org/10.1016/j.ejphar.2014.05.044
0014-2999/& 2014 Elsevier B.V. All rights reserved.
154
To characterize the effects of nalmefene against carfentanilinduced respiratory depression of arterial blood gases, rats were
injected with carfentanil (20 g/kg, i.v.), and then, 5-min later,
given different doses of nalmefene (9.4150.0 g/kg, i.m.) or
naloxone (150.0 g/kg, i.m.). At 0, 5, 15, and 30 min after injection
with carfentanil, arterial blood samples (1.0 ml) were drawn from
the abdominal aorta with a self-lling polypropylene syringe
containing 60 IU of dry, electrolyte-balanced heparin (PICO70;
Radiometer Medical; Copenhagen, Denmark). At the point time of
0 min, rats were anesthetized by ether before acquiring arterial
blood samples. Air pockets were expelled immediately after
sampling. Thereafter, samples were mixed gently until testing on
an ABL90 Flex Blood Gas Analyzer (Radiometer Medical). The
partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2) as
well as oxygen saturation (SO2) in arterial blood were recorded
and analyzed at the corresponding time point. Five animals per
time point were used for measurement of arterial blood gases.
2.1. Animals
For LORR and measurement of arterial blood gases, data are the
mean 7S.E.M. They were analyzed by two-way ANOVA followed
by Dunnett's t-test for multiple comparisons. The Student's t-test
was used if two independent groups were compared. P o0.05 was
considered signicant.
For determination of respiratory parameters, data are the
mean 7S.E.M. The value from stabilized rats before carfentanil
injection was considered to be the baseline. Analysis of two-way
ANOVA was used to compare differences, followed by Bonferroni
post-tests for comparisons between individual groups. P o0.05
was considered signicant for all tests.
3. Results
3.1. Nalmefene signicantly reduced the carfentanil-induced
duration of LORR
2.3. LORR
To study the effect of nalmefene on carfentanil-induced LORR,
rats were injected with carfentanil (10 g/kg, i.v.) according the
result of our preliminary experiment. Five minutes later, rats were
injected with varying doses of nalmefene (9.4150.0 g/kg, i.m.) or
naloxone (150.0 g/kg, i.m.). The duration of LORR was recorded
individually for each rat. It was calculated by subtracting the time
of onset of LORR from the time at recovery from carfentanilinduced sleep. The righting reex was deemed to be lost if an
animal did not right itself within 15 s of being placed on its back.
2.4. Determination of respiratory parameters
The dose-dependent effect of nalmefene against carfentanilinduced respiratory depression was studied in freely moving rats.
A Whole-body Plethysmograph (EMKA Technologies, Paris, France)
was used to measure respiratory parameters, i.e., respiratory rate
and enhanced pause (an index of airway obstruction). Rats were
stabilized in the plethysmograph chamber for 3040 min until
100
80
40
*
18.8
37.5
4. Discussion
20
120
60
0
Saline
9.4
75.0
40
% of saline control
30
20
10
0
0.
15
.0
75
37
.5
0
9.
4
18
.8
155
to determine its efcacy in countering respiratory depression. Carfentanil (20 g/kg) injection caused marked suppression of respiratory
rate (470% of baseline) and induced a striking increase in enhanced
pause (4300% of baseline) (Fig. 2). Specically, 10 min after administration of carfentanil, the respiratory rate decreased from
124.4714.1 bursts/min to 34.375.3 bursts/min (n5) and enhanced
pause increased from 0.370.1 to 1.170.1 (n5). Respiratory depression persisted for 1 h. Treatment with nalmefene (9.4150.0 g/kg,
i.m.) dose-dependently reversed the respiratory depression induced
by carfentanil. The respiratory rate after 60 min of nalmefene
(150.0 g/kg, i.m.) treatment increased from 34.375.3 bursts/min
(n 5) to 117.8718.9 bursts/min (n 5) and enhanced pause
decreased from 1.170.1 to 0.470.1 (n5), and these values were
close to those of normal rats. On the other hand, in rats treatment with
naloxone (150.0 g/kg, i.m.), the respiratory rate increased from
36.4711.3 bursts/min (n5) to 75.0711.1 bursts/min (n5) after
60 min of treatment, while, enhanced pause decreased from
1.370.1 to 0.770.1 (n5) after 30 min of treatment, followed by
an increase from 0.770.1 to 1.170.3 (n5) during the period of
3060 min treatment, and these values were close to those of rats
with carfentanil injection.
3.3. Nalmefene manifestly resumed arterial blood gas parameters
We studied the doseresponse effect of nalmefene treatment
upon changes in the parameters of arterial blood gases in carfentanilinjected rats. Injection of carfentanil caused a signicant reduction in
PaO2 (39.2% of 0 min) and SaO2 (25.4% of 0 min) and induced an
obvious increase in PaCO2 (252.8% of 0 min) after 5 min (Table 1).
Treatment with nalmefene (9.4150.0 g/kg, i.m.) dose-dependently
156
200
150
100
50
0
-20 -10
10
20
30
40
50
60
70
80
3.0
Carfentanil (20.0g/kg)
Nalmefene (150.0g/kg)
2.5
Nalmefene (75.0g/kg)
2.0
Nalmefene (37.5g/kg)
1.5
Nalmefene (18.8g/kg)
1.0
Nalmefene (9.4g/kg)
Naloxone (150.0g/kg)
0.5
##
0.0
-20 -10
10
20
30
40
50
60
70
80
157
Table 1
Treatment with nalmefene returned the changes in the parameters of arterial blood gases in carfentanil-injected rats to almost normal levels (drug doses are in g/kg).
PaO2 (mmHg)
0 min
5 min
15 min
30 min
Carfentanil 20.0
Naloxone 150.0
Nalmefene 9.4
18.8
37.5
75.0
150.0
94.9 73.60
94.2 74.39
96.0 73.20
94.3 73.81
94.4 73.94
94.17 3.62
95.9 73.40
37.2 7 14.86 a
49.3 715.95 a
36.17 16.93 a
35.47 16.52 a
43.8 7 2.00 a
35.77 16.73 a
35.07 16.14 a
31.4 7 18.23 a
72.6 7 5.81 b,c
60.5 73.75 b,c
78.4 77.09 b,c
88.17 2.84 b,c
98.2 712.34 b,c
97.4 7 14.40 b,c
48.47 5.60 a
83.2 7 5.07 b,c
69.7 7 7.06 b,c
72.5 7 12.73 b,c
94.0 7 2.41 b,c
95.9 7 8.37 b,c
97.8 710.46 b,c
PaCO2 (mmHg)
Carfentanil 20.0
Naloxone 150.0
Nalmefene 9.4
18.8
37.5
75.0
150.0
36.2 74.00
38.7 76.45
35.87 4.46
36.4 74.60
36.7 74.49
37.5 7 3.21
34.9 72.90
91.5 7 7.11 a
89.5 7 10.63 a
92.7 7 7.56 a
91.9 7 8.14 a
88.3 7 1.38 a
92.3 7 7.94 a
92.17 8.03 a
89.17 16.12 a
57.0 7 5.40 b,c
62.17 6.31 b,c
51.0 75.97 b,c
45.2 78.38 b,c
36.6 75.65 b,c
37.4 7 4.58 b,c
87.1 711.56 a
47.3 7 3.60 b,c
61.8 7 8.14 b,c
52.6 7 9.48 b,c
46.7 7 5.82 b,c
42.0 7 3.93 b,c
38.2 7 6.64 b,c
SaO2 (%)
Carfentanil 20.0
Naloxone 150.0
Nalmefene 9.4
18.8
37.5
75.0
150.0
93.7 71.33
93.2 72.35
94.17 1.22
93.7 71.54
93.4 71.21
93.5 71.34
94.17 1.31
23.8 7 10.20 a
20.8 7 4.28 a
23.3 7 11.70 a
23.0 7 11.60 a
28.2 7 3.28 a
23.2 7 11.65 a
21.5 7 10.20 a
21.2 7 11.97 a
58.3 78.60 b,c
60.6 74.92 b,c
78.2 73.59 b,c
87.8 7 4.60 b,c
90.4 73.93 b,c
91.8 7 3.68 b,c
33.478.76 a
72.8 7 18.54 b,c
68.77 14.66 b,c
81.1 77.94 b,c
90.4 7 2.89 b,c
92.4 7 2.23 b,c
93.3 7 3.36 b,c
Note: rats were injected with carfentanil (20 g/kg, i.v.) and then with nalmefene (9.4150.0 g/kg, i.m.) or naloxone (150.0 g/kg) 5-min later. At 0, 5, 15 and 30 min after
injection with carfentanil, the PaO2, PaCO2 and SaO2 of arterial blood were recorded and assessed.
a
b
c
P o0.05 relative to 0 min of the corresponding group (before carfentanil injection, n 5 per time point).
Po 0.05 relative to 5 min of the corresponding group (5 min after carfentanil injection and nalmefene or naloxone treatment, n 5, per time point).
Po 0.05 relative to the carfentanil-injection group (n 5, per time point).
Acknowledgment
Funding for this study was provided by National Science and
Technology Major Projects for Major New Drugs Innovation and
Development (2013ZX09J13104-01A) in China.
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