1
Cutaneous analgesia after subcutaneous injection of memantine
and amantadine and their systemic toxicity in rats
Yu-Wen Chena,b, Ja-Ping Shiehc, Yu-Chung Chend, Yuk-Man Leunge, Ching-Hsia
Hungf,*, Jhi-Joung Wangb
a
Department of Physical Therapy, China Medical University, Taichung, Taiwan
b
Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan
c
Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan
d
Division of Physical Therapy, Department of Physical Medicine and Rehabilitation,
Cheng Hsin General Hospital, Taipei, Taiwan
e
Graduate Institute of Neural and Cognitive Sciences, China Medical University,
Taichung, Taiwan
f
Institute & Department of Physical Therapy, National Cheng Kung University,
Tainan, Taiwan
Conflicts of interest: There is no conflict of interests for all authors.
*Corresponding Author:
Ching-Hsia Hung, Ph.D.
Associate Professor
National Cheng Kung University
Department of Physical Therapy
No.1 Ta-Hsueh Road, Tainan, Taiwan
Phone: 886-6-2353535 ext 5939
FAX: 886-6-2370411
Email: chhung@mail.ncku.edu.tw
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ABSTRACT
The purpose of the study is to find subcutaneous equianalgesic doses of memantine,
amantadine and bupivacaine and use these doses to quantify the cardiovascular and
central nervous system toxicity of these agents after intravenous administration.
Memantine, amantadine and bupivacaine, a local anesthetic, in a dose-related fashion
were determined for cutaneous analgesia by a block of the cutaneous trunci muscle
reflex in rats, and equipotent doses were calculated. Following rapid intravenous
infusion of equianalgesic bupivacaine, memantine, amantadine and saline (vehicle) in
rats, we observed the onset time of seizure, apnea and impending death, and
monitored mean arterial blood pressure and heart rate. Memantine and amantadine
elicited dose-dependent cutaneous analgesia. At the 50% effective dose (ED50), the
rank of potencies was bupivacaine [1.8 (1.7 – 2.0)] > memantine [19.1 (17.6 – 21.8)]
> amantadine [36.1 (32.0 – 40.3)] (P < 0.05). On ED25, ED50 and ED75 basis, the
duration caused by bupivacaine was similar to that caused by memantine or
amantadine. At equianalgesic doses, the infusion time of memantine or amantadine
required to induce seizure, impending death, and apnea was longer than that of
bupivacaine during rapid intravenous infusion (P < 0.01). The decreasing slope in
mean arterial blood pressure and heart rate was slower with memantine and
amantadine when compared with bupivacaine at equivalent doses (P < 0.01). Our data
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showed that memantine and amantadine (i) were equal to bupivacaine at producing
durations of cutaneous analgesia but (ii) were less likely than bupivacaine to cause
cardiovascular and central nervous system toxicity.
Key Words: memantine; amantadine; bupivacaine; cutaneous analgesia;
cardiovascular toxicity; central nervous system toxicity
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1. Introduction
Memantine, which is the derivative of amantadine (1-adamantanamine), is the
first in a novel class of Alzheimer's disease agents whose therapeutic utility is linked
to their ability to inhibit N-methyl-D-aspartate glutamate receptors (Otton et al., 2011;
Yeh et al., 2010). Amantadine is a drug that has approval for use both as an antiviral
and an antiparkinsonian drug by US Food & Drug Administration. Due to memantine
reversibly inhibited tetrodotoxin-resistant sodium currents (Brau et al., 2001),
memantine have a local anesthetic effect as infiltrative cutaneous analgesia in rats
(Chen et al., 2011c). However, cutaneous (local) analgesia following subcutaneous
injection of amantadine was not reported.
The German chemist, Albert Niemann, was successful in isolating an alkaloid
from the coca leaf and named it ‘cocaine’ in 1860 (Dillane and Finucane, 2010).
Despite those somewhat physical or chemical differences, they both cause
cardiovascular and central nervous system toxicity (Dillane and Finucane, 2010). For
instance, bupivacaine induces significant cardiovascular toxicity (Albright, 1979;
Khan et al., 2002). After clinical applications of the local anesthetics for more than
150 years, it is the right time to search a brand new local anesthetic whose chemical
structure totally differed from traditional local anesthetics.
Before considering the clinical use of memantine and amantadine as local
5
anesthetics, the systemic toxicity of these drugs should be examined. In this study,
memantine and amantadine were investigated in rats with two main objectives: (i) to
compare cutaneous analgesia of memantine and amantadine with that of bupivacaine
to obtain the equianalgesic doses and (ii) to investigate their cardiovascular and
central nervous system toxicity by rapid infusing equipotent doses of memantine,
amantadine and bupivacaine. Bupivacaine, a known and long-lasting local anesthetic,
was used as control.
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2. Materials and methods
2.1.Animals
Male Sprague-Dawley rats, 7–9 wk of age, weighting 285-335g, were purchased
from the National Laboratory Animal Centre (Taipei, Taiwan), and then housed in a
climate controlled room maintained at 21 ℃ with approximately 50% relative
humidity in the Animal Center of China Medical University (Taichung, Taiwan). The
lights were on a 12-h light/dark cycle (light on at 6:00 AM), with food and water
available ad libitum up to time of experiments. The Institutional Animal Care and Use
Committee of China Medical University (Taichung, Taiwan) approved this protocol
for animal experimentation.
2.2.Drugs
Memantine HCl, amantadine HCl, bupivacaine HCl and midazolam HCl were
obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All drugs were
dissolved in 0.9% NaCl (saline) before the subcutaneous and intraperitoneal injections
or intravenous infusions.
2.3.Experimental designs
The experimental protocol was divided into two parts. In Part 1, cutaneous
analgesia of memantine (50.6, 35.0, 26.7, 20.0, 13.3, 6.7, 3.5 μmol/kg), amantadine
(120.0, 100.0, 80.0, 40.0, 27.0, 20.0, 13.0 μmol/kg), bupivacaine (9.0, 5.3, 4.0, 2.0,
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1.3, 1.0, 0.7 μmol/kg) and saline was performed (n = 8 rats for each dose of each drug)
to obtain the equivalent potency 95% effective dose (ED95) of drugs; meanwhile, the
full recovery time (duration) of bupivacaine, memantine and amantadine was
evaluated at equianalgesic doses (ED25, ED50 and ED75) (n = 8 for each dose of each
drug). After these above experiments, one control group was added to exclude the
possibility of systemic analgesia of drugs. Animals (n = 8 rats for each dose of each
drug) received intraperitoneal injections of testing drugs (bupivacaine, memantine and
amantadine) at a dose of 2× ED95.
In Part 2, time to cause systemic toxicity (seizure, apnea, and cardiac arrest),
mean arterial blood pressure and heart rate were monitored after the equipotent dose
(ED95) of drugs (bupivacaine, memantine or amantadine) was infused into the rats (n
= 8 rats for each dose of each drug). Saline group (n = 8 rats) was used as control.
2.4.Part 1 - Skin Infiltrative analgesia
2.4.1. Subcutaneous Injections
An experienced investigator, who was blinded to the identity of the injected
drugs, was responsible for handling of animals and assessing the cutaneous analgesic
effect. Infiltrative analgesia of the skin was evaluated via the cutaneous trunci muscle
reflex (CTMR), characterized by the reflex movement of the skin over the back
produced by twitches of the lateral thoracospinal muscle in response to local dorsal
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cutaneous stimulation after subcutaneous injections of drugs (Chen et al., 2008; Hung
et al., 2010). In brief, the hair on the rat's dorsal surface of the thoracolumbar region
(10 cm × 6 cm) was mechanically shaved on the day before drug injection, and then
each drug solved in 0.6 ml saline was injected subcutaneously using a 30-gauge
needle into a naïve area of the shaved back of the un-anesthetized rats. Subcutaneous
injection produced a circular skin wheal, approximately 2 cm in diameter. Then the
wheal was marked with ink within 1 minute after subcutaneous injection. The cut end
of an 18-gauge needle was affixed to a von Frey filament (no. 15; Somedic Sales AB,
Stockholm, Sweden) to elicit a standardized noxious mechanical stimulus (191 g)
without making tissue damage (acute sharp pain) (Chen et al., 2011a; Chen et al.,
2007). In order to decrease the numbers of experimental animals used, the back of the
rat was further divided into right and left parts, either of which, after a washout period
of 1 week, received one drug injection.
2.4.2. Neurobehavioral Evaluation
After observing the animal’s normal reaction to stimuli applied outside the wheal
and on the contralateral side, we applied 6 stimuli at 6 different points within each
wheal, with a frequency of 0.5-1 Hz, and scored the number to which the rat failed to
react. Each drug’s cutaneous analgesia was quantitatively evaluated as the number of
times the stimuli failed to elicit a response and was described as the percent of
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possible effect (% PE). For instance, the absence of any response after 6 stimuli was
defined as complete nociceptive/sensory block (100% PE), which was calculated as
follows:
% PE =100%  ((number of stimuli that provoked no response)/6)
During the duration of drug action, the maximum value of % PE was presented
as percent of maximal possible effect (% MPE). Stimulus testing was applied 5 min
before injection of drugs to confirm normal responses of rats, then every 5 min after
injection for the first 30 min and every 10-15 min thereafter, until the CTMR fully
recovered from the block (no more than 150 min). Each duration (full recovery time)
of drug action was defined as the time from drug injection (i.e., time=0) to full
recovery of CTMR (no analgesic effect or 0% MPE) (Chen et al., 2011c; Hung et al.,
2012).
2.4.5. The effective doses (EDs)
Rats were subcutaneously injected with 7 different doses of each drug (n = 8 rats
for each dose of each drug), and dose-response curves were constructed from the %
MPE of each dose of each drug. The curves were then fitted via SAS nonlinear (NLIN)
procedures (version 9.1, SAS Institute, Cary, NC), and this value of ED50, defined as
the dose that caused 50% cutaneous analgesia, was obtained (Chen et al., 2008; Leung
et al., 2010). The ED25, ED75 and ED95 of drugs were obtained via using the same
10
curve-fitting (SAS NLIN procedures) that was used to derive the ED50 (Chen et al.,
2011b; Hung et al., 2011).
2.5. Part 2 - Neurological and cardiovascular effects
The rats were anesthetized with pentobarbital sodium at the dose of 50 mg/kg
intraperitoneally and the right femoral artery and vein were cannulated with
polyethylene catheters (PE-50), which were filled with heparin saline (35 U/mL). The
free end of the catheter was threaded through an 18-gauge needle and then tunneled
subcutaneously. The catheter was cut with 5 cm protruding from the skin at the
midline in the posterior cervical area and sealed via heating it with a match and
compressing it with a hemostat (Chen et al., 2011d; Hung et al., 2010).
On Day 2, the rats were sedated with midazolam 0.375 mg/kg (1 mg/ml,
midazolam HCl) intraperitoneally. Then, animals were placed in a small cage with an
open top to allow the lines to reach the animal and prevent the animal from chewing
on the lines. After 10 min, the tube in the right femoral vein was connected to an
infusion pump (Harvard Model 22 Infusion Pump, Harvard Apparatus Inc., Holliston,
MA) for delivery of the drugs. The tube in femoral artery was connected to a
transducer, and mean arterial blood pressure and heart rate were recorded using a
polygraph (MP36, BIOPAC Systems Inc, Goleta, CA, USA) (Hung et al., 2012;
Srinivasa et al., 2003). The investigator (YWC) was blinded to the drugs under the
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experiment. After rapid intravenous infusions of either 1) bupivacaine at 8
μmol/kg/min, memantine at 34 μmol/kg/min, or amantadine at 112 μmol/kg/min or 2)
normal saline at a rate of 400 μL/kg/min, the onset time of seizure, respiratory arrest
(apnea), time to cause impending death, mean arterial blood pressure and heart rate
were recorded. The onset time of seizures was defined as the time when the first
convulsion occurred and respiratory arrest when apnea occurred for 15 seconds by
observation of chest movement. The time to impending death was defined as the time
it took for the heart rate decreased to 0 per minute (Chen et al., 2011d; Hung et al.,
2010).
2.6. Statistical Analysis
Data are presented as mean ± S.E.M. or ED25, ED50, and ED75 values with 95%
confidence interval (95% CI). The differences in potencies (ED50; Table 1), baseline
data, %MPE (Fig. 2), and the time to cause toxicity (Fig. 4) between medications
were evaluated using 1-way analysis of variance (ANOVA) and then the pairwise
Tukey's honestly significant difference (HSD) test. The differences in duration (Fig. 3)
among drugs were evaluated by 2-way ANOVA followed by pairwise Tukey's HSD
test. Analysis of variance with repeated measures followed by Duncan’s
multiple-range test was used for post hoc multiple comparisons of means on mean
arterial blood pressure and heart rate (Fig. 5). SPSS for Windows (version 17.0) was
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used for all statistical analyses. Statistical significance was set at P < 0.05.
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3. Results
3.1.Part 1 - Cutaneous analgesia
The memantine and amantadine, as well as long-acting local anesthetic
bupivacaine elicited dose-dependent cutaneous analgesia in rats (Fig. 1). The ED50s of
these drugs are shown in Table 1. On the ED50 basis, the relative potency of these
drugs was found to be bupivacaine > memantine > amantadine (P<0.05; Fig. 1 and
Table 1). At maximum testing dose of 9 μmol/kg for bupivacaine, 51 μmol/kg for
memantine and 120 μmol/kg for amantadine, all these drugs caused 100%
nociceptive/sensory blockade with durations of actions of 120±10, 96±11 and 109±8
min, respectively (Fig. 2). At equianalgesic doses (ED25, ED50 and ED75), the block
duration caused by memantine or amantadine was similar to that caused by
bupivacaine as infiltrative cutaneous analgesic (Fig. 3). Also, neither the
subcutaneous injections of saline (Fig. 2) nor the intraperitoneal injections of testing
drugs (data not shown) produced cutaneous analgesia. All rats recovered completely
after subcutaneous injections of drugs.
3.2.Part 2 – Systemic toxicity
Before experiments, we confirmed those baseline data of body weight, mean
arterial blood pressure and heart rate demonstrated no significant differences among
these four groups (data not shown). At equianalgesic doses (ED95), the infusion time
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of memantidine and amantidine required to induce seizure, impending death and
apnea (respiratory arrest) was longer than that of bupivacaine (Fig. 4; P<0.01). The
rats in all drug groups eventually succumbed to seizure, apnea and asystole
(impending death). There were no systemic toxicities in any of the animals during the
infusion period in the saline group. In all drug groups, the mean arterial blood
pressure and heart rate showed a tendency to decrease before cardiovascular collapse
(Fig. 5). The declines in mean arterial blood pressure and heart rate were slower in the
amantadine (P<0.01) or memantine (P<0.01) group when compared with the
bupivacaine group (Fig. 5). The rapidity of decline of the mean arterial blood pressure
and heart rate occurred in the following order: bupivacaine > amantadine >
memantine (Fig. 5).
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4. Discussion
We found subcutaneous dosages of memantine and amantadine which produced
similar durations of skin analgesia as bupivacaine, while continuous intravenous
administration of memantine and amantdine appeared to lead to the later onset of
serious toxic cardiovascular and central nervous system effects.
Local anesthetics block voltage-gated Na+ channels (Borgeat and Aguirre, 2010).
Because memantine shows the blockade of tetrodotoxin-resistant sodium currents at
2-Hz stimulation in small dorsal root ganglion neurons (Brau et al., 2001), it has a
local anesthetic effect (Chen et al., 2011c). Additionally, the inhibition of Na+
channels is a well precedented mechanism used in therapy epilepsy as well as other
hyperexcitability disorders (Large et al., 2011). Furthermore, memantine and
amantadine in the case of a systemic administration are capable of blocking the
N-methyl-D-aspartate receptors and producing the anticonvulsant effects (Gmiro and
Serdiuk, 2000). Although the pharmacological mechanisms of amantadine are largely
unclear in this study, inhibiting the voltage-dependent Na+ channel may be one of the
principle mechanisms of amantadine to display a local anesthetic effect, which is
worth studied in the future.
Infiltrative analgesia of skin is an acceptable option for management of surgical
analgesia and postoperative pain, because it is relatively free of side effects (Khan et
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al., 2002). Currently, bupivacaine and lidocaine are commonly used in this field
(McLure and Rubin, 2005). It has been known that subcutaneous memantine
displayed more potent than lidocaine (Chen et al., 2010), but less potent than
bupivacaine as cutaneous analgesia. Besides, we showed for the first time that
amantadine produced dose-dependent cutaneous analgesia in rats. Furthermore, on an
equianalgesic basis (ED25, ED50 and ED75), the sensory block duration of memantine
or amantadine was similar to that long-acting local anesthetic bupivacaine. These
findings suggest that there may be a potential for the use of memantine and
amantadine as long-action local anesthetics in the clinical setting, provided that the
cardiovascular and central nervous system toxicity is investigated.
The local anesthetics through accidental intravenous injection may induce
cardiovascular and central nervous system toxicity and even result in death (Dony et
al., 2000; Srinivasa et al., 2003). Using a local anesthetic animal model, we examined
the local anesthetic effects of memantine, amantadine and bupivacaine as infiltrative
cutaneous analgesic to estimate the equianalgesic doses of these drugs. Alternatively,
we selected the animal model with the spontaneously breathing rats for a clinical
scenario when local analgesia is performed on humans. Nevertheless, this model has
its own limitations. First, the concomitant administration of midazolam may have
influenced the threshold doses of toxic manifestations (e.g., seizure activity). Second,
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in contrast with bolus injection, the slow infusion rate allows the administration of the
large dose of local anesthetics before the toxic symptoms arise. On an equipotent
basis, we demonstrated that infusion of memantine and amantadine displayed a
delayed onset of cardiovascular and central nervous system toxicity when compared
with bupivacaine. However, the grades of toxicities were the same once toxicity
occurred (Fig. 4). Furthermore, we also noticed that the declines in mean arterial
blood pressure and heart rate were longer in the memantine and amantadine groups
when compared with bupivacaine group. Overall, our results suggest that memantine
and amantadine are less “toxic” and may feature a safer systemic toxicity profile than
bupivacaine following continuous intravenous administration.
Ropivacaine has been shown to have less cardiac depression than bupivacaine,
although recent investigations reported that ropivacaine is at least 40% less potent
than bupivacaine (McDonald et al., 1999; Polley et al., 1999). Ohmura et al. clearly
(Ohmura et al., 2001) demonstrated that, at equivalent doses, the cumulative doses of
local anesthetic required to cause asystole was 2.7-folds larger in ropivacaine (108 ±
27 mg/ kg) group than in the bupivacaine group (40 ± 9 mg/kg). By contrast, we
revealed that the infusion time of memantidine (20.3 ± 6.4 min) and amantidine (12.1
± 2.9 min) required to induce impending death (asystole) was 5.8- and 3.5-folds
greater than that of bupivacaine (3.5 ± 0.1 min) at equianalgesic doses, respectively.
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In addition, there is also an antidote for local anesthetic toxicity, namely Intralipid,
that improves their safety (Espinet and Emmerton, 2009; Weinberg et al., 2008).
Toxicities arising from other effects of these agents (N-methyl-D-aspartate block,
anticholinergic, noradrenergic, dopaminergic, serotonergic) may be more likely to
occur but are difficult to assess in this model.
We observed that the molecular structures of memantine and amantadine also
contain hydrophilic and hydrophobic groups and do not resemble that of bupivacaine,
which holds a benzene ring. Furthermore, the observations that memantine and
amantadine have long-acting local anesthetic properties and possibly produce their
toxic effects later than bupivacaine may be clinically relevant.
We did not evaluate whether memantine and amantadine induce local toxicity in
the injected tissues, however, it is noteworthy that in this study we detected no
apparent side effects after subcutaneous injection. All rats recovered completely. Our
data suggest that memantine and amantadine may have features that make them as a
valuable alternative cutaneous analgesic or anesthetic, although further studies on
sciatic or spinal nerve block and the possibility of nerve (tissue) damage from drug
injection are important issues that need further investigation.
The previous experiment reported memantine elicits local analgesia against
cutaneous nociceptive stimuli, is more potent than lidocaine, and has a longer duration
19
of action (Chen et al., 2011c). This preclinical study reported that (i) amantadine and
memantine produce skin infiltrative analgesia in dose-related manners and their block
durations are similar to that of long-lasting local anesthetic bupivacaine. And (ii)
intravenous equianalgesic doses of memantine and amantadine are better tolerated to
induce central nervous system and cardiovascular system toxicity than bupivacaine.
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Acknowledgements
The authors gratefully acknowledge the financial support provided for this study
by the National Science Council (NSC 100-2314-B-039-017-MY3) of Taiwan.
21
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Table 1. The 50% effective dose (ED50), ED25 and ED75 of memantine, amantadine and bupivacaine for infiltrative cutaneous analgesia in rats.
Drug
ED25 ( 95% CI )
ED50 ( 95% CI )
ED75 ( 95% CI )
ED95
Bupivacaine
1.2 (1.1 – 1.4) ab
1.8 (1.7 – 2.0) ab
3.0 (2.9 – 3.2) ab
8
Memantine
9.1 (7.7 – 12.6) a
19.1 (17.6 – 21.8) a
29.5 (27.9 – 32.5) a
34
Amantadine
25.0 (23.1 – 27.9)
36.1 (32.0 – 40.3)
62.4 (59.5 – 67.7)
112
The ED50 of drugs (μmol/kg) were obtained from Fig. 1. CI = confidence interval. Symbols (a,b) indicated P < 0.05 when drugs was compared
with amantadine and memantine, respectively, using a one-way ANOVA and pairwise Tukey’s HSD test for paired comparisons.
%MPE (maximal possible effect)
27
100
80
60
40
20
Bupivacaine
Memantine
Amantadine
0
1
10
100
Dose ( mol/kg )
Fig. 1.
28
Bupivacaine (9 mol/kg)
Amantadine (120 mol/kg)
Memantine (51 mol/kg)
Saline
%PE (possible effect)
100
80
60
40
20
0
0
15
30
45
60
75
90 105 120 135 150
Time (min)
Fig. 2.
Full recovery time ( min )
29
Bupivacaine (B)
Memantine (M)
Amantadine (A)
50
40
30
20
10
B=M=A
0
25
50
75
ED ( effective dose )
Fig. 3.
Time to cause toxicity (min)
30
25
Seizure
Apnea
Impending death
**
20
**
15
* *
10
5
0
ND ND ND
line caine tadine antine
a
S
a
piv Aman Mem
u
B
Fig. 4.
Heart rate (bpm)
Mean arterial blood pressure (mmHg)
31
160
120
Saline
Memantine
Amantadine
Bupivacaine
80
40
0
0
5
10
15
20
*
25
30
35
40
*
Time (min)
400
300
200
100
0
0
5
10
15
20
*
25
30
35
*
40
Time (min)
Fig. 5.
32
Figure Legends
Fig. 1. The dose—response curves of bupivacaine, memantine and amantadine as
infiltrative cutaneous analgesia in rats (n = 8 at each testing point). Data are shown as
mean±S.E.M.
Fig. 2. Time courses of bupivacaine (9 μmol/kg), amantadine (120 μmol/kg),
memantine (51 μmol/kg) and saline (vehicle) as infiltrative cutaneous analgesia in rats.
Data are presented as mean±S.E.M.; each group, n=8.
Fig. 3. Full recovery time (duration) of cutaneous analgesia of bupivacaine,
memantine and amantadine at ED25, ED50 and ED75 (n = 8 at each testing point). Data
are mean±S.E.M. The difference in duration was evaluated via using two-way
ANOVA followed by pairwise Tukey's HSD test.
Fig. 4. Time to cause toxicity with equipotent (ED95) bupivacaine (8 μmol/kg/min),
memantine (34 μmol/kg/min) or amantadine (112 μmol/kg/min) at the onset of seizure,
respiratory arrest (apnea) and time to cause impending death. Saline group was not
detected (ND) the toxicity symptoms. The symbol (*) indicates P < 0.01 when
memantine or amantadine compared with bupivacaine. Data are presented as
meanS.E.M.; each group, n=8; 95% effective dose, ED95.
Fig. 5. Mean arterial blood pressure and heart rate change during infusion of either 1)
bupivacaine at 8 μmol/kg/min, memantine at 34 μmol/kg/min, or amantadine at 112
33
μmol/kg/min or 2) normal saline in the volume of 400 μL/kg/min (the same volume
given to the animals in the drug group) as infusion; 0 min is the start of infusion.
Infusion was stopped when the time to cause impending death was reached. The
symbol (*) indicates P < 0.01 for memantine or amantadine compared with
bupivacaine. Data are presented as meanS.E.M.; each testing group contained eight
rats.