1
Subcutaneous L-tyrosine elicits cutaneous analgesia in response
to local skin pinprick in rats
Ching-Hsia Hung1,2, Chong-Chi Chiu3,4, Kuo-Sheng Liu5, Yu-Wen Chen6,7,*,
Jhi-Joung Wang7
1
Department of Physical Therapy, College of Medicine, National Cheng Kung
University, Tainan, Taiwan
2
Institute of Allied Health Sciences, College of Medicine, National Cheng Kung
University, Tainan, Taiwan
3
Department of General Surgery, Chi Mei Medical Center, Tainan and
Liouying, Taiwan
4
Department of Electrical Engineering, Southern Taiwan University of Science and
Technology, Tainan, Taiwan
5
Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan,
Taiwan
6
Department of Physical Therapy, College of Health Care, China Medical University,
Taichung, Taiwan
7
Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan
Conflicts of interest: There is no conflict of interests for all authors.
*Corresponding author:
Yu-Wen Chen, Ph.D.
Department of Physical Therapy,
College of Health Care,
China Medical University,
No.91 Hsueh-Shih Road, Taichung 40402, Taiwan
Tel: 886-4-22053366 ext 7313
Fax: 886-4-22065051
E-mail: cywhwok@mail.cmu.edu.tw
2
ABSTRACT
The purpose of the study was to estimate the ability of L-tyrosine to induce cutaneous
analgesia and to investigate the interaction between L-tyrosine and the local
anesthetic lidocaine. After subcutaneously injecting the rats with L-tyrosine and
lidocaine in a dose-dependent manner, cutaneous analgesia (by blocking the
cutaneous trunci muscle reflex - CTMR) was evaluated in response to the local
pinprick. The drug-drug interaction was analyzed by using an isobolographic method.
We showed that both L-tyrosine and lidocaine produced dose-dependent cutaneous
analgesia. On the 50% effective dose (ED50) basis, the rank of drug potency was
lidocaine (5.09 [4.88－5.38] μmol) > L-tyrosine (39.1 [36.5－41.8] μmol) (P<0.05).
At the equipotent doses (ED25, ED50, ED75), the duration of cutaneous analgesia
caused by L-tyrosine lasted longer than that caused by lidocaine (P<0.01). Lidocaine
co-administered with L-tyrosine exhibited an additive effect on infiltrative cutaneous
analgesia. Our pre-clinical study demonstrated that L-tyrosine elicits the
local/cutaneous analgesia, and the interaction between L-tyrosine and lidocaine is
additive. L-tyrosine has a lower potency but much greater duration of cutaneous
analgesia than lidocaine. Adding L-tyrosine to lidocaine preparations showed greater
duration of cutaneous analgesia compared with lidocaine alone.
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Keywords: L-tyrosine; lidocaine; cutaneous analgesia; isobologram
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1. Introduction
Tyrosine is transported into nerve terminals for the synthesis of catecholamines,
such as epinephrine and norepinephrine (Palmiter, 2011; Rasmussen et al., 1983).
Recently, epinephrine (Chen et al., 2008) and norepinephrine (Shieh et al., 2009) have
been shown to produce a local anesthetic effect as infiltrative cutaneous analgesia in
rats. Because tyrosine is a precursor to epinephrine (Palmiter, 2011), we presume that
tyrosine is likely to produce the cutaneous analgesic effect.
Skin local anesthetic infiltration is routinely used during laparoscopic surgery
(Carbonell et al., 2003) and for postoperative pain relief following inguinal hernia
(Suraseranivongse et al., 2003) because it is relatively free of side effects (Khan et al.,
2002). However, the technique is limited by the shortened duration of analgesia or
anesthesia (Cameron and Cross, 1985). Fixed combinations of two drugs are
frequently utilized in anesthetic practice with a view of enhancing their effects or
reducing their adverse effects. For example, the eutectic mixture of 2.5% lidocaine
and 2.5% prilocaine (5% EMLA) is the first commercially-developed topical
transdermal anesthetic that provides a fast and lasting local anesthetic effect (Shaikh
et al., 2009).
It has been well-established that the local anesthetic lidocaine as the
most widely used agent is characterized by a rapid onset of action and intermediate
5
duration of efficacy (Fozzard et al., 2005; McLure and Rubin, 2005). Additionally,
isobolographic analysis may provide a basis for examining if the biological response
produced by the mixture of two drugs is greater, equal or lesser than that of drugs
administered alone (Tallarida et al., 1989). Using an isobologram method, we
demonstrated that the local anesthetic bupivacaine combined with epinephrine
produced a synergistic action in intensifying cutaneous analgesia in rats (Chen et al.,
2008).
Nevertheless, the cutaneous analgesic effect of L-tyrosine administered alone or
in combination with lidocaine is unknown. Based on the studies, we (1) queried
whether subcutaneous L-tyrosine produced cutaneous analgesia and (2) evaluated
using isobolographic analysis, whether L-tyrosine as an adjuvant to lidocaine
produced an antagonist, synergistic, or additive cutaneous analgesia.
common local anesthetic, was used as a control drug.
Lidocaine, a
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2. Materials and methods
2.1. Animals
Male Sprague-Dawley rats (202 to 252 g) were obtained from National Cheng
Kung University (Tainan, Taiwan) and were kept in the animal housing facilities at
the same university, with controlled humidity (approximately 50% relative humidity),
a 12 h on/12 h off light/dark cycle (6:00 AM to 6:00 PM), and room temperature
(22C). The investigative procedures were approved by the Institutional Animal Care
and Use Committee of National Cheng Kung University and were followed based on
the recommendations and policies of the International Association for the Study of
Pain (IASP).
2.2. Drugs
L-tyrosine disodium salt hydrate and lidocaine HCl monohydrate were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Before the subcutaneous
injection, all experimental drugs were weighed and solved into normal saline (0.9%
NaCl) freshly.
2.3. Experimental Designs
Five studies were carried out (n = 8 for each group). First, cutaneous analgesia of
L-tyrosine (90, 60, 45, 30, 15 µmol) and lidocaine (13.2, 7.5, 4.5, 2.0 µmol) in a
dose-related fashion was evaluated. Second, the %MPE (percent of maximal possible
7
effect), area under the curves (AUCs), and duration of L-tyrosine (90 μmol), lidocaine
(13.2 μmol), and saline (vehicle) on infiltrative cutaneous analgesia were compared.
Third, the duration of cutaneous analgesia produced by L-tyrosine was compared with
that produced by lidocaine at the equipotent doses [50% effective dose (ED50), ED25
and ED75]. Fourth, the effect of lidocaine co-administered with L-tyrosine as an
infiltrative anesthetic was analyzed by isobologram. Then, the %MPE and AUCs of
lidocaine (2xED50) alone, L-tyrosine (2xED50) alone, and the combination of
lidocaine (ED50) and L-tyrosine (ED50) as an infiltrative anesthetic were evaluated.
Lastly, two control groups were performed to exclude the possible systemic effect of
drugs from cutaneous analgesia. One group underwent intraperitoneal injection of
co-administration of lidocaine (ED50) and L-tyrosine (ED50), whereas another group
received intraperitoneal injection of drug (L-tyrosine or lidocaine) at a higher dose of
2ED75.
2.4. Infiltrative Cutaneous Analgesia
For consistency, one trained experimenter, who was blinded to the experimental
groups, was responsible for handling of all behavioral assessments. Before the
subcutaneous injections, animals were handled twice a day up to 5-7 days to reduce
the stress and to improve their performance during the experiment. Cutaneous
analgesia was performed as previously reported (Hung et al., 2014; Tzeng et al., 2014)
8
and was evaluated by the cutaneous trunci muscle reflex (CTMR), characterized by
the reflex movement of the skin over the back produced through twitches of the lateral
thoracospinal muscles in response to local dorsal cutaneous stimulation (Chen et al.,
2014a; Chen et al., 2014c). The duration of drug action was ranked by the period from
drug injection (i.e., time=0) to full recovery of CTMR (0% PE or no analgesic effect)
(Hung et al., 2012; Leung et al., 2013a). The cutaneous analgesic effect of each drug
was calculated quantitatively as the number of times the pinprick failed to produce a
response. For instance, the complete absence of six responses was portrayed as a full
nociceptive/sensory block (100% of possible effect; 100% PE) (Chen et al., 2012c;
Chen et al., 2012d). The maximum block during a time course of cutaneous analgesia
with drugs was described as the %MPE.
2.5. The ED50 and AUCs
After injecting the rats with 4-5 doses of each drug subcutaneously, the
dose-response curves were constructed from the % MPE of each dose of each drug.
The curves were then fitted by using SAS Nonlinear Procedures (version 9.1; SAS
Institute, Cary, NC), and the value of ED50, which defined as the dose that elicited
50% cutaneous analgesia, was obtained (Chen et al., 2012a; Leung et al., 2013b;
Minkin and Kundhal, 1999). The ED25 or ED75 was calculated by using the same
curve-fitting (SAS NLIN Procedures) system, which was used to derive the ED50
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(Chen et al., 2011; Chen et al., 2014b). The values of AUCs of nociceptive/sensory
block were calculated by using the Kinetica version 2.0.1 software (InnaPhase
Corporation, Philadelphia, PA).
2.6. Isobologram
Isobolographic methods (version 1.27, Pharm Tools Pro, McCary Group,
Wynnewood, PA) were used to examine the drug–drug interactions (Chen et al., 2010;
Tallarida et al., 1989). The isobologram is a non-mechanistic method of
characterizing the effect resulting from the administration of equieffective
concentrations of individual drugs (Tallarida et al., 1989).
In order to calculate the
experimental value of the ED50, the dose–response curve of combined drugs at four
equipotent doses (i.e., lidocaine combined with L-tyrosine under a ratio of ED50 vs.
ED50 [ED50+ED50, 1/2ED50+1/2ED50, 1/4ED50+1/4ED50, 1/6ED50+1/6ED50]) was
performed and calculated by using the nonlinear least-squares regression. The ED50 of
the combined drugs was evaluated through the isobologram method described by
Tallarida (Tallarida et al., 1989). In brief, the theoretical additive line was calculated
using the ED50s of L-tyrosine and lidocaine on the Y and X axes, respectively. Then,
the ED50 of combined drugs was plotted against the theoretical additive line. The
difference between the theoretical ED50 value (calculated from the theoretical additive
line by computer simulation) and the experimental ED50 value (calculated from the
10
dose–response curve of combined drugs) was analyzed (Chen et al., 2012b; Leung et
al., 2014).
2.7. Statistical Analysis
The values are shown as mean  S.E.M. or ED50 values with 95% confidence
interval (95% CI) and are analyzed using the Student’s t-test and one-way or two-way
analysis of variance (ANOVA) followed by pairwise Tukey’s honest significance
difference (HSD) test. The statistical software, SPSS for Windows (version 17.0,
SPSS, Inc, Chicago, IL, USA), was used, and the difference between groups was
considered to be significant at a value of 𝑃<0.05.
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3. Results
3.1. The dose-dependent effect of L-tyrosine as an infiltrative anesthetic
L-tyrosine, as well as lidocaine produced dose-dependent effects of cutaneous
analgesia in rats (Fig. 1). The ED25s, ED50s, and ED75s of L-tyrosine and lidocaine are
shown in Table 1. On the equipotent (ED50) basis, the rank of drug potency was
lidocaine greater than L-tyrosine (P<0.05; Table 1).
In Fig. 2, both L-tyrosine (90
μmol) and lidocaine (13.2 μmol) elicited a complete block of sensory/nociception
(100% MPE), whereas the subcutaneous injection of saline (vehicle) did not produce
cutaneous analgesia.
3.2. Duration of cutaneous analgesia produced by L-tyrosine and lidocaine
L-Tyrosine at a dose of 90 µmol approached its peak effect (100% MPE) with a
complete block time about 19 ± 3 min (Fig. 2 and Table 2). Lidocaine
dose-dependently (2.0-13.2 µmol) produced cutaneous analgesia (Fig. 1) and reached
its peak effect (100% MPE) with a complete block time about 22 ± 5 min (Fig. 2 and
Table 2). Furthermore, L-tyrosine displayed greater duration (full recovery time) than
lidocaine (P<0.001; Table 2), and the AUC value of L-tyrosine was greater than that
of lidocaine (P<0.01; Table 2). On an equianesthetic basis (ED25, ED50, ED75), the
nociceptive/sensory block duration elicited by L-tyrosine was longer than that
produced by lidocaine (P<0.01; Fig. 3). Also, neither intraperitoneal injection of
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drugs (L-tyrosine or lidocaine) at 2ED75 nor intraperitoneal injection of the
co-administration of lidocaine (ED50) and L-tyrosine (ED50) produced cutaneous
analgesia (data not shown).
3.3. The isobolographic analysis of the drug-drug interaction
The isobologram method was used to analyze the lidocaine and L-tyrosine
interaction (Fig. 4). The dose–response curve of combined drugs was constructed (Fig.
1) to obtain the experimental value of ED50, while the difference between the
theoretical additive value of ED50 and the experimental value of ED50 was not
significant (Table 3). The co-administration of lidocaine (ED50) with L-tyrosine (ED50)
produced a 96% nociceptive block, whereas lidocaine (2xED50) and L-tyrosine
(2xED50) elicited 98% and 100% nociceptive blocks, respectively (Fig. 5 and Table 2).
The AUC value of co-administration of lidocaine with L-tyrosine (3641 ± 192) or
L-tyrosine alone (4427 ± 301) was greater than that of lidocaine alone (2924 ± 211)
(P<0.05; Fig. 5 and Table 2). All rats recovered completely after subcutaneous
injection of drugs.
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4. Discussion
For the first time, our results demonstrated that L-tyrosine elicited a
dose-dependent local anesthetic effect as infiltrative cutaneous analgesia. It was less
potent than lidocaine, but the duration of sensory block produced by L-tyrosine was
greater than that induced by lidocaine. Furthermore, co-administration of lidocaine
with L-tyrosine produced an additive effect.
Assuredly, the blockade of the voltage-gated Na+ channels is an important
feature of the local anesthetic agents that accounts for the generations of infiltrative
cutaneous analgesia, sciatic nerve blockade, and epidural/spinal anesthesia (Borgeat
and Aguirre, 2010; Fozzard et al., 2005). Because lidocaine is a Na+ channel blocker
(Borgeat and Aguirre, 2010), it produces the sciatic nerve block (Hou et al., 2006),
spinal anesthesia (Hung et al., 2011), and skin infiltration anesthesia (Hung et al.,
2014). Although the pharmacological action (i.e., blocking Na+ currents) of L-tyrosine
in local anesthesia is largely unknown, we revealed that L-tyrosine elicited cutaneous
analgesia in a dosage-dependent fashion.
Furthermore, a possible mechanism by
which L-tyrosine could also elicit its effects, which acts as an agonist at alpha 1- and
alpha 2-adrenergic receptors on peripheral nociceptors.
In addition, chloroprocaine (2-chloroprocaine), a local anesthetic with a very
short half-life, has been found to be a candidate drug which could replace lidocaine in
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some short procedures (Goldblum and Atchabahian, 2013). Chloroprocaine is also a
favorable evolution of spinal anesthesia for ultra-short outpatient procedures
(Camponovo, 2014). Therefore, we compared the duration of L-tyrosine with that of
lidocaine. Although L-tyrosine was less potent than lidocaine in cutaneous analgesia,
it possessed a greater duration of nociceptive/sensory block than lidocaine. The
duration of cutaneous analgesia of L-tyrosine (AUC) was approximately 1.64-folds
greater than that of lidocaine (Table 2). At the equianesthetic doses (ED25, ED50 and
ED75), the block duration caused by L-tyrosine was longer than that caused by
lidocaine (Fig. 3).
Moreover, it has been known that the systemic administration of lidocaine
induced a central anti-nociceptive effect in both acute and chronic pain models
(Muth-Selbach et al., 2009). In order to exclude the possible systemic effect of drugs
from cutaneous analgesia, two control groups were carried out. Thus, our resulting
data showed that neither intraperitoneal injection of lidocaine (ED50) combined with
L-tyrosine (ED50) nor intraperitoneal injection of a large dosage of drugs (L-tyrosine
or lidocaine) produced the cutaneous analgesic effect. These data supported that
cutaneous analgesia of the drug alone or the combination of both drugs was due to
their peripheral action on the skin instead of systemic analgesia.
In our previous experiment, we demonstrated that epinephrine increased the
15
potency of bupivacaine as an infiltrative anesthetic in a rat model of cutaneous trunci
muscle reflex (CTMR) (Chen et al., 2008). Tyrosine is transported into the nerve
terminals for dopamine, epinephrine and norepinephrine syntheses (Palmiter, 2011).
In this case, we showed that its co-administration with lidocaine was found to be
additive along with the combination falling somewhere in the middle on the potency
scale between lidocaine and L-tyrosine alone. Therefore, this concludes that the
combination of the two compounds benefited L-tyrosine more than lidocaine. This
possibly suggests two assumptions: 1). these compounds may work via different
physiological mechanisms that do not potentiate with each other and 2). it is likely
that L-tyrosine does not improve the lidocaine function due to lidocaine's Na+ channel
blocking effect, which consists a superior blocking mechanism of the neural
transmission in the periphery (or L-tyrosine simply has a mechanism of action that
does not depend on Na+ channels).
Additionally, the use of having more than one agent to accomplish the desired
effects has been a frequent, regular method in clinical and pharmacological studies
(Tallarida et al., 1989). For instance, the co-administration of analgesics is commonly
prescribed with a view to enhancing pain relief and decreasing the side effects
(Tallarida et al., 1989). It has been well-established that combining two local
anesthetics (i.e., EMLA) was performed to increase the depth and duration of local
16
anesthesia (Ohzeki et al., 2008; Shaikh et al., 2009). Furthermore, the
lidocaine-and-prilocaine combination was used for regional nerve blockades with the
inhibition of the nerve endings in the superficial layers of the skin (Gupta et al., 2007).
In the present study, the advantage of using a combination of L-tyrosine and lidocaine
is that the combination was found to be longer acting compared to lidocaine alone.
Instead, it has been shown that intrathecal 10% lidocaine (approximately 6.9 mg/kg)
elicited persistent functional impairment and neurotoxicity (Sakura et al., 2005;
Takenami et al., 2005). In order to decrease the side effects, L-tyrosine as an adjuvant
might work as an alternative to prolong the duration of skin infiltration analgesia by
injecting local anesthetics (i.e., lidocaine) rather than increasing extra doses of local
anesthetics.
This study has some limitations. Several local anesthetics may cause both
cardiovascular and central nervous system toxicities because of their similar chemical
structures (Zink and Graf, 2008). We did not know whether L-tyrosine induced
systemic toxicity or neurotoxicity at the injection site, and further experiments on a
local nerve block or related neural and systemic toxicities will be considered.
Moreover, it has been confirmed that the low toxicity of L-tyrosine with an LD50 of
>5000 mg/kg was shown in formal acute dose toxicity studies in rats and mice
following a single intramuscular or subcutaneous injection (Baldrick et al., 2002). The
17
mechanisms other than local anesthetic (L-tyrosine) block of sodium channels,
including several dopamine receptors, could be evaluated in further studies.
In summary, we concluded that L-tyrosine and lidocaine dose-dependently
produced cutaneous analgesia. L-tyrosine was less potent in producing cutaneous
analgesia but possessed a greater duration of action when compared with lidocaine as
an infiltrative anesthetic. The co-administration of L-tyrosine with lidocaine created
an additive effect.
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Acknowledgments
This work was supported by the grants of the Ministry of Science and
Technology (MOST 104-2314-B-039-017-MY3) in Taiwan.
19
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26
TABLE 1. The 50% effective doses (ED50s), ED25s, and ED75s of drugs on infiltrative cutaneous analgesia in rats
Drug
Lidocaine
L-Tyrosine
ED25 ( 95% CI )
2.78 (2.59 – 3.09)
25.0 (22.5 – 28.6)
ED50 ( 95% CI )
5.09 (4.88 – 5.38)
39.1 (36.5 – 41.8)
ED75 ( 95% CI )
7.86 (7.70 – 8.10)
52.4 (50.6 – 54.2)
The EDs of drugs (μmol) were obtained from Fig. 1. CI = confidence interval. The potency of drug (ED50) was lidocaine > L-tyrosine (P<0.05,
for each comparison) by using one-way analysis of variance and pairwise Tukey honestly significant difference test.
27
TABLE 2. The percent of maximal possible effect (%MPE), duration, and area under the curves (AUCs) of L-tyrosine (90 μmol), lidocaine
(13.2 μmol), and saline (vehicle) on infiltrative cutaneous analgesia in rats
Duration (min)
%MPE
AUCs (%MPEmin)
Complete blockade time
Full recovery time
L-Tyrosine
100 ± 0
19 ± 3
109 ± 5b
5938 ± 347a
Lidocaine
100 ± 0
22 ± 5
59 ± 7
3621 ± 469
―
―
―
―
Saline
The values are presented as mean  S.E.M. (n = 8 in each group). The symbols (a, b) indicate P<0.01 and P<0.001, respectively, when
L-tyrosine compared with lidocaine by using the Student’s t-test.
28
TABLE 3. Isobolographic analysis of the drug-drug interaction
Drug combination
L-Tyrosine + Lidocaine
ED50 (95% CI)
Theoretical value
Experimental value
22.14 (12.02–40.79)
15.57 (13.92–17.43)
The value (μmol) was obtained from Fig. 1.
Abbreviations: CI, confidence interval; ED50, 50% effective dose.
There is no significant different between the theoretical and experimental values by
using the Student’s t-test
29
TABLE 4. The percent of maximal possible effect (%MPE) and area under the curves
(AUCs) of L-tyrosine (2xED50) alone, lidocaine (2xED50) alone, and the combination
of lidocaine (ED50) and L-tyrosine (ED50) (lidocaine+L-tyrosine) on infiltrative
cutaneous analgesia in rats
%MPE
AUCs (%MPEmin)
L-Tyrosine
100 ± 0
4427 ± 301b
Lidocaine
98 ± 2
2924 ± 211
Lidocaine+L-Tyrosine
96 ± 3
3641 ± 192a
Data are expressed as mean  S.E.M. (n = 8 in each group). The ED50 means 50%
effective dose. The symbols (a, b) indicate P<0.05 and P<0.01, respectively, when
compared with lidocaine by using one-way analysis of variance and pairwise Tukey
honestly significant difference test.
%MPE (maximal possible effect)
30
Lidocaine
Lidocaine+L-Tyrosine
L-Tyrosine
100
80
60
40
20
0
1
10
100
Dose ( mol )
Fig. 1.
31
%PE (possible effect)
100
L-Tyrosine
Lidocaine
Saline
80
60
40
20
0
0
15
30
45
60
75
90
105
120
Time (min)
Fig. 2.
32
Full recovery time (min)
60
L-Tyrosine
Lidocaine
45
30
15
0
25
50
75
ED ( % of effective dose )
Fig. 3.
33
L-Tyrosine (mol)
50
40
30
20
10
0
0
1
2
3
4
5
6
Lidocaine (mol)
Fig. 4.
34
%PE (possible effect)
100
Lidocaine (2xED50)
L-Tyrosine (2xED50)
80
Lidocaine (ED50) + L-Tyrosine (ED50)
60
40
20
0
0
15
30
45
60
75
90
105
Time (min)
Fig. 5.
35
FIGURE LEGENDS
Fig. 1. The dose-response curves after subcutaneous L-tyrosine alone, lidocaine alone,
and the combination of L-tyrosine and lidocaine (4-5 doses in each group) on
infiltrative cutaneous analgesia. Data are expressed as mean  S.E.M.; n = 8 rats for
each dose of each drug.
Fig. 2. Time courses of cutaneous analgesia of L-tyrosine (90 μmol), lidocaine (13.2
μmol), and saline (vehicle). Data are expressed as mean  S.E.M.; n=8 rats in each
group of the time course experiment.
Fig. 3. Full recovery time (duration) of L-tyrosine and lidocaine action (n = 8 at each
testing point) as an infiltrative anesthetic at ED25, ED50, and ED75. Values are mean 
S.E.M. The difference in duration among drugs was estimated using two-way
ANOVA followed by pairwise Tukey's HSD test.
Fig. 4. The isobolographic analyses of the drug-drug interactions on infiltrative
cutaneous analgesia. Data points on the X and Y axes mean the 50% effective doses
(ED50s) of drugs alone while the oblique line between the X and Y axes is the
theoretical additive line. N=8 rats in each group of the experimental and theoretical
ED50s studies. The symbols (△, ●), respectively, indicate the experimental and
theoretical ED50s with 95% confidence interval of combined drugs (data are expressed
as mean  S.E.M.).
36
Fig. 5. The time course of lidocaine (2xED50) alone, L-tyrosine (2xED50) alone, or
co-administration of lidocaine (ED50) and L-tyrosine (ED50) on infiltrative cutaneous
analgesia. Values are mean  S.E.M.; n=8 rats in each group of the time course
experiment.