Nisoxetine produces local but not systemic analgesia against cutaneous nociceptive stimuli in the rat

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Title: Nisoxetine produces local but not systemic analgesia against cutaneous nociceptive stimuli in the rat

Article Type: Research Paper

Section/Category: Neuropharmacology and analgesia

Keywords: nisoxetine; MK-801; lidocaine; additive effect; cutaneous analgesia Corresponding Author: Associate Professor Ching-Hsia Hung, Ph.D.

Corresponding Author's Institution: National Cheng Kung University First Author: Yu-Wen Chen, PhD

Order of Authors: Yu-Wen Chen, PhD; Chin-Chen Chu, MD, PhD; Yu-Chung Chen, MS; Jhi-Joung Wang, MD, PhD; Ching-Hsia Hung, Ph.D.; Dong-Zi Shao, PhD

Abstract: The aim of this study was to evaluate the local anesthetic effect of nisoxetine on infiltrative cutaneous analgesia. After rats were injected subcutaneously with nisoxetine, dose—response curves were constructed. The cutaneous analgesic effect of nisoxetine or MK-801 (dizocilpine) was compared with lidocaine, a traditional local anesthetic. We found that nisoxetine and MK-801 acted like lidocaine and elicited dose-related cutaneous (local) analgesia. The relative potency was nisoxetine > MK-801 > lidocaine (P < 0.01) on infiltrative cutaneous analgesia. On an equianalgesic doses (20% effective dose [ED20], ED50, and ED80), nisoxetine produced longer action of cutaneous analgesia than that of lidocaine or MK-801 (P < 0.01). Coadministration of nisoxetine or lidocaine with MK-801 showed an additive effect on infiltrative cutaneous analgesia. Neither local injection of a large dose of nisoxetine, MK-801 or lidocaine in the thigh area produced cutaneous analgesia (data not shown). In conclusion, nisoxetine had a local anesthetic effect on infiltrative cutaneous analgesia with durations of actions longer than that of lidocaine or MK-801. That N-methyl-D-aspartate receptors may not contribute to the cutaneous (local) analgesic effect of nisoxetine or lidocaine.

Nisoxetine produces local but not systemic analgesia against

cutaneous nociceptive stimuli in the rat

Yu-Wen Chena,b, Ph.D., Chin-Chen Chub, M.D., Ph.D., Yu-Chung Chenc, M.S., Jhi-Joung Wangb, M.D., Ph.D., Ching-Hsia Hungd,*, Ph.D., Dong-Zi Shaoe, Ph.D.

a

Department of Physical Therapy, China Medical University, Taichung, Taiwan; b

Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan; c

Division of Physical Therapy, Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan;

d

Institute & Department of Physical Therapy, National Cheng Kung University, Tainan, Taiwan;

e

Department of Cosmetics Application and Management, Chung Hwa University of Medical Technology, Tainan, Taiwan

Conflicts of interest: There is no conflict of interests for all authors.

*Address correspondence and reprint requests to: Ching-Hsia Hung, PhD, Institute & Department of Physical Therapy, National Cheng Kung University, No.1 Ta-Hsueh Road, Tainan 701, Taiwan

Tel: 886-6-2353535 ext 5939 Fax: 886-6-2370411

E-mail: [email protected]

Manuscript

ABSTRACT

The aim of this study was to evaluate the local anesthetic effect of nisoxetine on

infiltrative cutaneous analgesia. After rats were injected subcutaneously with

nisoxetine, dose—response curves were constructed. The cutaneous analgesic effect

of nisoxetine or MK-801 (dizocilpine) was compared with lidocaine, a traditional

local anesthetic. We found that nisoxetine and MK-801 acted like lidocaine and

elicited dose-related cutaneous (local) analgesia. The relative potency was nisoxetine

> MK-801 > lidocaine (P < 0.01) on infiltrative cutaneous analgesia. On an

equianalgesic doses (20% effective dose [ED20], ED50, and ED80), nisoxetine

produced longer action of cutaneous analgesia than that of lidocaine or MK-801 (P <

0.01). Coadministration of nisoxetine or lidocaine with MK-801 showed an additive

effect on infiltrative cutaneous analgesia. Neither local injection of a large dose of

nisoxetine, MK-801 or lidocaine in the thigh area produced cutaneous analgesia (data

not shown). In conclusion, nisoxetine had a local anesthetic effect on infiltrative

cutaneous analgesia with durations of actions longer than that of lidocaine or MK-801.

That N-methyl-D-aspartate receptors may not contribute to the cutaneous (local)

analgesic effect of nisoxetine or lidocaine.

1. Introduction

Nisoxetine, a potent inhibitor of norepinephrine reuptake (Yokogawa et al.,

2002), has been known to treat for affective disorders (Mongeau et al., 1997) and

suppress the nicotine-evoked increase of hippocampal noradrenaline release in a

dose-dependent manner (Bolden-Watson and Richelson, 1993; Wong et al., 1995) by

influencing the function of nicotinic acetylcholine receptors (Hennings et al., 1999).

In addition, nisoxetine shows an inhibition of the fast tetrodotoxin (full)-sensitive

inward Na+ currents in rat superior cervical ganglia (Hennings et al., 1999). The

blockade of Na+ channels is an essential activity of local anesthetics (Fozzard et al.,

2005). With this activity, local anesthetics produce infiltrative cutaneous analgesia,

spinal/epidural anesthesia, and peripheral neural blockades (Fozzard et al., 2005).

Because nisoxetine has a Na+ channel blocking effect (Hennings et al., 1999),

theoretically, it may have a local anesthetic effect, e.g., cutaneous (local) analgesia.

However, this was never tested.

The local anesthetic lidocaine is thought not only to block Na+ channels

(Yanagidate and Strichartz, 2007) but also to interact with various receptors

(Muth-Selbach et al., 2009). Also, there is a study to show that lidocaine, a well

studied Na+ channel blocker, decreases experimental pain behaviors via NMDA

investigate the cutaneous (local) analgesic effect of nisoxetine when compared with

lidocaine, a common used local anesthetic. Furthermore, a selective non-competitive

NMDA antagonist (MK-801) was used to evaluate lidocaine- or nisoxetine-elicited

2. Materials and methods

2.1. Animals

Male Sprague-Dawley rats 5–6 weeks of age (200-250g) were obtained from the

National Laboratory Animal Centre, Taipei, Taiwan. Then animals were housed in

groups of three, with food and water freely available until the time of testing. The climate controlled room maintained at 22 ℃ with approximately 50% relative

humidity on a 12-h light/dark cycle (6:00 AM–6:00 PM). The experimental protocols

were approved by the Institutional Animal Care and Use Committee of China Medical

University, Taiwan, and conformed to the recommendations and policies of the

International Association for the Study of Pain (ISAP).

2.2. Drugs

Nisoxetine HCl, (+)-MK-801 hydrogen maleate, and lidocaine HCl were

purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All drugs were

freshly prepared in saline (0.9% NaCl) as solution before drug injections.

2.3. The experimental protocol

Five experiments were carried out. In experiment 1, the dose-response curves of

nisoxetine, MK-801, and lidocaine on infiltrative cutaneous analgesia were evaluated.

In experiment 2, the cutaneous analgesic effect of nisoxetine was compared with that

doses (ED20, ED50, and ED80), the duration of drug action on infiltrative cutaneous

analgesia was obtained and compared. In experiment 4, the cutaneous analgesic effect

of coadministration of nisoxetine (1.50 µmol) and MK-801 (2.24 µmol) was

compared with nisoxetine (1.50 µmol) or MK-801 (2.24 µmol) alone. The cutaneous

analgesic effect of coadministration of lidocaine (6.05 µmol) and MK-801 (2.24 µmol)

was compared with lidocaine (6.05 µmol) or MK-801 (2.24 µmol) alone. In

experiment 5, one control group was further added into the study to rule out the

possibility of systemic effect of drugs on infiltrative cutaneous analgesia. Rats (n=8

rats for each group) received subcutaneous injection of testing drug (nisoxetine,

MK-801 or lidocaine) in the thigh area with a dose of 2ED80.

2.4. Infiltrative cutaneous analgesia

Before drug injection, rats were handled daily up to 7 days to domesticate them

with the investigator, the experimental environment, and the specific experimental

procedures. On the day before subcutaneous injections, the hair on the rats' dorsal

surface of the thoracolumbar region (6×10 cm2) was mechanically removed.

Subcutaneous injections of drugs were performed as reported previously (Chen et al.,

2011b; Chen et al., 2011c). In brief, the drugs were subcutaneously injected 0.6 mL

via a 30-gauge needle in unanesthetized rats at the dorsal surface of the thoracolumbar

approximately 2 cm in diameter occurred. The wheal was marked with ink within 30

seconds after injection. For consistency, one experienced investigator who was

blinded to the drugs injected was responsible for evaluating the cutaneous analgesic

effect. The drugs were prepared and injected by another investigator.

2.5. Neurobehavioral evaluation

The cutaneous (local) analgesic effect was evaluated via the cutaneous trunci

muscle reflex (CTMR), characterized by the reflex movement of the skin over the

back produced (Chen et al., 2011a; Hung et al., 2010). A Von Frey filament (No.15;

Somedic Sales AB, Stockholm, Sweden), to which the cut end of an 18-gauge needle

was affixed, was used to perform the standardized nociceptive stimulus (19±0.5 g). After observing an animal’s normal reaction to pinpricks applied outside the wheal and on the contralateral side, we applied six pinpricks with a frequency of 0.5-1.0 Hz

inside the wheal and scored the number to which the rat failed to react. The cutaneous

anesthetic effect of each drug was evaluated quantitatively as the number of times the

pinprick failed to elicit a response, with, for example, the complete absence of six

responses was defined as complete nociceptive block (100% of possible effect; 100%

PE). The test of six pinpricks was applied 5 min before drug injection, then every 5

min after injection for the first 30 min and every 10-15 min thereafter until the CTMR

of maximal possible effect (% MPE) during the test. The duration of action of each

drug was defined as the time from drug injection (i.e., time=0) to full recovery of

CTMR (no anesthetic effect was found or 0% MPE recorded) (Chen et al., 2008).

2.6. The 50% effective dose (ED50)

After rats were injected with 4-5 different doses of each drug (n = 8 for each

dose of each drug) subcutaneously, dose-response curves were constructed. The

curves were then fitted using SAS Nonlinear (NLIN) Procedures (version 9.1; SAS

Institute, Cary, NC), and the values of ED50, defined as the doses that caused 50%

blockades, were obtained (Chen et al., 2010;Leung et al., 2010). The ED20 and ED80

of drugs were obtained using the same curve fitting (SAS Nonlinear analysis) that was

used to derive the ED50. Furthermore, the area under curves (AUCs) of

nociceptive/sensory blockades of drugs was estimated using Kinetica version 2.0.1

(InnaPhase Corporation, Philadelphia, PA).

2.7. Statistical analysis

Data are presented as mean ± SEM or ED50 values with 95% confidence interval

(95% CI). The differences in potencies (ED50s) (Table 1) between medications and

the full recovery time, %MPE, and AUCs of drugs (Table 2) were evaluated by 1-way

analysis of variance (ANOVA) and then the pairwise Tukey's honestly significant

evaluated by 2-way ANOVA followed by the pairwise Tukey’s HSD test. SPSS for

Windows (version 17.0) was used for all statistical analyses. Statistical significance

3. Results

3.1. Dose-dependent effects of nisoxetine, MK-801, and lidocaine on infiltrative

cutaneous analgesia

The nisoxetine and MK-801, as well as local anesthetic lidocaine produced

dose-dependent effects of cutaneous analgesia in rats (Fig. 1). The ED50s of drugs are

shown in Table 1. The relative potency of these drugs was found to be nisoxetine >

MK-801 > lidocaine (P<0.01 for the differences between drugs; Table 1). All rats

recovered completely after each subcutaneous injection.

3.2. The cutaneous analgesic effects of nisoxetine, MK-801, and lidocaine

Nisoxetine at the dose of 3.0 μmol showed 96% of blockades (% MPE) with

duration of action of about 146 min (Fig. 2). At the same given dose, MK-801 elicited

65% of blockades (% MPE) with duration of action of about 22 min. Lidocaine at 3.0 μmol displayed 10% of blockades (% MPE) with duration of action of about 4 min. The full recovery time and AUCs of cutaneous analgesic effect of nisoxetine are

significantly greater than those of lidocaine or MK-801 (P<0.001 for the differences

between drugs; Fig. 2 and Table 2).

On an equipotent basis (ED20, ED50, and ED80), the blockade duration for

nisoxetine was longer than that for lidocaine or MK-801 on infiltrative cutaneous

injection of drugs (2ED80) in the thigh area produced no cutaneous analgesia, sedation

or loss of motor activity (data not shown).

3.3. Co-administration of nisoxetine or lidocaine with MK-801

The co-administration of nisoxetine with MK-801 produced similar %MPE to

the aggregate of nisoxetine alone and MK-801 alone on infiltrative cutaneous

analgesia (Fig. 4A and Table 3). The co-administration of lidocaine with MK-801 also

showed similar results (Fig. 4B and Table 3). These results reported that

co-administration of nisoxetine or lidocaine with MK-801 produced an additive effect

4. Discussion

Our study showed that nisoxetine, MK-801, and lidocaine elicited dose-related

cutaneous (local) analgesia. Nisoxetine was more potent and longer drug action at

producing cutaneous analgesia than lidocaine or MK-801. Coadministration of

MK-801 with nisoxetine or lidocaine displayed an additive effect on infiltrative

cutaneous analgesia.

Lidocaine is a local anesthetic agent that produces neural blockade via a direct

blocking effect on the voltage-gated Na+ channels of the nervous tissues (Fozzard et

al., 2005; Yanagidate and Strichartz, 2007). Because nisoxetine has a Na+ channel

blocking effect (Hennings et al., 1999), theoretically it may have a local anesthetic

effect. In this study, we did find that nisoxetine has a local anesthetic effect on

infiltrative cutaneous analgesia in rats. Furthermore, MK-801, a potent

non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, elicited

dose-related cutaneous analgesia, and we speculated that the local anesthetic property

of MK-801 is due to its characteristic of Na+ channel blockade (Halliwell et al.,

1989).

We showed that nisoxetine, lidocaine, and MK-801 have local anesthetic effects

as infiltrative cutaneous analgesia. Furthermore, nisoxetine was more potent at

Table 1). Nisoxetine had almost 1.5- and 4.0-folds higher potency than MK-801 and

lidocaine on infiltrative cutaneous analgesia, respectively. Combined administration

of a dose (ED50) of MK-801 with nisoxetine or lidocaine displays an additive

analgesic effect. The cutaneous analgesic effects of adding MK-801 to nisoxetine or

lidocaine are similar to the combinations of other anesthetics with nisoxetine or

lidocaine. Our study suggested that these drugs act in a similar manner, for example

Na+ channel blockades.

The long-acting local anesthetics are frequently practiced for surgery and the

management of postoperatic pain (Hung et al., 2009;Job et al., 1979). In this study,

nisoxetine produced longer duration of action than lidocaine or MK-801 at the same

dose of 3.0 µmol (Fig. 2 and Table 2). Additionally, the duration of action caused by

nisoxetine was longer than that caused by lidocaine or MK-801 at equianalgesic doses

(ED20, ED50, and ED80) (Fig. 3). An extra experiment was added to the study to rule

out the possibility of systemic effects by drugs. Systemic administration of a large

dose of the test drugs produced no cutaneous analgesia. These results supported the

local action of testing drugs on skin and nisoxetine elicits local (cutaneous) but not

systemic analgesia.

Meanwhile, it remains unclear whether nisoxetine cause toxicity to the

experiments. Our data suggest that nisoxetine may have features that make it a

valuable alternative cutaneous analgesia, although the possibility of nerve (tissue)

damage from nisoxetine injection remains an open question for further investigations.

This preclinical study reported that nisoxetine had a local anesthetic effect as

infiltrative cutaneous analgesia in rats. Nisoxetine elicited more potent and longer

action than lidocaine or MK-801 in providing cutaneous analgesia. NMDA receptors

Acknowledgements

The financial support provided for this study by the National Science Council of

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Table 1. The 50% effective doses (ED50s), ED20s, and ED80s of nisoxetine, MK-801, and lidocaine on infiltrative cutaneous analgesia in rats

Drug ED20 ( 95% CI ) ED50 ( 95% CI ) ED80 ( 95% CI ) Nisoxetine 0.47 (0.38–0.57) 1.50 (1.40–1.63) 2.73 (2.54–3.02) MK-801 1.20 (1.10–1.33) 2.24 (2.13–2.36) 4.37 (4.16–4.78) Lidocaine 3.51 (3.35–3.79) 6.05 (5.83–6.43) 9.35 (9.18–9.66) ED50s of drugs (μmol) were obtained from Figure 1. CI = confidence interval. The potency of drug (ED50) was nisoxetine > MK-801 > lidocaine (P<0.01, for each comparison).

Table 2. The percent of maximal possible effect (%MPE), time to full recovery, area under curves (AUCs) of

drugs on infiltrative cutaneous analgesia in rats

Drug %MPE Time to full recovery AUCs (%min)

Nisoxetine 96±4*** 146±25*** 8126±1729***

MK-801 65±5††† 22±3††† 775±164†††

Lidocaine 10±7 4±2 60±40

Saline – – –

The %MPE, duration, and AUCs for nisoxetine, MK-801, and lidocaine (meanSEM) at the same dose of 3.00 μmol (n = 8). Saline group was used as a control. Symbols (***) indicate P < 0.001 when nisoxetine compared with lidocaine or MK-801. Symbols (†††) indicate P < 0.001 when MK-801 compared with lidocaine.

Table 3. The percent of maximal possible effect (%MPE) of co-administration of

nisoxetine or lidocaine with MK-801 on infiltrative cutaneous analgesia in rats %MPE Nisoxetine with MK-801 Nisoxetine 50±12 Nisoxetine+MK-801 86±4 MK-801 56±7 Lidocaine with MK-801 Lidocaine 52±11 Lidocaine+MK-801 96±2 MK-801 50±10

Values are mean±SEM. The doses for injections were ED50 (50% effective dose) for a single drug or ED50 for drugs in combination. The values of % MPE were derived from Fig. 4A and B after calculation.

Dose (



mol )

%MPE (maxima

l possible effe

ct)

Fig. 1.

Time ( min )

0

20 40 60 80 100 120 140 160 180 200 220 240

% PE

( possible effect )

0

20

40

60

80

100

Nisoxetine

MK-801

Lidocaine

Saline

Fig. 2.

ED ( effective dose )

Tim

e

to

Full

Rec

ove

ry

(

m

in

)

Fig. 3.

A

Time ( min )

% P

E ( possib

le e

ffect )

Nisoxetine (1.50 mol) + MK-801 (2.24 mol) Nisoxetine (1.50mol) MK801 (2.24 mol)

B

Time ( min )

% P

E (

possibl

e effect )

Lidocaine (6.05 mol) + MK-801 (2.24 mol) Lidocaine (6.05 mol)

MK-801 (2.24 mol)

Figure Legends

Fig. 1. The dose—response curves of nisoxetine, MK-801, and lidocaine on

infiltrative cutaneous analgesia in rats (n = 8 at each testing point). Data are shown as mean±SEM.

Fig. 2. Time courses of cutaneous analgesia of nisoxetine, MK-801, and lidocaine at

the same dose of 3.0 μmol in rats. The saline group is as the control. Values are expressed as meanSEM. Each testing point of the time course study contained eight rats.

Fig. 3. Time to full recovery (duration) of drug effect on infiltrative cutaneous

analgesia at doses of ED20, ED50, and ED80 (n = 8 at each testing point). Data are mean±SEM. The differences in duration were evaluated using 2-way ANOVA and then the pairwise Tukey's HSD test.

Fig. 4. The time course (A) of nisoxetine at 1.50 µmol, MK-801 at 2.24 µmol or

coadministration of nisoxetine at 1.50 µmol and MK-801 at 2.24 µmol on infiltrative cutaneous analgesia in rats. The time course (B) of lidocaine at 6.05 µmol, MK-801 at 2.24 µmol or coadministration of lidocaine at 6.05 µmol and MK-801 at 2.24 µmol on infiltrative cutaneous analgesia in rats. Values are expressed as meanSEM. For each group of the time course study, n=8 rats.