Clonidine intensifies memantine cutaneous analgesia in response to local skin noxious pinprick in the rat

Clonidine intensifies memantine cutaneous analgesia in

response to local skin noxious pinprick in the rat

Background: The purpose s of this study were to evaluate the co-administration of clonidine with memantine and to determine whether it has a peripheral action in intensifying cutaneous analgesia.

Methods: Cutaneous analgesia was examined through inhibition of the cutaneous trunci muscle reflex in response to the local noxious pinprick in rats. Effect of the added subcutaneous clonidine to memantine on infiltrative cutaneous analgesia was assessed and compared with the local anesthetic lidocaine.

Results: On the 50% effective dose (ED50) basis, the rank of drug potency was memantine [4.05 (3.95 – 4.18) μmol] > lidocaine [5.81 (5.70 – 5.98) μmol] (p < 0.01). Clonidine at a dose of 0.12 µmol did not elicit cutaneous analgesia. Mixtures of clonidine (0.12 μmol) with drug (memantine or lidocaine) at ED50 or ED95 prolonged the duration of action and enhanced the potency as infiltrative cutaneous analgesia Clonidine enhanced the lidocaine cutaneous analgesia in which had a better effect than added to memantine.

Conclusions: Our resulting data showed that memantine displayed more potent cutaneous analgesia than lidocaine. Co-administration of memantine or lidocaine with clonidine increased the potency and duration of the cutaneous analgesia.

added to memantine.

Key words: Clonidine, Memantine, Lidocaine, Infiltrative cutaneous analgesia

Introduction

Memantine is the first novel class of Alzheimer's disease drugs whose

glutamate receptors [1,2]. Due to the inhibition of tetrodotoxin-resistant Na+ _currents [3], memantine also produced a local anesthetic effect in rats [4,5]. Infiltration

anesthesia through a local anesthetic injection is used for managing laparoscopic surgery [6] and postoperative pain relief after inguinal hernia repair [7], because it is relatively free of side effects [8]. However, this technique has a short duration of anesthesia or analgesia [9].

Research has been shown that memantine elicited skin infiltrative analgesia in a dose-related fashion [4] and its block duration was similar to that of the long-term local anesthetic bupivacaine [5]. Moreover, at the equianesthetic doses, intravenous memantine tolerated better to induce the cardiovascular system and central nervous system toxicity than bupivacaine [5]. The clinical value of memantine is worth y of being studied.

In general, clonidine is a commonly - used adjuvant to the local anesthetic agents. Moreover, the clonidine-analgesic mechanism goes through its α2-adrenoreceptor properties when administered intrathecally or epidurally [10,11]. Our previous study demonstrated that clonidine enhanced the sensory blocking effect and duration of bupivacaine [12]. It is widely suggested that clonidine improves the potency and duration of the local anesthetic blockade and diminishes the postoperative analgesic requirement [13]. However, the administration of clonidine impacting to wards the

local anesthetic memantine for peripheral nerve block remains unclear. Therefore, t he purpose s of this study were to assess the effect of

co-administration of clonidine with memantine and to determine whether it produces a peripheral action in intensifying the quality and duration of cutaneous analgesia, when compared with lidocaine. The local anesthetic lidocaine was used as a control agent.

Materials and Methods

Animals

The experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University (Taichung, Taiwan) in accordance to the recommendations and policies of the International Association for the Study of Pain (IASP). One hundred and seventy-six male Sprague-Dawley rats, each weighing 200

to 250 g, were purchased from the National Laboratory Animal Cent er (Taipei, Taiwan) and kept in the animal housing facilities at China Medical University, with controlled humidity (approximately 50% relative humidity), room temperature (22C), and a 12 hour on/ 12 hour off light / dark cycle (light on at 6:00 AM ) .

Drugs

Memantine HCl, lidocaine HCl monohydrate, and clonidine HCl were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA. All drugs were dissolved in saline (0.9% NaCl) before subcutaneous injection.

Groups and design

Three experiments were performed. In experiment 1, the cutaneous analgesia of memantine (12.0, 6.0, 3.0, 1.5 µmol) and lidocaine (15.0, 7.5, 5.0, 3.0 µmol) in a dose-related fashion was performed (n = 8 for each group). In experiment 2, the %MPE (percent of maximal possible effect), duration, and area under the curves (AUCs) of the drug (ED50 or ED95) alone or co-administration of the drug (ED50 or ED95) and clonidine (0.12 µmol) were evaluated on infiltrative cutaneous analgesia (n = 8 for each group). Subcutaneous injection of clonidine at 0.12 µmol elicited no cutaneous analgesia. In experiment 3, two control groups were tested to rule out the possible systemic effect of drugs on cutaneous analgesia. One group (n = 8 for each group) received intraperitoneal injection of drugs (memantine or lidocaine) at 2ED95

or clonidine at 0.24 µmol. Another group (n = 8 for each group) received intraperitoneal injection of co-administration of clonidine (0.12 µmol) and drug (memantine or lidocaine) at ED95.

Subcutaneous injection

Animals were handled daily for a week to minimize the stress on the rats during the experiment and generally improve d their experimental performance s . On the day before the injection, the hair on the rats' dorsal surface of the thoracolumbar region (10×6 cm2_{) was mechanically shaved. The subcutaneous injection procedure was} performed as previously reported [14,15]. In brief, the drugs, which were dissolved in saline, were injected subcutaneously in un - anesthetized rats at the dorsal surface of the thoracolumbar region by using a 30-gauge needle. The entire injection volume was 0.6 ml. After the subcutaneous injection, a wheal occurred approximately 2 cm in diameter as a circular elevation of the skin. It was marked with ink within one minute after injection. The cutaneous analgesia was assessed by the cutaneous trunci muscle reflex (CTMR) of subcutaneous muscles in response to the pinpricks. CTMR is a reaction from the noxious skin stimulus involving the local contraction of skeletal muscle beneath the skin with parallel movement s of the nearby skin over the rat’s dorsum [5,16].

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 produce the standardized nociceptive stimulation (19±1 g) without producing skin damage. After observing a normal reaction to pinpricks applied outside the wheal and on the contralateral side, we applied six pinpricks with a frequency of 1 Hz inside the wheal and selected the number to which the rat failed to react. The c utaneous analgesia of each drug was calculated quantitatively as the number of times the pinprick failed to elicit a response. For example, the complete absence of six responses demonstrated to be a full nociceptive block (100% of possible effect; 100% PE) [17,18].

For consistency, an experienced investigator, who was blinded to inject the drugs, was responsible for the neurobehavioral examinations. The six-pinprick test was applied at 0, 2 and 5 min utes after injection. First, the test was assessed e very five

min utes after the injection for the first 30 min utes. Second, it happened again every 10 min utes after the injection for 30-60 min utes , and then every 15-60 min utes until the CTMR fully recovered from the block. During the test, the maximal

blockade during the time course of cutaneous analgesia of the drug was described as the %MPE. The duration of the drug action started from the injection (i.e., time=0) to the full recovery moment of CTMR (no analgesic effect or 0% MPE) [19,20].

After subcutaneously injecting the rats with four doses of each drug (n = 8 for each dose of each drug), the dose-response curves were obtained from the % MPE for every drug dose . The value s of ED50 and ED95, as defined as the dose that caused 50% and 95% cutaneous analgesic effect, respectively, were obtained by a SAS NLIN analysis (SAS Institute Inc., Carey, NC) [21-23]. The AUCs of the sensory block of drugs were obtained by using the Kinetica version 2.0.1 (InnaPhase Corporation, Philadelphia, PA) program.

Statistical analysis

Data in Table 1 are presented as ED50 or ED95 values with 95% confidence interval (95% CI) and analyzed by the one-way analysis of variance (ANOVA) followed by the pairwise Tukey’s honest significance difference (HSD) test. The v alues in Tables 2 and 3 are displayed as mean  SEM with a secondary data spread shown even though a normal distribution was not assumed. The c omparisons between the drug alone and the co-administration of drug with clonidine in between

memantine and lidocaine groups were mentioned in each group, using nonparametric statistics (Mann–Whitney U test). A statistical software, SPSS for Windows (version 17.0, SPSS, Inc, Chicago, IL, USA), was used, and a P value less than 0.05 was considered statistically significant.

Results

Cutaneous analgesia of memantine and lidocaine

The subcutaneous injection s of memantine and lidocaine exhibited dose-dependent cutaneous analgesia in rats (Fig. 1). The ED50s and ED95s of memantine and lidocaine constructed from Fig.1 were shown in Table 1. On an ED50 basis, the rank of drug potency was memantine greater than lidocaine (Table 1).

Cutaneous analgesia of co-administration of clonidine with drugs (memantine and lidocaine) at ED50

Memantine and lidocaine at the same dose of ED50 showed 53% and 45% of blockade (%MPE), respectively, in Figure 2. The s ubcutaneous injection of clonidine alone at 0.12 µmol produced no cutaneous analgesia (Fig. 2A). When the drugs at ED50 were co-injected with clonidine (0.12 µmol), the complete nociceptive/sensory blockade (100% MPE) in the lidocaine (8 of 8 rats) group occurred instead of the memantine (3 of 8 rats) group (Fig. 2). Drugs (ED50) co-injected with 0.12 µmol clonidine, the %MPE, full recovery time, and AUCs were increased in the memantine and lidocaine groups (p < 0.001) compared with drugs at ED50 alone (Table 2).

Cutaneous analgesia of co-administration of clonidine with drugs (memantine and lidocaine) at ED95

At a dose of ED95, memantine and lidocaine exhibited 92% and 93%

nociceptive/sensory blockade, respectively (Fig. 2). After the drugs at ED95 were co-injected with clonidine (0.12 µmol), both memantine and lidocaine elicited a 100% sensory blockade (100% MPE), respectively (Fig. 2). The duration and AUCs of drugs (ED95) associated with clonidine were greater (p < 0.05) than the drugs (ED95) alone in Table 3. Neither the intraperitoneal injection of clonidine (0.12 µmol) combined with drug (memantine or lidocaine) nor the intraperitoneal dosage administration (2ED95) of the drug produced the cutaneous analgesia. All of the animals recovered completely after the drug was injected subcutaneously in them.

Discussion

In this report, we demonstrated for the first time that memantine was more potent as an infiltrative cutaneous analgesia than lidocaine. In addition, clonidine markedly improved the sensory blockade and duration of memantine and the local anesthetic lidocaine.

Previously, c linical and scientific experiments have provided evidence s for the mechanism of the clonidine action as a local anesthetic adjuvant and suggested the local anesthetic-like properties of clonidine itself [24,25]. However, the s edative effects of clonidine and motor block should be prevented after central neuraxial block .

When the high incidence of motor block elicited via clonidine, the clinical relevance of the prolonged sensory block in certain situations may overall limited its effect [26]. Given the dose-limiting central side effects, clonidine may benefit for the peripheral application. In this current study, we showed that a low dose of clonidine (0.12 μmol) produced no cutaneous analgesia. Moreover, we determined that the infiltrative cutaneous analgesia was defined as adding clonidine to local anesthetics . Therefore, o ur resulting data demonstrated that the co-administration of clonidine (0.12 μmol) with memantine or lidocaine intensified the cutaneous analgesia and prolonged the duration of its action. This result is similar to the previous study ’s, which reported that clonidine has a marked peripheral action in prolonging the local anesthetic duration of the subcutaneous co-infiltration with lidocaine [27].

In this experiment, the cutaneous analgesia of the co-administration of clonidine (0.12 μmol) and drug (memantine or lidocaine) at ED50 was approximately 2.9- and 13.7-folds, respectively, greater than the drug alone. Likewise, the cutaneous analgesia of the co-administration of clonidine (0.12 μmol) and drug (memantine or lidocaine) at ED95 was approximately 1.5- and 6.2-folds greater than the drug alone, respectively. In fact, i nfiltration analgesia of skin is a receivable method for managing postoperative pain and surgical analgesia [8], including both bupivacaine and

anesthesia with regards to its long duration of the analgesi c effect [29]. The administration of lidocaine with clonidine displayed longer duration than the co-administration of clonidine and memantine on infiltrative cutaneous analgesia. The benefit to use the combination of clonidine and memantine (or lidocaine) as a pain killer remains an open question for further investigations.

After the subcutaneous injection of clonidine at 1.2 mg/kg, rats , induced with clonidine, resulted in delayed tactile hypersensitivity 24–34 hours [30]. In this present study, the subcutaneous injection of clonidine (0.12µmol; 0.14mg/kg) did not elicit any cutaneous analgesia to the local noxious pinprick of the skin . However, adding clonidine to memantine or lidocaine produced a peripheral action in

prolonging and intensifying the cutaneous analgesia. This resemblance compares to our previous study that clonidine markedly improves the sensory blocking effect s and duration s of bupivacaine, dextrorphan, and oxybuprocaine [12].

Clonidine, used in peripheral nerve blockades [27,31] or combined with local anesthetics, [32,33] enhances anesthesia. It is well - established that clonidine was added to the preparation of the local anesthetic clinically to extend its duration of its action through three possible mechanisms. First, clonidine may induce local

vasoconstriction [10,34] to minimize the local anesthetic spread and removal that surrounds the nerves [10,33]. Second, clonidine blocked the Aδ and C fib er s [25,31]

as a result of an increase in potassium conductance in the isolated neuron [35,36] and an increased effect of lidocaine on C-fiber action potential [31]. Lastly, clonidine can activate the alpha-2 receptors. Specifically, the a lpha -2 receptors are expressed by the nociceptive primary sensory neurons [37]. The a ctivation of the alpha-2 adrenoceptors by the topical clonidine (50-75 mg/mL) expressed on the peripheral terminals of cutaneous nociceptors, inhibit s the acute thermal nociception [38]. In fact, we present an ineffective dose of subcutaneous clonidine (53.31 μg/mL) combining with

memantine and lidocaine becomes effective. Additionally, the activated peripheral alpha-2 receptors by clonidine can evoke several effects including the blockade of adenylyl cyclase activity. Thus, it possibly increases the nociceptive thresholds by blocking cAMP in the peripheral tissues [38,39].

On the contrary, t here is a limitation in our study. According to our present data, we cannot explain why the AUC of co-administration of clonidine with lidocaine is overly increased than with the more potent memantine. Therefore, the c o-

administration of drug (memantine or lidocaine) with the receptor (i.e., N-methyl-D-aspartate, α-adrenergic or β-adrenergic) antagonists or agonists would be

recommended for future testing.

Conclusions

In summary, memantine produced a more potent cutaneous analgesia when compared with lidocaine. On an equipotent basis (ED50), the effect of the co-administration of clonidine withmemantine or lidocaine exhibited a prominent peripheral action in intensifying and prolonging cutaneous analgesia. Indeed, c lonidine enhanced the effects of lidocaine , which was better than clonidine was an adjuvant to memantine.

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Table 1. The 50% effective doses (ED50s) and ED95s of memantine and lidocaine as infiltrative cutaneous analgesic

Drug ED50s ( 95% CI ) ED95s ( 95% CI )

Memantine 4.05 (3.95 – 4.18) 12.1 (11.0 – 13.7) Lidocaine 5.81 (5.70 – 5.98) 20.3 (19.3 – 21.6)

ED50s and ED95s of drugs (μmol) were obtained from Figure 1. CI = confidence interval. The potency of drug (ED50) was memantine > lidocaine (p < 0.01, for each comparison using one-way ANOVA followed by pairwise Tukey’s HSD test.

%MPE Duration (min) AUCs (%MPEmin) Complete blockade time Time to full recovery

Drug (ED50)

Memantine 53 ± 5 ― 41 ± 3f _{1216 ± 134}e

Lidocaine 45 ± 7 ― 24 ± 2 557 ± 93

Drug (ED50) + Clonidine

Memantine 85 ± 5a _{2 ± 1 77 ± 3}b _{3529 ± 276}c

Lidocaine 100 ± 0b,d _{55 ± 5}f _{99 ± 4}c,f _{7638 ± 318}c,f

Clonidine alone ― ― ― ―

Data are presented as mean  SEM (n = 8 in all groups). The symbols (a, b, c) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, when drug alone compared with the co-administration of drug and clonidine using nonparametric statistics (Mann–Whitney U test). The symbols (d, e, f) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, when memantine compared with lidocaine using nonparametric statistics (Mann–

%MPE Duration (min) AUCs (%MPEmin) Complete blockade time Time to full recovery

Drug (ED95)

Memantine 92 ± 3 5 ± 2 83 ± 3e _{3819 ± 197}d

Lidocaine 93 ± 3 5 ± 2 58 ± 2 3183 ± 218

Drug (ED95) + Clonidine

Memantine 100 ± 0 14 ± 1b _{109 ± 4}a _{5694 ± 218}b

Lidocaine 100 ± 0 161 ± 6c,f _{263 ± 8}c,f _{19775 ± 376}c,f

Data are presented as mean  SEM (n = 8 in all groups). The symbols (a, b, c) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, when drug alone compared with the co-administration of drug and clonidine using nonparametric statistics (Mann–Whitney U test). The symbols (d, e, f) indicate p < 0.05, p < 0.01, and p < 0.001, respectively, when memantine compared with lidocaine using nonparametric statistics (Mann–

Dose ( mol )

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Figure Legends

Fig.1. The dose-response curves of cutaneous analgesia after subcutaneous injection

of memantine and lidocaine (four doses in each group) in rats. Values are expressed as mean  SEM; n = 8 rats for each drug dose.

Fig.2. The addition of clonidine with memantine or lidocaine (ED50 or ED95) on infiltrative cutaneous analgesia in rats. Clonidine (CL) at a dose of 0.12 µmol elicited no cutaneous analgesia. Values are expressed as mean  SEM; n = 8 rats for each drug dose. The ED50 andED95 means 50% and 95% effective dose, respectively.