Caramiphen-induced block of sodium currents and spinal
anesthesia
Yuk-Man Leunga, PhD, Jann-Inn Tzengb,c, MD, Chi-Li Gongd, PhD, Yu-Wen Chene,f,*, PhD, Jhi-Joung Wangf, MD, PhD
a Graduate Institute of Neural and Cognitive Sciences, China Medical University, Taichung, Taiwan;
b Department of Anesthesiology, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan;
c Department of Food Sciences and Technology, Chia Nan University of Pharmacy and Science, Jen-Te, Tainan City, Taiwan;
d Department of Physiology, China Medical University, Taichung, Taiwan; e Department of Physical Therapy, China Medical University, Taichung, Taiwan; f Department of Medical Research, Chi-Mei Medical Center, Yong Kang, Tainan City,
Taiwan
Conflicts of interest: There is no conflict of interests for all authors.
*Address correspondence and reprint requests to: Yu-Wen Chen, Ph.D.
Department of Physical Therapy China Medical University
No.91 Hsueh-Shih Road, Taichung, Taiwan Tel: 886-4-22053366 ext 7313
Fax: 886-4-22065051
E-mail: [email protected]
ABSTRACT
The underlying mechanisms for the action of caramiphin used in local anesthesia are not well understood. The purpose of this study was to evaluate the block of
caramiphen on voltage-gated Na+ channels and in spinal anesthesia. We investigated the effect of caramiphen on voltage-gated sodium channels in differentiated neuronal NG108-15 cells as well as on rat motor function, proprioception, and pain behavior (when administered intrathecally). In in vitro studies, lidocaine produced
concentration- and state-dependent effects on tonic block of voltage-gated Na+ currents (IC50 of 58.8 and 238.3 µM at holding potentials of -70 and -100 mV,
respectively). Caramiphen did not show significant state-dependence of block (IC50 of 82.0 and 99.5 µM at holding potentials of -70 and -100 mV, respectively). Lidocaine showed a much stronger frequency-dependence of block than caramiphen: with high frequency stimulation (3.33Hz), 50µM caramiphen elicited an additional 20%
blockade, whereas the same concentration of lidocaine produced 50% more block. In
in vivo studies, caramiphen with a more sensory-selective action over motor blockade
was more potent than lidocaine (P < 0.05) in spinal anesthesia. On an equipotent basis (25% effective dose (ED25), ED50, and ED75), the duration of caramiphen at producing spinal anesthesia was longer than that of lidocaine (P < 0.01). These data revealed that caramiphen had a more potent, prolonged spinal blockade with a more
Spinal anesthesia with caramiphen could be by the suppression of voltage-gated Na+ currents.
Keywords: caramiphen; lidocaine; sodium currents; intrathecal; spinal block; duration
1. Introduction
Caramiphen, a centrally acting non-narcotic antitussive (cough suppressant agent), was first introduced into the management for diseases of the basal ganglia
(Sciarra et al., 1949) and was successfully used against the organophosphorous (OP) poisoning (Essig et al., 1950). Moreover, caramiphen was available as an antitussive agent in Europe since 1950 (Snyder, 1953). Recently, caramiphen has been shown to have an antidepressant-like effect (Kawaura et al., 2010), the anticonvulsive and neuroprotective properties against soman-induced seizures and neuropathology (Figueiredo et al., 2011) as well as the local anesthetic effects (Chen et al., 2010; Hung et al., 2012).
In in vitro studies, caramiphen reversibly attenuated NMDA-, but not AMPA- or kainate-evoked currents under the whole cell voltage-clamp study (Fletcher et al., 1995). In cultured rat hippocampal pyramidal neurons-exposed to high K+
concentrations, examination of the activity of caramiphen (40 μM) showed a marked effect on the evoked rise in intracellular [Ca2+] (Church et al., 1991). Blockade of voltage-gated Na+ channels, which is one of the major mechanisms of local anesthesia, produces sciatic nerve block, spinal anesthesia, and skin infiltration anesthesia (Borgeat and Aguirre, 2010; Vegh et al., 2006).
It has been shown that caramiphen has a local anesthetic effect on spinal anesthesia (Chen et al., 2010) and skin infiltrative anesthesia (Hung et al., 2012) in rats. Block duration of caramiphen was similar to long-acting local anesthetic bupivacaine in providing infiltrative cutaneous/local anesthesia (Hung et al., 2012),
whereas continuing intravenous administration of equipotent caramiphen was less toxic than bupivacaine to the central nervous system and cardiovascular system (Hung et al., 2012). It is reasonable to presume that caramiphen can produce local anesthesia through blocking Na+ currents, while no reports are available about the effects of caramiphen on voltage-gated Na+ currents so far.
The aim of this study was to test whether caramiphen inhibited Na+ currents and produced spinal anesthesia. Spinal anesthesia is known to be a relatively simple method, which supports suitable surgical conditions via injection of a small amount of local anesthetics with easy landmarks (Hung et al., 2009; Vandermeersch et al., 1991). In in vitro experiments, we examined whether caramiphen could suppress Na+ currents using the patch-clamp method; in in vivo experiments, we evaluated the duration of caramiphen spinal block in motor function, proprioception, and
nociception following intrathecal injection. Lidocaine, a traditional local anesthetic, was used as a control.
2. Materials and methods
2.1. Experimental designs
Five specific experiments were designed. The experimenters were blind for animal assignment to different experimental groups. In experiment 1, spinal blockade
with caramiphen and lidocaine in a dosage-dependent fashion were estimated. Intrathecal 5% dextrose (vehicle) did not elicit spinal anesthesia and was used as a negative control. In experiment 2, the percent of maximal possible effect (%MPE), duration, and area under the curves (AUCs) of the spinal blockades with caramiphen (1.5 µmol) were compared with those of lidocaine (3 μmol). In experiment 3, on an equipotent basis [50% effective dose (ED50), ED25, and ED75], the duration of
caramiphen on spinal anesthesia was compared with that of lidocaine. In experiment 4, blockade of Na+ currents by caramiphen and lidocaine was evaluated in a dose-dependent manner. In experiment 5, use-dose-dependent block of Na+ currents by
caramiphen and lidocaine at different frequencies (0.33 Hz, 1.00 Hz and 3.33 Hz) was performed.
Part 1 - in vivo studies
2.2. Animals
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 (IASP). Male Sprague-Dawley rats (300 to 350 g) were obtained from the National Laboratory Animal Centre (Taipei, Taiwan) and kept in the animal housing facilities
at China Medical University, with controlled humidity (approximately 50% relative humidity), room temperature (22C), and a 12-hour (6:00 AM to 6:00 PM) light/dark cycle.
2.3. Drugs
Caramiphen HCl and lidocaine HCl monohydrate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Before intrathecal injection, all drugs were freshly prepared in 5% dextrose as solution.
2.4. Intrathecal injection
Spinal anesthesia through intrathecal drug injection was performed on conscious rats as previously described (Gerner et al., 2000; Leung et al., 2013b). All animals were injected once in this experiment. Before intrathecal injection, each 50-µl of 0.5% lidocaine was injected into the right- and left-side of paraspinal space (0.5 cm in depth) which was 0.5 cm away from the mid-point of the longitudinal line of the lumbar 4-5 (L4-L5) intervertebral space. Two minutes later, a 27-gauge needle attached to a 100-µl syringe (Hamilton, Reno, Nevada) was inserted into the mid-line of the L4-L5 intervertebral space and 50-μl of drugs (carmiphen and lidocaine) or vehicle (5% dextrose) was injected. Rats were observed for the development of spinal anesthesia, as indicated by paralysis of both hind limbs (Chen et al., 2012c; Leung et
al., 2013a). For consistency, one trained investigator, who was blinded to the identity of the injected drug, was responsible for handling of all animals and behavioral evaluations.
2.5. Neurobehavioral measurements
Three neurobehavioral evaluations, which consisted of examinations of motor function, proprioception, and nociception were conducted after intrathecal injection (Chen et al., 2012a; Chen et al., 2012b). Animals were assessed before medication and at 1, 3, 5, 7, 10, 15, and 20 min afterwards, then again at 10-min interval until 1 h, at 15 min interval until 2 h and at 30 min interval until 3 h. The magnitude of spinal blockade in motor function, proprioception, and nociception was described as the percentage of possible effect (% PE). The maximum block in a time course of spinal anesthesia with drugs was described as the percentage of maximal possible effect (% MPE).
Motor function was evaluated through measuring 'the extensor postural thrust' of the right hind limb of the rats. The extensor thrust was measured as the gram force, which resisted contacting the platform through the rat heel applied to a digital platform balance (Mettler Toledo, PB 1502-S, Switzerland). The reduction in the force, representing decreased extensor muscle tone, was considered as a deficit of
motor function and expressed as a percentage of the control force. The pre-injection control value was considered as 0% MPE or 0% motor block. A force less than 20 g (also referred to as the weight of the 'flaccid limb') was interpreted as the absence of extensor postural thrust or a 100% MPE or100% motor block (Gerner et al., 2000; Thalhammer et al., 1995). The % PE is calculated by the equation:
% PE = (Gm-Gp)/(Gm-20) 100%
where Gm is the peak muscle force (g) of each rat before drug injection and Gp is the peak muscle force (g) of each rat after intrathecal injection (Leung et al., 2014; Leung et al., 2013a).
Proprioception was based on the resting posture (‘tactile placing’ and ‘hopping’) and postural reactions. Hopping response was examined through lifting the front half of the animal off the ground and lifting one hind limb at a time off the ground so that the animal was standing on just one limb. Then, the rat was moved laterally, which normally elicited a prompt hopping response with the weight-bearing limb in the direction of movement to prevent the animal from falling. A predominantly motor impairment exhibited a prompt but weaker than normal response. Conversely, with a predominantly proprioceptive block, delayed hopping was followed through greater lateral hops to prevent falling over or, in this case of complete block, no hopping at all. The functional deficit was quantified as 3 (normal or 0% MPE), 2 (slightly
impaired), 1 (severely impaired), and 0 (completely impaired or 100% MPE) (Hung et al., 2011a; Hung et al., 2011b).
Nociceptive reaction was assessed through the withdrawal reflex or vocalization elicited by pinching a skin fold over each rat's back at 1 cm from the proximal part of the tail, the lateral metatarsus of bilateral hind limbs, and the dorsal part of the mid-tail. At each testing time, only one pinch was given to each of the four testing sites, and the time interval between stimulations at different sites was around 2 s. The blockade of nociception was quantified as 4 (normal or 0% MPE), 3 (25% MPE), 2 (50% MPE), 1 (75% MPE), and 0 (absent or 100% MPE) (Chen et al., 2011a; Chen et al., 2012a).
2.6. The ED50, duration, and AUCs
After intrathecally injecting the rats with 4 different doses of each drug (n = 8 for each dose of each drug), the dose—response curves were constructed. The curves were then fitted using SAS Nonlinear (NLIN) Procedures (SAS Institute Inc., Carey, NC), and the value of ED50, defined as a dose that produced 50% spinal blockade, were obtained (Chen et al., 2011b; Minkin and Kundhal, 1999). Time to full recovery, defined as the interval from drug injection to full recovery (0% block or 0% MPE), produced by each drug was recorded. Furthermore, the AUCs of spinal anesthesia
with drug was obtained using Kinetica v 2.0.1 (MicroPharm International, USA).
Part 2 - in vitro studies
2.7. Cell culture
NG108-15 cells were cultured at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum(Invitrogen, Carlsbad, CA) and penicillin-streptomycin (100 units/ml, 100 µg/ml)(Invitrogen). NG108-15 cells were induced to differentiate into more mature neurons by being incubated in the above medium with 0.1 % fetal bovine serum, 10 μM retinoic acid and 30 μM forskolin for 3 days.
2.8. Measurement of voltage-gated Na+ currents
The electrophysiological measurements were carried out as previously described (Leung et al., 2010). Cells were voltage-clamped in the whole-cell mode. Borosilicate glass tubes (OD 1.5 mm, ID 1.10mm, Sutter Instrument, Novato, CA) were prepared with a micropipette puller (P-87, Sutter Instrument), and then fire polished by a microforge (Narishige Instruments, Inc., Sarasota,FL). The pipette resistance, when filled with intracellular solution, containing (mM): 120 CsCl; 20 TEA-Cl; 8 NaCl; 1 MgCl2; 1 EGTA; 10 HEPES and 5 MgATP (pH 7.25 adjusted with CsOH), was approximately 4-6 MΩ. The extracellular bathsolution contained (mM): 140 NaCl,
4 KCl, 1 MgCl2, 2 CaCl2, 10HEPES (pH 7.4 adjusted with NaOH). Membrane currents were recorded with an EPC-10 amplifier with Pulse 8.60 acquisition software and analyzed by Pulsefit 8.60 software (HEKA Electronik, Lambrecht, Germany). Data filtering and sampling frequencies were set at 2 and 10 kHz respectively. Once a whole-cell configuration was established, the membrane potential was held at -70 mV or -100 mV. Voltage-gated Ca2+ currents were not detected possibly due to their small amplitude and rapid rundown after establishment of whole-cell mode (Leung et al., 2012). To measure tonic block, Na+ currents were stimulated by -10 mV
depolarizing pulses every 20 s and the cells were treated with different concentrations of caramiphen or lidocaine. To assay frequency-dependence of block, the rates of -10 mV stimulations were set at 0.33, 1 or 3.33 Hz. Experiments were carried out at room temperature (~25ºC).
Concentration-inhibition curves are fitted by the Hill equation:
Idrug/Icontrol = 1/{1+([drug]/Kd)n}
where Idrug is the maximum current in the presence of drug, Icontrol is the maximum current in the absence of drug, [drug] is the extracellular drug concentration, Kd is the apparent dissociation constant, and n is the Hill coefficient.
2.9. Statistical analysis
Values are presented as mean ± S.E.M., IC50, or ED50 values with 95% confidence interval (95% CI). The differences between doses were analyzed using the one-way analysis of variance, followed by the Newman-Keuls test. Differences (Table 2) in %MPE, duration, and AUCs of drugs were evaluated by the Student's t-test. The differences in duration (Fig. 2) were evaluated by using 2-way ANOVA followed by pairwise Tukey’s HSD 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.
3. Results
3.1. Spinal anesthesia with caramiphen and lidocaine
Intrathecal caramiphen, as well as the local anesthetic lidocaine produced dose-dependent spinal blockade in motor, proprioception, and nociception in rats (Fig. 1). The ED50s of caramiphen and lidocaine obtained from their dose-response curves are shown in Table 1. On an ED50 basis, the rank of potencies of spinal blockade in proprioception, motor function, and nociception was caramiphen > lidocaine (P <
0.05; Table 1). Moreover, caramiphen, but not lidocaine, showed more sensory/nociceptive block (ED50) than motor block (P < 0.05; Table 1).
3.2. The %MPE, duration, and AUCs of caramiphen and lidocaine on spinal anesthesia
Caramiphen at a dose of 1.5 μmol showed 100, 100, and 100% of blockade (% MPE) in motor, proprioception, and nociception with duration of action of about 64, 86, and 133 min, respectively (Fig. 1), whereas intrathecal injection of 5% dextrose (vehicle) elicited no spinal blockade (Fig. 1). At a dose of 3.0 μmol, lidocaine exhibited 100, 100, and 100% of blockade in motor, proprioception, and nociception with duration of action of about 33, 34, and 44 min, respectively (Fig. 1). The %MPE, complete blockade time, time to full recovery, and AUCs of spinal anesthesia with caramiphen were markedly greater than those of lidocaine (P < 0.01; Table 2). Furthermore, caramiphen produced greater duration of sensory blockade than that of motor blockade (P < 0.01; Table 2).
3.3. The duration of spinal blockade with caramiphen and lidocaine
On the equianesthetic basis (ED25, ED50, ED75), the block duration caused by caramiphen was longer than that caused by lidocaine (P < 0.01; Fig. 2). Moreover,
(P < 0.01; Fig. 2). In in vivo studies, all rats recovered completely after intrathecal injection.
3.4. Block of Na+ currents by caramiphen did not exhibit state-dependence
NG108-15 cells express voltage-gated Na+ currents upon depolarization (Fig. 3A; (Gong et al., 2012)). The Na+ currents were blocked by 300 µM caramiphen (Fig. 3A) and 100 µM lidocaine (traces not shown). With a holding potential of -70 mV, at which voltage-gated Na+ channels of NG108-15 cells are partially inactivated (Gong
et al., 2012), the concentration-response curve of caramiphen inhibition gives an IC50 of 82.0 µM and a Hill coefficient of 0.97; lidocaine was slightly more potent in block, with an IC50 of 58.8 µM and a Hill coefficient of 1.0 (Fig. 3B). There is no significant difference between their IC50s (Fig. 3B). When the membrane potential was held at -100 mV, at which voltage-gated Na+ channels of NG108-15 cells are fully available (Gong et al., 2012), caramiphen block had an IC50 of 99.5 µM and a Hill coefficient of 1.2, while lidocaine block had an IC50 of 238.3 µM and a Hill coefficient of 1.0 (Fig. 3C). Thus, while lidocaine had a reduced potency at a hyperpolarized holding potential (that is, exhibiting state-dependence of block), caramiphen did not have a significant state-dependence of block. Thus, at -100 mV holding potential (Fig. 3C), caramiphen was more potent than lidocaine; however, at -70 mV holding potential
(which is close to resting membrane potential), potencies of block were indeed comparable for caramiphen and lidocaine (Fig. 3B).
3.5. Inhibition of Na+ currents by caramiphen has weak frequency-dependence Inhibition of Na+ currents by lidocaine displays frequency-dependence, such that the blockade increases with the frequency of stimulation (Hille, 2001; Leung et al., 2010). As shown in Fig. 4, the cumulative block by lidocaine augmented with
successive pulses and stimulation frequencies (0.33 to 3.33 Hz), caramiphen showed a much weaker frequency-dependence of block than lidocaine.
4. Discussion
This study showed for the first time that caramiphen, but lidocaine had more sensory/nociceptive block (ED50) than motor block. Although it has been known that caramiphen elicited a higher potency than lidocaine in spinal anesthesia (Chen et al., 2010), in this current study we also demonstrated that the duration produced by caramiphen was greater than that produced by lidocaine on an equipotent basis. The effect of caramiphen and lidocaine on voltage-gated Na channels was investigated and it was shown that they inhibited the currents with similar potency at -70 mV holding
Moreover, caramiphen block had a weak frequency-dependence compared with lidocaine block.
It has been well established that the preferential sensory versus motor block is a feature claimed for local anesthesia with several local anesthetic agents (Markham and Faulds, 1996; McLure and Rubin, 2005). Interestingly, our data showed that the potency (ED50) of caramiphen in nociceptive blockade was 1.33-fold greater than that in motor blockade. In addition, caramiphen also produced a longer duration of sensory block than that of motor block. It is in concordance with the clinical choice that bupivacaine is the local anesthetic of selection when a more potent sensory than motor block is required (Gurlit et al., 2004; Hung et al., 2009; Nau et al., 2000).
Most local anesthetics currently in clinical applications were absorbed rapidly
via blood vessels, therefore, and their benefits in local anesthesia or providing
postoperative analgesia might limit due to their short duration of local anesthesia. Our study demonstrated that caramiphen (1.5 μmol) produced a complete sensory block and had a longer duration of spinal anesthesia when compared with lidocaine (3.0 μmol). At equipotent doses (ED25, ED50, ED75), the duration of spinal anesthesia caused by caramiphen was longer than that caused by lidocaine. The effect may be beneficial for patients who require long-acting spinal analgesia. More efforts on peripheral/sciatic nerve blockade as well as related neural toxicities will be warranted
in the future before further consideration of caramiphen as a local anesthetic for clinical trials.
Injection of the local anesthetics (i.e. lidocaine) produced sciatic nerve blockade, infiltrative skin anesthesia, and spinal anesthesia according to the characteristic of blockade of voltage-gated Na+ channels in the nervous tissues (Borgeat and Aguirre,
2010; Vegh et al., 2006). Because caramiphen has been shown to have a local anesthetic effect, it may block voltage-gated Na+ channels. Our study showed that caramiphen, unlike lidocaine, exhibited a very shallow state-dependence of block (Fig. 3); its block was only slightly more potent at -70 mV (when Na+ channels are partially inactivated) than at -100 mV (complete channel availability) (Gong et al., 2012). This implies that caramiphen binds to the partially inactivated state of Na+ channels with only slightly higher affinity. For the experiments performed to construct the concentration- inhibition curves, a very low frequency (0.05 Hz) was used to stimulate the cells before and during drug addition. Thus, caramiphen and lidocaine elicited tonic channel block. Caramiphen was more potent than lidocaine at -100 mV holding potential, whilst the two drugs were almost equi-potent at -70 mV holding potential.
Voltage-dependent Na+ channels are the main targets for analgesic and anesthetic drugs. These substances show two types of block, namely, tonic (or closed channel)
and use-dependent (frequency-dependent or phasic) block (Chernoff and Strichartz, 1989). In our in vivo experiments, caramiphen at 1.5 μmol and lidocaine at 3 μmol produced complete (100%) spinal blockade of motor function, proprioception, and nociception. In our in vitro experiments, we demonstrated that caramiphen and lidocaine at a concentration of 300 μM exhibited 100% and 60% blockade, respectively, of Na+ currents at holding potential of –100 mV.
The use-dependence of Na+ channel blockade suggests that one of the drug binding sites is within the channel internal cavity, which becomes accessible to local anesthetics upon opening of the cytoplasmic activation gate (Catterall, 2000; Ragsdale et al., 1994). In this present study lidocaine has a stronger frequency-dependent block of Na+ currents, whereas caramiphen displays a much weaker frequency-dependence of block. Caramiphen anticonvulsant activity has been hypothesized to be due to high-affinity binding to sigma recognition sites in brain (DeHaven-Hudkins et al., 1995). Hanner et al. (Hanner et al., 1996) previously stated that the sigma1-receptor is widely located within the rat central nervous system, particularly neuronal cell bodies and dendrites of brain stem, hypothalamus, and hippocampus, but not axon fibers or terminals, while the sigma-1 receptor on the other hand is known to be a facilitator of nociception. For example, the direct activation of spinal sigma-1 receptors via
responses to both mechanical and thermal stimulation (Roh et al., 2008). Furthermore, spinal sigma-1 receptors may play an important pro-nociceptive modulatory role in spinally mediated pain sensation by PKA- and PKC-dependent phosphorylation of the N-methyl-D-aspartate receptor subunit 1 (Kim et al., 2008). Here we could not exclude, that spinal caramiphen elicited more potent spinal blockades that lidocaine, from caramiphin binding to the sigma1-receptor.
In summary, our resulting data demonstrated that caramiphen produced dose-dependent spinal blockade with a more sensory-selective action over motor blockade and elicited a longer duration of action when compared with lidocaine. The local (spinal) anesthetic properties of caramiphen are likely attributable to its ability to inhibit voltage-gated Na+ currents.
Acknowledgments
The authors gratefully acknowledge the financial support provided for this study by the National Science Council of Taiwan (NSC 100-2314-B-039 -017 -MY3).
References
Borgeat, A., Aguirre, J., 2010. Update on local anesthetics. Curr Opin Anaesthesiol 23, 466-471.
Catterall, W.A., 2000. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26, 13-25.
Chen, Y.W., Chu, C.C., Chen, Y.C., Hung, C.H., Wang, J.J., 2011a. Intrathecal propranolol displays long-acting spinal anesthesia with a more sensory-selective action over motor blockade in rats. Eur J Pharmacol 667, 208-214.
Chen, Y.W., Chu, C.C., Chen, Y.C., Kan, C.D., Wang, J.J., 2012a. Promazine and chlorpromazine for prolonged spinal anesthesia in rats. Neurosci Lett 521, 115-118.
prolonged spinal blockades of sensory and motor functions in rats. Eur J Pharmacol 693, 31-36.
Chen, Y.W., Chu, C.C., Chen, Y.C., Leung, Y.M., Wang, J.J., 2012c. Spinal blockades of class I antiarrythmic drugs with bupivacaine by isobolographic analysis in rats. Neurosci Lett 528, 46-50.
Chen, Y.W., Chu, C.C., Chen, Y.C., Wang, J.J., Hung, C.H., 2010. Isobolographic analysis of caramiphen and lidocaine on spinal anesthesia in rats. Neurosci Lett 469, 174-178.
Chen, Y.W., Chu, C.C., Chen, Y.C., Wang, J.J., Hung, C.H., 2011b. The local anesthetic effect of memantine on infiltrative cutaneous analgesia in the rat. Anesthesia and analgesia 113, 191-195.
Chernoff, D.M., Strichartz, G.R., 1989. Tonic and phasic block of neuronal sodium currents by 5-hydroxyhexano-2',6'-xylide, a neutral lidocaine homologue. J Gen Physiol 93, 1075-1090.
Church, J., Shacklock, J.A., Baimbridge, K.G., 1991. Dextromethorphan and
phencyclidine receptor ligands: differential effects on K(+)- and NMDA-evoked increases in cytosolic free Ca2+ concentration. Neurosci Lett 124, 232-234. DeHaven-Hudkins, D.L., Allen, J.T., Hudkins, R.L., Stubbins, J.F., Tortella, F.C.,
1995. Anticonvulsant activity of caramiphen analogs. Life Sci 56, 1571-1576. Essig, C.F., Hampson, J.L., et al., 1950. Effect of parpanit on brain wave changes
induced by diisopropyl fluorophosphate. Science 111, 38.
Figueiredo, T.H., Aroniadou-Anderjaska, V., Qashu, F., Apland, J.P., Pidoplichko, V., Stevens, D., Ferrara, T.M., Braga, M.F., 2011. Neuroprotective efficacy of caramiphen against soman and mechanisms of action. Br J Pharmacol. Fletcher, E.J., Church, J., Abdel-Hamid, K., MacDonald, J.F., 1995. Blockade by
sigma site ligands of N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurones. Br J Pharmacol 116, 2791-2800. Gerner, P., Nakamura, T., Quan, C.F., Anthony, D.C., Wang, G.K., 2000. Spinal
tonicaine: potency and differential blockade of sensory and motor functions. Anesthesiology 92, 1350-1360.
Gong, C.L., Wong, K.L., Cheng, K.S., Kuo, C.S., Chao, C.C., Tsai, M.F., Leung, Y.M., 2012. Inhibitory effects of magnolol on voltage-gated Na+ and K+ channels of NG108-15 cells. Eur J Pharmacol 682, 73-78.
Gurlit, S., Reinhardt, S., Mollmann, M., 2004. Continuous spinal analgesia or opioid-added continuous epidural analgesia for postoperative pain control after hip replacement. Eur J Anaesthesiol 21, 708-714.
Hanner, M., Moebius, F.F., Flandorfer, A., Knaus, H.G., Striessnig, J., Kempner, E., Glossmann, H., 1996. Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci U S A 93, 8072-8077.
Isoflurane for spinal anesthesia in the rat. Neurosci Lett 501, 138-142.
Hung, C.H., Chu, C.C., Chen, Y.C., Chen, Y.W., Li, Z.Y., Wang, J.J., 2011b. Spinal anesthesia with diphenhydramine and pheniramine in rats. Eur J Pharmacol 673, 20-24.
Hung, C.H., Chu, C.C., Chen, Y.C., Liu, K.S., Chen, Y.W., Wang, J.J., 2012.
Cutaneous analgesia and systemic toxicity of carbetapentane and caramiphen in rats. Reg Anesth Pain Med 37, 34-39.
Hung, C.H., Wang, J.J., Chen, Y.C., Chu, C.C., Chen, Y.W., 2009. Intrathecal oxybuprocaine and proxymetacaine produced potent and long-lasting spinal anesthesia in rats. Neurosci Lett 454, 249-253.
Kawaura, K., Miki, R., Shima, E., Honda, S., Soeda, F., Shirasaki, T., Takahama, K., 2010. Antidepressant-like effect of centrally acting non-narcotic antitussive caramiphen in a forced swimming test. Neurosci Lett 481, 188-190.
Kim, H.W., Roh, D.H., Yoon, S.Y., Seo, H.S., Kwon, Y.B., Han, H.J., Kim, K.W., Beitz, A.J., Lee, J.H., 2008. Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice. Br J Pharmacol 154, 1125-1134.
Leung, Y.M., Chu, C.C., Kuo, C.S., Chen, Y.W., Hung, C.H., Wang, J.J., 2014. Isobolographic analysis of interaction between nisoxetine- and mepivacaine-induced spinal blockades in rats. Fundamental & clinical pharmacology 28,
88-94.
Leung, Y.M., Chu, C.C., Kuo, C.S., Chen, Y.W., Wang, J.J., 2013a. Nisoxetine blocks sodium currents and elicits spinal anesthesia in rats. Pharmacol Rep 65, 350-357.
Leung, Y.M., Tzeng, J.I., Kuo, C.S., Chen, Y.W., Chu, C.C., Wang, J.J., 2013b. The use of carbetapentane for spinal anesthesia and use-dependent block of sodium currents. Eur J Pharmacol 714, 366-372.
Leung, Y.M., Wu, B.T., Chen, Y.C., Hung, C.H., Chen, Y.W., 2010. Diphenidol inhibited sodium currents and produced spinal anesthesia. Neuropharmacology 58, 1147-1152.
Markham, A., Faulds, D., 1996. Ropivacaine. A review of its pharmacology and therapeutic use in regional anaesthesia. Drugs 52, 429-449.
McLure, H.A., Rubin, A.P., 2005. Review of local anaesthetic agents. Minerva Anestesiol 71, 59-74.
Minkin, S., Kundhal, K., 1999. Likelihood-based experimental design for estimation of ED50. Biometrics 55, 1030-1037.
Nau, C., Wang, S.Y., Strichartz, G.R., Wang, G.K., 2000. Block of human heart hH1 sodium channels by the enantiomers of bupivacaine. Anesthesiology 93, 1022-1033.
determinants of state-dependent block of Na+ channels by local anesthetics. Science 265, 1724-1728.
Roh, D.H., Kim, H.W., Yoon, S.Y., Seo, H.S., Kwon, Y.B., Kim, K.W., Han, H.J., Beitz, A.J., Lee, J.H., 2008. Intrathecal administration of sigma-1 receptor agonists facilitates nociception: involvement of a protein kinase C-dependent pathway. J Neurosci Res 86, 3644-3654.
Sciarra, D., Carter, S., Merritt, H.H., 1949. Caramiphen hydrochloride in the
treatment of diseases of the basal ganglions. J Am Med Assoc 141, 1226-1229. Snyder, G.G., 1953. An evaluation of caramiphen ethane disulfonate as an antitussive
agent. Laryngoscope 63, 1008-1013.
Thalhammer, J.G., Vladimirova, M., Bershadsky, B., Strichartz, G.R., 1995. Neurologic evaluation of the rat during sciatic nerve block with lidocaine. Anesthesiology 82, 1013-1025.
Vandermeersch, E., Kick, O., Mollmann, M., de Gouw, N., Van Aken, H., 1991. [CSE--the combination of spinal and epidural anesthesia]. Reg Anaesth 14, 108-112.
Vegh, V., Cizmarik, J., Hahnenkamp, K., 2006. Is there a place for local anesthetics structurally different from classical amid or ester local anesthetics? Curr Opin Anaesthesiol 19, 509-515.
Table 1. The 50% effective dose (ED50) of spinal anesthesia with caramiphen and lidocaine in the rat
Drug
ED50 (95% CI)
Motor Function Proprioception Nociception Caramiphen 0.80 (0.73–0.90)a 0.64 (0.57–0.73)a 0.60 (0.53–0.69)a,b Lidocaine 1.05 (0.95–1.20) 1.02 (0.95–1.14) 0.90 (0.81–1.02) The ED50s (μmol) of caramiphen and lidocaine were constructed from Fig. 1 using SAS Nonlinear (NLIN) Procedures. The symbol (a) indicates P < 0.05 when caramiphen compared with lidocaine. The symbol (b) indicates P < 0.05 when nociception compared with motor function.
Table 2. The percentage of maximal possible effect (%MPE), duration, area under the curves (AUCs) of spinal anesthesia with caramiphen, lidocaine, and vehicle (5% dextrose) in rats
%MPE Duration (min) AUCs (%MPE x min)
Complete blockade time Time to full recovery
Motor Function Caramiphen 100 ± 0 18 ± 3b 64 ± 3b 3637 ± 269b Lidocaine 100 ± 0 10 ± 3 34 ± 3 1705 ± 196 5% dextrose － － － － Proprioception Caramiphen 100 ± 0 26 ± 4a 86 ± 5b 4935 ± 322b Lidocaine 100 ± 0 12 ± 2 36 ± 3 1840 ± 102 5% dextrose － － － － Nociception Caramiphen 100 ± 0 49 ± 7a,c 133 ± 13b,d 9059 ± 898b,d Lidocaine 100 ± 0 16 ± 3 39 ± 3 2159 ± 221 5% dextrose － － － －
Spinal anesthesia (meanS.E.M.) with caramiphen at 1.5 μmol (n = 8) and lidocaine at 3.0 μmol (n = 8) were obtained from Fig. 1. These symbols (a, b) indicate P < 0.01 and P < 0.001 when caramiphen compared with lidocaine, respectively. Those symbols (c, d) indicate P < 0.01
A. Motor B. Proprioception C. Nociception 0 15 30 45 60 75 90 105 120 135 150 165 180 % P E ( po ss ib le e ffe ct ) 0 20 40 60 80 100 1.50 mol Caramiphen 1.13 mol Caramiphen 0.75 mol Caramiphen 0.50 mol Caramiphen 5% dextrose 0 15 30 45 60 75 90 105 120 135 150 165 180 % P E ( po ss ib le e ffe ct ) 0 20 40 60 80 100 Time (min) 0 15 30 45 60 75 90 105 120 135 150 165 180 % P E ( po ss ib le e ffe ct ) 0 20 40 60 80 100 D. Motor E. Proprioception F. Nociception 0 5 10 15 20 25 30 35 40 45 50 % P E ( po ss ib le e ffe ct ) 0 20 40 60 80 100 3.00 mol Lidocaine 1.65 mol Lidocaine 1.00 mol Lidocaine 0.60 mol Lidocaine 0 5 10 15 20 25 30 35 40 45 50 % P E ( po ss ib le e ffe ct ) 0 20 40 60 80 100 Time (min) 0 5 10 15 20 25 30 35 40 45 50 % P E ( po ss ib le e ffe ct ) 0 20 40 60 80 100
Fig. 1.
25 50 75 F ul l R ec ov er y T im e (m in ) 0 10 20 30 40 Caramiphen Lidocaine ED ( effective dose ) 25 50 75 F ul l R ec ov er y T im e (m in ) 0 10 20 30 40 25 50 75 F ul l R ec ov er y T im e (m in ) 0 10 20 30 40
Motor
Proprioception
Nociception
Caramiphen > Lidocaine Caramiphen > Lidocaine Caramiphen > LidocaineFig. 2.
Figure Legends
Fig. 1. Time courses of spinal anesthesia (%PE) with caramiphen (1.5-0.5 μmol; A-C) and lidocaine (3.0-0.5 μmol; D-F) in rats. The control group is the group that injects 5% dextrose (vehicle) intrathecally. Carmiphen and lidocaine produced spinal anesthesia in dose-dependent manners. Intrathecal injection of 50 μL caramiphen at 1.5 μmol (A-C) elicited a complete nociceptive block (100% PE) lasting for
approximately 49 min. The spinal block recovered fully after 3 h. With 50 μL of lidocaine at 3.0 μmol, the complete nociceptive block lasted for approximately 20 min and recovered fully after approximately 50 min (D-F).The % possible effect (%PE) was measured at various intervals as described in the Methods section. At each drug concentration, eight rats were investigated and their %PE S.E.M. was plotted against the corresponding time interval.
Fig. 2. Full recovery time of action of caramiphen and lidocaine on spinal blockades of motor, proprioception, and nociception at equipotent doses [50% effective dose (ED50), ED25, and ED75] (n = 8 at each testing point). Values are expressed as mean ± S.E.M. The differences in duration were evaluated by using 2-way ANOVA followed by pairwise Tukey’s HSD test.
Fig. 3. Block of voltage-gated sodium currents by caramiphen and lidocaine in differentiated NG108-15 cells. (A) Depolarization (-10 mV)-triggered sodium currents in the absence and presence of 300M caramiphen. (B) Concentration-response curves constructed for caramiphen and lidocaine block of sodium currents are fitted with the Hill equation. (see Methods). Membrane potential was held at -70
block of sodium currents are fitted with the Hill equation. (see Methods). Membrane potential was held at -100 mV. Each data point represents mean ± S.E.M. from 3-6 cells.
Fig. 4. Differentiated NG108-15 cells were stimulated with -10 mV pulses at different frequencies (0.33 Hz, 1 Hz and 3.33 Hz; A-C respectively) in the absence of drug, 50M caramiphen or 50M lidocaine. The peak current amplitudes of the second to tenth pulses (I) are normalized with the peak current amplitude of the first pulse (Iinitial) and plotted against the number of pulse. Each data point represents mean ±S.E.M. from 4-6 cells.
