N-Phenylethyl Amitriptyline in Rat Sciatic Nerve Blockade

Background: The antidepressant amitriptyline is commonly used orally for the treatment of chronic pain, particularly neuropathic pain, which is thought to be caused by high-frequency ectopic discharge. Among its many properties, amitriptyline is a potent Na+ channel blocker in vitro, has local anesthetic properties in vivo, and confers additional blockade at high stimulus-discharge rates (use-dependent blockade). As with other drug modifications, adding a phenylethyl group to obtain a permanently charged quaternary ammonium derivative may improve these advantageous properties.

Methods: The electrophysiologic properties of N-phenylethyl amitriptyline were assessed in cultured neuronal GH3 cells with the whole cell mode of the patch clamp technique, and the therapeutic range and toxicity were evaluated in the rat sciatic nerve model.

Results: In vitro, N-phenylethyl amitriptyline at 10 [mu]m elicits a greater block of Na+ channels than amitriptyline (resting block of approximately 90%vs. approximately 15%). This derivative also retains the attribute of amitriptyline in evoking high-degree use-dependent blockade during repetitive pulses. In vivo, duration to full recovery of nociception in the sciatic nerve model was 1,932 +/- 72 min for N-phenylethyl amitriptyline at 2.5 mm (n = 7) versus 72 +/- 3 min for lidocaine at 37 mm (n = 4; mean +/- SEM). However, there was evidence of neurotoxicity at 5 mm.     

Amitriptyline versus Bupivacaine in Rat Sciatic Nerve Blockade

Background: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro.

Methods: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated.

Results: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mm, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mm, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mm, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mm) and 656 +/- 27 min (10 mm) versus 155 +/- 9 min for bupivacaine (15.4 mm). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 [mu]m) at -150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 [mu]m) at -60 mV. High-frequency stimulation at 3 [mu]m caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline.     

Local anesthetic properties of prenylamine.

BACKGROUND: Local anesthetics that produce analgesia of long duration with minimal impairment of autonomic functions are highly desirable for pain management in the clinic. Prenylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels have not been studied thus far. METHODS: The authors characterized the tonic and use-dependent prenylamine block of native Na(+) channels in cultured rat neuronal GH3 cells during whole cell voltage clamp conditions and the local anesthetic effect of prenylamine by neurologic evaluation of sensory and motor functions of sciatic nerve during neural block in rats. RESULTS: Prenylamine elicits both use-dependent block of Na(+) channels during repetitive pulses (3 microm prenylamine produced 50% block at 5 Hz) and tonic block for both resting and inactivated Na(+) channels. The 50% inhibitory concentration for prenylamine was 27.6 +/- 1.3 microm for resting channels and 0.75 +/- 0.02 microm for inactivated channels. Furthermore, in vivo data show that 10 mm prenylamine produced a complete sciatic nerve block of motor function, proprioceptive responses, and nociceptive responses that lasted approximately 27, 34, and 24 h, respectively. Rats injected with 15.4 mm bupivacaine, a known local anesthetic currently used for pain management, had a significantly shorter duration of blockade (< 2 h) compared with rats injected with prenylamine. CONCLUSIONS: The data presented here demonstrate that prenylamine possesses local anesthetic properties in vitro and elicits prolonged local anesthesia in vivo.     

Spinal tonicaine: potency and differential blockade of sensory and motor functions

BACKGROUND: Long-acting local anesthetics are beneficial for the management of postoperative pain and chronic pain. The authors recently reported that a single injection of N-beta-phenylethyl-lidocaine (tonicaine), a quaternary lidocaine derivative, effectively blocks rat sciatic nerve function four to nine times longer than lidocaine, with a predominance of sensory versusmotor blockade. The purposes of this study were to measure directly the potency of this charged drug by internal perfusion of cultured neuronal cells, and to evaluate the differential blockade of sensory versus motor function via spinal route in rats. METHODS: The tonic and additional use-dependent blockade of Na+ currents by internal tonicaine was assayed in cultured GH3 cells during whole cell voltage-clamp conditions. In addition, tonicaine was injected into the intrathecal space of rats at intervertebral space L4-L5, and the proprioceptive, motor, and sensory functions, and tissue integrity, subsequently were evaluated. RESULTS: Internal application of tonicaine in GH3 cells revealed that it was approximately 80 times more potent in blocking Na+ currents than was externally applied lidocaine. In vivotesting in a rat neuraxial anesthesia model showed that tonicaine at 0.5 mm produced blockade that lasted much longer than that produced by bupivacaine even at approximately a 55 times higher concentration (28.8 mm). Tonicaine spinal block also produced a longer duration of sensory than motor blockade (112.5 +/- 16.3 min vs. 45.8 +/- 7.1 min). Evidence of neurotoxicity was seen at a concentration of 1.0 mm. CONCLUSION: In vitro testing shows that tonicaine displays a higher affinity for the local anesthetic binding site than does lidocaine; in vivotesting indicates that tonicaine elicits sensory blockade of a duration significantly longer than that elicited by bupivacaine. Tonicaine, however, has a narrow therapeutic index, with substantial neurotoxicity at 1 mm in rats, and may have limited clinical value.     

Assessment of differential blockade by amitriptyline and its N-methyl derivative in different species by different routes

BACKGROUND: Increasing the duration of local anesthesia and/or creating greater differential blockade (i.e., selective block of pain-transmitting nerve fibers) has been attempted by modifying currently available agents. Most drugs show a different profile depending on the model or species studied. This study was designed to investigate the differential nerve-blocking properties of amitriptyline and its quaternary ammonium derivative in rats and sheep. METHODS: The Na+ channel-blocking properties of N-methyl amitriptyline were determined with the patch clamp technique in cultured GH(3) cells. Various functions (motor, nociception, proprioception-ataxia) were compared in rats (spinal and sciatic nerve blockade) and sheep (spinal blockade) with amitriptyline, N-methyl amitriptyline, lidocaine, and bupivacaine (partially from historical data). RESULTS: In vitro testing revealed N-methyl amitriptyline to be a potent Na+ channel blocker similar to amitriptyline but with a much longer duration of action. All drug concentrations tested in both the sciatic nerve model and the spinal block model produced no significant differential blockade in rats. Three of six rats in the 20-mM N-methyl amitriptyline group showed residual blockade 4 days after sciatic nerve injection. However, in the sheep spinal model, amitriptyline and in particular N-methyl amitriptyline displayed significant differential blockade at most time points. Sheep data for lidocaine and bupivacaine seemed to be more comparable to the clinical experience in humans than did rat data. CONCLUSIONS: Amitriptyline and N-methyl amitriptyline are potent Na+ channel blockers and show greater differential blockade in sheep than in rats. This differential blockade in sheep is greater than that produced by lidocaine or bupivacaine.     

Local anesthetic properties of a novel derivative, N-methyl doxepin, versus doxepin and bupivacaine

Among various tricyclic antidepressants, doxepin and amitriptyline are also long-acting local anesthetics. We synthesized a new compound, N-methyl doxepin, and investigated whether this derivative possesses local anesthetic properties. N-methyl doxepin and doxepin were tested in a rat sciatic nerve model at 2.5, 5.0, and 10 mM. Proprioceptive, motor, and nociceptive blockade were evaluated and compared with those induced by 0.5% bupivacaine. Block of Na(+) channels by N-methyl doxepin and doxepin was assessed in cultured pituitary tumor cells under voltage clamp conditions. N-methyl doxepin elicited complete nociceptive blockade that generally lasted longer than that caused by doxepin (e.g., approximately 7.4 h versus 5.3 h at 10 mM). Significant differences were observed for full recovery of function at all concentrations and for the duration of complete blockade except at 2.5 mM. Bupivacaine at 0.5% (15.4 mM) was less effective in producing complete blockade (approximately 1.5 h) than N-methyl doxepin and doxepin. Both doxepin and N-methyl doxepin were potent Na(+) channel blockers, although N-methyl doxepin displayed a slower wash-in rate. No morphological alterations were detected in cross-sectioned sciatic nerve specimens with these three drugs. We conclude that N-methyl doxepin is a potent Na(+) channel blocker and a long-acting local anesthetic for rat sciatic nerve blockade. IMPLICATIONS: N-methyl doxepin and doxepin are both potent Na(+) channel blockers; they elicit rat sciatic nerve block lasting longer than that induced by bupivacaine and seem to be nontoxic to peripheral nerves at concentrations up to 10 mM.     

Assessment of Differential Blockade by Amitriptyline and Its N-Methyl Derivative in Different Species by Different Routes

Background: Increasing the duration of local anesthesia and/or creating greater differential blockade (i.e., selective block of pain-transmitting nerve fibers) has been attempted by modifying currently available agents. Most drugs show a different profile depending on the model or species studied. This study was designed to investigate the differential nerve-blocking properties of amitriptyline and its quaternary ammonium derivative in rats and sheep.

Methods: The Na+ channel-blocking properties of N-methyl amitriptyline were determined with the patch clamp technique in cultured GH3 cells. Various functions (motor, nociception, proprioception-ataxia) were compared in rats (spinal and sciatic nerve blockade) and sheep (spinal blockade) with amitriptyline, N-methyl amitriptyline, lidocaine, and bupivacaine (partially from historical data).

Results: In vitro testing revealed N-methyl amitriptyline to be a potent Na+ channel blocker similar to amitriptyline but with a much longer duration of action. All drug concentrations tested in both the sciatic nerve model and the spinal block model produced no significant differential blockade in rats. Three of six rats in the 20-mm N-methyl amitriptyline group showed residual blockade 4 days after sciatic nerve injection. However, in the sheep spinal model, amitriptyline and in particular N-methyl amitriptyline displayed significant differential blockade at most time points. Sheep data for lidocaine and bupivacaine seemed to be more comparable to the clinical experience in humans than did rat data.     

Spinal Tonicaine: Potency and Differential Blockade of Sensory and Motor Functions

Background: Long-acting local anesthetics are beneficial for the management of postoperative pain and chronic pain. The authors recently reported that a single injection of N-[beta]-phenylethyl-lidocaine (tonicaine), a quaternary lidocaine derivative, effectively blocks rat sciatic nerve function four to nine times longer than lidocaine, with a predominance of sensory versus motor blockade. The purposes of this study were to measure directly the potency of this charged drug by internal perfusion of cultured neuronal cells, and to evaluate the differential blockade of sensory versus motor function via spinal route in rats.

Methods: The tonic and additional use-dependent blockade of Na+currents by internal tonicaine was assayed in cultured GH3 cells during whole cell voltage-clamp conditions. In addition, tonicaine was injected into the intrathecal space of rats at intervertebral space L4-L5, and the proprioceptive, motor, and sensory functions, and tissue integrity, subsequently were evaluated.

Results: Internal application of tonicaine in GH3 cells revealed that it was ~80 times more potent in blocking Na+ currents than was externally applied lidocaine. In vivo testing in a rat neuraxial anesthesia model showed that tonicaine at 0.5 mm produced blockade that lasted much longer than that produced by bupivacaine even at ~a 55 times higher concentration (28.8 mm). Tonicaine spinal block also produced a longer duration of sensory than motor blockade (112.5 +/- 16.3 min vs. 45.8 +/- 7.1 min). Evidence of neurotoxicity was seen at a concentration of 1.0 mm.     

Effect of drugs used for neuropathic pain management on tetrodotoxin-resistant Na(+) currents in rat sensory neurons

BACKGROUND: Tetrodotoxin-resistant Na(+) channels play an important role in generation and conduction of nociceptive discharges in peripheral endings of small-diameter axons of the peripheral nervous system. Pathophysiologically, these channels may produce ectopic discharges in damaged nociceptive fibers, leading to neuropathic pain syndromes. Systemically applied Na(+) channel--blocking drugs can alleviate pain, the mechanism of which is rather unresolved. The authors investigated the effects of some commonly used drugs, i.e., lidocaine, mexiletine, carbamazepine, amitriptyline, memantine, and gabapentin, on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglia. METHODS: Tetrodotoxin-resistant Na(+) currents were recorded in the whole-cell configuration of the patch-clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Half-maximal blocking concentrations were derived from concentration-inhibition curves at different holding potentials (-90, -70, and -60 mV). RESULTS: Lidocaine, mexiletine, and amitriptyline reversibly blocked tetrodotoxin-resistant Na(+) currents in a concentration- and use-dependent manner. Block by carbamazepine and memantine was not use-dependent at 2 Hz. Gabapentin had no effect at concentrations of up to 3 mm. Depolarizing the membrane potential from -90 mV to -60 mV reduced the available Na(+) current only by 23% but increased the sensitivity of the channels to the use-dependent blockers approximately fivefold. The availability curve of the current was shifted by 5.3 mV to the left in 300 microm lidocaine. CONCLUSIONS: Less negative membrane potential and repetitive firing have little effect on tetrodotoxin-resistant Na(+) current amplitude but increase their sensitivity to lidocaine, mexiletine, and amitriptyline so that concentrations after intravenous administration of these drugs can impair channel function. This may explain alleviation from pain by reducing firing frequency in ectopic sites without depressing central nervous or cardiac excitability.     

Amitriptyline versus bupivacaine in rat sciatic nerve blockade

BACKGROUND: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro. METHODS: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated. RESULTS: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mM, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mM, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mM, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mM) and 656 +/- 27 min (10 mM) versus 155 +/- 9 min for bupivacaine (15.4 mM). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 microM) at - 150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 microM) at -60 mV. High-frequency stimulation at 3 microM caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline. CONCLUSION: Amitriptyline is a more potent blocker of neuronal Na+ channels than bupivacaine in vivo and in vitro. These findings suggest that amitriptyline could extend its clinical usefulness for peripheral nerve blockade.     

Local Anesthetic Properties of Prenylamine

Background : Local anesthetics that produce analgesia of long duration with minimal impairment of autonomic functions are highly desirable for pain management in the clinic. Prenylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels have not been studied thus far.

Methods : The authors characterized the tonic and use-dependent prenylamine block of native Na+ channels in cultured rat neuronal GH3 cells during whole cell voltage clamp conditions and the local anesthetic effect of prenylamine by neurologic evaluation of sensory and motor functions of sciatic nerve during neural block in rats.

Results : Prenylamine elicits both use-dependent block of Na+ channels during repetitive pulses (3 [mu]m prenylamine produced 50% block at 5 Hz) and tonic block for both resting and inactivated Na+ channels. The 50% inhibitory concentration for prenylamine was 27.6 +/- 1.3 [mu]m for resting channels and 0.75 +/- 0.02 [mu]m for inactivated channels. Furthermore, in vivo data show that 10 mm prenylamine produced a complete sciatic nerve block of motor function, proprioceptive responses, and nociceptive responses that lasted approximately 27, 34, and 24 h, respectively. Rats injected with 15.4 mm bupivacaine, a known local anesthetic currently used for pain management, had a significantly shorter duration of blockade (< 2 h) compared with rats injected with prenylamine.     

Cutaneous analgesia after transdermal application of amitriptyline versus lidocaine in rats

Amitriptyline, a tricyclic antidepressant, has potent local anesthetic properties. However, there is no report of cutaneous analgesic effects after transdermal application. We report here that transdermally applied amitriptyline is more potent than lidocaine in providing cutaneous analgesia in rats. Solutions of amitriptyline base in 50, 100, and 500 mM concentrations were applied as a patch to rats, and their effects were compared with those of lidocaine base at the same concentrations and of the vehicle alone (45% water, 45% isopropyl alcohol, and 10% glycerin). Rats in each test group developed a concentration-dependent cutaneous analgesic block in the areas to which the drugs were applied; however, amitriptyline produced a longer block than lidocaine at the same concentration. The development of amitriptyline as a longer-lasting topical analgesic may improve our ability to treat chronic pain, such as neuropathic pain and neuralgia, and to prevent pain in procedures such as venipuncture. IMPLICATIONS: The tricyclic antidepressant amitriptyline, often used perorally for the management of chronic pain, is shown here to be more potent than lidocaine in providing cutaneous analgesia when applied transdermally with an occlusive dressing in rats.     

The effects of class Ic antiarrhythmics on tetrodotoxin-resistant Na+ currents in rat sensory neurons

IV or oral administration of antiarrhythmics has been reported to be effective for relieving neuropathic pain. Recent reports have indicated that tetrodotoxin-resistant (TTX-R) Na(+) channels play important roles in the nerve conduction of nociceptive sensation. In the present study, we investigated the effects of flecainide, pilsicainide (class Ic antiarrhythmics), and lidocaine (a class Ib drug) on TTX-R Na(+) currents in rat dorsal root ganglion neurons using the whole-cell patch-clamp method. Flecainide, pilsicainide, and lidocaine reversibly blocked the peak amplitude of TTX-R Na(+) currents in a concentration-dependent manner with half-maximum inhibitory concentration values of 8.5 +/- 6.6 microM (n = 7), 78 +/- 6.9 microM (n = 7), and 73 +/- 6.8 microM (n = 7), respectively. Each drug shifted the inactivation curve for the TTX-R Na(+) currents in the hyperpolarizing direction and caused a use-dependent block. We also studied an interaction between these antiarrhythmics on TTX-R Na(+) channels. Additional application of flecainide or pilsicainide to lidocaine resulted in an additive increase of tonic and use-dependent block. These results suggest that the inhibition of TTX-R Na(+) currents of dorsal root ganglion neurons by such antiarrhythmics is attributable, at least partly, to their antinociceptive effects.     

The Quaternary Lidocaine Derivative, QX-314, Produces Long-lasting Local Anesthesia in Animal Models In Vivo

Background: QX-314 is a quaternary lidocaine derivative considered to be devoid of clinically useful local anesthetic activity. However, several reports document that extracellular QX-314 application affects action potentials. Hence, the authors tested the hypothesis that QX-314 could produce local anesthesia in animal models in vivo.

Methods: The authors tested QX-314 (10, 30, and 70 mm) in three standard in vivo local anesthetic animal models, using a randomized, blinded experimental design with negative (placebo) and positive (70 mm lidocaine) controls. The guinea pig intradermal wheal assay (n = 29) was used to test for peripheral inhibition of the cutaneous trunci muscle reflex, the mouse tail-flick test (n = 30) was used to test for sensory blockade, and the mouse sciatic nerve blockade model (n = 45) was used to test for motor blockade.

Results: In all three animal models, QX-314 concentration-dependently and reversibly produced local anesthesia of long duration, at concentrations equivalent to those clinically relevant for lidocaine. In the guinea pig intradermal wheal assay, QX-314 produced peripheral nociceptive blockade up to 6 times longer than lidocaine (650 +/- 171 vs. 100 +/- 24 min [mean +/- SD]; n = 6 per group; P < 0.0001). In the mouse tail-flick test, QX-314 produced sensory blockade up to 10 times longer than lidocaine (540 +/- 134 vs. 50 +/- 11 min; n = 6 per group; P < 0.0001). Finally, in the mouse sciatic nerve model, QX-314 produced motor blockade up to 12 times longer compared with lidocaine (282 +/- 113 vs. 23 +/- 10 min; n = 9 or 10 per group; P < 0.0001). The onset of QX-314-mediated blockade was consistently slower compared with lidocaine. Animals injected with saline exhibited no local anesthetic effects in any of the three models.     

Effect of Drugs Used for Neuropathic Pain Management on Tetrodotoxin-resistant Na+ Currents in Rat Sensory Neurons

Background: Tetrodotoxin-resistant Na+ channels play an important role in generation and conduction of nociceptive discharges in peripheral endings of small-diameter axons of the peripheral nervous system. Pathophysiologically, these channels may produce ectopic discharges in damaged nociceptive fibers, leading to neuropathic pain syndromes. Systemically applied Na+ channel-blocking drugs can alleviate pain, the mechanism of which is rather unresolved. The authors investigated the effects of some commonly used drugs, i.e., lidocaine, mexiletine, carbamazepine, amitriptyline, memantine, and gabapentin, on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglia.

Methods: Tetrodotoxin-resistant Na+ currents were recorded in the whole-cell configuration of the patch-clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Half-maximal blocking concentrations were derived from concentration-inhibition curves at different holding potentials (-90, -70, and -60 mV).

Results: Lidocaine, mexiletine, and amitriptyline reversibly blocked tetrodotoxin-resistant Na+ currents in a concentration- and use-dependent manner. Block by carbamazepine and memantine was not use-dependent at 2 Hz. Gabapentin had no effect at concentrations of up to 3 mm. Depolarizing the membrane potential from -90 mV to -60 mV reduced the available Na+ current only by 23% but increased the sensitivity of the channels to the use-dependent blockers approximately fivefold. The availability curve of the current was shifted by 5.3 mV to the left in 300 [mu]m lidocaine.     

Ephedrine Blocks Rat Sciatic Nerve In Vivo and Sodium Channels In Vitro

Background: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na+ channels.

Methods: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na+ currents from human embryonal kidney cells stably transfected with Nav1.4 channels.

Results: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 [mu]m, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics.     

Ephedrine blocks rat sciatic nerve in vivo and sodium channels in vitro

BACKGROUND: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na channels. METHODS: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na currents from human embryonal kidney cells stably transfected with Nav1.4 channels. RESULTS: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 mum, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics. CONCLUSIONS: Because ephedrine's properties are at least partly due to Na channel blockade, detailed histopathologic investigations are justified to determine the potential of ephedrine as an adjuvant to clinically used local anesthetics.     

Lidocaine and octanol have different modes of action at tetrodotoxin-resistant Na(+) channels of peripheral nerves

Local anesthetics and alcohols block impulse conduction in peripheral nerves by inhibiting Na(+) currents. In small peripheral nerve fibers, tetrodotoxin-resistant (TTX-r) Na(+) channels play an important role in impulse generation. We investigated the effects of lidocaine and the alcohol octanol on TTX-r Na(+) channels. Currents were recorded with the whole-cell patch-clamp method from enzymatically isolated rat dorsal root ganglion cells (data evaluation: nonlinear least-squares fitting). Lidocaine and octanol blocked the TTX-r Na(+) current in a reversible and concentration-dependent manner (50% inhibitory concentration values: 177 +/- 25 and 455 +/- 25 microM, respectively). Lidocaine additionally produced a strong use-dependent block. Both drugs showed a strong dynamic block (i.e., block developed during the time course of current activation and inactivation). Double-pulse protocols showed a slow dissociation of lidocaine from the channel during repolarization (time constant: 1763 +/- 63 ms; 300 microM). The dissociation of octanol was too quick to be distinguished from normal current repriming kinetics of 2.2 ms. Lidocaine and octanol acted noncompetitively in the Na(+) channel. Lidocaine and octanol have different blocking properties on the TTX-r Na(+) current and bind to different channel sites. IMPLICATIONS: Lidocaine and octanol have different inhibitory effects on the function of tetrodotoxin-resistant Na(+) channels in rat dorsal root ganglion cells, as well as noncompetitive modes of action, as investigated by the whole-cell patch-clamp method, and therefore are likely to have different binding sites on the channel.     

The quaternary lidocaine derivative, QX-314, produces long-lasting local anesthesia in animal models in vivo

BACKGROUND: QX-314 is a quaternary lidocaine derivative considered to be devoid of clinically useful local anesthetic activity. However, several reports document that extracellular QX-314 application affects action potentials. Hence, the authors tested the hypothesis that QX-314 could produce local anesthesia in animal models in vivo. METHODS: The authors tested QX-314 (10, 30, and 70 mm) in three standard in vivo local anesthetic animal models, using a randomized, blinded experimental design with negative (placebo) and positive (70 mm lidocaine) controls. The guinea pig intradermal wheal assay (n = 29) was used to test for peripheral inhibition of the cutaneous trunci muscle reflex, the mouse tail-flick test (n = 30) was used to test for sensory blockade, and the mouse sciatic nerve blockade model (n = 45) was used to test for motor blockade. RESULTS: In all three animal models, QX-314 concentration-dependently and reversibly produced local anesthesia of long duration, at concentrations equivalent to those clinically relevant for lidocaine. In the guinea pig intradermal wheal assay, QX-314 produced peripheral nociceptive blockade up to 6 times longer than lidocaine (650 +/- 171 vs. 100 +/- 24 min [mean +/- SD]; n = 6 per group; P < 0.0001). In the mouse tail-flick test, QX-314 produced sensory blockade up to 10 times longer than lidocaine (540 +/- 134 vs. 50 +/- 11 min; n = 6 per group; P < 0.0001). Finally, in the mouse sciatic nerve model, QX-314 produced motor blockade up to 12 times longer compared with lidocaine (282 +/- 113 vs. 23 +/- 10 min; n = 9 or 10 per group; P < 0.0001). The onset of QX-314-mediated blockade was consistently slower compared with lidocaine. Animals injected with saline exhibited no local anesthetic effects in any of the three models. CONCLUSION: In a randomized, controlled laboratory study, the quaternary lidocaine derivative, QX-314, concentration-dependently and reversibly produced long-lasting local anesthesia with a slow onset in animal models in vivo. The authors' results raise the possibility that quaternary ammonium compounds may produce clinically useful local anesthesia of long duration in humans and challenge the conventional notion that these agents are ineffective when applied extracellularly.     

Bulleyaconitine A isolated from aconitum plant displays long-acting local anesthetic properties in vitro and in vivo

BACKGROUND: Bulleyaconitine A (BLA) is an active ingredient of Aconitum bulleyanum plants. BLA has been approved for the treatment of chronic pain and rheumatoid arthritis in China, but its underlying mechanism remains unclear. METHODS: The authors examined (1) the effects of BLA on neuronal voltage-gated Na channels in vitro under the whole cell patch clamp configuration and (2) the sensory and motor functions of rat sciatic nerve after single BLA injections in vivo. RESULTS: BLA at 10 microm did not affect neuronal Na currents in clonal GH3 cells when stimulated infrequently to +50 mV. When stimulated at 2 Hz for 1,000 pulses (+50 mV for 4 ms), BLA reduced the peak Na currents by more than 90%. This use-dependent reduction of Na currents by BLA reversed little after washing. Single injections of BLA (0.2 ml at 0.375 mm) into the rat sciatic notch not only blocked sensory and motor functions of the sciatic nerve but also induced hyperexcitability, followed by sedation, arrhythmia, and respiratory distress. When BLA at 0.375 mm was coinjected with 2% lidocaine (approximately 80 mm) or epinephrine (1:100,000) to reduce drug absorption by the bloodstream, the sensory and motor functions of the sciatic nerve remained fully blocked for approximately 4 h and regressed completely after approximately 7 h, with minimal systemic effects. CONCLUSIONS: BLA reduces neuronal Na currents strongly at +50 mV in a use-dependent manner. When coinjected with lidocaine or epinephrine, BLA elicits prolonged block of both motor and sensory functions in rats with minimal adverse effects.