Commercially available bupivacaine is a racemic mixture of S(−) and R(+) enantiomers. Bupivacaine toxicity is attributed primarily to the R(+) enantiomer (1–3), with in vivo studies demonstrating greater cardio- and neurotoxicity than found in S(−) bupivacaine (levobupivacaine) (1,2). Ropivacaine is a pure S(−) enantiomer, recently introduced into clinical practice, that is structurally similar to bupivacaine but less toxic to the heart (4) and central nervous system (5). S(−) bupivacaine is a new anesthetic now undergoing clinical trials.
Many studies of anesthetic potency compare the effects of bupivacaine with those of either S(−) bupivacaine or ropivacaine. In sheathed frog sciatic nerve, racemic bupivacaine and ropivacaine appear to have equivalent phasic (use-dependent) blocking effect (6) and an R(+) bupivacaine equivalent or greater effect than S(−) bupivacaine (1,7). Investigations of the neuronal sodium channel kinetics of S(−) and R(+) bupivacaine indicate more potent block with R(+) bupivacaine, because of slower dissociation from neuronal sodium channels than that observed with the S(−) enantiomer (7). Comparison of the anesthetic action of ropivacaine and S(−) bupivacaine has focused on in vivo studies in the rat (8,9). These studies indicated that blocking potencies of S(−) bupivacaine and ropivacaine are different between sensory and motor block in peripheral nerve block (8) and in epidural or intrathecal block (9).
Under physiological conditions, local anesthetics exist in equilibrium between the ionized, protonated state and the unionized, neutral state, depending on the dissociation constants and the local pH. Both the ionized and neutral forms can inhibit sodium channels. The neutral form crosses the axonal membrane to interact with an intramembranous functional site, i.e., a sodium channel, by direct access (10,11). On reaching the cytoplasm, the neutral form becomes ionized, then plays a large role in blocking the excitable membrane (10,12,13). Generally, the ionized form of local anesthetics has a markedly low lipid solubility compared with the neutral form. Above all, the ionized forms of bupivacaine show high hydrophobicity against the other hydrophobic local anesthetics (14). The hydrophobicity of ionized ropivacaine is a quarter of that of ionized bupivacaine, however, it is relatively high compared with the other local anesthetics (14). We hypothesized that the intracellular ionized forms of these anesthetics have a large contribution to local anesthetic action.
Anesthetic potency of S(−) and R(+) bupivacaine and ropivacaine was compared by assessing the evoked action potential amplitude and maximal rate of rise of the action potential (dV/dtmax) in crayfish giant axon in vitro. By using specially designed local anesthetic-sensitive microelectrodes (15), the intracellular concentration of the ionized forms of S(−) bupivacaine, R(+) bupivacaine, and ropivacaine was measured to determine the correlation between intracellular cationic local anesthetic concentration and anesthetic action.
Abdominal nerve bundles from crayfish (weight, 24–51 g;n = 78) were isolated under hypothermic narcosis. The four giant axons in each bundle were exposed by removal of the nerve sheath between the second and fourth ganglia. Axons were treated for 30 min in 15 mM HEPES-added van Harrevelds’ solution containing 1 mg/mL collagenase (Wako Pure Chemical, Osaka, Japan) to ease microelectrode insertion. The preparation was mounted in a recording chamber (2.5-mL volume) and perfused at 5 mL/min with bicarbonate-free HEPES-van Harrevelds’ solution (BCF-HH) containing 190 mM NaCl, 5.4 mM KCl, 13.5 mM CaCl2, 2.6 mM MgCl2, and 5 mM HEPES. Three local anesthetic test solutions were prepared in BCF-HH: 1.0 mM S(−) bupivacaine HCl (levobupivacaine), R(+) bupivacaine HCl, and ropivacaine HCl (Chiroscience R & D Ltd, Cambridge, UK). The pH in these solutions was adjusted to 7.0 by adding NaOH.
A conventional glass microelectrode was filled with 4M KCl and inserted into one of the two median giant axons at the lesion between the second and fourth ganglia (Figure 1). The membrane potential (Em) was amplified by a high impedance and continuously monitored by using a storage oscilloscope. Action potential was evoked by a square wave pulse of supramaximal intensity (a two-fold suprathreshold voltage of 0.3 ms at 0.1 Hz or 5 Hz) by using an electrical stimulator and converted to a differential waveform (dV/dt) by a differentiator. The action potential, Em, and dV/dt were recorded continuously. The dV/dtmax was calculated because it is a more sensitive index than action potential amplitude for evaluating local anesthetic action (16).
After a conventional Em electrode was inserted into the axon, BCF-HH solution was perfused for 30 min at 0.1 Hz. For observation of use-dependent (phasic) block, BCF-HH was perfused for 30 min at 0.1 Hz, then for 5 min at 5 Hz before exposure to local anesthetic solutions. Local anesthetic solutions were then perfused for 15 min at 0.1 Hz or 5 Hz. Baseline Em, action potential amplitude, and dV/dtmax were defined by the value obtained just before the change to a solution containing local anesthetic. The percentage decrease in dV/dtmax after perfusion of local anesthetic was calculated as a percentage of the predrug baseline value. To avoid hypersensitivity of the experimental preparation, local anesthetic effects (Em, action potential amplitude, dV/dt:n = 36, 6 per group) and intracellular local anesthetic concentrations (n = 42, 7 per group) were measured in different nerves. After exposure to local anesthetic test solutions, nerves were continuously washed with the BCF-HH solution for 1 h. All experiments were performed at room temperature (25 ± 1°C).
Local anesthetic-sensitive microelectrodes were constructed on details described by Ibusuki et al. (15). As the dissociation constants of bupivacaine enantiomers and ropivacaine are near physiologic pH levels (17), the pH of the experimental solutions was set at 7.0 (at which bupivacaine and ropivacaine exist primarily in cationic form) to facilitate measurement of intracellular local anesthetic concentration. Each microelectrode contained 98.2 weight/%o-nitrophenyl octyl ether, 1.8 weight/% polyvinylchloride, 2 mM tungstophosphoric acid hydrate, and 2 mM of S(−) and R(+) bupivacaine HCl or ropivacaine HCl.
Local anesthetic sensors were calibrated with various concentrations of each anesthetic dissolved in a solution containing 100 mM KCl, 10 mM NaCl, 5 mM HEPES at pH 6.8 (Figure 2). Local anesthetic sensor response was linear over a concentration range of 0.06 to 1 mM. The slope of the electrode responses was determined (mean ± SD), as 61.1 ± 5.6 mV/log [S(−) bupivacaine], 63.5 ± 5.7 mV/log [R(+) bupivacaine], or 63.6 ± 5.7 mV/log [ropivacaine] by regression analysis of the linear portion of the calibration curves. The potentials at 1.0 mM of the calibration curves of S(−) bupivacaine, R(+) bupivacaine, and ropivacaine were 138.9 ± 36.2, 131.6.± 21.5, and 126.3 ± 18.5, respectively. Repeated measurement of electrode potentials with different concentrations of local anesthetics indicated a coefficient of variance ranging from 0.57% to 1.76% (mean, 1.19%). The influence of pH on the response of the local anesthetic sensor was negligible (data not shown), as determined by previously described methods (15). Selectivity coefficients, which express the ability of the local anesthetic-sensitive microelectrode to distinguish between different ions, were determined by using the fixed interference method (15). The selectivity coefficients values for Na +, K +, and Ca2+ were 5.66 × 10−8, 1.34 × 10−7, and 9.47 × 10−7, respectively, in the range of 0.004 mM to 1 mM.
Intracellular recording with microelectrodes allowed us to compare the local anesthetic effect under the same experimental system used to measure the intracellular concentration of cationic species. Both a local anesthetic-sensitive microelectrode and a conventional glass Em microelectrode were inserted into the same axon (Figure 1). The Em and the potential of the local anesthetic-sensitive microelectrode (ELA) (where ELA = ES(−)-bup or ER(+)-bup or Erop) were then monitored continuously by using the storage oscilloscope. The perfused solution and duration were established by using the same protocol described to evaluate local anesthetic action. Local anesthetic solutions were perfused for 15 min while Em and ELA were recorded. The intracellular local anesthetic concentration was calculated by the following equation (15): [LAi] = 10 [(ΔE-C)/S], where [LAi] is intracellular local anesthetic expressed in mM, ΔE is the change in the difference between ELA and Em during exposure to local anesthetic, C is relative ELA at 1 mM local anesthetic from 0 mM, and S is slope of the linear portion of the calibration curve. Because ELA is the sum of the Em and the potential produced by each local anesthetic cation, the Em measurement must be subtracted from ELA.
All values were expressed as the mean ± SD. Study groups were compared by using one-way analysis of variance, followed by the Scheffé test. A difference of P < 0.05 was considered statistically significant.
A typical recording of Em, action potential, and dV/dt is shown in Figure 3. When electrical stimuli were conducted to the axon at 0.1 Hz, there was no difference in depression of action potential amplitude and dV/dtmax among the three anesthetics. However, at 5-Hz stimulation, the mean percentage decrease in dV/dtmax was significantly greater in S(−) bupivacaine than ropivacaine, R(+) bupivacaine than ropivacaine, and S(−) bupivacaine than R(+) bupivacaine (Table 1). Use-dependent block did not occur with ropivacaine at 5 Hz, but was apparent with S(−) and R(+) bupivacaine and most potent with S(−) bupivacaine in the desheathed crayfish axon. The Em change in drug perfusion was 1–4 mV, which was not significantly different among three anesthetics. The action potential amplitude and dV/dtmax showed no significant difference after washout for 15 min among the three local anesthetics at 5 Hz. The action potential amplitude and dV/dtmax completely returned to baseline values within 60 min after drug application of all local anesthetics.
Figure 3B (bottom panel) shows a typical recording of ropivacaine potential (Erop). The intracellular local anesthetic concentration after 15 min perfusion of 1-mM local anesthetic solutions at 0.1-Hz stimulation was not significantly different. There was no significant difference in the intracellular local anesthetic concentration at 5 Hz among the three anesthetic, and the stimulation frequency did not alter the intracellular concentration with each (Figure 4). The intracellular local anesthetic was 52%–68% recovered after 5-min washout from the values of 15-min local anesthetic application, and 83%–89% after 15-min washout. The rate of washout of anesthetic from cytoplasm showed no difference among the groups studied.
Our findings in crayfish giant axon indicate equivalent potency for tonic block with S(−) bupivacaine, ropivacaine, or R(+) bupivacaine, but more intense phasic (use-dependent) block with the S(−) bupivacaine when applied in an equal dose. These results agree with the studies that local anesthetic effects are frequency-dependent (10,11). The intracellular cationic local anesthetic concentrations of the three anesthetics did not differ after extracellular application of concentrations of 1.0 mM.
These results in crayfish axons support the fact that the phasic block tends to show more stereoselectivity than the tonic block (18). The comparison of the potency of neuronal sodium channel block between S(−) bupivacaine and ropivacaine has not been published. Previous in vivo studies suggest that S(−) bupivacaine has an equivalent or greater sensory and motor-blocking effect than ropivacaine in peripheral nerve block and epidural or spinal block in the rat (8,9). This report indirectly demonstrated that S(−) bupivacaine has a more potent sodium channel block than ropivacaine in the crayfish giant axon. Two studies comparing racemic bupivacaine and ropivacaine also suggest that bupivacaine has a comparable or more potent blocking effect than ropivacaine in isolated nerves in vitro (6,19).
We obtained more potent phasic blocking effects of S(−) bupivacaine than R(+) bupivacaine, which is opposed to the generally stereoselective potency ratio [R(+) > S(−)] of local anesthetics. Greater potency of the R(+) enantiomer has been demonstrated in investigations of compound action potential in desheathed frog sciatic nerve and of stereoselective block of sodium channels in neuronal tissue (7,20). Alternatively, similar potency of the R(+) and S(−) enantiomers has been reported in studies of inhibition of the compound action potential in frog sciatic nerve (1).
One basis for these differences may be conformational and state-dependent drug-channel interactions. Although sodium channels appear to have major functional similarities across animal species (21), the properties of local anesthetic block of these channels may differ by species. Phasic block is related to the change in association and dissociation of drug to the closed channels. As the differences in the potency of enantiomers in vitro result from a stereoselective interaction with sodium channels, the site(s) of stereoselective action may differ for the sodium channels of crayfish axon. An additional consideration may be differences between species in resting membrane potential. Specifically, differences in membrane potential induce a difference in stereoselectivity that can invert the stereoselectivity ratio with R(+) and S(−) bupivacaine (22). Assuming an inverted ratio resulting from a difference in the extent of resting inactivation would account, in part, for different experimental outcomes.
Independent of potency, the intracellular local anesthetic concentrations of the three anesthetics in crayfish axon were similar and unrelated to stimulation frequency. This finding indicates that the cytoplasmic cation concentration of the specific anesthetic per se does not relate to the local anesthetic blocking effect, although the depression of dV/dtmax paralleled an increase in intracellular cation concentration (see Figure 3). The neutral forms of hydrophobic local anesthetics have high hydrophobicity and greater lipid permeability, the ionized species in axoplasm then account for the rapid increase in local anesthetic concentration after perfusion at a relatively low pH. Accordingly, the blocking property of the functional sites of the sodium channel is more important for action potential inhibition.
The concentration of intracellular ionized bupivacaine and ropivacaine was measured by using cation-sensitive microelectrodes specific to each anesthetic. When the surrounding pH of the sensor electrode is altered, the cation:base ratio of bupivacaine or ropivacaine changes. However, we held intracellular pH constant at 6.8 by using a pH-sensitive microelectrode perfused extracellularly with BCF-HH at pH 7.0. Because bupivacaine and ropivacaine exist primarily in cationic form at physiologic pH, we were thus able to maintain a constant 94%–95% of the cationic form of these anesthetics, thereby enhancing the accuracy of the microelectrode measurements. Intracellular local anesthetic concentrations and local anesthetic effects were not simultaneously measured, as there was concern that the use of two electrodes might desensitize the nerve preparations, although pilot experiments had shown minimal or no differences in measurements obtained independently.
We found that the intracellular local anesthetic concentration is approximatley one fourth of the concentration of extracellular local anesthetics in this study. This is a reasonable value because intracellular local anesthetic in BCF-HH is lower than that in normal bicarbonate HEPES-van Harrevelds’ solution at the same extracellular pH (15).
In conclusion, compared with ropivacaine, S(−) bupivacaine has a more potent phasic blocking effect in crayfish giant axon in vitro. Our finding of an acceptable anesthetic effect in these models, combined with previous reports of lower systemic toxicity, suggest that the S(−) enantiomer of bupivacaine warrants further study to determine clinical use. The intracellular local anesthetic concentrations of S(−) and R(+) bupivacaine and ropivacaine were not significantly different regardless of differences in blocking effect and stimulation frequency. This finding suggests that the properties governing local anesthetic block of functional sites of the neuronal sodium channel are more important for action potential inhibition than intracellular cation concentration per se.
The authors thank Chiroscience R & D Ltd, Cambridge for drug supply, and Ms. Winifred von Ehrenburg for editorial assistance.
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© 2000 International Anesthesia Research Society
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