Naturally occurring phenolic monoterpenes, e.g. thymol, eugenol and carvacrol, are the active ingredients in the essential oils of plants occurring in the Mediterranean flora [1-5]. Several beneficial effects have been ascribed to these essential oils, among others their strong bactericidal and antifungal activity [2,6-9] and their anti-inflammatory properties [1,10-12]. In addition, aromatic alcohols with intact phenolic groups and different phenol derivatives have been shown to act as scavengers of reactive oxygen species, thus protecting neurons effectively against oxidative damage and cell death . Dietary supplementation with thyme oil even tended to prevent age-related changes in the phospholipid composition in several organs in rats [14,15].
Antinociceptive and local anaesthetic effects have long been described for L-menthol , eugenol and thymol ; however, a precise molecular target site for these effects has not yet been identified. It has recently been shown that phenol derivatives, among others the anaesthetic propofol (2,6-diisopropylphenol), are potent blockers of heterologously expressed voltage-operated sodium channels in vitro[17-19]. The close structural relationship between the anaesthetic propofol (2,6-diisopropyl-phenol) and the naturally occurring phenol derivative thymol (5-methyl-2-isopropyl-phenol) gave rise to the hypothesis that the antinociceptive effects described for this compound might be mediated via blockade of voltage-operated sodium channels analogous to the effects describe for other phenol derivatives and local anaesthetics (Fig. 1).
We investigated the effects of thymol (5-methyl-2-isopropyl-phenol) and its structural homologue menthol (5-methyl-2-isopropyl-cyclohexanol) on heterologously expressed α-subunits of rat brain IIA and human skeletal muscle (hSkM1) sodium channels as possible targets contributing to the local anaesthetic and antinociceptive activity of the drugs. The α-subunit is the primary pore-forming subunit of voltage-operated sodium channels and it functions as an ion channel when expressed alone . α-Subunits of rat brain IIA and hSkM1 sodium channels show normal gating characteristics (with respect to experiments in native tissue) when expressed in a mammalian cell line [21,22]. The rat brain IIA form is most abundant in adult brain , hSkM1 is the principal voltage-gated sodium channel expressed in adult human skeletal muscle .
Transfection and cell culture
Stably transfected HEK 293 cell lines expressing either the α-subunit of rat brain IIA or hSkM1 sodium channels were a gift from Professor Lehmann-Horn, Ulm, Germany. The expression vector pRc/CMV® (Invitrogen, San Diego, CA, USA) was used for mammalian transfection . Transfection was performed using calcium phosphate precipitation . Permanent expression was achieved by selection for resistance to the aminoglycoside antibiotic geneticin G418® (Life Technology, Eggenstein, Germany) . Successful channel expression was verified electrophysiologically. The clones have been used in several investigations [22,25,27].
Thymol and menthol were prepared as a 1 mol stock solution in ethanol, light protected and stored in glass vessels at −20°C. The stock solution was dissolved directly in a bath solution immediately before the experiments. Drug-containing vials were vigorously vortexed for 60 min. The solution was applied via a glass-polytetrafluoroethylene perfusion system and a stainless-steel superfusion pipette. The bath solution contained NaCl 140 mmol, MgCl2 1 mmol, KCl 4 mmol, CaCl2 2 mmol, Hepes 5 mmol and dextrose 5 mmol. Patch electrodes contained CsCl2 130 mmol, MgCl2 2 mmol, EGTA 5 mmol and Hepes 10 mmol. All solutions were adjusted to 290 mOsm by addition of mannitol, and to pH 7.4 by addition of CsOH.
Standard whole cell voltage-clamp experiments  were performed at 20°C. Each experiment consisted of test recordings with the drug present at only one concentration, and of drug-free control recordings before and after the test. Each cell was exposed to one test concentration only. At least four experiments were performed at each concentration. The effects of the diluent ethanol corresponding to higher drug concentrations were tested separately up to a maximum ethanol concentration of 17.4 mmol corresponding to a drug concentration of 1000 μmol.
For data acquisition and further analysis, we used the EPC9® digitally controlled amplifier in combination with Pulse® and Pulse Fit® software (HEKA Electronics, Lambrecht, Germany). The EPC9® provides automatic subtraction of capacitive and leakage currents by means of a prepulse protocol. The data were filtered at 10 kHz and digitized at 20 μs per point. Input resistance of the patch pipettes was at 1.8-2.5 MΩ, cells capacitances ranged between 9 and 15 pF; and residual series resistance (after 50% compensation) was 1.2-2.5 MΩ. Experiments with a rise in series resistance were rejected. Voltage-activated currents were studied by applying different voltage-clamp protocols, as described in the Results or in the appropriate Figure legends.
Hyperpolarizing shifts in the voltage dependence of steady state availability as a function of the recording time, which may reach −6 mV after 10 min of wholecell recording , may pose a problem when considering drug effects on current-voltage relationships. To avoid a biasing of the test results, all test experiments were performed within 5 min of patch rupture. During the first 5 min of whole-cell recording, time-dependent hyperpolarizing shifts in control conditions were less than −2 mV . Washout of drug effects on the voltage dependence of channel availability was considered successful when the drug-induced shift changed its direction during washout and when the difference in the mid-point of the availability curve between the starting value and that obtained during washout did not largely exceed −6 mV.
Drug effects on the peak current amplitude were assessed at a holding potential of −70 mV, which is close to the resting potential found in single muscle fibres of frog semitendinosus muscle after equilibration with an external potassium concentration of 5.0 mmol in vitro (−76 ± 2 mV) , and at hyperpolarized holding potentials (−100 and −150 mV). The residual sodium current (INa+) in the presence of drug (with respect to the current amplitude in control solution) was plotted against the applied concentration of each drug [C]. As each cell was exposed to one test concentration only, each datum point represents the mean derived from at least four different experiments, error bars are SD. Fits of the Hill equation: EQUATION 1
to the averaged data yielded the concentration for half-maximum channel blockade (IC50) and the Hill coefficient nH.
A one-sample t-test was applied to analyse the significance of changes in gating parameters in the presence of drug with respect to the starting value. To avoid problems related to multiple hypotheses testing, we applied the method of multiple comparisons with a priori-ordered hypotheses . The test is based on the assumption that if the null hypothesis is rejected, there is a positive monotonic relation between concentration and effect. Consequently, the hypotheses to be tested could be ordered in advance, starting with the highest concentration. If the test results differed significantly from the control data, the effects of the next, lower concentration were evaluated. The evaluation was stopped as soon as the first insignificant result was obtained. The advantage of this procedure, compared with other approaches to multiple testing, is that the level of Type I error is kept at α = 0.05 for each statistical test. The null hypothesis was rejected when P < 0.05.
Resting state affinity
Seventy-five cells were included in the study. Average currents in the control experiments following depolarization from −100 to 0 mV were −2.9 ± 1.2 nA for the skeletal muscle isoform and −3.7 ± 1.4 nA for the neuronal isoform. Maximum inward currents elicited by 10 ms pulses from either −150, −100 or −70 to 0 mV were reversibly suppressed by thymol and menthol in a concentration-dependent manner. Figure 2 shows representative current traces in the absence and presence of either 100 μmol thymol or 500 μmol menthol. Normalized currents (with respect to control), derived from at least four different experiments for each drug concentration, were averaged to establish concentration-response plots. The amount of block achieved by either thymol or menthol strongly depended on the holding potential from which the depolarization was started. Hill fits to the averaged data obtained for thymol (Fig. 3) yielded IC50 of 149 and 104 μmol at −70 mV, 366 and 222 μmol at −100 mV, and 410 and 245 μmol at −150 mV holding potential for the neuronal and skeletal muscle isoform, respectively.
Fitted Hill coefficients nH were 1.13 and 1.09 (−70 mV), 1.56 and 1.49 (−100 mV), and 2.87 and 1.62 (−150 mV). Menthol was less potent than thymol, the half-maximum blocking concentrations being 571 and 376 μmol at −70 mV, 1150 and 783 μmol at −100 mV, and 1215 and 792 μmol at −150 mV holding potential for the neuronal and skeletal muscle isoform, respectively.
Blocking effects were generally reversible on washout, when washout was incomplete, currents in the presence of drug were normalized to the current amplitude during washout.
At a concentration of 17.4 mmol, corresponding to a drug concentration of 1000 μmol, the diluent ethanol had a small effect on the peak current amplitude at −150 mV holding potential. The peak current was −2.2 ± 0.1 nA in the controls and −2.3 ± 0.1 nA in the presence of 8.7 mmol (n = 3), and −2.8 ± 0.2 in the controls and −2.3 ± 0.1 nA in the presence of 17.4 mmol ethanol (n = 3). This means that in drug concentrations > 1000 μmol, the diluent ethanol contributes to the peak current suppression induced by the test solution by approximately 16%.
Inactivated state affinity
The increase in blocking potency at a holding potential of −70 mV, where some of the channels are inactivated, compared with −150 mV, where all channels are expected to be in the resting state, suggests that the amount of block achieved by either thymol or menthol depends on the membrane potential and is increased with an increased fraction of inactivated channels with respect to resting channels. The voltage dependence of block and affinity for the inactivated state was further assessed by applying a double-pulse protocol. After brief depolarizations, sodium channels enter a fast-inactivated state from which they cannot readily reopen. Currents elicited by test pulses (Itest) starting from varying prepulse potentials (from −150 to −5 mV) to 0 mV, normalized to the current elicited at the most hyperpolarized prepotential (−150 mV), represent the relative fraction of channels that have not been inactivated during the 50 ms inactivating prepulse. Boltzmann fits to the resulting current-voltage plots yield the membrane potential at half-maximum channel availability (V0.5) and the slope factor k:EQUATION 2
In control conditions, the parameters of the Boltzmann fits reflect the voltage dependence of the distribution between resting and fast-inactivated channels.
Summarized control data showed that half of the channels were unavailable at −43.4 ± 3.2 mV (rat IIA isoform) and at −59.6 ± 3.7 mV (hSkM1) because of fast inactivation. The slope factors k were 7.9 ± 1.1 and 6.9 ± 1.1, respectively. With exposure to either thymol or menthol, V0.5 was shifted considerably in the direction of more negative prepulse potentials; the degree of alteration showed concentration dependence. The averaged data for the thymol effects are depicted in Figure 4. Drug effects on the voltage dependence of channel availability were reversible during washout, taking into account that a shift of about −6 mV is expected to occur as a function of the recording time until completion of the washout experiments. For example, 200 μmol thymol shifted the mid-point of the availability curve from −39.1 ± 3.9 mV (rat IIA) and −60.2 ± 5.8 mV (hSkM1) to −51.8 ± 4.6 and −74.8 ± 6.8 mV. The values obtained during washout were −44.3 ± 3.1 and −69.1 ± 1.4 mV, respectively. The slope factors k remained unchanged in the presence of drug (Fig. 4). The drug-induced hyperpolarizing shifts reflect an additional reduction of channel availability induced by both drugs in the voltage range of channel inactivation compared with −150 mV. To estimate the dissociation constant (Kd) of thymol and menthol for the fast-inactivated state of both channel isoforms, we employed a model developed by Bean and colleagues  for the example of lidocaine effects on Purkinje fibres. The model is based on the assumption that the higher amount of channel block achieved with consecutive membrane depolarization, revealed by the drug-induced hyperpolarizing shift, is determined by the apportionment of channels between resting and fast-inactivated states as well as the different binding affinities for the two channel states. The concentration dependence of the shift in the mid-point was well described by the equation: EQUATION 3
where ΔV0.5 is the shift in the mid-point in each drug concentration (mean, n > 4), k is the mean of the slope factor derived from Boltzmann fits to the current-voltage plots (see above), [C] is the applied concentration of drug, IC−15050 is the concentration for half-maximum effect derived from the concentration-response plots at −150 mV membrane potential described above, and Kd the dissociation constant for the drug from the inactivated state.
For thymol, the estimated Kd derived from that fit were 22 and 23 μmol for the neuronal and skeletal muscle isoform (Fig. 4b). For menthol, Kd was 106 and 97 μmol.
When applied instead of test solution at 17.4 mmol, corresponding to a drug concentration of 1000 μmol, the diluent ethanol did not shift the steady-state voltage dependence in the hyperpolarizing direction. In the presence of ethanol, a small positive shift of 5.0 ± 0.2 mV (n = 3) was observed.
Time-course of channel inactivation and recovery from an inactivated channel block in the presence of thymol
As other phenol derivatives have been shown to accelerate the time course of sodium channel inactivation during a depolarization, and to delay the time-course of recovery from fast inactivation , we studied the possible effects of thymol on inactivation-gating parameters of the neuronal and skeletal muscle isoform.
The time constant of channel inactivation τh was obtained by fitting a single exponential to the decay of current during a 40 ms depolarization from −100 to 0 mV at a holding potential of −100 mV: EQUATION 4
At least three experiments were evaluated for each concentration tested. In control conditions, τh was 0.72 ± 0.2 ms (n = 15) for rat brain IIA and 0.58 ± 0.09 ms (n = 18) for hSkM1 sodium channels. Thymol did not significantly accelerate the current decay. τh obtained in the presence of drug were: 0.61 ± 0.1 and 0.48 ± 0.1 ms in 200 μmol thymol and 0.66 ± 0.1 and 0.54 ± 0.06 ms in 100 μmol thymol in the neuronal and skeletal muscle isoform, respectively.
After inactivation, channel reopenings are impossible until the channels recover from inactivation, a process that requires several milliseconds after membrane repolarization. Further information about drug effects on the stability of the fast-inactivated state or the kinetics of drug dissociation from the fast-inactivated state can be derived from the rate at which the channels recover from inactivation in the presence of the drug. The time of membrane repolarization required to remove fast inactivation was assessed at −100 mV by a two-pulse protocol with varying time intervals (up to 100 ms) between the inactivating prepulse and the test pulse. The time constants of recovery, τrec, were derived from biexponential fits to the fractional current after recovery from inactivation plotted against the time interval between the inactivating prepulse and the test pulse: EQUATION 5
At least three experiments were evaluated for each concentration tested. Without drug, recovery time constants obtained for the neuronal (n = 17) and skeletal muscle isoform (n = 16) were τrec1 2.2 ± 0.7 and 2.6 ± 1.0 ms with a slow component τrec2 of 19.9 ± 0.9 and 19.6 ± 1 ms, which comprised 9 and 11% of the current amplitude. Thymol did not significantly increase the time constants of recovery from fast inactivation, indicating that the time-course of drug unbinding from inactivated channels is fast enough to parallel the time-course of channel gating. τrec1 in the presence of 200 μmol thymol was 2.9 ± 1.5 and 5.3 ± 0.7 ms; τrec2 was 18.3 ± 1.6 and 25.0 ± 7.1 ms. However, the slow component of recovery of about 20 ms increased in amplitude up to 30 ± 14% in 200 μmol thymol in both isoforms. This means that recovery from an inactivated channel block at a hyperpolarized holding potential (−100 mV) should be too fast to accumulate relevant frequency-dependent block at stimulating frequencies <50 Hz.
The accumulation of block during trains of depolarizing pulses indicates that the interval between pulses is too short to allow recovery of sodium-channel availability. To derive an estimate of the kinetics of drug binding and unbinding during the interpulse interval, we applied series of 1-10 ms depolarizing pulses from −100 to 0 mV at high frequencies (10, 50, 100 Hz). Frequency-dependent block was defined as the additional reduction in INa+ for the last pulse relative to the first pulse in a test train in the presence of drug. In control conditions, the amplitude of the last pulse relative to the first pulse in a test train was 99 ± 2% at 10 and 50 Hz, and 86 ± 6% at 100 Hz due to incomplete recovery from inactivation at very high stimulating frequencies. As expected, thymol did not induce frequency-dependent block over 5% at 10 and 50 Hz. Only at 100 Hz did thymol produce a small amount of frequency-dependent block. During a 100-Hz train, the additional fall relative to the first pulse was 29 ± 11% (rat brain IIA) and 30 ± 8% (hSkM1) in 200 μmol thymol.
Voltage-gated sodium channels are membrane-spanning proteins responsible for the initiation and propagation of action potentials in nerve and muscle cells. In response to membrane depolarization, the channels open from the resting, closed state and then inactivate spontaneously. The main result of the study was that thymol, and to a much lesser extent menthol, blocks voltage-gated neuronal and skeletal muscle sodium channels in a concentration-dependent manner in resting and inactivated states. The blocking potency of thymol is comparable with the blocking potency of the local anaesthetic lidocaine . This effect provides the molecular basis for potential analgesic and antinociceptive effects of the compound. In vitro, the sodium channel blocking potency of thymol is higher than the potency of menthol. These results conflict with a recent study showing that local anaesthetic activity of menthol, assessed in vivo in the rabbit conjunctival reflex test and in vitro in a rat phrenic nerve hemidiaphragm preparation, was superior to thymol . Thus, in the case of menthol, local anaesthetic activity in vivo might rely on other mechanisms besides blockade of voltage-operated sodium channels. Alternatively, discrepancies concerning the activity of thymol might be explained by differences in storage and application. Analogous to the structurally closely related anaesthetic propofol, thymol should be light protected and stored in glass vessels to avoid loss of activity .
The block of sodium channels by thymol and menthol shares many attributes with sodium channel blockade by local anaesthetic and antidysrrhythmic drugs. In analogy to lidocaine-like local anaesthetics, the blocking potency of thymol and menthol strongly depends on the kinetic state of the channel [32,36]. The affinity of both compounds for the resting state assessed at an extremely hyperpolarized holding potential (−150 mV) was more than one order of magnitude below the estimated affinity for the fast-inactivated state. Consequently, the blocking effects of both compounds were determined by the fraction of inactivated channels, which in turn depended on the respective holding potential. The maintenance of a normal resting potential of about −70 to −80 mV strongly depends on the potassium gradient , and thus, a proper function of the energy-dependent Na+-K+-ATPase. As a consequence, stronger effects of either thymol, menthol or lidocaine should be expected in ischaemic or hypoxically damaged tissue, where a normal resting potential cannot be maintained and membranes are more depolarized, increasing the fraction of inactivated (=high-affinity) channels.
However, following membrane repolarization, recovery from an inactivated channel block is faster in the presence of thymol compared with lidocaine (τrec(slow) 20 versus >100 ms in case of lidocaine ), reducing the risk of prolonged channel blockade. Thus, the integrative function of sodium channels in normal tissue can be supposed to be maintained in the presence of thymol.
Channel-gating kinetics in voltage-operated sodium channels from different tissues are similar when concerned with activation and inactivation time constants, but differ markedly in the voltage dependence of these processes . Differences in the voltage dependence of inactivation between channel isoforms determine the sensitivity to blocking drugs with stronger binding affinities to inactivated compared with resting channels at a given membrane potential [38,39]. The estimated dissociation constants Kd of thymol and menthol for the inactivated state of the channel were the same for both channel isoforms, indicating that differences in blocking potency between neuronal and skeletal muscle sodium channels are explained by differences in the apportionment between resting and inactivated states at a given membrane potential, rather than differences in the inactivated state binding site. The membrane potential of half-maximum channel inactivation was −59 mV for skeletal muscle and −43 mV for the neuronal isoform in this study; thus, in the voltage range between −60 and −150 mV, the neuronal isoform should be less sensitive due to a higher fraction of resting (=low-affinity) with respect to inactivated (=high-affinity) channels.
The use of virtually all local anaesthetics is limited due to central nervous toxicity occurring with higher brain concentrations. Local anaesthetic-induced convulsions are especially attributed to the depression of inhibitory circuits in the central nervous system (CNS) [40-42]. Both local anaesthetics, phenol and its derivatives p-cresol and benzyl alcohol have been shown to inhibit voltage-operated potassium channels [43,44], one mechanism that may cause axonal hyperexcitability and convulsions in vivo. It is conceivable that the phenol derivative thymol and menthol affect potassium channels in a similar way and, thus, possess proconvulsive activities. However, while thymol and local anaesthetics both block voltage-operated ion channels, they differ profoundly in their effects on the major receptor for inhibitory neurotransmission in the mammalian brain, the γ-aminobutyric acid (GABAA-) receptor. Local anaesthetics like lidocaine, bupivacaine, procaine, benzocaine and cocaine have been shown to inhibit GABA-induced currents, one mechanism likely to contribute to central nervous toxicity of local anaesthetics [40-42]. In contrast, thymol, as well as other ortho-alkylated phenol derivatives, activates Cl− currents through GABAA receptors in the absence of GABA , a mechanism that might contribute to the sedative activity of ortho-alkylated phenol derivatives revealed by animal experiments . Results from this study suggest that the naturally occurring phenol derivative thymol might represent an interesting alternative to local anaesthetics as it can be expected to combine lidocaine-like actions due to inhibition of voltage-operated sodium channels with sedative-anticonvulsant effects due to activation of inhibitory circuits in the CNS and neuroprotective effects due to its radical scavenging properties . However, further in vivo studies are required to confirm the pharmacological effects postulated from these in vitro results.
Up to now, the use of thymol in clinical practice did not take advantage of its potential analgesic or local anaesthetic effects. Thymol is still in use as halothane-stabilizing agent. High concentrations may be reached in irregularly drained vaporizers . However, no reports on plasma concentrations reached in patients during halothane anaesthesia or a potential minimum alveolar concentration (MAC)-reduction in thymol-containing halothane preparations with respect to thymol-free halothane are available at present. In combination with talc, when thymol iodine was used in the treatment of recurrent malignant and non-malignant pleural effusion providing pleurodesis when installed into a chest tube, no adverse effect was observed in the treatment group [49,50].
The authors are indebted to Professor Lehmann-Horn, Ulm, for providing them with transfected cells, to Dr Hans-Peter Reiffen, Department of Anaesthesiology, Hannover, for help with software problems, to Jobst Kilian and Andreas Niesel, Department of Neurology, Hannover, for technical support, and to W. Heyde, Clinical Pharmacy, Hannover, for providing stock solutions of thymol.
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Keywords:© 2002 European Society of Anaesthesiology
ANTI-INFECTIVE AGENTS, LOCAL, thymol; CHLORHEXANOLS, menthol; ION CHANNELS, sodium channel; TERPENES, menthol, thymol