Use of long-acting local anesthetics that elicit complete neural blockade for more than 3 h often is desirable in pain management. Unfortunately, clinically available local anesthetics are in general not suitable for prolonged analgesia. This report describes the organic synthesis and functional testing of a lidocaine derivative that appears to fulfill the criteria of long-acting local anesthetics.

Methods: A lidocaine derivative, N-beta-phenylethyl lidocaine quaternary ammonium bromide, was synthesized, and its ability to inhibit Sodium sup + currents in cultured rat neuronal GH3cells was tested in vitro under whole-cell voltage clamp conditions. Neurologic evaluation of sciatic nerve block of sensory and motor functions in vivo was subsequently performed in rats.

Results: N-beta-phenylethyl lidocaine was found to be a potent Sodium sup + channel blocker in vitro. It produced both tonic and use-dependent blocks of Sodium sup + currents that exceeded lidocaine's effects by a factor of > 2 (P < 0.05). In vivo, N-beta-phenylethyl lidocaine elicited a prolonged and complete sciatic nerve block of the motor function and the withdrawal response to noxious pinch that was 3.6- and 9.3-fold longer than that of lidocaine (P < 0.001), respectively.

Conclusions: In an attempt to elicit prolonged local anesthesia, a quaternary ammonium derivative of lidocaine containing a permanent charge and an additional hydrophobic component was synthesized. Complete sciatic neural blockade of more than 3 h was achieved with this derivative. Of note, sensory blockade was prolonged to a greater extent than motor blockade. The approach used in this study may prove useful for developing new drugs applicable in pain management. (Key words: Anesthetics, local: N-beta-phenylethyl lidocaine.)

DURING the last two decades, the search has been on for new local anesthetics that produce analgesia of long duration, minimal impairment of autonomic function, and low toxicity. Such drugs are highly desirable for pain management. Attempts to develop this new class of local anesthetics have so far yielded mixed results. For example, both bupivacaine and etidocaine are considered to be long-acting local anesthetics; the duration of the major nerve block by these two local anesthetics is about 3–12 h. Unfortunately, both local anesthetics are also highly cardiotoxic. Amino amide local anesthetic cyclization was invented in the early 1970s, but this type of local anesthetic has not evolved for clinical use, probably because cyclization requires a haloalkyl amine component that is also a cancer-causing agent and because the chemical reaction of intramolecular cyclization often occurs before penetration of the nerve membrane. Finally, alkyl triethyl quaternary ammonium (QA) ions were reported to produce sensory block of rat infraorbital nerves for several days or weeks after injection. Later studies, however, revealed that this type of compound gives rise to severe morphologic damage, loss of myelinated axons, and axonal edema within 4 weeks of treatment. .

What are the structural determinants of local anesthetics that cause reversible blockade of neural functions? Traditional local anesthetics, exemplified by lidocaine (Figure 1), contain a tertiary amine separated at a distance of 6–9 Angstrom from a benzene ring by an intermediate chain. The intermediate chain is usually linked by either an ester (-COO-) or an amide (-CONH-) bond to the benzene ring. As a tertiary amine, lidocaine can be hydrophilic when it is protonated and can be hydrophobic when it is in its neutral form. If lidocaine is made permanently charged, such as its derivative QX-314 (F1-20), the compound becomes inactive when applied externally. Internal application of QX-314, however, elicits profound block of Sodium sup + channels, an indication that the charged form of lidocaine is an active form. This result also suggests that external QX-314 cannot readily cross the cell membrane to reach its receptor site on the Sodium sup + channel.

Tertiary amine local anesthetics including lidocaine produce both tonic and use-dependent block of Sodium sup + channels. Tonic block is defined as the block of Sodium sup + channels in their resting state while the nerve is stimulated infrequently, whereas use-dependent block is the additional block of Sodium sup + channels in their activated states while the nerve is stimulated repetitively (usually greater or equal to 1 Hz). The underlying mechanism for these two types of block has been intensively studied under voltage clamp conditions. Hille proposed a Modulated Receptor Hypothesis to explain these two types of Sodium sup + channel block elicited by local anesthetics. He hypothesized that the local anesthetic receptor site on the Sodium sup + channels is modulated by depolarization. The local anesthetic receptor site changes its configuration as functions of membrane potential and time (i.e., voltage- and time-dependent conformational changes). At different states, the local anesthetic receptor is thought to have different affinity toward local anesthetics.

The ability of local anesthetics to block Sodium sup + currents therefore is relevant to the topology of the local anesthetic receptor in the Sodium sup + channel, because the higher the affinity of Sodium sup + channel blocker, the stronger the interaction with its receptor. Several additional findings on the structure-activity relationships of local anesthetics have now emerged from these studies. First, there are two hydrophobic local anesthetic binding domains in the Sodium sup + channel. Each binding domain can accommodate up to a 12-hydrocarbon chain. External amphipathic QAs apparently can cross the membrane barrier to reach their internal receptor site, albeit the rate of cross is slower than that of the traditional local anesthetics. Second, the neutral form of tertiary amine is in general far less potent. Third, the permanently charged amphipathic local anesthetics, such as the products of cyclization of a haloalkyl amine local anesthetic compound, are easily "trapped" within the cell after external application. These basic findings may permit us to develop potent Sodium sup + channel blockers. The purpose of this study was (1) to synthesize a long-acting lidocaine derivative, and (2) to test this drug both in vivo and in vitro.


Lidocaine base was purchased from Sigma Chemical Co. (St. Louis, Mo); 1-bromododecane and (2-bromoethyl)benzene were from Aldrich, Chemical Company, Inc. (Milwaukee, WI). QX-314 chloride was donated by Astra Pharmaceutical Products (Worcester, MA). Silica gel G was obtained from Brinkmann Instruments, Inc. (Westbury, NY). All other chemicals were reagent grade from commercial sources.

Synthesis of Tonicaine

The conventional method for QA synthesis was used to modify the lidocaine structure. Tonicaine was synthesized from lidocaine (base) and (2-bromoethyl)benzene. A 2:1 molar ratio of lidocaine and (2-bromoethyl)benzene were refluxed at 80–90 degrees Celsius in absolute ethanol for 5 days. Excess ethanol was evaporated. The product was washed several times each with 25 ml warm hexane (60 degrees Celsius). Residual hexane was removed under vacuum. The product, N-beta-phenylethyl lidocaine bromide [diethyl-(2,6-dimethylanilinocarbonyl)methyl-beta-phenylethyl ammonium bromide], was purified by silica gel column chromatography. The mobile phases were chloroform/ethyl acetate (13/1, vol/vol) followed by chloroform/ethanol (80/20, vol/vol). Tonicaine was eluted in the chloroform/ethanol. The eluted fractions containing the product were pooled and the solvents were removed under vacuum. The product was > 98% pure as judged by thin layer chromatography systems. Thin layer chromatography systems employed in the synthesis were normal phase thin layer chromatography plates (Fisher Scientific, PA) developed with ethanol, 96% ethanol/0.8 M NH4Cl (80/20, vol/vol), or chloroform/ethyl acetate (13/1, vol/vol). The practical yield was 39%. Structural analysis of tonicaine by mass spectrometry yielded a molecular mass of 339.2, which is consistent with the structure of tonicaine cation shown in F1-20.

Synthesis of N-dodecyl Lidocaine Quaternary Ammonium

N-dodecyl lidocaine was synthesized from lidocaine (base) and 1-bromododecane by the method similar to that for tonicaine synthesis (see above). The product was > 98% pure as judged by thin layer chromatography systems. Structural analysis of N-dodecyl lidocaine QA by mass spectrometry yielded a molecular mass of 403.2, which agreed with the structure of N-dodecyl lidocaine QA shown in F1-20.

Cell Culture

Rat clonal pituitary GH3cells were purchased from the American Type Culture Collection (Rockville, MD) and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Labs, Logan, UT), as described by Cota and Armstrong. For Sodium sup + current recording, cells were grown in a 35-mm culture dish, which was then used as a recording chamber.

Whole-cell Voltage Clamp

The whole-cell variant of the patch-clamp method was used to measure Sodium sup + currents in GH3cells. The external solution contained (in mM) 150 choline Cl, 0.2 CdCl2, 2 CaCl2, and 10 hydroxyethylpiperazineethane sulfonic acid adjusted to pH 7.4 with tetramethyl hydroxide. Micropipettes were fabricated and had a tip resistance of approximately 1 M Omega when filled with an Sodium sup + solution containing (in mM) 100 NaF, 30 NaCl, 10 EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), and 10 hydroxyethylpiperazineethane sulfonic acid adjusted to pH 7.2 with CsOH. The junction potential of electrodes was nulled before seal formation. A liquid junction potential of 1.6 mV for the 0-mM-Sodium sup + external solutions was not corrected in this report. After the rupture of the patch membrane, the cell was allowed to equilibrate with the pipette solution for at least 15 min at the holding potential of -100 mV. Under these reversed Sodium sup + gradient conditions, outward Sodium sup + currents were activated at approximately -30 mV. The advantages of using the reversed Sodium sup + gradient have been discussed by Cota and Armstrong. Tonicaine, N-dodecyl lidocaine QA, and QX-314 at appropriate concentrations, were applied to cells with a flow rate of about 0.12 ml/min via a series of narrow-bored capillary tubes positioned within 200 micro meter of the cell. Washout of drugs was performed via a tube containing the external solution without drug present. Voltage-clamp protocols were created with pClamp software (Axon Instruments, Inc., Foster City, CA). Leak and capacitance were subtracted by a leak and capacity compensator as described by Hille and Campbell. Additional compensation was achieved by the patch clamp device (EPC7, List-Electronic, Darmstadt/Eberstadt, Germany). All experiments were performed at room temperature (23 plus/minus 2 degrees Celsius). At the end of the experiments, the drift in the junction potential was generally < 2 mV.

Neurologic Evaluation of Sciatic Nerve Block in the Rat

The following protocols have received approval from the Harvard Medical Area Standing Committee on Animals. The observations of all sciatic nerve functions were made under free behavior conditions. The integrity of neurologic functions was examined in handled rats. Handling reduces stress during neurologic examination. Measurements of functional impairment were performed by comparison of functions before and after injection. Suitable anatomic landmarks (greater trochanter and ischial tuberosity ) easily located by palpation make it possible to administer the studied drug with precision. Changes of function were estimated as percentage of maximal possible effect (% MPE) before and at different times after drug administration. Complete block of function was considered 100% MPE, no change in function 0% MPE. Motor function, proprioception, and nociception were evaluated at 10 min before, at 5, 10, 30, and at every 30 min until full recovery after injection of 0.1 ml tonicaine (n = 14, over a period of 9–12 h) and at 10 min before and at 1, 5, 10, 20, 30, and every 15 minutes until full recovery after injection of 0.1 ml lidocaine (n = 6, over a period of 3 h) at the sciatic notch. Six of the rats were injected with lidocaine (1%) and three with isotonic saline at the contralateral side and 24–36 h before the injection of tonicaine. The molar concentration was identical for tonicaine and lidocaine at 42.67 mM in saline. Throughout the experiment all animals were observed for abnormalities in mental status and free motor behavior, e.g., alertness, responsiveness to environment, motor activity, gait, and resting posture. The observer was blinded to the type of drug or saline solution injected. Details of the functional evaluation can be found in Thalhammer et al. .

Proprioception. The evaluation was based on resting posture, gait, and postural reactions such as "hopping" and "tactile placing" and measured by scores from 3 (normal posture and gait) to 0 (full absence of postural reactions). Full absence of postural reactions was considered 100% MPE.

Tactile Placing. The toes of one foot were ventroflexed and their dorsi were placed onto the supporting surface, while the animal was kept in normal resting posture. The ability to reposition the toes was evaluated.

Hopping. The front half of the animal was lifted off the ground to allow the body's weight to be supported by their hind limbs. Then one hind limb at the time was lifted off the ground surface and the animal's body was moved laterally. The ability of the animal to follow the lateral movement of the body by hopping with the weight-supporting limb was evaluated.

Motor function of hind limbs was evaluated by the "extensor postural thrust." The rat was held upright so that the hind limbs were extended and the body's weight was supported by the distal foot. The force necessary to bring the heel in contact with the platform of a balance was measured (in grams). The reduction in force, resulting from reduced extensor muscle tone, was considered motor deficit. A force < 15 g was considered absence of extensor postural thrust or 100% motor block. .

Nociception was evaluated by measuring the latency (heat) or amplitude (pinch) of withdrawal response to noxious heat and noxious mechanical stimulation. Care was taken to avoid tissue injury resulting in hyperanalgesia by properly spacing the stimulations and by a 10-s cutoff time for heat stimulation. .

Withdrawal Response to the Heat Stimulation (Heat WR). Latency measurement of the withdrawal response to the application of a hot (51.0 plus/minus 0.5 degree Celsius) handheld metal probe through which hot water was circulated to the dorsolateral surface of the metatarsus. Absence of withdrawal to 10-s stimulation was considered 100% MPE.

Withdrawal Response to Pinch (Pinch WR). The fifth toe was pinched (to 300 g) with a force-calibrated serrated forceps for 2 s and the WR was graded as 4 (normal, brisk generalized motor reaction, withdrawal of the stimulated hind limb, attempts to bite forceps, and vocalization); 3 (like 4, but slower than on the control side); 2 (like 3, but with one of the responses lacking, e.g., no vocalization or no general motor reaction, only turning of the head); 1 (only weak attempt to withdraw); or 0 (no response).

Statistical Analysis

Results of analyses from voltage clamp experiments are presented as mean plus/minus SE. An unpaired Student's t test and a one-way analysis of variance (SigmaStat, Jandel Scientific Software, San Rafael, CA) were used to evaluate the significance of changes produced by the drugs on the tonic and the use-dependent block, respectively. P < 0.05 was considered statistically significant.

Magnitude of functional changes from neurologic evaluations is expressed in % MPE, mean plus/minus SE. Duration of functional changes is expressed in minutes, mean plus/minus SE. Validity of differences was tested by an unpaired Student's t test.

Tonicaine as a Potent Sodium sup + Channel Blocker

Like lidocaine, tonicaine is a Sodium sup + channel blocker but with a potency far greater than that of its parent compound. Tonicaine when applied externally to cultured neuronal GH3cells tonically inhibited Sodium sup + currents by 55.0 plus/minus 7.0%(n = 5) at the concentration of 100 micro Meter for 30 min, whereas lidocaine usually inhibited currents by 27.1 plus/minus 6.8%(n = 7, Figure 2(A)). As for most local anesthetics, the block of Sodium sup + currents by lidocaine reached its steady state within 1.5 min. In contrast, the time course of the block by tonicaine was much slower than that by lidocaine at 100 micro Meter (F2-20(B and C)). Because of its permanently positive charge, it is likely that tonicaine does not cross the cell membrane as easily as lidocaine, which can be in a neutral form at physiologic pH. Tonicaine continued to inhibit Sodium sup + currents by 80–90% after 1 h of treatment. Due to this slow on-rate of tonicaine, we were unable to perform the conventional dose-response study, because the time required for tonicaine to reach steady state effect at lower concentrations would be prohibitively long for the voltage clamp experiments. Preincubation of cells with this drug, unfortunately, would prevent us from determining the current amplitude without drug.

Trapping the Charged Compound within the Cell

The tonic block elicited by tonicaine was only partially and slowly reversed after continuous perfusion of a drug-free external solution. Figure 3(B and C) shows that Sodium sup + current was inhibited by about 80% by 100 micro Meter tonicaine for over 45 min and recovered to about 55% of the control value after a 30-min wash. In contrast, the blocking effect of lidocaine at 100 micro Meter was quickly and often completely reversed by washing (F3-20(A and C)). The half times of washout for lidocaine and tonicaine were < 30 s and approximately 30 min, respectively.

Use-dependent Inhibition of Sodium sup + Currents by Tonicaine

In addition to tonic inhibition of Sodium sup + currents (when the nerve was stimulated infrequently), tonicaine also elicited use-dependent inhibition of Sodium sup + currents when the nerve was stimulated at 2 Hz. Figure 4(B) shows that after a 5-min treatment an additional 50–60% of Sodium sup + currents were blocked by tonicaine at 100 micro Meter at a frequency of 2 Hz. At the same concentration, lidocaine produced about 20% of use-dependent block (also see ), which was significantly (P < 0.05) greater than that produced by the control in the absence of local anesthetics (approximately 8%). The rate of use-dependent block by lidocaine was relatively fast with a time constant of 1.7 pulse sup -1, whereas the rate of block by tonicaine was again much slower (P < 0.05) with a time constant of 17.2 pulse sup -1 (F4-20(B and C)). Thus, tonicaine appears to retain the use-dependent characteristic of local anesthetics but with a slower on-rate kinetic.

Neurologic Evaluation of Sciatic Nerve Block by Tonicaine

In no instance was death or any visible impairment of mental status, ataxia, or posture abnormality in the noninjected limbs observed. All neurobehavioral changes were completely reversible.

Onset of Functional Block

As soon as 1.0 min after injection of lidocaine (n = 6) motor function was impaired; impairment of heat WR occurred at 1.7 plus/minus 0.7 min and pinch WR at 3.0 plus/minus 0.9 min. Tonicaine (n = 14) induced impairment of motor function and heat WR by 5.0 min and pinch WR by 5.7 plus/minus 0.5 min. Lidocaine fully abolished motor function and heat WR at 5.0 min and pinch WR at 9.2 plus/minus 2.4 min, tonicaine at 6.4 plus/minus 0.6 min for motor, at 5.4 plus/minus 0.4 min for heat WR and at 8.2 plus/minus 0.7 min for pinch WR (Figure 6). No significant differences were found among these onset times.

Duration of Complete Block

The absence of functions lasted 57.5 plus/minus 4.0 min for motor, 48.3 plus/minus 6.3 min for heat WR, and 34.2 plus/minus 7.1 min for pinch WR after injection of lidocaine (Figure 5). Motor block after lidocaine was not significantly different from the block of heat WR in duration but did outlast pinch WR (P < 0.05). The block of functions induced by tonicaine was 209.1 plus/minus 19.9 min for motor, 373.0 plus/minus 22.9 min for heat WR, and 319.1 plus/minus 29.4 min for pinch WR (F5-20). The duration was shortest for motor block, which was shorter than block of heat WR (P < 0.001) and block of pinch WR (P < 0.005). Thus, tonicaine produced blocks 3.6 times longer for motor (P < 0.001), 7.7 times for heat WR (P < 0.001) and 9.3 times (P < 0.001) for pinch WR than lidocaine.

Recovery of Functions

After lidocaine injection pinch WR was fully recovered at 56.7 plus/minus 6.4 min, heat WR at 59.3 plus/minus 9.8 min, and motor at 77.5 plus/minus 4.6 min. There was no significant difference in duration of partial impairment (i.e., in time of recovery) between motor and nociceptive function induced by lidocaine (P > 0.1). After injection of tonicaine all functions recovered in 9–24 h in all animals (F6-20). Whenever nociception was blocked completely with lidocaine, all other functions were absent. To the contrary, with tonicaine, full block of nociception outlasted full block of motor and proprioception, and during recovery the % MPEs of WR to heat and pinch were higher than those of motor and proprioception (P < 0.05). At 300 min after injection, when MPE was 100% for heat WR, MPE of pinch WR was 86 plus/minus 9%, motor 70 plus/minus 7%, and proprioception 65 plus/minus 7%.

Structure/Function Relationship.

To determine the role of the additional hydrophobic arm of tonicaine, in vivo injection of QX-314 and N-dodecyl lidocaine QA was subsequently performed in three and four rats, respectively. QX-314 at 1% lidocaine equivalent concentration elicits only incomplete and brief sensory and motor block. Complete block of proprioceptive, motor, and heat and pinch WR functions was not achieved.

Likewise, N-dodecyl lidocaine QA elicited no functional block of the sciatic nerve in rats. None of the functions of proprioceptive, motor, heat, and pinch WR characteristics were impaired by this compound at the 1% lidocaine equivalent concentration (42.67 mM). In contrast to this in vivo result, N-dodecyl lidocaine QA is a strong Sodium sup + channel blocker in vitro. At 25 micro Meter, N-dodecyl lidocaine QA slowly blocked all Sodium sup + currents within 40 min of external application (data not shown). This tonic block was not reversible by washing with an external drug-free solution for 30 min. N-dodecyl lidocaine QA also elicited strong use-dependent block when the cell was stimulated repetitively. In general, the results from N-dodecyl lidocaine QA were very similar to those from the trimethyl C14-QA compound described previously in GH3cells. Together they demonstrate that in vitro results cannot always be extrapolated to the in vivo environment. Neural sheaths, myelin sheath, and surrounding tissues may hinder some potent Sodium sup + channel blockers from reaching target sites in vivo.

This report demonstrates that tonicaine is a potent Sodium sup + channel blocker under voltage-clamp conditions in vitro and a long-acting local anesthetic when injected into rats in vivo. An unexpected characteristic of tonicaine is that it elicits differential block of sensory and motor functions. The importance of these findings is discussed later.

Tonicaine as a Long-acting Local Anesthetic

The design of local anesthetics with a long duration of analgesia requires detailed understanding of the local anesthetic receptor topology; of the physicochemical properties of drugs, the lipid membrane, the myelin sheath, and of the diffusion/adsorption of local anesthetics by the surrounding tissues. Our approach starts at the receptor site of the Sodium sup + channel, which has been shown to contain two large hydrophobic local anesthetic binding domains. The reason to use N-beta-phenylethyl modification is the unexpected failure of -C12H sub 25 modification in sciatic nerve block. We surmise that N-dodecyl-lidocaine QA ions dwell in the cell membrane and/or the myelin sheath too long and therefore may be unable to cross the cell membrane to reach the cytoplasm at a sufficient concentration. It is also possible that N-dodecyl-lidocaine QA ions form micelle vesicles, because dodecyl trimethyl QA ions do. As a result, free N-dodecyl-lidocaine QA ions could be rather low in concentration in vivo (i.e., << 42.67 mM). In contrast, ethyl-modification of lidocaine yields a rather hydrophilic compound, QX-314, that does not penetrate the membrane efficiently, and hence the drug is nearly inactive when applied externally (also see Strichartz, 1973). Our results indicate that the receptor topology and the physicochemical properties of the drug are both important in determining the local anesthetic potency in vivo. A permanently charged compound is not necessarily disadvantageous; this positive charge may be shielded by surrounding hydrophobic arms of the drug, as indicated here for tonicaine, and may still be able to cross the membrane. In fact, a charged compound could be trapped within the cell and therefore may exert its action for a longer duration. These basic principles, which differ from those of short-acting local anesthetics (containing a tertiary amine component), should be applicable to the design of other long-acting compounds.

Differential Block of Sensory and Motor Functions

In medical practice, the often observed ability of bupivacaine to block the motor functions to a lesser extent than other local anesthetics during regional anesthesia makes this agent particularly valuable for obstetric analgesia. However, such a differential block has so far not been conclusively demonstrated in vitro. As for veratridine, which was shown in vitro to block the C-fibers more efficiently than the A-fiber in vagus nerve, the in vivo experiments show no such differential block of sensory and motor functions in the sciatic nerve. It appears that tonicaine can elicit a significant differential block of sensory and motor functions in vivo during recovery (with motor function and proprioception recovering to a greater extent than nociception). These results suggest that tonicaine may be of greater clinical use when differential block of sensory and motor function is desirable during regional anesthesia. Furthermore, tonicaine may provide a tool to dissect the underlying mechanisms of differential block, which until now remain elusive.

In Vivo Versus In Vitro Block

We have shown that tonicaine acts more slowly than lidocaine at 100 micro Meter in blocking Sodium sup + currents in GH3cells (it takes longer than 1 h to reach steady-state concentration), possibly because of its permanent charge, which may hinder its ability to cross the cell membrane quickly. In vivo injection of tonicaine at 1% lidocaine equivalent concentration (42.67 mM), however, elicits complete functional block for pinch WR within 10 min. Perhaps the difference in effect between the two preparations is the result of differences in the effective concentration of tonicaine present in neuronal tissue between the in vivo and in vitro models. Other factors such as the permeability of the neural sheath by these compounds and the adsorption/diffusion of these compounds in the surrounding tissues are not clear and remain to be examined.

The authors thank Dr. G. Richard Arthur for his assistance on statistical analysis, Dr. Lin-Er Lin for her initial screening of lidocaine derivatives, Ms. Rachel Abrams for secretarial services, and Ms. Jaylyn Olivo for editorial services.

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