Introduction: Local anesthetics act by blocking voltage-gated sodium channels in the neural membrane, consequently interrupting signal transmission along the nerve. To reach their target site, they must pass through the membrane of the nerve cell. However, as weak bases with an acid dissociation constant (pKa) between 7 and 10, at physiological pH, they exist mostly in their protonated, less lipophilic form, which has a lower affinity to the membrane. The explanations regarding the influence of environmental pH and physicochemical characteristics of local anesthetics on their pharmacokinetic properties vary widely. Notably, their behavior within the entire complex environment of the nerve cell, with aqueous and lipophilic compartments, remains poorly understood. Additionally, while there is evidence that local anesthetics have antioxidant effects, it is unclear whether they act as direct radical scavengers or have an indirect impact on the antioxidant system. This doctoral research aims to investigate the pharmacokinetic properties of local anesthetics using a combination of experimental and computational methods and to explore the connection with their physicochemical properties.
Methods: Computational chemistry methods were employed to calculate the free energies of transfer (ΔG) for local anesthetics between the aqueous environment (extracellular fluid and axoplasm) and the lipid membrane, enabling the construction of equilibrium concentration profiles. The influence of varying pH conditions in the extracellular fluid and axoplasm on membrane transfer was assessed. Additionally, a diffusion model was established to investigate the rate of local anesthetic permeation through biological membranes. Numerical solutions of the Smoluchowski equation were employed to analyze temporal changes in local anesthetic concentrations within distinct regions of the nerve. A simplified nerve cell model, based on ordinary differential equations, was developed to simulate the local anesthetic time of onset and duration of action. Furthermore, multiple laboratory methods were utilized to assess the antioxidant activity of local anesthetics, and computational chemistry methods were employed to simulate the most plausible reactions with free radicals.
Results: We found that only the solvation model based on density (SMD) successfully reproduced experimental octanol-water partition coefficients for neutral and protonated species. Most local anesthetics exhibited membrane transfer distribution coefficients exceeding 1, corresponding to negative free energies of transfer. Under physiological conditions, equilibrium concentrations of all local anesthetics were 1.86-fold higher in the axoplasm compared to the extracellular fluid. However, under local acidosis, the axoplasmic concentration was reduced to half that of the extracellular fluid. Numerical solution of the Smoluchowski equation revealed that lidocaine and bupivacaine attained equilibrium concentration within the nerve membrane on a microsecond timescale, with lidocaine exhibiting a faster rate of equilibration compared to bupivacaine. The simplified four-compartment nerve cell model predicted equilibrium establishment times of approximately 4 ms and 40 ms for lidocaine and bupivacaine, respectively. Simulated onset of action times were 167 s and 186 s, with the corresponding durations of action of 41 min and 328 min for lidocaine and bupivacaine, respectively. Notably, under local acidosis, both the time to reach equilibrium concentration and the half-life of model emptying were reduced for both anesthetics. Lidocaine, bupivacaine, and ropivacaine demonstrated the capacity to scavenge the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS•+), with lidocaine being the most effective. However, the half-maximal inhibitory concentration (IC50) of lidocaine was 200-fold higher than that of vitamin C, indicating that local anesthetics are relatively weak radical scavengers. The most thermodynamically favorable reaction, and the only one deemed plausible, involved a hydrogen atom transfer between the free radical and the -C-H group adjacent to the carbonyl group of the anesthetic molecule. Notably, the antioxidant activity of all tested local anesthetics was negligible within lipid environments, a finding supported by quantum chemical calculations.
Conclusions: Our nerve model demonstrated a preferential partitioning of local anesthetics into the membrane and Schwann cell environment, with an energy barrier for membrane transfer comparable to substances known to readily cross the blood-brain barrier. These findings suggest that the biological membrane poses a negligible barrier to the passage of local anesthetics from the extracellular fluid to their axoplasmic receptor, and thus likely does not significantly influence the onset time of their anesthetic effect. Local anesthetics exhibit preferential diffusion from regions of higher pH to those of lower pH, driven by the more favorable solvation free energy of the protonated species. This phenomenon, as captured by our model, explains the diminished efficacy of local anesthetics in inflamed tissues. Furthermore, the duration of action of local anesthetics is influenced by the balance between their accumulation within lipophilic nerve compartments and their rate of clearance through capillary networks. Notably, this accumulation capacity is diminished under conditions of local acidosis. While local anesthetics demonstrate moderate free radical scavenging ability in aqueous environments, with lidocaine being the most potent, their antioxidant activity within lipid environments appears negligible.
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