20.500.12556/RUL-168588 Dinamika prenosa lokalnih anestetikov skozi membrano nevrona in njihovo antioksidativno delovanje Dynamics of neural membrane transfer of local anesthetics and their antioxidative activity Uvod: Lokalni anestetiki delujejo tako, da inhibirajo napetostno odvisne natrijeve kanale v membrani živčnih vlaken in s tem prenos signala vzdolž aksona. Da dosežejo tarčno mesto, morajo prehajati skozi membrano živčne celice. Kot šibke baze z vrednostmi disociacijske konstante kisline (pKa) med 7 in 10 so pri fiziološkem pH večinoma v protonirani, torej manj lipofilni obliki, ki ima manjšo afiniteto do membrane. V literaturi najdemo precej različnih pojasnil glede vpliva kemijsko-fizikalnih lastnosti lokalnih anestetikov in pH okolice na njihovo farmakokinetiko, saj njihovo obnašanje v celostnem, zapletenem okolju živčnih celic s hidrofilnimi in lipofilnimi predelki ni bilo nikoli dosledno raziskano. Obstajajo dokazi, da imajo lokalni anestetiki antioksidativni učinek, vendar ni jasno, ali delujejo kot neposredni lovilci radikalov ali imajo posreden vpliv na antioksidativni sistem. Namen doktorske naloge je raziskati farmakokinetične lastnosti lokalnih anestetikov s kombinacijo eksperimentalnih in računskih metod ter poiskati povezave med njihovimi farmakokinetičnimi in fizikalno-kemijskimi lastnostmi. Metode: Z uporabo metod računske kemije smo določili prosto energijo prehoda (?G) med vodnim okoljem (zunajcelična tekočina in aksoplazma) ter lipidno membrano. Izdelali smo koncentracijski profil lokalnih anestetikov v ravnotežnem stanju. Upoštevali smo vpliv različnih pH-vrednosti zunajcelične tekočine in aksoplazme na membranski prenos. Z modelom difuzije lokalnih anestetikov, ki smo ga ustvarili, smo proučili njihovo hitrost prehoda skozi biološko membrano. Za dinamično modeliranje prehoda skozi živčno vlakno smo uporabili numerično reševanje enačbe Smoluchowskega, ki je omogočila analizo spreminjanja časovno odvisne koncentracije anestetikov v različnih delih živca. Razvili smo poenostavljeni model živčne celice z uporabo navadnih diferencialnih enačb, s katerim smo lahko simulirali čas do začetka delovanja (angl. onset of action) in trajanje delovanja (angl. duration of action) lokalnih anestetikov. Uporabili smo več različnih laboratorijskih metod za določanje antioksidativne aktivnosti lokalnih anestetikov. Najbolj verjetne reakcije s prostimi radikali smo simulirali z metodami računske kemije. Rezultati: Ugotovili smo, da je samo solvatacijski model, ki temelji na elektronski gostoti (angl. solvation model based on density, SMD), uspel poustvariti eksperimentalne vrednosti porazdelitvenega koeficienta za nevtralne in protonirane zvrsti. Večina lokalnih anestetikov je imela ravnotežno konstanto prehoda iz vodne okolice v membrano večjo od 1. Posledično je bila prosta energija za prehod pri večini primerov negativna. Koncentracija vseh lokalnih anestetikov v fizioloških pogojih je bila v ravnotežnem stanju v aksoplazmi 1.86-krat večja kot v zunajcelični tekočini. Ob lokalni acidozi je bila koncentracija lokalnih anestetikov v aksoplazmi za polovico nižja kot v zunajcelični tekočini. Rešitev difuzijske enačbe je pokazala, da lidokain v membrani živčne celice doseže ravnotežno koncentracijo malo hitreje kot bupivakain, oba pa jo dosežeta v podobnem velikostnem redu, in sicer v milisekundah. Poenostavljeni model živčne celice s štirimi predelki je podal čas do vzpostavitve ravnovesnih koncentracij – približno 4 ms pri lidokainu in 40 ms pri bupivakainu. Simulirani čas do začetka delovanja je bil 167 s za lidokain in 186 s za bupivakain. Simulirano trajanje delovanja je bilo 41 min za lidokain in 328 min za bupivakain. Ob lokalni acidozi sta se pri obeh proučevanih anestetikih skrajšala tako čas do dosega ravnovesne koncentracije kot razpolovni čas praznjenja modela. Vsi trije lokalni anestetiki so pokazali sposobnost za lovljenje radikala 2,2'-azino-bis-3-etilbenzotiazolin-6-sulfonske kisline (ABTS·+), pri čemer je bil lidokain najučinkovitejši. V primerjavi z vitaminom C je lidokain pokazal 200-krat višjo polovično maksimalno inhibitorno koncentracijo (angl. half-maximal inhibitory concentration, IC50), kar pomeni, da so lokalni anestetiki dvesto krat šibkejši lovilci radikalov kot vitamin C. Termodinamično najugodnejša in tudi edina verjetna reakcija je prenos vodikovega atoma med prostim radikalom in skupino -C-H, ki se nahaja poleg karbonilne skupine. Ugotovili smo, da je antioksidativna aktivnost vseh testiranih lokalnih anestetikov v lipofilnem okolju zanemarljiva, kar so dodatno potrdili kvantnokemijski izračuni. Zaključki: Naš model živčnega vlakna je pokazal, da se skoraj vsi lokalni anestetiki kopičijo v membrani in Schwannovih celicah ter da je energijska pregrada za prehod skozi membrano primerljiva z učinkovinami, ki dobro prehajajo krvno-možgansko pregrado. Biološka membrana je torej zanemarljiva kinetična ovira pri prehodu lokalnega anestetika iz zunajcelične tekočine do njegovega receptorja v aksoplazmi in najverjetneje nima vpliva na čas do začetka delovanja anestetika. Lokalni anestetiki difundirajo iz predelkov z višjim pH v predelke z nižjim pH zaradi ugodne solvatacijske proste energije protoniranih zvrsti. Naš model pojasni manjšo učinkovitost lokalnih anestetikov v vnetem tkivu. Trajanje delovanja lokalnih anestetikov je odvisen od razmerja med nakopičenim lokalnim anestetikom v lipofilnih delih živca in hitrostjo izplavljanja lokalnega anestetika s kapilarami. Pri lokalni acidozi je v lipofilnih delih živca nakopičenega precej manj lokalnega anestetika, zato je zato učinek šibkejši. Lokalni anestetiki kažejo zmerno aktivnost pri lovljenju prostih radikalov v vodnem okolju, pri čemer je lidokain pokazal največjo aktivnost. Zdi se, da je njihova antioksidativna aktivnost v lipidnem okolju zanemarljiva. 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. lokalni anestetiki šibka baza prehod skozi membrano blokada živčnega signala metode računske kemije model difuzije prosta energija prehoda čas do začetka delovanja trajanje delovanja antioksidativna aktivnost lovljenje prostih radikalov local anesthetics weak base membrane transfer nerve signal block computational chemistry methods diffusion model free energy of transfer onset of action duration of action antioxidant activity free radical scavenging true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2025-04-18 07:15:54 2025-04-18 07:16:05 2025-04-19 04:36:57 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 30580 6745.pdf 6745.pdf 1 EF18F2D617D12CAA3E69370DA710EEB0 f8a0fec2f1c8c4aa699b99d22e169c0e4a2aaedce979e45012b0fdf5b031f66f 194d9055-1c13-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=209464 Medicinska fakulteta 0 0 0