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Orientador: Eneida de Paula Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Os anestésicos locais (AL) são capazes de interromper a condução nervosa, impedindo o influxo de sódio através dos canais voltagem-dependentes das membranas excitáveis. A etidocaína (EDC) é um anestésico local de longa duração, cuja suposta toxicidade restringiu seu uso clínico. Os lipossomas podem prolongar o tempo de analgesia e reduzir a toxicidade dos anestésicos locais. Lipossomas de gradiente iônico (IGL) foram propostos para aumentar a capacidade de carregamento e liberação sustentada de fármacos, dos lipossomas. Nesta tese sintetizamos o cloridrato de etidocaína (EDC) para posterior encapsulação em IGL. Três tipos de vesículas lipossomais foram preparadas contendo 250 mM de (NH4)2SO4 no interior aquoso, para encapsulamento de 0,5% de EDC: com fosfatidilcolina de ovo:colesterol: "alfa" tocoferol e com fosfatidilcolina hidrogenada de soja:colesterol (10 e 20 mM), essas últimas otimizadas por planejamento fatorial. Dispersão dinâmica de luz, rastreamento de nanopartículas, ressonância magnética nuclear de hidrogênio (diffusion ordered spectroscopy, saturation-transfer difference), calorimetria diferencial de varredura, microscopia eletrônica de transmissão e ressonância paramagnética eletrônica foram usados para caracterizar: o tamanho, a polidispersão e o potencial elétrico de superfície das nanopartículas, sua concentração, morfologia, fluidez da membrana e capacidade de encapsulação. Testes de cinética da liberação do fármaco, testes de toxicidade in vitro (em células de Schwann e fibroblastos gengivais) e in vivo (em C. elegans e em ratos) também foram realizados. Para determinar o efeito anestésico, foi usado o teste de pressão da pata (PWPT) em ratos Wistar. A formulação otimizada, IGLEDC 20 mM, apresentou população de partículas com diâmetro médio de 450 ± 10 nm, baixa polidispersão (PDI < 0,25), concentração média de 8,5 ± 0,4x1013 partículas/mL, potencial zeta negativo (-25,1 ± 0,7 mV) e esses parâmetros permaneceram estáveis durante armazenamento a 37oC por 180 dias. A eficiência de encapsulação foi de 41% e a formulação induziu liberação sustentada do fármaco (ca. 24 h), reduzindo a toxicidade da EDC sobre células de Schwann in vitro e C. elegans, in vivo. Em ratos, o tempo de anestesia alcançado com etidocaína 0,5% encapsulada em IGL foi 1,5 x maior (130 min) que a induzida pelo fármaco livre. Uma nova formulação farmacêutica é proposta para promover liberação sustentada e redução da toxicidade da etidocaína. IGLEDC pode vir a ser uma ferramenta para reintroduzir a etidocaína no uso clínico Abstract: Local anesthetics (LA) stop nerve conduction by preventing the influx of sodium ions through the voltage-gated channels of excitable membranes. Etidocaine (EDC) is a long-lasting local anesthetic, which alleged toxicity has restricted its clinical use. Liposomes can prolong the analgesia and reduce the toxicity of local anesthetics. Ionic gradient liposomes (IGL) have been proposed to increase the upload and prolong drug release from liposomes. In this thesis, etidocaine was synthesized for further encapsulation into liposomes. Three kinds of liposomes, containing 250 mM (NH4)2SO4 in their inner aqueous core were prepared for the encapsulation of 0.5% EDC: with egg phosphatidylcholine:cholesterol: "alfa"-tocopherol or with hydrogenated soy phosphatidylcholine:cholesterol (10 and 20 mM), the last ones optimized through factorial design. Dynamic light scattering, nanotracking analysis, nuclear magnetic resonance (diffusion ordered spectroscopy, saturation-transfer difference), differential scanning calorimetry, transmission electron microscopy and electron paramagnetic resonance were used to characterize nanoparticles size, polydispersity, zeta potential, concentration, morphology, membrane fluidity and encapsulation efficiency. Release kinetics, in vitro (over Schwann and gingival fibroblasts cells) and in vivo (C. elegans and rats) toxicity tests were also performed. To determine the anesthetic effect, the Paw Withdrawal Threshold to Pressure test (PWPT) was used in Wistar rats. The optimized formulation (IGLEDC 20 mM) showed liposomes with 450 ± 10 nm average diameters, low polydispersity (< 0.25), mean concentration of 8.5 ± 0.4x1013 particles/mL and negative zeta potentials (-25.1 ± 0.7 mV), these parameters remaining stable during 180 days of storage at 37ºC. The encapsulation efficiency was 41% and the formulation induced sustained drug release (ca. 24h), while reducing EDC toxicity to Schwann cells in vitro and to C. elegans in vivo. The total anesthesia time achieved with 0.5% etidocaine in IGL was 1.5 times longer (130 min) than that induced by EDC in solution. A novel pharmaceutical formulation is proposed, to promote sustained release and reduced toxicity of etidocaine. IGLEDC can come to be a tool to reintroduce etidocaine in the clinical practice Doutorado Bioquímica Doutora em Biologia Funcional e Molecular CNPQ 141169/2015-0

Local anesthetics have revolutionized surgery and many more medical subspecialties. The first local anesthetic ever used was cocaine, which remained for many years the only active substance and also the standard until procaine was synthesized. Local anesthetics are used for surface, infiltration, regional and plexus blocks, epidural, and intrathecal anesthesias. The aim of local anesthesia is to make the skin and mucous membranes, as well as deeper tissues, insensitive to pain. Topical anesthetics are a help for venipuncture, vaccinations, some very superficial procedures, such as curettage, shave excisions, and laser treatments, and a variety of other procedures. Topical anesthetics have gained wide acceptance in dermatologic surgery, but also in pediatrics and wherever there is unbalanced fear of a needle prick. Lidocaine is certainly the most commonly used local anesthetic in dermatologic surgery and has an excellent safety report. Etidocaine is a long-acting local anesthetic that is well tolerated not only for nerve blocks but also for infiltration anesthesia.

Etidocaine (EDC) is a long lasting local anesthetic, which alleged toxicity has restricted its clinical use. Liposomes can prolong the analgesia time and reduce the toxicity of local anesthetics. Ionic gradient liposomes (IGL) have been proposed to increase the upload and prolong the drug release, from liposomes. First, a HPLC method for EDC quantification was validated. Then, large unilamellar vesicles composed of hydrogenated soy phosphatidylcholine:cholesterol with 250 mM (NH4)2SO4 - inside gradient - were prepared for the encapsulation of 0.5% EDC. Dynamic light scattering, nanotracking analysis, transmission electron microscopy and electron paramagnetic resonance were used to characterize: nanoparticles size, polydispersity, zeta potential, concentration, morphology and membrane fluidity. Release kinetics and in vitro cytotoxicity tests were also performed. IGLEDC showed average diameters of 172.3 ± 2.6 nm, low PDI (0.12 ± 0.01), mean particle concentration of 6.3 ± 0.5 × 1012/mL and negative zeta values (−10.2 ± 0.4 mV); parameters that remain stable during storage at 4°C. The formulation, with 40% encapsulation efficiency, induced the sustained release of EDC (ca. 24 h), while reducing its toxicity to human fibroblasts. A novel formulation is proposed for etidocaine that promotes sustained release and reduces its cytotoxicity. IGLEDC can come to be a tool to reintroduce etidocaine in clinical use.

In October 1979, Albright wrote an editorial warning of the risks of cardiac toxicity due to bupivacaine and etidocaine. The editorial followed a report by Prentiss concerning a cardiac arrest in a patient receiving caudal anesthesia with etidocaine. In that case, cardiovascular collapse followed the rapid onset of convulsions after possible intravascular (iv) injection of etidocaine. To Albright’s knowledge, this was the sixth anecdotal case of sudden cardiovascular collapse following presumed iv injection of bupivacaine (0.5% or 0.75%) or etidocaine. He suggested that inadvertent iv administration of clinical doses of etidocaine and bupivacaine, in comparison with other local anesthetics (LAs), could result in almost simultaneous central nervous system and cardiovascular system toxicity without preceding hypoxia. Albright also noted that resuscitation was difficult, prolonged, and, in most situations, the outcome was poor. Subsequently, many additional cases of cardiovascular collapse were reported to the Federal Drug Administration (FDA) in the United States. A worrying number of these cases occurred in young previously healthy women who received epidural analgesia/anesthesia for childbirth. Albright’s editorial and the reports to the FDA prompted a letter to anesthesiologists in 1984 stating that 0.75% bupivacaine was no longer recommended for obstetric anesthesia because of the risk of cardiac arrest. Following this letter, the manufacturers of bupivacaine sent similar correspondence to Canadian anesthesiologists that caused considerable controversy and debate. In other countries, caution was urged in using 0.75% bupivacaine, although many anesthesiologists felt that the problem of cardiotoxicity was confined to the United States. In this issue of the Journal, Dillane and Finucane review the systemic toxicity of LAs, describing the mechanisms underlying central nervous system and cardiovascular toxicity associated with elevated plasma levels of LAs. They point out that systemic toxicity of LAs frequently results from inadvertent iv injection or from rapid absorption of the LA from the injection site with subsequent high plasma levels, and they suggest that there are benefits from the use of ultrasound during the performance of regional anesthesia. Ultrasound not only facilitates the identification of anatomical structures, hence avoiding iv injection, but also, it can facilitate more targeted administration of smaller volumes of LAs, thereby increasing the safety of regional anesthesia. What can we learn from this review article and other similar articles? First, the authors reiterate the history and the known facts regarding this potentially fatal complication. Second, by so doing, they encourage practicing anesthesiologists to examine their own practices and to consider whether LA administration is safe in light of the evidence presented. Although Albright and others implicated bupivacaine’s greater toxicity as the direct cause of cardiac arrest, this assessment was not universally accepted. As most drugs that are used for anesthesia are toxic if used in the wrong dose or wrong space, some anesthesiologists urged caution in blaming bupivacaine. These anesthesiologists questioned the anesthetic technique, as several of the obstetric cases presented to the FDA did not reflect what was considered good anesthetic practice at the time. In some of these cases, a large volume of a high concentration of LA was injected through the epidural J. Shannon, FCARCSI J. Douglas, MD (&) Department of Anesthesia, British Columbia’s Women’s Hospital and Health Centre and University of British Columbia, Room 1Q72, 4500 Oak Street, Vancouver, BC V6H 3N1, Canada e-mail: jdouglas@cw.bc.ca

A double-blind study has been made comparing etidocaine 1% (Duranest®) and lidocaine 2% (Xylocain®). The investigation comprised 92 patients with senile cataract, operated under retrobulbar anesthesia and facial nerve block. During the normal interval between the injection and the operation, lidocaine seemed to be more effective than etidocaine, both as an analgetic and kinetic agent, while etidocaine had a longer analgetic effect. The relative high adrenaline (epinephrine) content in lidocaine 2% is discussed.

The uptake of lidocaine, methyllidocaine, bupivacaine, etidocaine was studied in rat lung slices at different pH-values. The accumulation of the quaternary analogue, methyllidocaine, was not changed in the pH interval 7.0--8.0. The uptake of the three other substances was about 3--4 times lower at pH 7.0 than at pH 8.0. The rank order of distribution at a fixed cation/base ratio was bupivacaine greater than etidocaine greater than lidocaine. Interactions between lidocaine and other substances were studied in lung slices and in isolated perfused lungs. Bupivacaine and nortriptyline counteracted the accumulation of 14C-lidocaine in lung slices in a dose-dependent manner. Nortriptyline was more effective than bupivacaine. In isolated perfused lung, bolus injections of nortriptyline and lidocaine rapidly displaced 14C-lidocaine from the tissue. In this study we suggest that the base form of local anaesthetics accumulate in the lung tissue, while the cationic form binds to accessible binding sites in the cell membranes.

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, (2)H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S(mol)) and dynamics (T(1)) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel.

A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with methanol. Etidocaine served as the internal standard. After microscale equilibrium-dialysis, measurement of free ropivacaine levels was performed after direct injection of the dialysate into the chromatograph. The system used a Zorbax eclipse XD8 C8 analytical column packed with 5 microm diameter particles as the stationary phase. The mobile phase consisted of a 15-min gradient (mobile phase A: 0.05% (v/v) trimethylamine in acetonitrile, mobile phase B: 2mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/z 275 for ropivacaine and m/z 277 for etidocaine. The drug/internal standard peak area ratios (plasma) or peak areas (dialysate) were linked via a quadratic relationship to concentrations. Precision ranged from 1 to 7.6% accuracy was between 92.6 and 109%. The lower limits of quantitation were 1 microg/l in plasma and 2 microg/l in the dialysate. This method was found suitable for the analysis of plasma samples collected during a clinical trial performed in 30 infants undergoing epidural anaesthesia or continuous psoas compartment block.

Local anesthetics provoke reversible blockade of nerves by interaction with sodium channels in membranes of nerves. The uncharged molecular configuration of the local anesthetic penetrates the membrane from the outside and the charged configuration then interacts with the sodium channel from the inside. The potency of a local anesthetic is determined mainly by lipid solubility, the time of onset by the pK(a) of the substance and the duration of action by protein binding. Local anesthetic molecules consist of a hydrophilic tertiary amine and a lipophilic aromatic system combined by an ester or amide linkage. Local anesthetics may therefore be classified as aminoester or aminoamide compounds. Aminoesters are hydrolysed by plasmacholinesterase whereas aminoamides undergo metabolisation in the liver. Aminoamides cause fewer allergic reactions. Local anesthetics show dose-dependent CNS and cardiac toxicity. Reports of toxicity, mainly involving bupivacaine and etidocaine initiated the development of ropivacaine which is the first aminoamide marketed as a pure S-enantiomer. Recently introduced was levo-bupivacaine, the S-enantiomer of bupivacaine.

An editorial in 1979 by George Albright about sudden cardiac arrest after regional anesthesia spawned an era of intense research focusing on what local anesthetics do to the heart and how they do it. The ultimate goal of the research was to bring to the clinician long-acting local anesthetics that are less cardiotoxic than ones available before 1979, bupivacaine and etidocaine, in particular. In this article, I will review results of studies of local anesthetic cardiotoxicity using the intact mammalian heart in vitro published after the Albright editorial through 2001.

1. The structural features that determine the state-dependent interaction of local anaesthetics with voltage-operated sodium channels are still a matter of debate. We have studied the blockade of sodium channels by 2,6-dimethylphenol, a phenol derivative which resembles the aromatic tail of lidocaine, etidocaine, and bupivacaine. 2. The effects of 2,6-dimethylphenol were studied on heterologously (HEK 293) expressed rat neuronal (rat brain IIA) and human skeletal muscle (hSkM1) sodium channels using whole-cell voltage-clamp experiments. 3. 2,6-Dimethylphenol was effective in blocking whole-cell sodium inward currents. Its potency was comparable to the potency of lidocaine previously obtained with similar protocols by others. The IC(50) at -70 mV holding potential was 150 and 187 microM for the skeletal muscle and the neuronal isoform, respectively. In both isoforms, the blocking potency increased with the fraction of inactivated channels at depolarized holding potentials. However, the block achieved at -70 mV with respect to -150 mV holding potential was significantly higher only in the skeletal muscle isoform. The estimated dissociation constant K(d) from the inactivated state was 25 microM and 28 microM in the skeletal muscle and the neuronal isoform, respectively. The kinetics of drug equilibration between resting and inactivated channel states were about 10 fold faster compared with lidocaine. 4. Our results show that the blockade induced by 2,6-dimethylphenol retains voltage-dependency, a typical feature of lidocaine-like local anaesthetics. This is consistent with the hypothesis that the 'aromatic tail' determines the state-dependent interaction of local anaesthetics with the sodium channel.

Alanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Na(v)1.2 channel alpha subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na(+) currents at the end of 30-ms depolarizations, indicating a role for these residues in Na(+) channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6.

A spectrophotometric method was developed for the determination of etidocaine hydrochloride (EH) in injectable pharmaceutical preparation. The proposal of this work was to develop a rapid, simple, inexpensive, precise and accurate visible spectrophotometric method. The method is based on the formation of the ion-pair complex by the EH reaction with bromocresol green in the pH 4.6 which after chloroform extraction gives a yellow color that in basic medium change to blue color and exhibits a maximum absorbance at 625 nm. The calibration graph was linear over the range 2.0–6.0 μg ml −1 EH calculated on the final yellow solution. The R.S.D. of the slope of the four lines was 0.73%. This method can be applied to injectable pharmaceutical preparation dosage studied.

In 1850, about three centuries after the conquest of Peru by Pizzaro, the Austrian von Scherzer brought a sufficient quantum of coca leaves to Europe to permit the isolation of cocaine. As suggested by his friend Sigmund Freud, descriptions of the properties of the coca prompted the Austrian Koller to perform in 1884 the first clinical operation under local anesthesia, by administration of cocaine on the eye. The use of cocaine for local and regional anesthesia rapidly spread throughout Europe and America. The toxic effects of cocaine were soon identified resulting in many deaths among both patients and addicted medical staff. Local anesthesia was in a profound crisis until the development of modern organic chemistry which led to the synthesis of pure cocaine in 1891. New amino ester local anesthetics were synthesized between 1891 and 1930, such as tropocaine, eucaine, holocaine, orthoform, benzocaine, and tetracaine. In addition, amino amide local anesthetics were prepared between 1898 and 1972 including nirvaquine, procaine, chloroprocaine, cinchocaine, lidocaine, mepivacaine, prilocaine, efocaine, bupivacaine, etidocaine, and articaine. All of these drugs were ostensibly less toxic than cocaine, but they had differing amounts of central nervous system (CNS) and cardiovascular (CV) toxicity. Bupivacaine is of special interest because of its long duration of action and history of clinical application. Synthesized in 1957, the introduction of bupivacaine on the market in 1965 paralleled the progressive and cumulative reports of CNS and CV toxicity, leading to the restriction of its use and the identification of a special therapy-resistant CV toxicity. Numerous experimental studies were conducted to identify the fine cellular mechanism of this toxicity, which refines our understanding of the action of local anesthetics. The identification of optically active isomers of the mepivacaine family led to the selection of ropivacaine, a pure S-(-) enantiomer, whose toxicology was selectively and extensively studied before its introduction on the market in 1996. During the rapid and extensive use of ropivacaine in the clinic, unwanted side-effects have been found to be very limited.

Mutations of amino acid residues in the inner two-thirds of the S6 segment in domain III of the rat brain type IIA Na(+) channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an alpha-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na(+) channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na(+) channels for the anticonvulsant 4030w92 and etidocaine by 3- and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.

The local anesthetic lidocaine was determined in hair by hydrolysis of the samples with 4% NaOH in the presence of excessive Na2SO4 and subsequent headspace solid-phase microextraction with a 65-pro Carbowax/divinylbenzene fiber, and gas chromatography-mass spectrometry measurement with etidocaine as the internal standard. The calibration curve was linear between 0.1 and 1000 ng/mg. The detection and quantitation limits were 0.1 and 0.4 ng/mg, respectively. The method was applied to hair samples of 49 drug fatalities, and positive results were obtained in 32 cases with lidocaine concentrations between 0.4 and 400 ng/mg and 675 ng/mg in one extreme case. For comparison, morphine, 6-acetylmorphine, codeine, dihydrocodeine, methadone, cocaine, and benzoylecgonine were also determined by usual methods. From segmental investigations in four of the cases and from comparison with the hair concentrations of the other drugs, it follows that lidocaine was consumed for a longer period of time as an adulterant of cocaine and heroin preparations. case of death after repeated use of this anesthetic during episodes of self mutilation. Such high concentrations suggest a very efficient deposition of this drug into hair.