Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)(2+), with the detection limits of 2.4 x 10(-7) and 2.0 x 10(-8) mol/L (S/N=3), respectively. The Michaelis constant K-m value was 1.73 x 10(-4) mol/L and the maximum velocity V-max was 1.62 x 10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24 x 10(-3) and 2.94 x 10(-4) mol/L.

Ion pair stabilization effects on a series of procaine structural analogs

In this work, a series of 10 structural procaine analogs have been synthesized in order to investigate the structural features affecting the stability of ion pair formation and its influence on the lipophilicity of ionizable compounds. The structural variation within this series was focused on the terminal nitrogen substituents and on the intermediate chain linkage nature. The hydrophobic parameters log P(n) and log P(i) (partition coefficient of the neutral and ionic species, respectively), as well as the ionization constants pK(a) and pK(a)(oct), were obtained from log D-pH profiles measured at pH values ranging from 2 to 12. The difference between log P(i) and log P(n) values (i.e. difflog P) of each prepared compound was considered a measure of the stability of ion pair formation. In this set, the difflog P values varied nearly over one log unit, ranging from -2.40 to -3.37. It has been observed that the presence of hydrogen bonding groups (especially donor) and low steric hindrance around the terminal amine ionizable group increases the relative lipophilicity of the ionic species as compared to the corresponding neutral species. These results were interpreted as due to the increased stability of ion pairs of the compounds bearing these structural features. (C) 2010 Elsevier B.V. All rights reserved.

Flow injection amperometric determination of procaine in pharmaceutical formulation using a screen-printed carbon electrode

A rapid and simple method for procaine determination was developed by flow injection analysis (FIA) using a screen-printed carbon electrode (SPCE) as amperometric detector. The present method is based on the amine/hydroxylamine oxidation from procaine monitored at 0.80 V on SPCE in sodium acetate solution pH 6.0. Using the best experimental conditions assigned as: pH 6.0, flow rate of 3.8 mL min(-1), sample volume of 100 mu L and analytical path of 30 cm it is possible to construct a linear calibration curve from 9.0 x 10(-6) to 1.0 x 10(-4) mol L-1. The relative standard deviation for 5.0 x 10(-5) mol L-1 procaine (15 repetitions using the same electrode) is 3.2% and detection limit calculated is 6.0 x 10(-6) mol L-1. Recoveries obtained for procaine gave a mean values from 94.8 to 102.3% and an analytical frequency of 36 injections per hour was achieved. The method was successfully applied for the determination of procaine in pharmaceutical formulation without any pre-treatment, which are in good accordance with the declared values of manufacturer and an official method based on spectrophotometric analysis. (c) 2006 Elsevier B.V. All rights reserved.

Lipophilicity as a determinant of binding of procaine analogs to rat alpha(3)beta(4) nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structureactivity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the a3 beta 4 nAChR heterologously expressed in KXa3 beta 4R2 cells. Affinity values (log[1/IC50]) of these compounds for the a3 beta 4 nAChR were determined by their competition with [3H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structureactivity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity. (c) 2012 Wiley Periodicals, Inc.

Neuralgia of the glossopharyngeal nerve in a patient with posttonsillectomy scarring: recovery after local infiltration of procaine-case report and pathophysiologic discussion

We describe a patient with a three-year history of severe progressive left-sided glossopharyngeal neuralgia (GPN) that failed to adequately respond to various drug therapies. The application of lidocaine spray to the posterior pharyngeal wall provided no more than short-term relief. Apart from a large hypertrophic tonsillectomy scar on the left side all clinical and radiologic findings were normal. In terms of therapeutic local anaesthesia, the hypertrophic tonsillectomy scar tissue was completely infiltrated with the local anaesthetic (LA) procaine 1%. The patient has been almost completely pain-free ever since, and the lidocaine spray is no longer needed. Six weeks after the first treatment a repeat infiltration of the tonsillectomy scar led to the complete resolution of all symptoms. The patient has become totally symptom-free without the need to take any medication now for two and a half years. This is the first report of a successful therapeutic infiltration of a tonsillectomy scar using an LA in a patient with GPN that has been refractory to medical treatment for several years. A possible explanation may be that the positive feedback loop maintaining neurogenic inflammation is disrupted and "sympathetically maintained pain" resolved by LA infiltration.

Study of ion movements in isolated chicken retinas during spreading depression

Spreading depression (SD) is a phenomenon observed in several sections of vertebrate central nervous system. It can occur spontaneously or be evoked by a variety of stimuli, and consists of a wave of depression of the normal electrical activity of the nervous tissue which spreads slowly in all directions in the tissue. This wave of depression is accompanied by several concomitants including ion movements. All the concomitants of SD can be explained by an increase in the sodium permeability of the plasma membranes of cellular elements involved in this phenomenon.

In the chicken retina, SD is accompanied by a transparency change which can be detected with the naked eye. The isolated retina is a thin (0.1 mm) membrane in which the extracellular fluid quickly and completely equilibrates with the incubation solutions. This preparation was therefore used to study the ion movements during SD by measuring and comparing the ion contents and the extracellular space (ECS) of retinas incubated in various solutions of which some inhibited SD, whereas others allowed this phenomenon to occur.

The present study has shown that during SD there is a shift of extracellular sodium into the intracellular compartment of the retina, a release of intracellular K and a decrease in the magnitude of ECS. These results are in agreement with previous postulates about SD, although the in vitro experimental condition makes the ion movements appear larger and the loss of ECS smaller than observed in the intact cortical tissue. The movements of Na and K, in opposite directions, are reversible. The development and magnitudes of SD is very little affected by deprivation of the oxygen supply.

It was established that the inward sodium shift is not a consequence of an arrest of the Na-pump. It can be prevented, together with SD by the membrane stabilizers, magnesium and procaine. Spreading depression and the ion movements are incompletely inhibited by tetrodotoxin, which blocks the sodium influx into nerve fibers during the action potential. The replacement of Na in the bathing solution by Li does not prevent SD, which is accompanied by Li accumulation in the intracellular compartment. From these experiments and others it was concluded that the mechanism underlying SD and the ion shifts is an increase in the sodium permeability of cell membranes.

Investigation of the role of the nucleus accumbens in amphetamine-induced 50 kHz ultrasonic vocalizations in the rat

There is extensive evidence that the mesolimbic dopamine system underlies the production of 50 kHz ultrasonic vocalizations in rats. In particular, the shell of the nucleus accumbens is associated with generation of frequency modulated 50 kHz calls (a specific type of 50 kHz call which can be subdivided into various subtypes). There is also evidence that amphetamine administered systemically preferentially increases the proportion of trill and step calls compared to other frequency modulated 50 kHz subtypes. The purpose of this study was to investigate the effect of drug administration route and the role of the nucleus accumbens shell in amphetamine-induced 50 kHz call profile in the rat. Three experiments investigated this by using subcutaneous and intra-accumbens microinjections of amphetamine, as well as procaine (a local anesthetic) blockade of the nucleus accumbens. Ultrasonic vocalizations were recorded digitally from 24 rats and were analysed for sonographic structure based on general call parameters. The results of the three experiments were partially supportive of the hypotheses. Systemic amphetamine was found to induce greater bandwidth in 50 kHz calling compared to spontaneous calls in a vehicle condition. Systemic amphetamine was also found to preferentially increase the proportion of trill and step subtypes compared to vehicle. Moreover, there was no difference in the proportions of 50 kHz subtypes resulting from intracerebral or systemic application of amphetamine. There was, however, a significant difference for bandwidth, with systemic amphetamine inducing greater bandwidth over intraaccumbens application. Procaine blockade of the nucleus accumbens shell paired with subcutaneous amphetamine produced no difference in bandwidth of calls compared with those after a vehicle pre-treatment similarly paired. There was no reduction in the proportions of trill and step 50 kHz subtypes as well, with the procaine condition showing significantly greater proportion of step calls. The results of the study support a role for the iii nucleus accumbens shell in the amphetamine-induced changes on 50 kHz call profile. They also indicate there are more regions and pathways involved in generating 50 kHz calls than the projections from the ventral tegmental area to the nucleus accumbens. The implications of this work are that frequency modulated 50 kHz subtypes may be generated by distinct neurophysiological mechanisms and may represent a profitable avenue for investigating different circuits of 50 kHz call categories in the rat.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Preparation and Characterization of Graphite-Epoxy Composite Modified with Zinc Hexacyanoferrate and Their Electrochemical Behaviour in Presence of Substituted Anilines

The growth of zinc hexacyanoferrate (ZnHCF) hybrid film on the surface of graphite-epoxy composite (GEC) electrodes was demonstrated by cyclic voltammetry. Surface morphology of the hybrid film was investigated by using scanning electron microscopy. The effect of the type of monovalent cations on the redox behaviour of hybrid film was also studied. This effect indicated that the radius of the hydrated cation mainly determines the ion permeability of the film.The electrochemical behavior of the substituted anilines (procaine and sulfamerazine) in 1 M KCl of the modified GEC electrode showed a decrease of the cathodic currents while increasing the concentration of these analytes. The developed sensor also showed excellent stability for long time usage, higher sensitivity and cost-effective fabrication.

Silver Nanocomposite Electrode Modified with Hexacyanoferrate. Preparation, Characterization and Electrochemical Behaviour Towards Substituted Anilines

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Study of zinc hexacyanoferrate-modified platinum electrodes using electrochemical quartz crystal microbalance

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Physicochemical parameters involved in the lethal toxicity of N,N-[(dimethylamino)ethyl]-4-substituted benzoate hydrochlorides: A QSAR study

A set of sixteen para-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides structurally related to procaine was synthesized. The apparent partition coefficients were determined by either shake-flask or HPLC methods and were taken as hydrophobic parameters. The IR stretching frequencies of the carbonyl group were determined in chloroform and taken as one of the electronic parameters. Additional physicochemical parameters were either taken from the literature: pi, sigma, T and R, MR4, or calculated: log P. The lethal potency was determined in the mouse via the LD50. In order to verify the nature and the relative contributions of the physicochemical parameters to lethal toxicity, QSAR equations were derived using regression analysis. A major contribution of hydrophobicity to ether with a smaller but still significant contribution of electronic or polar properties was found to a describe the toxicity within this set of compounds.