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Biochimica Et Biophysica Acta.... Jun 2019Mitochondrial membranes are pointed out as the site of cardiotoxic action of local anaesthetics. Its three main phospholipids components are phosphatidylcholine,...
Mitochondrial membranes are pointed out as the site of cardiotoxic action of local anaesthetics. Its three main phospholipids components are phosphatidylcholine, phosphatidylethanolamine and cardiolipin. Cardiolipins, in eukaryotes, are only found in mitochondria and are essential for the maintenance of its integrity and dynamics. Fluorescence and nuclear magnetic resonance spectroscopy were used to study the interactions of a local anaesthetics, Dibucaine (DBC), with different mitochondrial membrane models constituted by combinations of its three main lipid components in which cardiolipin was a natural extract (CL). Both CL presence/absence and its percentage in the model membranes were evaluated. Fluorescence spectroscopy showed that DBC lowered the transition temperature of all membrane models understudy. DBC partition showed to be dependent of CL presence and phosphatidylethanolamine:CL ratio. Furthermore, the maximum emission wavelength (λ) exhibited a notorious decreased with increasing phospholipid to DBC ratio, in all the membrane models containing CL. Nevertheless, it remained approximately the same in the membrane without CL. This indicates a differential membrane localization of the anaesthetics, dependent on the membrane models used. NMR results showed that DBC interaction and location in the membrane models is mainly influenced by CL presence, and DBC can significant alter lipid systems properties e.g. percentage and type of lipid phase present. Taken all together it was shown that DBC interaction and location are largely dependent on the membrane model system. Furthermore, DBC is able to produce significant changes in the lipidic systems which might help to explain its high toxicity.
Topics: Anesthetics, Local; Binding Sites; Cardiolipins; Dibucaine; Mitochondrial Membranes; Models, Biological; Phosphatidylethanolamines; Temperature
PubMed: 30840858
DOI: 10.1016/j.bbamem.2019.02.011 -
Pharmaceutics Nov 2018Dibucaine (DBC) is among the more potent long-acting local anesthetics (LA), and it is also one of the most toxic. Over the last decades, solid lipid nanoparticles (SLN)...
Dibucaine (DBC) is among the more potent long-acting local anesthetics (LA), and it is also one of the most toxic. Over the last decades, solid lipid nanoparticles (SLN) have been developed as promising carriers for drug delivery. In this study, SLN formulations were prepared with the aim of prolonging DBC release and reducing its toxicity. To this end, SLN composed of two different lipid matrices and prepared by two different hot-emulsion techniques (high-pressure procedure and sonication) were compared. The colloidal stability of the SLN formulations was tracked in terms of particle size (nm), polydispersity index (PDI), and zeta potential (mV) for 240 days at 4 °C; the DBC encapsulation efficiency was determined by the ultrafiltration/centrifugation method. The formulations were characterized by differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR), and release kinetic experiments. Finally, the in vitro cytotoxicity against 3T3 fibroblast and HaCaT cells was determined, and the in vivo analgesic action was assessed using the test in rats. Both of the homogenization procedures were found suitable to produce particles in the 200 nm range, with good shelf stability (240 days) and high DBC encapsulation efficiency (~72⁻89%). DSC results disclosed structural information on the nanoparticles, such as the lower crystallinity of the lipid core vs. the bulk lipid. EPR measurements provided evidence of DBC partitioning in both SLNs. In vitro (cytotoxicity) and in vivo () experiments revealed that the encapsulation of DBC into nanoparticles reduces its intrinsic cytotoxicity and prolongs the anesthetic effect, respectively. These results show that the SLNs produced are safe and have great potential to extend the applications of dibucaine by enhancing its bioavailability.
PubMed: 30441802
DOI: 10.3390/pharmaceutics10040231 -
Indian Journal of Anaesthesia Jul 2017Dibucaine, a potent and toxic local anaesthetic, although currently withdrawn by the United States Food and Drug Administration for use as a spinal anaesthetic,...
Dibucaine, a potent and toxic local anaesthetic, although currently withdrawn by the United States Food and Drug Administration for use as a spinal anaesthetic, continues to remain available in many over-the-counter topical formulations. Systemic toxicity following oral ingestion of local anaesthetics is rare. We report a case of accidental ingestion of dibucaine (ear drops) in a 7-year-old child who developed diplopia, giddiness, ventricular premature contractions and a right bundle branch block. We also present a brief discussion on the pharmacologic and toxicity profile of dibucaine, the Naranjo algorithm for assessing causality in case of adverse drug reactions and a review of current guidelines on the management of local anaesthetic systemic toxicity.
PubMed: 28794532
DOI: 10.4103/ija.IJA_166_17 -
Antimicrobial Agents and Chemotherapy Mar 2017The efficacy of antimicrobial drugs against , an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic...
The efficacy of antimicrobial drugs against , an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in infected macrophages. Specifically, weak bases with moderate activity against (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.
Topics: Ammonium Chloride; Anesthetics, Local; Antitubercular Agents; Biological Transport; Clofazimine; Dibucaine; Fluoxetine; Humans; Hydrogen-Ion Concentration; Macrolides; Macrophages; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Protons; Selective Serotonin Reuptake Inhibitors; Sertraline
PubMed: 28052847
DOI: 10.1128/AAC.01639-16 -
European Journal of Hospital Pharmacy :... Jul 2016This work aimed to evaluate the quality of non-sterile formulations compounded at Centro Hospitalar Cova da Beira (Covilhã, Portugal) immediately after preparation and...
OBJECTIVES
This work aimed to evaluate the quality of non-sterile formulations compounded at Centro Hospitalar Cova da Beira (Covilhã, Portugal) immediately after preparation and up to the defined 'beyond-use date'.
METHODS
Microbiological quality control tests were performed in accordance with monograph 5.1.4 of the European Pharmacopoeia 8.0. Samples of compounded products were collected from January to December 2014 after preparation and were analysed immediately and reanalysed after storage under the established conditions, for each preparation.
RESULTS
In the test period, 392 preparations were analysed, corresponding to 24 different formulations (8 intermediate preparations, 11 oral solutions/suspensions and 5 topical preparations). All preparations were in accordance with the pharmacopoeia specifications immediately after preparation. However, for the formulations 'prednisolone oral solution (5 mg/mL)' and 'nitroglycerine and cinchocaine ointment (0.25%/0.5%)', the microbial counts of some batches exceeded the defined limits after storage up to the beyond-use date.
CONCLUSIONS
These results show that the compounding practices implemented at this pharmacy department are able to ensure the microbiological quality of compounded products. This microbiological quality control methodology also allowed identification of the need to replace formulations shown not to be stable throughout the storage period. On the basis of these results, a monthly routine of microbiological control of a random sample of compounded medicines was established in order to ensure their quality and safety for use.
PubMed: 31156854
DOI: 10.1136/ejhpharm-2015-000769 -
The West Virginia Medical Journal 2016Prolonged paralysis due to a quantitative or qualitative deficiency of pseudocholinesterase activity is an uncommon but known side effect of succinylcholine. We describe...
Prolonged paralysis due to a quantitative or qualitative deficiency of pseudocholinesterase activity is an uncommon but known side effect of succinylcholine. We describe a patient who experienced prolonged paralysis following administration of succinylcholine for general anesthesia and endotracheal intubation for an emergent cesarean section despite laboratory evidence of normal enzyme function. The patient required mechanical ventilation in the intensive care unit for several hours following surgery. The patient was extubated following return of full muscle strength and had a good outcome. The enzyme responsible for the metabolism of succinylcholine, pseudocholinesterase, was determined to be low in quantity in this patient but was functionally normal. This low level, by itself, was unlikely to be solely responsible for the prolonged paralysis. The patient likely had an abnormal pseudocholinesterase enzyme variant that is undetectable by standard laboratory tests.
Topics: Adult; Anesthesia, General; Anesthetics, Local; Butyrylcholinesterase; Cesarean Section; Dibucaine; Emergencies; Female; Humans; Intensive Care Units; Intubation, Intratracheal; Neuromuscular Depolarizing Agents; Obstetric Labor Complications; Paralysis; Pregnancy; Respiration, Artificial; Succinylcholine; Time Factors; Treatment Outcome
PubMed: 27025119
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy May 2016Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We...
Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.
Topics: Antiviral Agents; Carbazoles; Carrier Proteins; Clopenthixol; Dibucaine; Enterovirus; Fluoxetine; Formoterol Fumarate; HeLa Cells; Humans; Rhinovirus; Viral Nonstructural Proteins; Viral Proteins; Virus Replication
PubMed: 26856848
DOI: 10.1128/AAC.02182-15 -
PloS One 2015Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of...
Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.
Topics: Biological Assay; Butyrylcholinesterase; Butyrylthiocholine; Cholinesterase Inhibitors; Humans; Indicator Dilution Techniques; Pesticides
PubMed: 26444431
DOI: 10.1371/journal.pone.0139480 -
Biochimica Et Biophysica Acta Mar 2015Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM1)...
Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM1) using fluorescence spectroscopy. The fluorescence intensity of tetracaine showed a marked increase with the increasing molar ratio of the phospholipid to tetracaine, while that of dibucaine showed opposite effects. Steady state anisotropy and the wavelength of maximum emission (λmax) decreased with the increasing phospholipids to tetracaine ratio. The extent of such changes in anisotropy and λmax in the presence and absence of two important components of neuronal membranes, cholesterol and GM1 indicated differential membrane localization of the two local anesthetics. To understand the intercellular mode of action of local anesthetics, we have also studied the interactions of dibucaine and tetracaine with brain spectrin which indicate differential spectrin interactions with similar binding strength. Thermodynamic parameters associated with such binding reveal that binding is favored by entropy. Tetracaine brings about distinct structural changes in spectrin compared to dibucaine, as reflected in the tryptophan mean lifetime and far-UV CD spectra. Tetracaine also exhibits a detergent-like property inducing concentration dependent decrease in spectrin anisotropy, further indicating structural changes in brain spectrin with probable implications in its anesthetic potential.
Topics: Algorithms; Anesthetics, Local; Animals; Brain; Cell Membrane; Cholesterol; Circular Dichroism; Dibucaine; G(M1) Ganglioside; Kinetics; Membrane Lipids; Models, Chemical; Models, Molecular; Phospholipids; Protein Binding; Sheep; Spectrin; Spectrometry, Fluorescence; Tetracaine; Thermodynamics
PubMed: 25482358
DOI: 10.1016/j.bbamem.2014.11.022 -
PloS One 2014Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in...
Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.
Topics: Alleles; Apnea; Base Sequence; Biocatalysis; Butyrylcholinesterase; DNA Mutational Analysis; Family Health; Female; Humans; Infant, Newborn; Isoquinolines; Kinetics; Male; Mivacurium; Molecular Dynamics Simulation; Mutation, Missense; Neuromuscular Depolarizing Agents; Pedigree; Succinylcholine
PubMed: 25054547
DOI: 10.1371/journal.pone.0101552