Electron Paramagnetic Resonance and Small-Angle X-ray Scattering Characterization of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Dibucaine Encapsulation.

Dibucaine (DBC) is one of the most potent long-acting local anesthetics, but it also has significant toxic side effects and low water solubility. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been proposed as drug-delivery systems to increase the bioavailability of local anesthetics. The purpose of the present study was to characterize SLNs and NLCs composed of cetyl palmitate or myristyl myristate, a mixture of capric and caprylic acids (for NLCs only) plus Pluronic F68 prepared for the encapsulation of DBC. We intended to provide a careful structural characterization of the nanoparticles to identify the relevant architectural parameters that lead to the desirable biological response. Initially, SLNs and NLCs were assessed in terms of their size distribution, morphology, surface charge, and drug loading. Spectroscopic techniques (infrared spectroscopy and electron paramagnetic resonance, EPR) plus small-angle X-ray scattering (SAXS) provided information on the interactions between nanoparticle components and their structural organization. The sizes of nanoparticles were in the 180 nm range with low polydispersity and negative zeta values (-25 to -46 mV). The partition coefficient of DBC between nanoparticles and water at pH 8.2 was very high (>104). EPR (with doxyl-stearate spin labels) data revealed the existence of lamellar arrangements inside the lipid nanoparticles, which was also confirmed by SAXS experiments. Moreover, the addition of DBC increased the molecular packing of both SLN and NLC lipids, indicative of DBC insertion between the lipids, in the milieu assessed by spin labels. Such structural information brings insights into understanding the molecular organization of these versatile drug-delivery systems which have already demonstrated their potential for therapeutic applications in pain control.

Differential interactions of two local anesthetics with phospholipid membrane and nonerythroid spectrin: Localization in presence of cholesterol and ganglioside, 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.

History of T-cain: a local anesthetic developed and manufactured in Japan.

In many anesthesia textbooks written in English, lidocaine, tetracaine, bupivacaine, ropivacaine, and chloroprocaine are listed as useful local anesthetics for spinal anesthesia. In contrast, T-cain is not included in these lists, even though it has been reported to be suitable for spinal anesthesia in Japan. T-cain was developed as a local anesthetic in the early 1940s by Teikoku Kagaku Sangyo Inc. in Itami, Japan, by replacing a methyl group on tetracaine (Pantocaine(®)) with an ethyl group. T-cain was clinically approved for topical use in Japan in November 1949, and a mixture of dibucaine and T-cain (Neo-Percamin S(®)) was approved for spinal use in May 1950. Simply because of a lack of foreign marketing strategy, T-cain has never attracted global attention as a local anesthetic. However, in Japan, T-cain has been used topically or intrathecally (as Neo-Percamin S(®)) for more than 60 years. Other than the side effects generally known for all local anesthetics, serious side effects have not been reported for T-cain. In fact, several articles have reported that T-cain decreases the neurotoxicity of dibucaine. In this historical review, the characteristics of T-cain and its rise to become a major spinal anesthetic in Japan are discussed.

Role of viscosity in influencing the glass-forming ability of organic molecules from the undercooled melt state.

PURPOSE: Understanding the critical factors governing the crystallization tendency of organic compounds is vital when assessing the feasibility of an amorphous formulation to improve oral bioavailability. The objective of this study was to investigate potential links between viscosity and crystallization tendency for organic compounds from the undercooled melt state. METHODS: Steady shear rate viscosities of numerous compounds were measured using standard rheometry as a function of temperature through the undercooled melt regime. Data for each compound were fit to the Vogel-Tamman-Fulcher (VTF) equation; kinetic fragility via strength parameter (D) was determined. RESULTS: Compounds with high crystallization tendencies exhibited lower melt viscosities than compounds with low crystallization tendencies. A correlation was observed between rate of change in viscosity with temperature and crystallization tendency, with slowly crystallizing compounds exhibiting larger increases in viscosity as temperature decreased below T(m). Calculated strength parameters indicated all compounds were kinetically fragile liquids; thus, kinetic fragility may not accurately assess glass-forming ability from undercooled melt state. CONCLUSIONS: A link was observed between the viscosity of a compound through the undercooled melt regime and its resultant crystallization tendency, indicating viscosity is a critical parameter to fully understand crystallization tendency of organic compounds.

Selective effect of procaine, tetracaine and dibucaine on gold nanoparticles.

An aqueous colloid dispersion of gold nanoparticles (AuNPs) was prepared by reduction of gold(III) chloride and its interaction with three local anesthetics (procaine, dibucaine or tetracaine) was investigated. Optical spectra reveal the modifications in the absorption band of nanoparticles related to their self assembly mediated by anesthetic molecules and depending on the progress in time of the aggregation process. TEM images show the features of the self assemblies formed by the association of gold nanoparticles in presence of anesthetics, and reveal marked differences in the behavior of the AuNPs against the three anesthetics. The main effect of various anesthetics can be described in terms of electrostatic forces between the negatively charged metal nanoparticles and anesthetic molecules, existing in their cationic form at the working pH. Then, the anesthetics functionalized nanoparticles trigger specific interactions to form different self assemblies through a selective combination of hydrophobic and hydrogen bonding interactions between the coated nanoparticles and anesthetics molecular species.

Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study.

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Discovery of Quinoline Analogues as Potent Antivirals against Enterovirus D68 (EV-D68).

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC50 < 1 µM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes.

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.

Simultaneous Determination of Cinchocaine Hydrochloride and Betamethasone Valerate in Presence of Their Degradation Products.

Cinchocaine hydrochloride (CIN) and betamethasone valerate (BMV) are co-formulated in pharmaceutical formulations that could be used for local treatment of hemorrhoids. Both drugs are susceptible to hydrolytic degradation. Two sensitive and precise stability-indicating chromatographic methods were developed for the simultaneous determination of both active pharmaceutical ingredients. The developed methods were applied for quantitation of CIN and BMV in their pure forms, in presence of their corresponding degradation products and in their pharmaceutical formulation. The first method was a high performance liquid chromatographic (HPLC) one, separation and quantitation was achieved using a Waters Spheriosorb® 5 µm ODS2 C18 analytical column and an isocratic mobile phase formed of acetonitrile-acetate buffer (pH 6.5 ± 0.1) in a ratio of (55:45, v/v). The mobile phase was pumped at a flow rate of 1.2 mL/min. UV-detection was done at 240 nm using photodiode array detector. The second method was based on thin layer chromatography (TLC) fractionation coupled with densitometric determination. Separation was done on high performance thin layer chromatography (HPTLC) silica gel 60F254 plates using a developing system formed of chloroform-toluene-ethanol-acetic acid in a ratio of (4.5:4.5:1:1, by volume). The separated bands were scanned densitometrically at 240 nm. For the HPLC method, linearity was confirmed over concentration ranges of 4-300 and 4-350 µg/mL for CIN and BMV, respectively. For the HPTLC-densitometric method, the obtained ranges were 0.5-12 and 0.5-10 µg/band for CIN and BMV, respectively. The developed methods were optimized and validated according to the ICH guidelines. CIN acid degradation products were separated and identified by mass spectroscopy. The developed HPLC method was used to study the kinetics of acid and alkali degradation of the both drugs. The results obtained were statistically analyzed and compared with those obtained by applying the official methods for both drugs.

In vitro release of hydrophilic and hydrophobic drugs from liposomal dispersions and gels.

A method for determining the rate of hydrophilic and hydrophobic drugs release from different types of liposomal dispersions and gels using a dialysis method is described. Dibucaine base and 5-fluorouracil were used as model drugs for a hydrophobic and a hydrophilic drug, respectively. A dialysis technique was employed. Release rates were affected by the rate of rotation of the paddles of the tablet dissolution tester, temperature, and the volume of release medium. The method was used to evaluate the in vitro drug release from hydrophilic and hydrophobic drugs from liposomal dispersions and gels. The in vitro release study of dibucaine base showed no burst effect, while the in vitro release study of 5-fluorouracil showed a clear burst effect with an initial fast release phase followed by a sustained release phase.

Activity and polymorphisms of butyrylcholinesterase in a Polish population.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3'UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5'UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

Charge- and pH-dependent binding sites for dibucaine in ionic micelles: a fluorescence study.

Binding of micromolar concentrations of the local anesthetic dibucaine to micelles of cationic, zwitterionic and anionic detergents was studied using the fluorescence emission of dibucaine. Difference in quantum yields for charged and neutral dibucaine allowed to obtain shifts of pKa values due to binding. Estimates for the electrostatic potential affecting the tertiary amine of dibucaine were obtained from the pKa shifts. Change of fluorescence emission upon binding allowed to obtain the binding constants of both charged and neutral dibucaine to the micelles. The binding constant for the neutral form is essentially independent of micelle charge and of specific differences in detergent structure. Consistency between the ratio of neutral to cationic dibucaine binding constants and the measured pKa shift was tested. For LPC micelles complete agreement was found. For CTAC, however, the ratio of binding constants does not explain the pKa shift. The discrepancy between the results is used to estimate the errors involved upon neglecting non-coulombic electrostatic interactions of drugs to charged membrane surfaces. Fluorescence quenching with sodium iodide and nitroxide stearic acid derivatives allowed a depth profiling of the drug in the micelles.

Effect of cholesterol on interaction of dibucaine with phospholipid vesicles: a fluorescence study.

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid membranes.

Light-induced reversible local fusions of thylakoid membranes in the presence of dibucaine or tetracaine.

The dynamics of structural changes in pea chloroplasts in the presence of 25-50 microM dibucaine or tetracaine has been examined using electron microscopy. The light-induced uptake of anesthetic cations by thylakoids is attended by the appearance of local fusions of stroma-exposed thylakoid membranes. The first membrane protrusions and interthylakoid contacts are observed after 4 s illumination and they become numerous by 10 s. As a result, a network of anastomoses is formed which is maintained during at least 10 min. These effects are reversible in the dark and can be reproduced several times. The formation of membrane fusions is inhibited by the addition of protonophore. It is supposed that the energy-dependent uptake of protonated anesthetics by thylakoids leads to an increase in positive surface charge and thus a lateral pressure on the inner side of the thylakoid membrane. The appearance of membrane protrusions (crinkles) having the positive curvature of their inner surface may be considered as a way of compensating for lateral pressure. Presumably, anastomoses result from the fusion of crinkles to adjacent thylakoids.

Effect of various formulation variables on the encapsulation and stability of dibucaine base in multilamellar vesicles.

Formulation of local anesthetics in liposomal topical drug delivery system could provide a sustained and localized anesthesia. The aim of this study was to develop a liposomal dibucaine base (DB) local anesthetic delivery system. DB-loaded multilamellar vesicles (MLVs) were prepared through varying lipid composition, induced charge and pH of the hydration medium. Liposomes were characterized for morphology, size, entrapment efficiency (EE), in vitro drug release and stability including leakage stability. The percentage of drug entrapped in liposomes was found to be hydration medium pH dependent and charge dependent and more pronounced for negatively charged liposomes prepared using hydration medium of pH 9. In vitro release studies of liposomes have shown a sustained release of entrapped dibucaine compared to control solution. Results revealed that adjusting the various formulation variables of dibucaine base MLVs could yield stable and effective topical liposomal local anesthetic formulations.

Derivative spectrophotometry as a tool for the determination of drug partition coefficients in water/dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) liposomes.

The partition coefficients (K(p)) between lipid bilayers of dimyristoyl-L-alpha-phosphatidylglycerol (DMPG) unilamellar liposomes and water were determined using derivative spectrophotometry for chlordiazepoxide (benzodiazepine), isoniazid and rifampicin (tuberculostatic drugs) and dibucaine (local anaesthetic). A comparison of the K(p) values in water/DMPG with those in water/DMPC (dimyristoyl-L-alpha-phosphatidylcholine) revealed that for chlordiazepoxide and isoniazid, neutral drugs at physiological pH, the partition coefficients are similar in anionic (DMPG) and zwitterionic (DMPC) liposomes. However, for ionised drugs at physiological pH, the electrostatic interactions are different with DMPG and DMPC, with the cationic dibucaine having a stronger interaction with DMPG, and the anionic rifampicin having a much larger K(p) in zwitterionic DMPC. These results show that liposomes are a better model membrane than an isotropic two-phase solvent system, such as water-octanol, to predict drug-membrane partition coefficients, as they mimic better the hydrophobic part and the outer polar charged surface of the phospholipids of natural membranes.

Local anesthetic-phospholipid interactions. Effects of ionic strength, temperature, and phospholipid mixtures on the binding of dibucaine to phospholipids.

The nature of the interaction of amphipathic drugs, such as dibucaine, with phospholipid bilayer membranes was investigated using equilibrium dialysis. Profiles for the binding of cationic dibucaine to unilamellar vesicles were obtained at different temperature and ionic strengths, and for mixtures of neutral phospholipid dimyristylphosphatidylcholine (DMPC) with negatively charged dimyristylphosphatidylglycerol (DMPG). The degree of binding of the cationic drug at pH 5 was found to be higher at temperatures above the Tm of DMPC (24 degrees C) than below Tm. Also enhanced drug binding was found to occur as the concentration of monovalent salt was increased (0.01-0.85 M) and as the percentage of DMPG was increased. Using the Stern and Guoy-Chapman model, which takes into consideration electrostatic effects, we were able to simultaneously fit all our binding data with a minimum of fitting parameters. These parameters (for data at 45 degrees C) are an association constant, K, of 330 M-1, a maximum possible number of drug molecules absorbed per unit surface of vesicle, sigma m+, of 1.70 x 10(-2) A2, and a surface area per bound drug, gamma D, of 48 A2. The data were fitted equally well by an alternate model in which binding of the drug is described as a partitioning equilibrium, with factors included for electrostatic effects and surface expansion caused by drug intercalation between the fatty acid chains.

Fluorescence studies of a local anesthetic-phospholipid interaction.

Steady-state and time-resolved fluorescence data are reported for the local anesthetic dibucaine in the absence and presence of phospholipid vesicles. These vesicles were comprised of dimyristylphosphatidyl choline and approximately 10% dimyristylphosphatidyl glycerol. Solute quenching studies show the bound drug to be protected from collision with iodide ion. The fluorescence lifetime of dibucaine is not significantly changed upon binding to vesicles. The fluorescence anisotropy of dibucaine increases upon association with the vesicles. Anisotropy decay measurements show that the rotational correlation time, phi, of bound dibucaine is increased about one hundred fold over that for free dibucaine. This indicates that the rotational motion of bound dibucaine is slowed by its interaction with the phospholipids. However, we find no evidence that the rotational motion of bound dibucaine is anisotropic.

Pathways involved in trifluoperazine-, dibucaine- and praziquantel-induced hemolysis.

This work elucidates differences in the hemolytic pathway developed by the antipsychotic trifluoperazine (TFP), the local anesthetic dibucaine (DBC) and the antihelminthic praziquantel (PZQ). Their partition coefficients (P) were measured at pH 7.4 between n-octanol, microsomes, liposomes, erythrocyte ghosts and n-octanol/water. The effective drug:lipid molar ratios for the onset of membrane solubilization (ReSAT) and complete hemolysis (ReSOL) were calculated from the experimental P values and compared with a classical surface-active compound treatment Lichtenberg, D. Biochim. Biophys. Acta 821 (1985) 470-478[. The contribution of charged/uncharged forms of TFP and DBC for the hemolytic activity was also analyzed. In all cases the hemolytic phenomena could be related to the monomeric drug insertion into the membrane. Only for TFP at isosmotic condition lysis occurs at concentrations beyond the CMC of the drug, indicating that micellization facilitates TFP hemolytic effect, while DBC and PZQ reach a real membrane saturation at their monomeric form.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in 1H nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.