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Nanostructured lipid carriers (NLC) have emerged as innovative drug delivery systems, offering distinct advantages over other lipid-based carriers, such as liposomes and solid lipid nanoparticles. Benzocaine (BZC), the oldest topical local anesthetic in use, undergoes metabolism by pseudocholinesterase, leading to the formation of p -aminobenzoic acid, a causative agent for allergic reactions associated with prolonged BZC usage. In order to mitigate adverse effects and enhance bioavailability, BZC was encapsulated within NLC. Utilizing a 2 3 factorial design, formulations comprising cetyl palmitate (solid lipid), propylene glycol monocaprylate (liquid lipid), and Pluronic F68 as surfactants were systematically prepared, with variations in the solid/liquid lipid mass ratios (60:40-80:20%), total lipid contents (15-25%), and BZC concentrations (1-3%). The optimized formulation underwent characterization by dynamic light scattering, differential scanning calorimetry, Raman imaging, X-ray diffraction, small-angle neutron scattering, nanotracking analysis, and transmission electron microscopy (TEM)/cryo-TEM, providing insights into the nanoparticle structure and the incorporation of BZC into its lipid matrix. NLC BZC exhibited a noteworthy encapsulation efficiency (%EE = 96%) and a 1 year stability when stored at 25 °C. In vitro kinetic studies and in vivo antinociceptive tests conducted in mice revealed that NLC BZC effectively sustained drug release for over 20 h and prolonged the anesthetic effect of BZC for up to 18 h. We therefore propose the use of NLC BZC to diminish the effective anesthetic concentration of benzocaine (from 20 to 3% or less), thus minimizing allergic reactions that follow the topical administration of this anesthetic and, potentially, paving the way for new routes of BZC administration in pain management.

Possible application of incorporating a well-known drug (benzocaine) with cyanoacetamide function to get a powerful synthon ethyl 4-cyanoacetamido benzoate. This synthetic intermediate was used as a precursor for the synthesis of triazine, pyridone, thiazolidinone, thiazole and thiophene scaffolds containing the benzocaine core. Facile coupling, Michael addition, condensation and nucleophilic attack reactions were used to synthesize our targets. The structural features of the synthesized scaffolds were characterized using IR, 1 H NMR, 13 C NMR and mass spectroscopy. The antibacterial activities against Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) were evaluated using ampicillin as a reference drug. DNA/methyl-green colorimetric assay of the DNA-binding compounds was also performed. Theoretical studies of the newly synthesized compounds based on molecular docking and QSAR study were conducted. The molecular docking studies were screened by MOE software for the more potent antibacterial agent 28b and each native ligand against four of S. aureus proteins 1jij, 2xct, 2w9s and 3t07.

Benzocaine (BZC), an efficient and highly permeable anaesthetic and an active pharmaceutical ingredient of many commercially available drugs, was studied under high pressure up to 0.78 GPa. As a result, new BZC polymorph (IV) was discovered. The crystallization of polymorph (IV) can be initiated by heating crystals of polymorph (I) at a pressure of at least 0.45 GPa or by their compression to 0.60 GPa. However, no phase transition from polymorph (I) to (IV) was observed. Although polymorph (IV) exhibits the same main aggregation motif as in previously reported BZC polymorphs (I)-(III), i.e. a hydrogen-bonded ribbon, its molecular packing and hydrogen-bonding pattern differ considerably. The N-H...N hydrogen bonds joining parallel BZC ribbons in crystals at ambient pressure are eliminated in polymorph (IV), and BZC ribbons become positioned at an angle of about 80°. Unfortunately, crystals of polymorph (IV) were not preserved on pressure release, and depending on the decompression protocol they transformed into polymorph (II) or (I).

The skin is an effective barrier that prevents the influx of harmful substances from the environment and the efflux of body fluid. This barrier function is ascribed to the intercellular lipids present in the outermost layer of the skin referred to as the stratum corneum (SC). These lipids are composed mainly of ceramides (CERs), cholesterol, and free fatty acids (FFAs). Alterations in the SC lipid composition and barrier function impairment occur in several skin diseases including atopic dermatitis (AD). As the etiology of AD is multifactorial, establishing the relationship between the changes in SC lipid composition and barrier function impairment in the patients remains a challenge. Here, we employed model membrane systems to investigate the contribution of various anomalies in the SC CER and FFA composition observed in AD patients' skin to the barrier dysfunction. Using ethyl- p -aminobenzoate permeation and transepidermal water loss values as markers for barrier function, we determined that the alterations in SC lipid composition contribute to the impaired barrier function in AD patients. By the use of biophysical techniques, we established that the largest reduction in barrier capability was observed in the model with an increased fraction of short-chain FFAs, evident by the decrease in chain packing density. Modulations in the CER subclass composition impacted the lamellar organization while having a smaller effect on the barrier function. These findings provide evidence that AD therapies normalizing the FFA composition are at least as important as normalizing CER composition.

The solubility of butamben has been measured gravimetrically in pure methanol, 1-propanol, 2-propanol, 1-butanol, and toluene over the temperature range 268-298 K. Polymorph transition and melting temperatures, associated enthalpy changes, and the heat capacity of the solid forms and the supercooled melt have been measured by differential scanning calorimetry. Based on extrapolated calorimetric data, the Gibbs energy, enthalpy and entropy of fusion, and the activity of solid butamben (the ideal solubility) have been calculated from below ambient temperature up to the melting point. Activity coefficients of butamben at equilibrium in the different solvents have been estimated from solubility data and the activity of the solid, revealing that all investigated systems exhibit positive deviation from Raoult's law. Solubility data are well correlated by a semiempirical regression model. On a mass basis, the solubility is clearly higher in methanol than in the other solvents, but mole fraction solubilities are very similar across all 5 solvents. The 2 known polymorphs are enantiotropically related, and the transition point is located at 283 K. Polymorph interconversions occur within 0.3 K of the transition point even in the solid state, and the 2 forms exhibit strong similarities in investigated properties., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The failures of glutaraldehyde (GLUT) cross-linked bioprosthetic heart valves (BHVs) are mainly due to degeneration and calcification. In this study, we developed a new preparation strategy for BHVs named as "HPA/EDC/EGCG" that utilized 3,4-hydroxyphenylpropionic acid (HPA)-conjugated pericardium, epigallocatechin gallate (EGCG), and horseradish peroxidase (HRP)/hydrogen peroxide (H 2 O 2 ) enzymatic cross-linking. HPA-pericardium conjugation was done by carbodiimide coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then HPA-conjugated pericardium was cross-linked by HRP/H 2 O 2 enzyme-catalyzed oxidation. The feeding ratios of HPA and EGCG were optimized. The consumption of amino groups, collagenase and elastase degradation in vitro, biomechanics, extracellular matrix stability, and calcification of HPA-/EDC-/EGCG-treated pericardiums were characterized. We demonstrated that HPA-/EDC-/EGCG-treated pericardiums had better elastin stabilization and less calcification. EGCG and enzymatic cross-linking treated pericardiums showed improved mechanical properties. This new EGCG and enzymatic cross-linking strategy would be a promising method to make BHVs with better elastin stability and anti-calcification property. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1551-1559, 2019., (© 2018 Wiley Periodicals, Inc.)

We report on a mass-resolved IR spectrosopic study on propofol-benzocaine aggregates. This is a complex system due to the several conformational isomers that both monomers may adopt and to the combination of functional groups they present, which allow the molecules to interact in many possible ways. However, our results demonstrate that a single conformation is favored for each stoichiometry. In the heterodimer, propofol acts as a proton donor to the ester group of benzocaine, while the whole cluster is stabilized by dispersive forces. These dispersive forces account for an important part of the system's stabilization energy as the calculations suggest. Propofol does not show any affinity for the amino group of benzocaine, even when a second molecule of propofol is introduced. These results demonstrate the difficulty in anticipating the aggregation preferences of even small organic molecules.

In recent years, the development of hybrid drug delivery systems, such as hydrogels and nanoparticles, has gained considerable attention as new formulations for skin-delivery. Meanwhile, transdermal diffusion synthetic membranes have been used to assess skin permeability to these systems, providing key insights into the relationships between drug and nanoformulations. In this study, benzocaine-loaded poly-ε-caprolactone nanoparticles (BZC:NPs) were synthesized, characterized and incorporated into Poloxamer 407-based hydrogel (PL407). Benzocaine (BZC) was used as a drug model since has been commonly applied as a topical pain reliever in the last years. Hence, we developed a hybrid polymeric nanoparticle/thermosensitive hydrogels system and evaluated the in vitro permeation of the BZC, as well as nanoformulation tracking in an artificial membrane. In vitro permeation study was conducted in a vertical diffusion cell system using a Strat-M ® membrane model. BZC:NPs were prepared by coprecipitation method and their physicochemical stability measured before incorporating into the thermosensitive hydrogel. Also, viscosity measurements and sol-gel transition temperature were performed by rheological analysis. Different techniques, including microscopy, were used to tracking the nanoparticles on both receptor medium and synthetic membranes. Results showed high BZC encapsulation efficiency into NPs (93%) and good physicochemical stability before and after hydrogel incorporation. BZC in vitro permeation kinetics from NPs-loaded Poloxamer 407-based hydrogel presented slower permeation profile compared with the BZC: Poloxamer 407-based hydrogel. Also, NPs were observed into the diffusion cells receptor compartment after the in vitro permeation study. These results contribute to a better understanding the interaction between hydrogels, nanoparticles and synthetic membrane, as well as open perspectives for the development of new drug delivery systems for skin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the development of new pharmaceutical formulations it is important to consider the possible interactions between the active pharmaceutical ingredient (API) and excipients which is a well-known problem. The objective of the work presented here was to investigate such reactions by means of diffusion ordered NMR spectroscopy (DOSY). The known reaction of 5-aminosalicylic acid (5-ASA) and the excipient citric acid was studied. Three reaction products have been verified by DOSY, 1 H NMR and HPLC measurements. Despite a poor separation in the DOSY diagram, the reaction products could be assign due to the processing of thoughtful selected parts of the signals. The reaction of 5-ASA with formic acid and benzocaine with dibutyl phthalate was also studied by means of DOSY experiments., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The study was a pioneering attempt to assess the influence of the structural polymorphism (forms I, II, III) of benzocaine on its solubility, apparent solubility, and chemical stability, which are vital parameters for preformulation and formulation work. The impact of differences in the solubility of selected polymorphs of benzocaine on their permeability through artificial biological membranes (PAMPA system) was evaluated. The polymorphs of benzocaine were obtained by means of techniques commonly used for the preparation of various pharmaceutical dosage forms: ball milling, micro milling, and cryogenic grinding, which allowed for the appearance or preservation of form III, the initial conformation of benzocaine. Ball milling resulted in the conversion of form III to I, whereas micro milling yielded form II. As a result of cryogenic grinding, form III of benzocaine was preserved. The identification of all polymorphic forms of benzocaine was confirmed via X-ray powder diffraction (PXRD) supported by FT-IR spectroscopy coupled with density functional theory (DFT) calculations. The differences in solubility, dissolution, and permeability through artificial biological membranes resulting from the polymorphic forms of benzocaine were established by using chromatographic determinations. Accelerated stability tests indicated that all polymorphic forms were chemically stable at a required level.

Understanding the phase behavior of crystal forms is essential in drug formulation development, as physical stability of the active pharmaceutical ingredient (API) is critical to achieving the desired bioavailability. Solvents greatly impact the physical stability of crystalline solids, resulting in a variety of well-known phase transitions, such as hydrate/solvate formation. However, solvent incorporation may also result in the formation of a less-known crystalline solid solutions (CSSs). The identification and characterization of CSSs and their effect on API physicochemical properties have not been investigated. This is the first reported instance of a CSS for an API. An exhaustive study of the phase behavior of the enantiotropically related polymorphs, I and II, of Benzocaine in water and ethanol revealed that Form I formed a CSS with water below 294.5K. Construction of the phase diagrams of Forms I and II in water and ethanol revealed that CSS formation significantly decreased the phase transition temperature between Forms I and II in water. This change resulted from the increased disorder in the lattice of Form I due to the presence of water. This work demonstrates the importance of understanding the formation of CSSs on the thermodynamic behavior of crystalline pharmaceutical solids., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ethyl-4-aminobenzoate (Et-PABA) is currently used as a substitute for 4-aminobenzoate (PABA) in sunscreens and anesthetic ointments. Despite its widespread use and hydrophilicity, Et-PABA has never been found in environmental waters. This study, probed the occurrence of Et-PABA in both seawater and drinking water sources in Hong Kong, and evaluated its transformation products (TPs) and environmental fate via cumulative potency and photocatalytic profile analyses. Another 11 UV filters used in skin-care products were also studied. Et-PABA was not detected in any water sample. Four other UV filters were dominant at ng/L level in both seawater and drinking water sources. UHPLC-QTOF-MS was used to elucidate the structure of TPs. With high resolution accurate mass data and fragment rationalization, 11 Et-PABA TPs were characterized, including seven intermediates firstly proposed as TPs; two compounds were reported for the first time. It is proposed that photocatalysis induces transformation pathways of (de)hydroxylation, demethylation and molecular rearrangement. Luminescent bacteria tests showed decreasing toxicity with increasing irradiation of Et-PABA, suggesting that irradiation TPs are less toxic than the parent compound. Transformation of Et-PABA appears to explain why Et-PABA has not been detected in the natural environment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the in vitro cytotoxicity and the in vivo analgesic effect and local toxicity of the local anesthetic butamben (BTB) encapsulated in conventional or elastic liposomes incorporated in gel formulations. The results showed that both gel formulations of liposomal BTB reduced the cytotoxicity (p < 0.001; one-way ANOVA/Tukey's test) and increased the topical analgesic effect (p < 0.05; one-way ANOVA/Tukey's test) of butamben, compared to plain BTB gel. The gel formulations presented good rheological properties, and stability assays detected no differences in physicochemical stability up to 30 d after preparation. Moreover, histological assessment revealed no morphological changes in rat skin after application of any of the gel formulations tested.

AC electrokinetics (ACEK) has been shown to deliver certain drugs into human teeth more effectively than diffusion. However, using electrical wires to power intraoral ACEK devices poses risks to patients. The study demonstrates a novel interdigitated electrode arrays (IDE) assembly powered by inductive coupling to induce ACEK effects at appropriate frequencies to motivate drugs wirelessly. A signal generator produces the modulating signal, which multiplies with the carrier signal to produce the amplitude modulated (AM) signal. The AM signal goes through the inductive link to appear on the secondary coil, then rectified and filtered to dispose of its carrier signal, and the positive half of the modulating signal appears on the load. After characterizing the device, the device is validated under light microscopy by motivating carboxylate-modified microspheres, tetracycline, acetaminophen, benzocaine, lidocaine and carbamide peroxide particles with induced ACEK effects. The assembly is finally tested in a common dental bleaching application. After applying 35 % carbamide peroxide to human teeth topically or with the IDE at 1200 Hz, 5 Vpp for 20 min, spectrophotometric analysis showed that compared to diffusion, the IDE enhanced whitening in specular optic and specular optic excluded modes by 215 % and 194 % respectively. Carbamide peroxide absorbance by the ACEK group was two times greater than diffusion as measured by colorimetric oxidation-reduction and UV-Vis spectroscopy at 550 nm. The device motivates drugs of variable molecular weight and structure wirelessly. Wireless transport of drugs to intraoral targets under ACEK effects may potentially improve the efficacy and safety of drug delivery in dentistry.

An alkyl-radical loss and an alkene loss are two competitive fragmentation pathways that deprotonated aminobenzoate esters undergo upon activation under mass spectrometric conditions. For the meta and para isomers, the alkyl-radical loss by a homolytic cleavage of the alkyl-oxygen bond of the ester moiety is the predominant fragmentation pathway, while the contribution from the alkene elimination by a heterolytic pathway is less significant. In contrast, owing to a pronounced charge-mediated ortho effect, the alkene loss becomes the predominant pathway for the ortho isomers of ethyl and higher esters. Results from isotope-labeled compounds confirmed that the alkene loss proceeds by a specific γ-hydrogen transfer mechanism that resembles the McLafferty rearrangement for radical cations. Even for the para compounds, if the alkoxide moiety bears structural motifs required for the elimination of a more stable alkene molecule, the heterolytic pathway becomes the predominant pathway. For example, in the spectrum of deprotonated 2-phenylethyl 4-aminobenzoate, m/z 136 peak is the base peak because the alkene eliminated is styrene. Owing to the fact that all deprotonated aminobenzoate esters, irrespective of the size of the alkoxy group, upon activation fragment to form an m/z 135 ion, aminobenzoate esters in mixtures can be quantified by precursor ion discovery mass spectrometric experiments., (Copyright © 2016 John Wiley & Sons, Ltd.)

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid that finds an application as a local anesthetic, is found to adopt in its protonated form at least two populations of distinct structures in the gas phase, and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization process. Here, we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry to yield gas-phase IR spectra of simultaneously m/z and drift-time-resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species: the N- and O-protonated forms. Density functional theory calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution-phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation have been suggested.

Flephedrone (4-fluoromethcathinone, 4-FMC) was analysed using (1)H, (13)C, (15)N HMBC, and (19)F observe spectroscopy, gas chromatography-flame ionisation detection (GC-FID), and electrospray ionisation-mass spectrometry (ESI-MS). Analysis of four 4-FMC samples (from a Bristol nightclub in 2013) showed that they all contained benzocaine as the cutting agent present in different amounts from 5 to 12%. Using these methods, we successfully differentiated between flephedrone regioisomers and mephedrone in an analytical method validated for flephedrone as a substituted cathinone. The data show that these now illegal cathinone-derived stimulants (highs) are now being cut; users cannot be certain of the purity of the drug they are taking. Furthermore, there are risks from the pharmaceutically active cutting agents themselves., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study we demonstrate the potential of selective reagent ionisation-time of flight-mass spectrometry for the rapid and selective identification of a popular new psychoactive substance blend called 'synthacaine', a mixture that is supposed to imitate the sensory and intoxicating effects of cocaine. Reactions with H3O(+) result in protonated parent molecules which can be tentatively assigned to benzocaine and methiopropamine. However, by comparing the product ion branching ratios obtained at two reduced electric field values (90 and 170 Td) for two reagent ions (H3O(+) and NO(+)) to those of the pure chemicals, we show that identification is possible with a much higher level of confidence then when relying solely on the m/z of protonated parent molecules. A rapid and highly selective analytical identification of the constituents of a recreational drug is particularly crucial to medical personnel for the prompt medical treatment of overdoses, toxic effects or allergic reactions., (Copyright © 2015 John Wiley & Sons, Ltd.)

Purpose: The aim of this study was to investigate how factors such as temperature, relative humidity and particle size impact the extent of disproportionation (salt to free base conversion) in powder blends of miconazole, benzocaine or sertraline mesylate salts mixed with a basic additive., Method: Raman spectroscopy was used to quantitate the extent of disproportionation. The data was further analyzed by multivariate analysis with partial least squares (PLS) modeling., Results: It was found that salt disproportionation was significantly influenced by % weight gain due to moisture sorption both in terms of the kinetics and the conversion extent, suggesting a solution-mediated reaction. Temperature plays an important role in impacting the value of pHmax which in turn has a significant correlation to the amount of free base formed. The particle size and drug: additive ratio were also found to influence the extent of disproportionation., Conclusions: This study shows that the extent of salt disproportionation is influenced by multiple factors and the application of PLS modeling demonstrated the feasibility of utilizing multivariate analysis to generate a predictive model for estimating the extent of conversion and thus may serve as a tool for risk assessment.

We present a general framework to predict the excess solubility of small molecular solids (such as pharmaceutical solids) in binary solvents via molecular simulation free energy calculations at infinite dilution with conventional molecular models. The present study used molecular dynamics with the General AMBER Force Field to predict the excess solubility of acetanilide, acetaminophen, phenacetin, benzocaine, and caffeine in binary water/ethanol solvents. The simulations are able to predict the existence of solubility enhancement and the results are in good agreement with available experimental data. The accuracy of the predictions in addition to the generality of the method suggests that molecular simulations may be a valuable design tool for solvent selection in drug development processes.

Since computing resources have advanced enough to allow routine molecular simulation studies of drug molecules interacting with biologically relevant membranes, a considerable amount of work has been carried out with fluid phospholipid systems. However, there is very little work in the literature on drug interactions with gel phase lipids. This poses a significant limitation for understanding permeation through the stratum corneum where the primary pathway is expected to be through a highly ordered lipid matrix. To address this point, we analyzed the interactions of p-aminobenzoic acid (PABA) and its ethyl (benzocaine) and butyl (butamben) esters with two membrane bilayers, which differ in their fluidity at ambient conditions. We considered a dioleoylphosphatidylcholine (DOPC) bilayer in a fluid state and a ceramide 2 (CER2, ceramide NS) bilayer in a gel phase. We carried out unbiased (100 ns long) and biased z-constraint molecular dynamics simulations and calculated the free energy profiles of all molecules along the bilayer normal. The free energy profiles converged significantly slower for the gel phase. While the compounds have comparable affinities for both membranes, they exhibit penetration barriers almost 3 times higher in the gel phase CER2 bilayer. This elevated barrier and slower diffusion in the CER2 bilayer, which are caused by the high ordering of CER2 lipid chains, explain the low permeability of the gel phase membranes. We also compared the free energy profiles from MD simulations with those obtained from COSMOmic. This method provided the same trends in behavior for the guest molecules in both bilayers; however, the penetration barriers calculated by COSMOmic did not differ between membranes. In conclusion, we show how membrane fluid properties affect the interaction of drug-like molecules with membranes.

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

The aim of this work was to study the interaction between the local anesthetic benzocaine and p-sulfonic acid calix[n]arenes using NMR and theoretical calculations and to assess the effects of complexation on cytotoxicity of benzocaine. The architectures of the complexes were proposed according to (1) H NMR data (Job plot, binding constants, and ROESY) indicating details on the insertion of benzocaine in the cavity of the calix[n]arenes. The proposed inclusion compounds were optimized using the PM3 semiempirical method, and the electronic plus nuclear repulsion energy contributions were performed at the DFT level using the PBE exchange/correlation functional and the 6-311G(d) basis set. The remarkable agreement between experimental and theoretical approaches adds support to their use in the structural characterization of the inclusion complexes. In vitro cytotoxic tests showed that complexation intensifies the intrinsic toxicity of benzocaine, possibly by increasing the water solubility of the anesthetic and favoring its partitioning inside of biomembranes., (© 2013 John Wiley & Sons A/S.)

A number of isosteres (oxadiazoles, thiadiazoles, tetrazoles and diazines) of benzocaine were prepared and evaluated for their capacity to induce methemoglobinemia-with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed within each series, with 1,2,4-oxadiazole 3 (metHb%=61.0±3.6) and 1,3,4-oxadiazole 10 (metHb%=52.4±0.9) demonstrating the greatest activity. Of the 5 candidates (compounds 3, 10, 11, 13 and 23) evaluated in vivo, failure to induce a lethal end-point at doses of 120mg/kg was observed in all cases. Inadequate metabolic stability, particularly towards hepatic enzymes such as the CYPs, was postulated as one reason for their failure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Breast cancer remains one of the most common cancers for females. Drug delivery based on cancer nanotechnology could improve the performance of some chemotherapeutic medicines already used in clinic. The emergence of polymersomes provided the potential to encapsulate hydrophobic/hydrophilic drugs. By modifying the weight ratio of methoxy-poly (ethylene glycol) (mPEG) chain to ethyl-p-aminobenzoate (EAB) side group, a series of amphiphilic graft polyphosphazenes (PEPs) was prepared. PEP can be tuned from micelles to polymersomes with the decrease of mPEG content via dialysis. Either hydrophilic doxorubicin hydrochloride (DOX·HCl) or hydrophobic doxorubicin base (DOX) could be encapsulated into PEP polymersomes with high payload and high encapsulation efficiency due to the strong intermolecular interaction with PEP. Compared with free DOX·HCl administration, in vivo investigation in growth inhibition of MCF-7 xenograft tumors in nude mice demonstrated that PEP polymersomes could enhance life safety without compromise of therapeutic efficacy, especially DOX·HCl loaded delivery system., From the Clinical Editor: In this preclinical study, polymerosomes based on PEPs were investigated as doxorubicin delivery systems, demonstrating similar efficacy but less toxicity compared to standard delivery methods., (© 2014.)

Molecular dynamics simulations were used to examine the partitioning behaviour of the local anaesthetic benzocaine and the anti-epileptic phenytoin into lipid bilayers, a factor that is critical to their mode of action. Free energy methods are used to quantify the thermodynamics of drug movement between water and octanol as well as for permeation across a POPC membrane. Both drugs are shown to favourably partition into the lipid bilayer from water and are likely to accumulate just inside the lipid headgroups where they may alter bilayer properties or interact with target proteins. Phenytoin experiences a large barrier to cross the centre of the bilayer due to less favourable energetic interactions in this less dense region of the bilayer. Remarkably, in our simulations both drugs are able to pull water into the bilayer, creating water chains that extend back to bulk, and which may modify the local bilayer properties. We find that the choice of atomic partial charges can have a significant impact on the quantitative results, meaning that careful validation of parameters for new drugs, such as performed here, should be performed prior to their use in biomolecular simulations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Understanding the phase behavior of an active pharmaceutical ingredient in a drug formulation is required to avoid the occurrence of sudden phase changes resulting in decrease of bioavailability in a marketed product. Benzocaine is known to possess three crystalline polymorphs, but their stability hierarchy has so far not been determined. A topological method and direct calorimetric measurements under pressure have been used to construct the topological pressure-temperature diagram of the phase relationships between the solid phases II and III, the liquid, and the vapor phase. In the process, the transition temperature between solid phases III and II and its enthalpy change have been determined. Solid phase II, which has the highest melting point, is the more stable phase under ambient conditions in this phase diagram. Surprisingly, solid phase I has not been observed during the study, even though the scarce literature data on its thermal behavior appear to indicate that it might be the most stable one of the three solid phases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The benzocaine-toluene cluster has been used as a model system to mimic the interaction between the local anesthetic benzocaine and the phenylalanine residue in Na(+) channels. The cluster was generated in a supersonic expansion of benzocaine and toluene in helium. Using a combination of mass-resolved laser-based experimental techniques and computational methods, the complex was fully characterized, finding four conformational isomers in which the molecules are bound through N-H···π and π···π weak hydrogen bonds. The structures of the detected isomers closely resemble those predicted for benzocaine in the inner pore of the ion channels, giving experimental support to previously reported molecular chemistry models.

Fourier transform (FT)-Raman and infrared (IR) spectra of the nonlinear optical (NLO) material ethyl p-amino benzoate (EPAB) have been recorded and analyzed. The geometry and harmonic vibrational wavenumbers are calculated with the help of B3LYP density functional theory method. The detailed interpretation of the vibrational spectra has been carried out with the aid of normal coordinate analysis following the scaled quantum mechanical force field methodology. Stability of the molecule arising from hyperconjugative interactions leading to its NLO activity and charge delocalization have been analyzed using natural bond orbital (NBO) analysis. Employing the open-aperture z-scan technique, NLO absorption of the sample has been studied in two excitation regimes, using 100 fs and 5 ns laser pulses respectively. It is found that EPAB is a three-photon absorber for 100 fs pulses at the excitation wavelength of 800 nm. For ns pulses at 532 nm it exhibits strong optical limiting, indicating possible photonics applications., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rationale: Stable isotope analysis is a critical tool for understanding ecological food webs; however, results can be sensitive to sample preparation methods. To limit the possibility of sample contamination, freezing is commonly used to euthanize invertebrates and preserve non-lethal samples from vertebrates. For destructive sampling of vertebrates, more humane euthanasia methods are preferred to freezing and it is essential to evaluate how these euthanasia methods affect stable isotope results., Methods: Stable isotope ratios and elemental composition of carbon and nitrogen were used to evaluate whether the euthanasia method compromised the integrity of the sample for analysis. Specifically, the stable isotope and C:N ratios were compared for larval wood frogs (Rana sylvatica  =  Lithobates sylvaticus), an ectothermic vertebrate, that had been euthanized by freezing with four different humane euthanasia methods: CO2, benzocaine, MS-222 (tricaine methanesulfonate), and 70% ethanol., Results: The euthanasia method was not related to the δ(13)C or δ(15)N values and the comparisons revealed no differences between freezing and any of the other treatments. However, there were slight (non-significant) differences in the isotope ratios of benzocaine and CO2 when each was compared with freezing. The elemental composition was altered by the euthanasia method employed. The percentage nitrogen was higher in CO2 treatments than in freezing, and similar (non-significant) trends were seen for ethanol treatments relative to freezing. The resulting C:N ratios were higher for benzocaine treatments than for both CO2 and ethanol. Similar (non-significant) trends suggested that the C:N ratios were also higher for animals euthanized by freezing than for both CO2 and ethanol euthanasia methods., Conclusions: The euthanasia method had a larger effect on elemental composition than stable isotope ratios. The percentage nitrogen and the subsequent C:N ratios were most affected by the CO2 and ethanol euthanasia methods, whereas non-significant trends suggested that benzocaine and CO2 altered the stable isotope ratios. It appears that the use of MS-222 and freezing with dry ice are the most appropriate euthanasia methods for ectothermic vertebrates., (Copyright © 2013 John Wiley & Sons, Ltd.)

Many challenges arise during the development of new drug carrier systems, and paramount among them are safety, solubility and controlled release requirements. Although synthetic polymers are effective, the possibility of side effects imposes restrictions on their acceptable use and dose limits. Thus, a new drug carrier system that is safe to handle and free from side effects is very much in need and food grade polysaccharides stand tall as worthy alternatives. Herein, we demonstrate for the first time the feasibility of sodium iota-carrageenan fibers and their distinctive water pockets to embed and release a wide variety of drug molecules. Structural analysis has revealed the existence of crystalline network in the fibers even after encapsulating the drug molecules, and iota-carrageenan maintains its characteristic and reproducible double helical structure suggesting that the composites thus produced are reminiscent of cocrystals. The melting properties of iota-carrageenan:drug complexes are distinctly different from those of either drug or iota-carrageenan fiber. The encapsulated drugs are released in a sustained manner from the fiber matrix. Overall, our research provides an elegant opportunity for developing effective drug carriers with stable network toward enhancing and/or controlling bioavailability and extending shelf-life of drug molecules using GRAS excipients, food polysaccharides, that are inexpensive and non-toxic., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The aim of the present study was to characterize a liposome-based benzocaine (BZC) formulation designed for topical use on the oral mucosa and to evaluate its in vitro retention and permeation using the Franz-type diffusion cells through pig esophagus mucosa. To predict the effectiveness of new designed formulations during preclinical studies, a correlation between in vitro assays and in vivo efficacy was performed. Liposomal BZC was characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and morphology. Liposomal BZC (BL10) was incorporated into gel formulation and its performances were compared to plain BZC gel (B10) and the commercially available BZC gel (B20). BL10 and B10 presented higher flux and retention on pig esophagus mucosa with a shorter lag time, when compared to B20. BZC flux was strongly correlated with in vivo anesthetic efficacy, but not with topical anesthesia duration. The retention studies did not correlate with any of the in vivo efficacy parameters. Thus, in vitro permeation study can be useful to predict anesthetic efficacy during preclinical tests, because a correlation between flux and anesthetic efficacy was observed. Therefore, in vitro assays, followed by in vivo efficacy, are necessary to confirm anesthetic performance.

The insertion of local anaesthetics into a cell membrane is a key aspect for explaining their activity at a molecular level. It has been described how the potency and response time of local anaesthetics is improved (for clinical applications) when they are dissolved in a solution of sodium bicarbonate. With the aim of gaining insight into the physico-chemical principles that govern the action mechanism of these drugs at a molecular level, simulations of benzocaine in binary lipid bilayers formed by DPPC/DPPS were carried out for different ionic strengths of the aqueous solution. From these molecular dynamic simulations, we observed how the thermodynamic barrier associated with benzocaine insertion into the lipid bilayers diminished exponentially as the fraction of DPPS in the bilayer increased, especially when the ionic strength of the aqueous solution increased. In line with these results, we also observed how this thermodynamic barrier diminished exponentially with the phospholipid/water interfacial tension., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The crystallization kinetics of various active pharmaceutical ingredient/polyethylene glycol (API/PEG) solid dispersions has been investigated using wide-angle X-ray diffraction (XRD) and Raman spectroscopy. APIs with different physicochemical properties and crystallization tendency were employed to form solid dispersions with PEG. The crystallization rate of benzocaine (BZC) in BZC/PEG (20/80 wt %) solid dispersions was decreased substantially in comparison to that of the pure API, while the PEG matrix did not affect the crystallization behavior of haloperidol (HLP). The induction time for crystallization of ibuprofen (IBP) and fenofibrate (FNB) in a PEG matrix was decreased relative to the induction times for pure IBP and FNB. For the latter systems, it appears that crystalline PEG acted as a favorable heterogeneous nucleation site. The crystallization behavior of PEG in the API/PEG systems was also affected to different extents, depending on the API studied. These results suggest that PEG can delay, promote or have no influence on the crystallization kinetics of different APIs, and that any effects on crystallization behavior should be investigated in order to be able to produce a solid dispersion with consistent properties.

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.

As the primary step for 'drug repositioning', we evaluated the effect of 2000 drugs and drug candidates on the commitment of bi-potential mesenchymal precursor C2C12 cells into osteoblasts in the presence of bone morphogenetic protein (BMP)-2 and found that butamben enhanced BMP-2-stimulated induction of alkaline phosphatase, a biomarker of osteoblastogenesis. Investigating the underlying mechanism of its anabolic actions, we found anabolic action of its derivative (compound 4) relies on BMP-2 signaling and mRNA induction of BMPs and voltage-gated potassium channels., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C(BZC)/C(Lys)<9) and a combined quenching process at higher concentration of BZC (C(BZC)/C(Lys)>9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn(2+) on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Despite the general consensus concerning the role played by sodium channels in the molecular mechanism of local anesthetics, the potency of anaesthetic drugs also seems to be related with their solubility in lipid bilayers. In this respect, this work represents a thermodynamic study of benzocaine insertion into lipid bilayers of different compositions by means of molecular dynamics simulation. Thus, the free energy profiles associated with benzocaine insertion into symmetric lipid bilayers composed of different proportions of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine were studied. From the simulation results, a maximum in the free energy (ΔG) profile was measured in the region of the lipid/solution interface. This free energy barrier appears to be very much dependent on the lipid composition of the membrane. On the other hand, the minimum free energy (ΔG) within the bilayer remained almost independent of the lipid composition of the bilayer. By repeating the study at different temperatures, it was seen how the spontaneity of benzocaine insertion into the lipid bilayer is due to an increase in the entropy associated with the process., (© 2011 American Institute of Physics)

Purpose: The aim of this work was to investigate the influence of the oily nucleus composition on physico-chemical properties and anesthetic activity of poly (lactide-co-glycolide) nanocapsules with benzocaine., Methods: Nanocapsules containing benzocaine were prepared with three different oily nucleus composition and characterized by mean diameter, polydispersivity, zeta potential, pH and stability were investigated as a function of time. In vitro release kinetics were performed in a system with two compartments separated by a cellulose membrane. Intensity and duration of analgesia were evaluated in rats by sciatic nerve blockade., Results: The greatest stability, slower release profile and improvement in the local anesthetic activity of BZC were obtained with the formulation using USP mineral oil as component., Conclusions: Results from our study provide useful perspectives on selection of the primary materials needed to produce suspensions of polymeric nanocapsules able to act as carriers of BZC, with potential future application in the treatment of pain.

Benzocaine (ethyl 4-aminobenzoate, 4) and its derivatives ethyl 2-aminobenzoate, 2, and ethyl 3-aminobenzoate, 3, were found to form association complexes with supramolecular structures of micelles and cyclodextrins (CDs). The fluorescence emission of 2, 3 or 4 dissolved in the pseudo-micellar phase or included into α-, β-, or γ-CD cavity increases dramatically with respect to that observed in only water. High percentages of organic solvents like dioxane, acetonitrile, DMSO in the aqueous solution lead to a similar effect. The stability constants of the complexes formed between these drugs and cyclodextrins have been determined. In neutral or acid medium, a 1:1 stoichiometry for drug:CD complexes have been found, whereas in alkaline medium 1:2 stoichiometry was also detected in some cases. Kinetic studies of both the nitrosation of the amine group and the alkaline hydrolysis of the ester function was employed to infer the conformation of the complexes as well as to evaluate their stability constants. Theoretical calculations to optimize the molecular structure of 2, 3 and 4 allow us to propose possible geometries of the complexes that are in agreement with the experimental data.

In this work we describe the screening of four parameters in the preparation, by nanoprecipitation, of poly(epsilon-caprolactone) nanocapsules, used as a drug carrier system for the local anesthetic, benzocaine. A 2(4-1) factorial experimental design was used to study the influence of four different independent variables (polymer, oily phase, Span 60 and Tween 80) on nanocapsule characteristics (size, polydispersion index, zeta potential) and drug loading capability. Best results were obtained using an aqueous formulation comprising 100 mg of polymer, 200 mg of oily phase, 40 mg of Span 60 and 60 mg of Tween 80 in a final volume of 10 mL which produced a colloidal system with particle size of 188 nm, zeta potential -32 mV, polydispersion index 0.07, and benzocaine association efficiency > 87%. These findings open the way for future clinical studies using such formulations.

Background and Objective: The pattern of contact sensitization among patients with chronic leg ulcers depends on the local practice of wound treatment along with demographic and clinical confounders. The study was aimed at revealing the associations between chronic leg ulcers and contact sensitization., Material and Methods: Between 2006 and 2008, 35 patients with chronic leg ulcers and surrounding dermatitis and 59 patients with contact dermatitis of the lower leg or foot were prospectively recruited at the Department of Skin and Venereal Diseases, Lithuanian University of Health Sciences. Demographic and clinical data were collected in accordance with the "minimal data set" of the European Surveillance System on Contact Allergy. Patch testing was performed with the allergens of the European baseline series., Results: At least one positive patch test reaction was found in 28 (80%) of the patients with chronic leg ulcers and in 24 (41%) of the patients with dermatitis of the lower leg or foot (P<0.001). Sensitization to some of the most common allergens, namely colophony, Myroxylon pereirae resin, and methyldibromo glutaronitrile, was prevalent in both the groups of patients, whereas sensitization to benzocaine, p-phenylenediamine, and lanolin alcohol was associated with the presence of chronic leg ulcers. Benzocaine was found to be the leading allergen among patients with chronic leg ulcers (positive patch test reactions in 34.4% of the patients)., Conclusions: Contact sensitization to benzocaine, p-phenylenediamine, and lanolin was found to be associated with the presence of chronic leg ulcers.

Objectives: This study evaluated the efficacy of liposome-encapsulated 2% ropivacaine in topical anesthesia and its influence on pulpal response., Study Design: Forty volunteers received the following topical formulations in the buccal fold of the maxillary lateral incisors region (bilaterally): liposome-encapsulated 2% ropivacaine gel (RL2); 20% benzocaine gel (B20); liposomal placebo gel (LP); and placebo gel (P). Formulations were kept in place for 30 minutes, during which time the teeth were electric pulp tested every 10 minutes. After this procedure, a dental needle was inserted until periosteum contact in the same site of topical application and pain was rated by a visual analog scale. Duration of soft tissue anesthesia was assessed by pinprick test., Results: RL2 and B20 showed lower pain response to needle insertion and longer soft tissue anesthesia then P and LP (P = .0003 and P < .0001, respectively); however, RL2 was not different from B20 (P > .05) regarding those parameters. None of the formulations was able to induce pulpal anesthesia., Conclusion: RL2 was as effective as B20 in reducing pain during needle insertion and inducing soft tissue anesthesia; however, neither one was able to induce pulpal anesthesia after a 30-min application., (Copyright © 2010 Mosby, Inc. All rights reserved.)

The combined approach of cyclodextrin complexation and entrapment in liposomes was investigated to develop a topical formulation of local anaesthetics. For both benzocaine (BZC) and butamben (BTM), hydroxypropyl-beta-cyclodextrin (HPbetaCD) was a better partner than betaCD; drug-HPbetaCD coevaporated products showed the best solubility and dissolution properties, and were selected for loading into liposomes. Addition of stearylamine to the phosphatidylcholine-cholesterol mixture of the vesicle bilayer allowed obtainment of deformable liposomes with improved permeation and in vivo drug anaesthetic effect (P<0.05). Double-loaded deformable liposomes were obtained by adding the drug-HPbetaCD complex at its maximum aqueous solubility in the vesicles hydrophilic phase, and the remaining amount up to 1% as free drug in the lipophilic phase. The properties of double-loaded liposomes were compared with those of classic single-loaded ones, obtained by adding 1% free drug in the aqueous or lipophilic phase of the vesicles. Size, charge, morphology and entrapment efficiency of the different batches were investigated, respectively, by light scattering, confocal laser scanning microscopy and dialysis, while their therapeutic efficacy was evaluated in vivo on rabbits. For both drugs, double-loaded liposomes, exploiting the favourable effects of drug-CD complexation, allowed a significant (P<0.05) enhancement of intensity and duration of anaesthetic effect with respect to those single-loaded., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The benzocaine-phenol complex is proposed as a model system of the interaction between the local anaesthetic benzocaine and the tyrosine residue. The complex has been generated by supersonic expansion of benzocaine and phenol in helium and probed by 1- and 2-color mass-resolved laser spectroscopies. The electronic excitation spectrum of the 1 : 1 complex spans some approximately 700 cm(-1) and includes well resolved bands from at least two isomers, as demonstrated using UV-UV hole burning spectroscopy. The combination of ion dip infrared spectroscopy (IDIRS) and ab initio calculations shows that both isomers are stabilized by an OH...N hydrogen bond between the phenol hydroxyl group and the benzocaine amino moiety, differing only in the conformation adopted by the benzocaine monomer (trans and gauche). The application of the fragmentation threshold method to benzocaine-phenol suggests the existence of chemical reactions in the electronic excited state of the complex and/or in the ion. Such hypothesis is also supported by the calculated potential energy curves along the hydrogen bond coordinate.

Rationale: Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in Na(V)1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block., Objective: Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes., Methods and Results: The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed., Conclusions: These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.

A low-temperature structural phase transition has been observed for form (II) of benzocaine (BZC). Lowering the temperature doubles the b-axis repeat and changes the space group from P2(1)2(1)2(1) to P112(1) with gamma now 99.37 degrees. The structure is twinned, the twin rule corresponding to a 2(1) screw rotation parallel to a. The phase transition is associated with a sequential displacement parallel to a of zigzag bi-layers of ribbons perpendicular to b*. No similar phase transition was observed for form (I) and this was attributed to the different packing symmetries of the two room-temperature polymorphic forms.

The precise molecular mechanism of general anesthetics remains unknown. It is therefore important to understand where molecules with anesthetic properties localize within biological membranes. We have determined the free energy profile of a benzocaine molecule (BZC) across a biological membrane using molecular dynamics simulation. We use an asymmetric phospholipid bilayer with DPPS in one leaflet of a DPPC bilayer (Lopez Cascales et al. J. Phys. Chem. B 2006, 110, 2358-2363) to model a biological bilayer. From the free energy profile, we predict the zone of actuation of a benzocaine is located in the hydrocarbon region or at the end of the lipid head, depending of the presence of charged lipids (DPPS) in the leaflet. We observe a moderate increase in the disorder of the membrane and in particular an increase in the disorder of DPPS. Static and dynamic physicochemical properties of the benzocaine, such as its dipole orientation, translational diffusion coefficient, and rotational relaxation time were measured.