Characterization of the methemoglobin forming metabolites of benzocaine and lidocaine.

1. Topical anesthesia with benzocaine or lidocaine occasionally causes methemoglobinemia, an uncommon but potentially fatal disorder where the blood has a reduced ability to transport oxygen. Previous in vitro studies using human whole blood have shown that benzocaine causes more methemoglobin (MetHb) formation than lidocaine, and that both compounds require metabolic transformation to form the MetHb producing species. In the current investigation, the active species of benzocaine forming the MetHb was investigated. 2. HPLC analysis of benzocaine samples incubated with human hepatic S9 showed the formation of a peak with the same UV spectrum and retention time as benzocaine hydroxylamine (BenzNOH). To confirm the activity of BenzNOH, MetHb production following exposure to the compound was determined in whole human blood using an Avoximeter 4000 CO-oximeter. 3. BenzNOH produced MetHb in a concentration dependent manner without the need for metabolic activation. Benzocaine in the presence of metabolic activation required a concentration of 500 µM to produce a similar degree of MetHb formation as 20 µM BenzNOH without activation. Previous work suggested that two metabolites of lidocaine may also form MetHb; N-hydroxyxylidine and 4-hydroxyxylidine. Of these two metabolites 4-hydroxyxylidine produced the most MetHb in whole blood in vitro in the absence of metabolic activation, however BenzNOH produced up to 14.2 times more MetHb than 4-hydroxyxylidine at a similar concentration. 4. These results suggest that the ability of benzocaine to form MetHb is likely to be mediated through its hydroxylamine metabolite and that this metabolite is inherently more active than the potentially MetHb-forming metabolites of lidocaine.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

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.

Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

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.

Salt stability--effect of particle size, relative humidity, temperature and composition on salt to free base conversion.

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.

Molecularly imprinted microspheres synthesized by a simple, fast, and universal suspension polymerization for selective extraction of the topical anesthetic benzocaine in human serum and fish tissues.

A simple, fast, and universal suspension polymerization method was used to synthesize the molecularly imprinted microspheres (MIMs) for the topical anesthetic benzocaine (BZC). The desired diameter (10-20 µm) and uniform morphology of the MIMs were obtained easily by changing one or more of the synthesis conditions, including type and amount of surfactant, stirring rate, and ratio of organic to water phase. The MIMs obtained were used as a molecular-imprinting solid-phase-extraction (MISPE) material for extraction of BZC in human serum and fish tissues. The MISPE results revealed that the BZC in these biosamples could be enriched effectively after the MISPE operation. The recoveries of BZC on MIMs cartridges were higher than 90% (n = 3). Finally, an MISPE-HPLC method with UV detection was developed for highly selective extraction and fast detection of trace BZC in human serum and fish tissues. The developed method could also be used for the enrichment and detection of BZC in other complex biosamples.

More methemoglobin is produced by benzocaine treatment than lidocaine treatment in human in vitro systems.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro. Incubation of 500µM benzocaine with whole human blood and pooled human liver S9 over 5h resulted in MetHb levels equaling 39.8±1.2% of the total hemoglobin. No MetHb formation was detected for 500µM lidocaine under the same conditions. Because liver S9 does not readily form lidocaine hydrolytic metabolites based on xylidine, a primary metabolic pathway, 500µM xylidine was directly incubated with whole blood and S9. Under these conditions MetHb levels of 4.4±0.4% were reached by 5h. Studies with recombinant cytochrome P450 revealed benzocaine to be extensively metabolized by CYP 1A2, with 2B6, 2C19, 2D6, and 2E1 also having activity. We conclude that benzocaine produces much more MetHb in in vitro systems than lidocaine or xylidine and that benzocaine should be more likely to cause MetHba in vivo as well.

Microemulsion for simultaneous transdermal delivery of benzocaine and indomethacin: in vitro and in vivo evaluation.

This study investigated simultaneous transdermal delivery of indomethacin and benzocaine from microemulsion. Eucalyptus oil based microemulsion was used with Tween 80 and ethanol being employed as surfactant and cosurfactant, respectively. A microemulsion formulation comprising eucalyptus oil, polyoxyethylene sorbitan momooleate (Tween 80), ethanol and water (20:30:30:20) was selected. Indomethacin (1% w/w) and benzocaine (20% w/w) were incorporated separately or combined into this formulation before in vitro and in vivo evaluation. Application of indomethacin microemulsion enhanced the transdermal flux and reduced the lag time compared to saturated aqueous control. The same trend was evident for benzocaine microemulsion. Simultaneous application of the two drugs in microemulsion provided similar enhancement pattern. The in vivo evaluation employed the pinprick method and revealed rapid anesthesia after application of benzocaine microemulsion with the onset being 10 min and the action lasting for 50 min. For indomethacin microemulsion, the analgesic effect was recorded after 34.5 min and lasted for 70.5 min. Simultaneous application of benzocaine and indomethacin provided synergistic effect. The onset of action was achieved after 10 min and lasted for 95 min. The study highlighted the potential of microemulsion formulation in simultaneous transdermal delivery of two drugs.

Using lidocaine and benzocaine to link sodium channel molecular conformations to state-dependent antiarrhythmic drug affinity.

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.

EGCG and enzymatic cross-linking combined treatments for improving elastin stability and reducing calcification in bioprosthetic heart valves.

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 (H2 O2 ) 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/H2 O2 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.

Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ.

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) have been studied by visible absorption spectroscopy using native receptor-rich membranes from Discopyge tschudii electric tissue and liposomes obtained by aqueous dispersion of endogenous lipids extracted from the same tissue. The fact that merocyanine partitions into the membrane when this is in the liquid-crystalline state, exhibiting a characteristic peak at 567 nm, was exploited to obtain quantitative information about the physical state of the AChR-rich membrane. Spectra of MC540 revealed that this molecule was preferentially incorporated into AChR-rich membranes, with an affinity (Kdapp 30 microM) 10-fold higher than that in liposomes (Kdapp 290 microM). Changes were observed in the equilibrium dissociation constant of MC540 at different temperatures: the two-fold higher affinity at 8 degrees C than at 23 degrees C can be rationalized in terms of a higher value of the overall dimerization constant (Kdim) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher potency in AChR-rich native membranes than in liposomes made with endogenous lipids. This competition was found to be AChR concentration-dependent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent dissociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the benzocaine binding site is deduced from the competition experiments to be at the lipid annulus surrounding the nicotinic AChR protein.

Characterization of site I on human serum albumin: concept about the structure of a drug binding site.

Human serum albumin (HSA) possesses at least three sites or areas for high-affinity binding of drugs. Of these sites, site I was investigated by series of ultrafiltration and equilibrium dialysis experiments. Three ligands, acenocoumarol, dansyl-L-asparagine (DNSA) and n-butyl p-aminobenzoate (n-butyl p-ABE) were employed as marker ligands. Each ligand binds to a single high-affinity site on HSA, and binding studies with different pairs of the ligands revealed independent high-affinity binding. Preliminary displacement studies performed with the typical site I binding drugs warfarin, phenylbutazone and iodipamide showed different displacement patterns of the three marker ligands. These studies were followed by stringent competition experiments involving all possible combinations of the three test ligands themselves and of these and the three marker ligands. On the basis of the results obtained it seems that the acenocoumarol and DNSA binding regions correspond to the warfarin and azapropazone binding regions, respectively, of site I reported by others (Fehske, Schläfer, Wollert and Müller (1982) Mol. Pharmacol. 21, 387-393). The new binding region, represented by n-butyl p-ABE, is probably located adjacent to the acenocoumarol binding region but apart from that of DNSA. We have elaborated a model for binding site I in which we propose novel nomenclatures, region Ia, Ib, and Ic for the acenocoumarol, DNSA and n-butyl p-ABE binding regions, respectively. Furthermore, the relation between these regions and the high-affinity binding sites for other drugs have been discussed.

Binding of benzocaine in batrachotoxin-modified Na+ channels. State-dependent interactions.

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

Modeling of benzocaine analog interactions with the D4S6 segment of NaV4.1 voltage-gated sodium channels.

Local anesthetics (LAs) are compounds that inhibit the propagation of action potentials in excitable tissues by blocking voltage-gated Na+ channels. Mutagenesis studies have demonstrated that several amino acid residues are important sites of LA interaction with the channel, but these studies provide little information regarding the molecular forces that govern drug-binding interactions, including the binding orientation of drugs. We used computational methods to construct a simple model of benzocaine analog binding with the D4S6 segment of rat skeletal muscle (NaV4.1) sodium channels. The model revealed that four hydrophobic residues form a binding cavity for neutral LAs, and docking studies indicated that increasing hydrophobicity among the benzocaine analogs allowed a better fit within the binding cavity. The similarities between our simple model and published experimental data suggested that modeling of LA interactions with sodium channels, along with experimental approaches, could further enhance our understanding of LA interactions with sodium channels.

Putative binding sites for benzocaine on a human cardiac cloned channel (Kv1.5).

OBJECTIVES: It has been demonstrated that at nanomolar concentrations benzocaine increased, whereas at micromolar concentrations, it blocked hKv1.5 channels in a voltage-dependent manner and modified the voltage-dependence of channel activation. The present study was undertaken to localize the putative binding sites involved in the 'agonists' and blocking effects of benzocaine. METHODS: Experiments were carried out on wild-type and site directed mutated hKv1.5 channels stably expressed on Ltk(-) cells using the whole-cell patch-clamp. RESULTS: At 35 mM [K+](i) the voltage-dependent unblock produced by 500 microM benzocaine was preserved at both 4 and 140 mM [K+](o). Mutations located in the inner mouth of the pore (T477S, T505A, L508M and V512M) abolished the agonist but increased the blocking effects of benzocaine. Intracellular application of tetraethylammonium (3 mM) abolished the 'agonist' effects whereas the blocking effects of benzocaine remained unaltered. Block induced by benzocaine and intracellular tetraethylammonium was additive. In contrast, the combination of benzocaine and bupivacaine (>25 microM) produced less blockade than bupivacaine alone. However, mutation of the extracellular residue R485Y did not modify the effects of benzocaine. Extracellular application of tetraethylammonium (100 mM) did not modify the agonist effects of benzocaine, but abolished the voltage- and time-dependence of benzocaine-induced block. CONCLUSIONS: The results suggested that benzocaine binds with high affinity to an intracellular binding site to produce 'agonist' effects and to a low affinity subsite, which is also located in the inner mouth, to produce the blocking effects. Furthermore, benzocaine and extracellular K(+) interact to modify the voltage-dependence of channel opening.

Effects of the local anesthetic benzocaine on the human erythrocyte membrane and molecular models.

The interaction of the local anesthetic benzocaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphospatidylcholine (DMPC), and phospholipid multilayers of DMPC and dimyristoylphospatidyletanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy of human erythrocytes revealed that benzocaine induced the formation of echinocytes. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy showed that benzocaine interacted with the phospholipid bilayer polar groups and hydrophobic acyl chains. X-ray diffraction analysis of DMPC confirmed these results and showed that benzocaine had no effects on DMPE. The effect on sodium transport was also studied using the isolated toad skin. Electrophysiological measurements indicated a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of benzocaine, reflecting inhibition of active ion transport.

Vehicle effects in percutaneous absorption: in vitro study of influence of solvent power and microscopic viscosity of vehicle on benzocaine release from suspension hydrogels.

The release through silicone rubber membranes of benzocaine suspended in carbomer hydrogels containing different concentrations of low molecular weight polysols (ethylene glycol, propylene glycol, glycerol, and sorbitol) was studied to establish general principles and procedures for control of the effects on percutaneous absorption caused by changes in drug solubility and/or diffusivity in the vehicle. The effect of the additives on the release is expressed in terms of the relative released amount, i.e., the ratio, Q/Qw, of the amount of drug released from each additive-containing gel to the amount released at the same time from the additive-free gel. The experimental Q/Qw values are correlated with values calculated by a simple equation involving known or readily measurable parameters such as the drug concentration in the gel, the drug solubility in the pure liquid phase, and the viscosity of this phase. Derivation of such a relationship from a known equation describing the vehicle-controlled relase of suspended drugs was possible because an inverse proportionality was observed between drug diffusivity in the gels and the viscosity of the respective solvents. This relationship is interpreted with respect to current theories on drug diffusion in diluted gels.

Absorption and distribution of radioactivity from suppositories containing 3H-benzocaine in rats.

The effects of the suppository vehicle, drug concentration, and nonionic surfactants on in vitro benzocaine dialysis through a cellulose membrane and on rectal absorption in rats of total radioactivity following administration of 3H-benzocaine were investigated. In vitro dialysis correlated quite well with in vivo absorption, and drug release was greater from water-soluble vehicles than from oleaginous vehicles. Inclusion of a nonionic hydrophilic or lipophilic surfactant in cocoa butter resulted in a statistically significant increase for in vitro drug release, while a lipophilic surfactant showed little effect in vivo and a hydrophilic surfactant depressed release in vivo. Both types of surfactant had small effects on release from polyethylene glycol. In vitro release of benzocaine from some commercially available suppositories was compared with experimental preparations. Variation in blood radioactivity following administration of the same concentration of 3H-benzocaine in the same dosage form in male and female rats is reported.

Absorption of 3H-benzocaine from ointments following rectal administration in rats.

Total radioactivity in the blood of rats for 5 hr following rectal administration of 3H-benzocaine in oleaginous, absorption, emulsion (water-in-oil and oil-in water), and water-soluble ointment vehicles was measured. The release was greatest from the water-soluble vehicle and followed the same relative order as seen in an earlier in vitro experiment. No intact benzocaine was found in the blood using radiochromatography. In vitro hydrolysis of benzocaine by rat blood did not occur as determined with the techniques of this experiment.

Benzocaine diffusion from polyethylene glycol through human stratum corneum.

The diffusion, penetration, and surface effects of benzocaine incorporated in various polyethylene glycol ointment bases through human stratum corneum were studied. Benzocaine diffusion was measured by following the benzocaine concentration in the receiving compartment of a diffusion cell. The ointment was placed in the other cell compartment and was separated from the receiving compartment by sheets of human stratum corneum. Surface effects were monitored by scanning electron micrographs of the stratum corneum. Results showed a decrease in drug diffusion in the presence of relatively high amounts of the lower molecular weight portions of polyethylene glycol. Scanning electron microscope studies showed that both benzocaine and polyethylene glycol affect the surface structure of the stratum corneum. Thermal analysis indicated that benzocaine dissolves in polyethylene glycol.

Quantification of benzocaine and its metabolites in channel catfish tissues and fluids by HPLC.

Methods for extraction and gradient HPLC quantification were developed for benzocaine (BZ) and three of its metabolites to be used in conjunction with a reverse isotope technique. The metabolites were p-aminobenzoic acid (PABA), acetyl-p-aminobenzoic acid (AcPABA) and acetylbenzocaine (AcBZ). The matrixes studied were white muscle, red muscle, skin, liver, trunk kidney, head kidney, plasma and the bile of channel catfish. Analytes were validated for each of the compounds at 25 and 100 nmol per sample in the various tissues and fluids. The intraday variability (R.S.D.) was less than 13% in all tissues and fluids except for BZ in the liver. Recoveries varied from matrix to matrix for each analyte. The highest recoveries were obtained from plasma which ranged from 82.8-99.8% depending on the concentration. The average recovery of the compounds from tissues was between 50 and 78%, except for liver where the recovery of PABA and BZ was below 30%. Detection was by UV absorbance at 286 nm and the linear range was 2.5-15 nmol 100 ml-1 for all analytes. The method was selective; no interference peaks coeluted with the analytes.