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.

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.

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.

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.

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.

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.

Molecular dynamics simulation of the partitioning of benzocaine and phenytoin into a lipid bilayer.

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.

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.

Effect of the interfacial tension and ionic strength on the thermodynamic barrier associated to the benzocaine insertion into a cell membrane.

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.

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.

A novel in vitro percutaneous penetration model: evaluation of barrier properties with p-aminobenzoic acid and two of its derivatives.

PURPOSE: The objective of this study was to evaluate the utility of a stratum corneum substitute (SCS) as a novel in vitro percutaneous penetration model. The SCS consists of synthetic stratum corneum (SC) lipids (cholesterol, free fatty acids, and specific ceramides) applied onto a porous substrate. The composition, organization, and orientation of lipids in the SCS bear high resemblance to that of the intercellular barrier lipids in SC. METHODS: The barrier integrity of the SCS was evaluated by means of passive diffusion studies, using three model compounds with different lipophilicities. The effects of lipid layer thickness, permeant lipophilicity, and altered lipid composition on the barrier properties were investigated, using isolated human SC as a control sample. RESULTS: For all three model compounds, the permeability characteristics of the SCS with a 12-mum-thick lipid layer closely resemble those of human SC. Modification of the lipid composition, generating an SCS that lacks the characteristic long periodicity phase as present in SC, was accompanied by a 2-fold increased permeability. CONCLUSIONS: The SCS offers an attractive tool to predict solute permeation through human skin. Moreover, as its lipid composition can be modified, they may also serve as a suitable screening model for diseased skin.

Inhibition of the A-type K+ channels of dorsal root ganglion neurons by the long-duration anesthetic butamben.

n-Butyl-p-aminobenzoate (BAB; butamben) is a long-duration anesthetic used for the treatment of chronic pain. Epidural administration of BAB is thought to reduce the electrical excitability of dorsal root nociceptor fibers by inhibiting voltage-gated ion channels. To further investigate this mechanism, we examined the effects of BAB on the potassium currents of acutely dissociated neurons from the rat dorsal root ganglion (DRG). These neurons express a rapidly inactivating A-type K(+) current (I(A)) that is resistant to tetraethylammonium (20 mM) but inhibited by 4-aminopyridine (5 mM). At low concentrations, BAB (< or =1 microM) selectively inhibited the I(A) component of DRG K(+) current. The voltage dependence of activation and inactivation, kinetics of recovery from inactivation, and the pharmacology of the DRG I(A) were similar to those of the Kv4 family of K(+) channels. Reverse transcription-polymerase chain reaction was used to establish that the messages encoding for all three of the mammalian Kv4 channel subunits (Kv4.1-Kv4.3) were present in the rat DRG. BAB produced a high-affinity, partial inhibition of heterologously expressed Kv4.2 channels (K(D) = 59 nM) but did not alter the kinetics or voltage sensitivity of gating. Substituting polar threonines for conserved hydrophobic residues of the S6 segment weakened BAB binding but did not alter the voltage-dependent gating of the Kv4.2 channel. At physiological pH, BAB is uncharged, suggesting that hydrophobic interactions may contribute to drug binding. The data support a mechanism in which BAB binds near the narrow cytoplasmic entrance of Kv4 channels and inhibits current by a pore blocking mechanism.

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.

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.

Thermodynamics of partitioning of benzocaine in some organic solvent/buffer and liposome systems.

The thermodynamics of partitioning of benzocaine (BZC) were studied in octanol/buffer (ROH/W), isopropyl myristate/buffer (IPM/W), cyclohexane/buffer (CH/W), and dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC) liposome systems. In all cases the partition coefficients were greater than unity; therefore the free energies of transfer were negative, that is, the processes of transfer of BZC from aqueous media to organic systems were spontaneous. The partition coefficients were approximately three-fold higher in DMPC liposomes compared with the ROH/W system in the 30 degrees -40 degrees C temperature range. The enthalpies of transfer from aqueous media to ROH and IPM were negative, but positive for CH, while this property was negative for DMPC liposomes and positive for DPPC liposomes. The entropies of transfer were positive in almost all cases, except for DMPC. The results presented here confirm the lipophilic nature of BZC.

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 temperature on the elimination of benzocaine and acetylated benzocaine residues from the edible fillet of rainbow trout (Oncorhynchus mykiss).

The effect of temperature (7 degrees C and 16 degrees C) on the extent of accumulation and the elimination of benzocaine (BNZ) and its metabolite, acetylated benzocaine (AcBNZ), in the fillet tissue of rainbow trout was investigated. Residues were measured after bath exposure to an anesthetizing concentration of benzocaine (30 mg/l for 5 min) followed by a maintenance concentration (15 mg/l for 30 min). Immediately after exposure, the BNZ concentration in fillet tissue was approximately 27 micrograms/g at both temperatures; AcBNZ was 0.3 microgram/g at 7 degrees C and 0.6 microgram/g at 16 degrees C. The rates for elimination (alpha and beta) of BNZ and AcBNZ were not significantly different between the two temperatures. Terminal half-lives of elimination for BNZ were 1.62 h at 7 degrees C and 1.63 h at 16 degrees C; half-lives for AcBNZ were 2.36 h at 7 degrees C and 2.77 h at 16 degrees C.

Preparation of microcapsules for skin allergy testing by the solvent evaporation process.

Polyurethanes and polyvinyl alcohol modified by stearyl isocyanate are used as a matrix for microparticles made by a solvent evaporation process to encapsulate allergenic molecules, with petrolatum used as a neutral vehicle. The encapsulation yields, depending on the agent to be encapsulated, vary from 22 to 45%.

Lysine point mutations in Na+ channel D4-S6 reduce inactivated channel block by local anesthetics.

Voltage-gated Na+ channels are a primary target for local anesthetics (LAs). Open or inactivated Na+ channels usually have a severalfold higher affinity for LAs than do resting channels. Hille's modulated receptor hypothesis attributed the changes in LA affinity to state-dependent alterations in the conformation of the LA receptor. We expressed wild-type and mutant rat skeletal muscle (mu1) Na+ channels in human embryonic kidney cells to investigate the state-dependent modulation of LA receptor affinity. As an alternative approach to using alanine for point mutation, we substituted lysine (a hydrophilic residue) for native residues in the putative LA receptor located in D4-S6 of the mu1 Na+ channel. Lysine mutation at Y1586 did not alter resting channel affinity for cocaine but did reduce resting affinity at F1579K and N1584K by 2- and 3-fold, respectively. Compared with mu1, resting benzocaine block did not change at F1579K, decreased at N1584K, and increased at Y1586K. These effects on resting block could largely be accounted for by either steric/charge interference or cation-pi electron interactions between particular moieties on the LA and lysine. Surprisingly, lysine substitution at these residues allowed the channels to undergo steady state fast inactivation yet reduced inactivated channel block by cocaine by up to 27-fold and reduced the benzocaine-induced leftward shift in the h(infinity) curve by up to 22 mV. Our data suggest that transitions in channel state indeed invoke conformational changes in the LA receptor and that lysine mutations in the LA receptor region alter such conformational changes during the transition to the inactivated state.