Preparation and evaluation of novel microemulsion-based hydrogels for dermal delivery of benzocaine.

The purpose of the current research was to prepare and evaluate the potential use of microemulsion-based hydrogel (MBH) formulations for dermal delivery of benzocaine (BZN). The pseudoternary-phase diagrams were constructed for various microemulsions composed of isopropyl myristate (IPM) as oil phase, Span 20, Tween 20, Tween 80, cremophor EL and cremophor RH40 as surfactants, ethanol as cosurfactant and distilled water as aqueous phase. Finally, concentration of BZN in microemulsions was 2% (w/w). The physicochemical properties, such as conductivity, viscosity, pH, droplet size, polydispersity index and zeta potential of microemulsions, were measured. Carbopol 940 was used to convert BZN-loaded microemulsions into gel form without affecting their structure. Furthermore, excised rat abdominal skin was used to compare permeation and penetration properties of BZN loaded M3 and M3BHs with BZN solution. According to ex vivo study results, BZN-loaded M3BH1 showed highest flux values and high release rate values, and furthermore, this gel formulation had low surfactant content. Finally, in order to learn the localization of formulations within the dermal penetration, formulations and BZN solution were labeled with red oil O and subjected to fluorescence observation. In conclusion, BZN-loaded MBHs could be offered as a promising strategy for dermal drug delivery.

Rationalization of reduced penetration of drugs through ceramide gel phase membrane.

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.

Benzocaine polymorphism: pressure-temperature phase diagram involving forms II and III.

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.

Development, characterization, and in vitro and in vivo evaluation of benzocaine- and lidocaine-loaded nanostructrured lipid carriers.

The present study concerns the in vitro and in vivo evaluation of benzocaine (BENZO) and lidocaine (LIDO) topical delivery from nanostructured lipid carriers (NLCs). Morphology and dimensional distribution of NLCs have been, respectively, characterized by differential scanning calorimetry (DSC) and photon correlation spectroscopy. The release pattern of BENZO and LIDO from NLCs was evaluated in vitro determining drug percutaneous absorption through excised human skin. Radiant heat tail-flick test was carried out in mice to determine the antinociceptive effect of BENZO and LIDO from NLC. DSC studies revealed that the inner oil phase of NLC plays a significant role in stabilizing the particle architecture and increasing the drug solubility. In vitro evidences show that BENZO and LIDO, when incorporated in viscosized NLC dispersions, exhibited a lower flux with respect to formulations containing the free drugs in the aqueous phase. In vivo study enabled to demonstrate that BENZO and LIDO can be released in a prolonged fashion when incorporated into lipid carriers. The results obtained pointed out NLC capability to act as an effective drug reservoir, thus prolonging the anesthetic effect of BENZO and LIDO.

Bioadhesive films containing benzocaine: correlation between in vitro permeation and in vivo local anesthetic effect.

PURPOSE: The aim of this work was to develop anesthetic bioadhesive films containing benzocaine and study their in vitro skin permeation and in vivo performance, in comparison with commercial formulations. METHODS: Films containing 3% and 5% w/w of benzocaine were prepared and characterized by weight, drug content, thickness and morphology. In vitro permeation assays were performed in vertical diffusion cells using full-thickness pig ear skin as barrier. Intensity and duration of analgesia were evaluated in rats by tail-flick test, and skin histological analysis was carried out. RESULTS: Tail-flick test showed that the duration of benzocaine-induced analgesia was significantly prolonged with the films compared to commercial creams, in agreement with the higher in vitro permeation. Histological analysis of the rat tail skin did not reveal morphological tissue changes nor cell infiltration signs after application of the commercial creams or films. CONCLUSIONS: Results from our study indicate that the films developed in this work can be considered as innovative dermal/transdermal therapeutic systems for benzocaine local delivery.

Fish skin as a model membrane: structure and characteristics.

OBJECTIVES: Synthetic and cell-based membranes are frequently used during drug formulation development for the assessment of drug availability. However, most of the currently used membranes do not mimic mucosal membranes well, especially the aqueous mucous layer of the membranes. In this study we evaluated catfish (Anarichas lupus L) skin as a model membrane. METHOD: Permeation of hydrocortisone, lidocaine hydrochloride, benzocaine, diethylstilbestrol, naproxen, picric acid and sodium nitrate through skin from a freshly caught catfish was determined in Franz diffusion cells. KEY FINDINGS: Both lipophilic and hydrophilic molecules permeate through catfish skin via hydrated channels or aqueous pores. No correlation was observed between the octanol/water partition coefficient of the permeating molecules and their permeability coefficient through the skin. Permeation through catfish skin was found to be diffusion controlled. CONCLUSIONS: The results suggest that permeation through the fish skin proceeds via a diffusion-controlled process, a process that is similar to drug permeation through the aqueous mucous layer of a mucosal membrane. In addition, the fish skin, with its collagen matrix structure, appears to possess similar properties to the eye sclera.

Timely drug delivery from controlled-release devices: dynamic analysis and novel design concepts.

Design tools are provided to assist in the development of drug-release devices that can control the time to establish a steady-state flux in addition to a desired delivery rate. In this contribution, the primary focus is placed on passive, heat-aided, and electrically assisted transport through planar membranes. Approximate analytical methods, that describe process dynamics, were applied to appropriate mathematical models to derive relationships between the system properties (e.g., diffusion coefficient, temperature and voltage potential) and the flux response time. Three case studies were investigated to illustrate the theoretical results. A steady-state benzocaine flux through ethylene-vinyl acetate membranes was achieved in 40min as predicted by a first-moment time constant approach. It took 5h and 45min to reach a constant delivery rate of amitriptyline HCl across human skin for a donor cell concentration of 0.032M and an electric current of 0.4mA/cm(2). Based on the upper limit and range estimates of the first eigenvalue, the onset of steady-state flux occurred between 3.4 and 3.5min when the donor and receiver cells were maintained at 47 and 37 degrees C, respectively. These predictions were confirmed by simulation, experimental evidence and graphical examination of drug-release data.

Atomic determinants of state-dependent block of sodium channels by charged local anesthetics and benzocaine.

Molecular modeling predicts that a local anesthetic (LA) lidocaine binds to the resting and open Na(v)1.5 in different modes, interacting with LA-sensing residues known from experiments. Besides the major pathway via the open activation gate, LAs can reach the inner pore via a "sidewalk" between D3S6, D4S6, and D3P. The ammonium group of a cationic LA binds in the focus of the pore-helices macrodipoles, which also stabilize a Na(+) ion chelated by two benzocaine molecules. The LA's cationic group and a Na(+) ion in the selectivity filter repel each other suggesting that the Na(+) depletion upon slow inactivation would stabilize a LA, while a LA would stabilize slow-inactivated states.

Preparation and evaluation of bioadhesive benzocaine gels for enhanced local anesthetic effects.

This study was performed to develop new enhanced anesthetic benzocaine gels with a suitable bioadhesive property for local anesthetic effects. As the concentration of benzocaine in the HPMC gels increased up to 15%, the permeation of drug increased, thereafter slightly increased. The activation energy of drug permeation was 11.29 kcal/mol. Bioadhesive forces were also measured. The permeation rate of drug through the skin was studied using various enhancers, such as glycols, non-ionic surfactants or fatty acids. Among the enhancers used, diethylene glycol showed the most enhancing effects. Analgesic activity was examined using a tail-flick analgesimeter. According to the rat tail-flick test, the value of AUEC (0 - 360min) of 15% benzocaine gels containing diethylene glycol was 4662 +/- 200 s min, while that of gels without diethylene glycol was 3353 +/- 132 s min, showing about 1.39-fold increase in analgesic activity. Fifteen percentage of benzocaine gels containing diethylene glycol showed the most enhanced, prolonged analgesic effects, showing the maximum anesthetic effects at 240 min, while the gels without diethylene glycol showed maximum effect at 180 min.

Diffusion of n-butyl-p-aminobenzoate (BAB), lidocaine and bupivacaine through the human dura-arachnoid mater in vitro.

INTRODUCTION: Epidural administration of a suspension of n-butyl-p-aminobenzoate (BAB) to humans resulted in a selective, ultra-long lasting sensory block without motor function impairment. The absence of motor block was attributed to 'spatial' confinement of active concentrations of dissolved BAB within the epidural space. This study was designed to investigate the diffusion of BAB through the human dura-arachnoid membrane in vitro relative to lidocaine and bupivacaine and to quantify the influence of the composition of the suspension formulation on this flux. MATERIALS AND METHODS: Human dura-arachnoid specimens were mounted between the donor and the receiver compartment of a diffusion cell. Five concentrations of BAB, lidocaine and bupivacaine in phosphate-buffered saline, pH 7.4, were added to the donor compartment and the increase of the concentration of the agent in time in the receiver compartment was measured by automated UV-spectrometry. Fluxes and permeabilities were calculated. The influence of pH, polysorbate 80 (PS 80) and polyethylene glycol 3350 (PEG 3350) on the flux of BAB in solution and in suspension formulations were analyzed. RESULTS: The flux of both lidocaine and bupivacaine at pH 4 was considerably smaller than at pH 7.4. Permeabilities decreased in the order bupivacaine>lidocaine&z.Gt;BAB and at the level T12>T1. PS 80 at concentrations exceeding 0.025 mg/ml and PEG 3350 decreased the flux of BAB from BAB-solutions. Used in the preparation of the suspension, PS 80 and PEG 3350 did significantly reduce the permeability. DISCUSSION: The results of this study are consistent with the hypothesis that the selective action of epidurally applied BAB suspension can be attributed to the spatial confinement of active BAB-concentrations within the epidural space. Additives used in the preparation of the aqueous suspension formulation may substantially influence the local pharmacokinetics and by that the pharmacodynamic effects.

Mathematical modelling of drug transport in emulsion systems.

Two mathematical models for the prediction of drug transport in triphasic (oil, water and micellar) emulsion systems as a function of micellar concentration have been developed and these models were evaluated by comparing experimental and simulated data. Fick's first law was used to derive a transport model for hydrophilic drugs, assuming that the oil/water (o/w) partitioning process was fast compared with membrane transport and therefore drug transport was limited by the membrane. Consecutive rate equations were used to model transport of hydrophobic drugs in emulsion systems assuming that the o/w interface acts as a barrier to drug transport. Benzoic acid and phenol were selected as hydrophilic model drugs. Phenylazoaniline and benzocaine were selected as hydrophobic model drugs. Transport studies at pH 3.0 and 7.0 were conducted using side-by-side diffusion cells. According to the hydrophilic model, an increase in micellar concentration is expected to decrease drug transport rates. The effective permeability coefficients (Peff) of drugs were calculated using an equation relating Peff and the total apparent volume of drug distribution (determined experimentally using drug/membrane permeability and partition coefficient values). The hydrophobic model was fitted to the experimental data for the cumulative amount of model drug in the receiver cells using a weighted least-squares estimation program (PCNONLIN). The oil/continuous phase partitioning rates (k1) and the membrane transport rates (k2) were estimated. The goodness of fit was assessed from the correlation coefficients of plots of predicted versus experimental data. The predicted data were consistent with the experimental data for both the hydrophilic and hydrophobic models.

Effect of temperature on the pharmacokinetics of benzocaine in rainbow trout (Oncorhynchus mykiss) after bath exposures.

The pharmacokinetics of benzocaine during bath exposures at 1 mg/L were determined in rainbow trout acclimated at 6 degrees C, 12 degrees C or 18 degrees C for at least 1 month. Individual fish were exposed to benzocaine in a recirculating system for 4 h and pharmacokinetic parameters were estimated in a unique manner from the concentration of benzocaine in the bath water vs. time curve. Elimination from plasma was also determined after the 4 h exposure. The uptake clearance and metabolic clearance increased with increased acclimatization temperatures (uptake clearance 581 +/- 179 mL/min/kg at 6 degrees C and 1154 +/- 447 mL/min/kg at 18 degrees C; metabolic clearance 15.2 +/- 4.1 mL/min/kg at 6 degrees C and 22.3 +/- 4.2 mL/min/kg at 18 degrees C). The apparent volume of distribution had a trend for increasing with temperature that was not significant at the 5% level (2369 +/- 678 mL/kg at 6 degrees C to 3260 +/- 1182 mL/kg at 18 degrees C). The elimination half-life of benzocaine in plasma was variable and did not differ significantly with temperature (60.8 +/- 30.3 min at 6 degrees C to 35.9 +/- 13.0 min at 12 degrees C). Elimination of benzocaine from rainbow trout is relatively rapid and even more rapid at higher acclimatization temperatures based on calculated metabolic clearances and measured plasma concentrations, but was not evident by measurement of terminal plasma half-lifes.

Maintaining a near zero-order drug delivery from minidose reservoirs: simultaneous drug diffusion and binary vehicle evaporation.

The purpose of this study was to develop a new design for transdermal system that provides a nearly constant drug delivery rate for the desired period of time in the absence of a large excess of drug in the donor reservoir. Simultaneous drug diffusion and aqueous binary vehicle evaporation has been investigated as a means of maintaining constant drug delivery from minidose reservoirs. Benzocaine was used as the model drug and water-ethanol mixtures as the binary vehicles. Benzocaine is much more soluble in ethanol than in water. Simultaneous diffusion-evaporation experiments were conducted using the water-ethanol mixture saturated with benzocaine as the minidose-reservoir. The reservoir vehicle was allowed to evaporate through a permselective membrane so as to maintain drug saturation in the vehicle even though the drug mass in the donor reservoir was constantly decreasing. Saturation activity of drug is maintained during donor depletion of drug by the selective loss of alcohol from the donor solvent, which concomitantly lowers the solubility. The superiority of the evaporable binary vehicle over a conventional sealed system was demonstrated by achieving near zero-order drug delivery with minimum drug remaining. A theoretical model was developed to predict drug flux, drug amount delivered, reservoir solvent volume and composition, and drug solubility during the two simultaneous processes of drug release and preferential solvent evaporation. Experimental data are in good agreement with theoretical calculations.

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.

Topical liposomal system for localized and controlled drug delivery.

A liposomal reservoir system bearing the local anesthetic, benzocaine, was developed for controlled and localized delivery via topical route. The liposomal suspension was incorporated into an ointment and gel base. The developed systems were studied for various physical and kinetic attributes in vitro. The systems delivered the drug at a controlled rate over 24 h, whereas plain drug ointment showed a rapidly decreased release rate over 24 h, with more than 92% released. The drug delivery across human cadaver skin following liposomal ointment application was noted to be considerably slow. The in vivo study revealed a longer duration of action in the case of liposomal formulations. An effort was made to study the effect of ultrasound as a reversible means to effect pulsatile delivery of the drug from the liposomal depot. The work proved the potential of liposomes as a slow release vehicle which follows apparent zero order kinetics and its use in modulating drug input via skin.

Metabolism of benzocaine during percutaneous absorption in the hairless guinea pig: acetylbenzocaine formation and activity.

The effect of dose and enzymatic inhibition on the percutaneous absorption and metabolism of benzocaine was studied in vitro in the hairless guinea pig. At the dose level of 2 micrograms/cm2, benzocaine was rapidly absorbed and extensively metabolized (80%) by acetyltransferase. As the applied dose of benzocaine was increased to 40 and 200 micrograms/cm2, N-acetylation of benzocaine decreased to 44 and 34%, respectively, suggesting saturation of the acetyltransferase system. Total 14C absorption after benzocaine application was not significantly different between control and enzyme-inhibited skin and therefore does not appear to be affected by the extent of benzocaine metabolism during percutaneous penetration. Skin provides a significant first-pass metabolic effect for therapeutic doses of percutaneously absorbed benzocaine, and the primary metabolite formed, acetylbenzocaine, is biologically active.

Capillary gas chromatographic method for the determination of n-butyl-p-aminobenzoate and lidocaine in plasma samples.

A fast capillary gas chromatographic method with nitrogen-selective detection is described that allows selective and reproducible quantification of n-butyl-p-aminobenzoate (BAB) and lidocaine in plasma. The sampling and sample storage conditions are critical for the quantification of BAB. Diisopropyl fluorophosphate, an organo-phosphorus pesticide, has to be added during sampling to prevent the rapid decomposition of BAB by cholinesterases.

Ester hydrolysis and conjugation reactions in intact skin and skin homogenate, and by liver esterase of rabbits.

1. Procaine, 2-chloroprocaine, ethyl aminobenzoate and methyl salicylate were added at various concentrations to liver esterase, supernatant of skin homogenate, or single-pass perfused ears of rabbits. 2. Vmax of product formation by purified liver esterase correlated with the rank order of the distribution coefficients (n-octanol/buffer) of the substrates and ranged between 11 and 1100 pmol/min per micrograms protein. Km values were between 20 and 50 microM. 3. No correlation was observed when the apparent enzyme kinetics, calculated by non-linear adaptation, were compared with each other after substrate administration to skin, arterial influx, or incubation with skin homogenate. 4. An acid labile conjugate of ethyl 4-aminobenzoate was found, mainly during arterial perfusion and in supernatant of skin homogenate, after administration of ethyl 4-aminobenzoate. 5. Acetamidobenzoic acid was observed in quantities of about 10% of the free 4-aminobenzoic acid during dermal or arterial application of procaine. This metabolite was not found with ethyl 4-aminobenzoate. 6. The results from isolated rabbit ear perfusion differ quantitatively and qualitatively with those obtained from supernatant of skin homogenate or purified liver esterase.

In vitro skin absorption and metabolism of benzoic acid, p-aminobenzoic acid, and benzocaine in the hairless guinea pig.

The percutaneous absorption and metabolism of three structurally related compounds, benzoic acid, p-aminobenzoic acid (PABA), and ethyl aminobenzoate (benzocaine), were determined in vitro through hairless guinea pig skin. Benzocaine was also studied in human skin. Absorption of benzocaine was rapid and similar through both viable and nonviable skin. The absorption of the two acidic compounds, benzoic acid and PABA, was greater through nonviable skin. A small portion (6.9%) of absorbed benzoic acid was conjugated with glycine to form hippuric acid. Although N-acetyl-benzocaine had not been observed as a metabolite of benzocaine when studied by other routes of administration, both PABA and benzocaine were extensively N-acetylated during percutaneous absorption. Thus, the metabolism of these compounds should be considered in an accurate assessment of absorption after topical application.