Reduced graphene oxide as an efficient sorbent in microextraction by packed sorbent: Determination of local anesthetics in human plasma and saliva samples utilizing liquid chromatography-tandem mass spectrometry.

Herein, reduced graphene oxide (RGO) has been utilized as an efficient sorbent in microextraction by packed sorbent (MEPS). The combination of MEPS and liquid chromatography-tandem mass spectrometry has been used to develop a method for the extraction and determination of three local anesthetics (i.e. lidocaine, prilocaine, and ropivacaine) in human plasma and saliva samples. The results showed that the utilization of RGO in MEPS could minimize the matrix effect so that no interfering peaks at the retention times of the analytes or internal standard was observed. The high extraction efficiency of this method was approved by mean recoveries of 97.26-106.83% and 95.21-105.83% for the studied analytes in plasma and saliva samples, respectively. Intra- and inter-day accuracies and precisions for all analytes were in good accordance with the international regulations. The accuracy values (as percentage deviation from the nominal value) of the quality control samples were between -2.1 to 13.9 for lidocaine, -4.2 to 11.0 for prilocaine and between -4.5 to -2.4 for ropivacaine in plasma samples while the values were ranged from -4.6 to 1.6 for lidocaine, from -4.2 to 15.5 for prilocaine and from -3.3 to -2.3 for ropivacaine in human saliva samples. Lower and upper limit of quantification (LLOQ, ULOQ) were set at 5 and 2000 nmol L-1 for all of the studied drugs. The correlation coefficients values were ≥0.995. The limit of detection values were obtained 4 nmol L-1 for lidocaine and prilocaine, and 2 nmol L-1 for ropivacaine.

Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human In Vitro Systems.

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5-10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

Coating solid dispersions on microneedles via a molten dip-coating method: development and in vitro evaluation for transdermal delivery of a water-insoluble drug.

This study demonstrates for the first time the ability to coat solid dispersions on microneedles as a means to deliver water-insoluble drugs through the skin. Polyethylene glycol (PEG) was selected as the hydrophilic matrix, and lidocaine base was selected as the model hydrophobic drug to create the solid dispersion. First, thermal characterization and viscosity measurements of the PEG-lidocaine mixture at different mass fractions were performed. The results show that lidocaine can remain stable at temperatures up to ∼130°C and that viscosity of the PEG-lidocaine molten solution increases as the mass fraction of lidocaine decreases. Differential scanning calorimetry demonstrated that at lidocaine mass fraction less than or equal to 50%, lidocaine is well dispersed in the PEG-lidocaine mixture. Uniform coatings were obtained on microneedle surfaces. In vitro dissolution studies in porcine skin showed that microneedles coated with PEG-lidocaine dispersions resulted in significantly higher delivery of lidocaine in just 3 min compared with 1 h topical application of 0.15 g EMLA®, a commercial lidocaine-prilocaine cream. In conclusion, the molten coating process we introduce here offers a practical approach to coat water-insoluble drugs on microneedles for transdermal delivery.

Development of prilocaine gels for enhanced local anesthetic action.

Prilocaine, one of local anesthetics, has been used for regional pain relief. When applied as an ointments or creams, it is hard to expect their effects to last for long time, because they are easily removed by wetting, movement and contacting. For more comfortable and better application, we developed a prilocaine gel system using a bioadhesive polymer, carboxymethyl cellulose (CMC). For suitable bioadhesion, the bioadhesive force of various polymers was tested using an auto-peeling tester. The bioadhesive force of various types of CMC such as 100MC, 150MC and 300MC, was 0.0264, 0.0461 and 0.0824 N, at 1.5% concentration, respectively. The CMC-300MC gels showed the most suitable bioadhesive forces. The effect of drug concentration on drug release was studied from the prepared 1.5% CMC gels using a synthetic cellulose membrane at 37 ± 0.5°C. As the concentration of drug increased, the drug release increased. The effects of temperature on drug release from the 1.0% prilocaine gels were evaluated at 27, 32, 37 and 42°C. As the temperature of the drug gels increased, drug release increased. The enhancing effects of penetration enhancers such as pyrrolidones, non-ionic surfactants, fatty acids and propylene glycol derivatives were studied. Among the enhancers used, polyoxyethylene 2-oleyl ether was superior. The anesthetic effects were studied by a tail flick analgesic meter. In the rat tailflick test, 1.0% prilocaine gels containing polyoxyethylene 2-oleyl ether showed the most prolonged local analgesic effects. The results support the view that prilocaine gels with enhanced local anesthetic action could be developed using CMC bioadhesive polymer.

Dynamic in-situ eutectic formation for topical drug delivery.

OBJECTIVES: The relationship between the solution-state chemistry of eutectic systems and their transmembrane transport characteristics is difficult to define as these mixtures are sensitive to delivery vehicle-induced penetration enhancement. Through in-situ formation of a molten eutectic mixture using highly evaporative sprays this study aimed to gain an understanding of solution-state thermodynamic and chemical properties of eutectic combinations pertinent to transmembrane transport in the absence of a delivery vehicle. METHODS: In-situ molten lidocaine-prilocaine eutectics were formed using a hydroflouroalkane (HFA) propellant. Transport through silicone membranes and human skin in upright Franz diffusion cells was determined using in-house manufactured creams as controls. KEY FINDINGS: The application of the two drugs in an HFA spray produced a molten oil even when the melting point of the drug mixture was above the experimental temperature at the membrane surface. In the absence of vehicle effects, molecule presentation to the membrane interface was most effective using a lidocaine-rich mixture of 0.7% w/w lidocaine:prilocaine - 1985.06 ± 128.87 µg/h/cm(2). CONCLUSIONS: There appeared to be no link between melting point and transmembrane transport of lidocaine:prilocaine from a eutectic mixture. The rate of drug presentation to the membrane interface, which was highest in drug-rich, high-activity molten eutectic mixtures, was the driver for transmembrane transport in the absence of significant barrier interactions.

A fundamental investigation into the effects of eutectic formation on transmembrane transport.

Eutectic systems enhance the permeation of therapeutic agents across biological barriers, but the mechanism by which this occurs has not previously been elucidated. Using human skin it has proven difficult to isolate the fundamental effects of eutectic formation on molecule diffusion and partition from those that arise as a consequence of the simultaneous application of two agents. The aim of this work was to employ a model hydrophobic membrane to understand the fundamental permeation characteristics of two agents when applied as a eutectic mixture. Lidocaine and prilocaine were selected as model agents and infinite-dose permeation studies were carried out using pre-calibrated Franz diffusion cells with two thicknesses of silicone membrane. Membrane solubility was determined by HCl solution extraction and the membrane diffusion coefficients were calculated from the permeation lag-times. The maximum permeation enhancement was achieved using a eutectic mixture at a 0.7:0.3 prilocaine/lidocaine ratio. A higher solubility of both agents in silicone membrane, enhanced diffusivity of prilocaine and superior release of both drugs, all contributed to produce enhanced permeation from the eutectic mixtures. Deconvolution of the transmembrane transport process suggests that the eutectic enhancement phenomena is a consequence of more favorable permeation characteristics of the two molecules in the absence of a formulation vehicle which competes in the transport process.

Hemoglobin adducts of the human bladder carcinogen o-toluidine after treatment with the local anesthetic prilocaine.

Prilocaine, a widely used local anesthetic, is metabolized to o-toluidine which is classified as human carcinogen. We aimed to assess the impact of prilocaine-treatment on hemoglobin adducts from o-toluidine. Blood samples were obtained before and 24h after receiving prilocaine local anesthesia (Xylonest, 100mg) from 20 head and neck surgery patients and 6 healthy volunteers. Hemoglobin adducts of o-toluidine and 4-aminobiphenyl were determined by gas chromatography/mass spectrometry. Hemoglobin adducts of o-toluidine were significantly increased 24h after 100mg prilocaine-treatment by 21.6+/-12.8ng/g hemoglobin (mean+/-S.D., N=26; P<0.0001). This corresponds to a 6-360-fold increase of o-toluidine adduct levels in 25 patients from 0.54+/-0.95ng/g before treatment to 22.0+/-13.2ng/g 24h after surgery (mean+/-S.D.). Because of an extremely high background level the increase was only 1.6-fold in one patient (40.9ng/g before and 64.4ng/g 24h after prilocaine injection). Current smoking had no influence on background values and on the increase of o-toluidine adducts. No treatment-related differences were seen in mean hemoglobin adduct levels of 4-aminobiphenyl which were significantly higher in smokers, 0.149+/-0.096ng/g (mean+/-S.D., N=8) as compared to nonsmokers 0.036+/-0.035ng/g (mean+/-S.D., N=16; P<0.01). In conclusion, prilocaine anesthesia leads to a massive increase of hemoglobin adducts of the carcinogenic arylamine o-toluidine. This implies a carcinogenic risk which should be taken into account in preventive hazard minimization.

Protein binding of prilocaine in human plasma: influence of concentration, pH and temperature.

Protein binding of prilocaine was investigated in vitro under various conditions of changing pH, temperature and total plasma concentration by means of HPLC with UV-detection and ultrafiltration. Whereas changes in temperature (25 degrees C-40 degrees C) and pH (pH 5-pH 10) influenced protein binding markedly, rising plasma concentrations up to 16 micrograms/ml did not affect plasma protein binding significantly. This may be a possible explanation for clinical evidence of low toxicity associated with the use of prilocaine. Discussions concerning protein binding of local anaesthetics should always be based on defined ambient conditions (pH, temperature, concentration).

Prilocaine in arthroscopy: clinical pharmacokinetics and rational use.

Prilocaine pharmacokinetics were determined in 60 patients receiving the drug by two different routes of administration (intra-articular and subcutaneous) during arthroscopy under local anesthesia with controlled pressure irrigation. Resorption of prilocaine by subcutaneous tissues was slow and did not lead to high serum levels. On the contrary, prilocaine resorption by the synovium was fast and induced a sharp serum peak (265.8 +/- 163.5 ng/ml) in the hour after the end of the examination. The drug was completely eliminated from the blood after 24 hours, as the prilocaine t1/2 is about 5 hours. The first procedure was perfected to reduce the risk of methemoglobinemia, which occurred in four of 105 patients. Applied pressure was lowered to 100 mm Hg to prevent the escape of anesthetic solution into the soft tissue of the leg, the prilocaine concentration was reduced to 1 gm/L, and the arthroscope was only set up after a delay to allow the intra-articular anesthetic effect of prilocaine to become established. So far, 200 arthroscopies have been performed with this improved protocol without any problem.

Pharmacokinetic and clinical pharmacological studies with mepivacaine and prilocaine.

The tolerance and pharmacokinetic properties of mepivacaine and prilocaine were compared following i.v. infusion of 250 mg (0.88 and 0.97 mmol respectively) of each drug in five healthy volunteers. Side-effects were minor and occurred in only two subjects during the infusion of mepivacaine. Plasma concentrations of mepivacaine were greater in each subject than the corresponding values for prilocaine. The elimination half-life of mepivacaine was generally longer than that for prilocaine, whereas the total body clearance of prilocaine was consistently greater than the corresponding value for mepivacaine. For each subject the clearance of prilocaine substantially exceeded normal heptic blood flow and therefore an extra-hepatic site of metabolism of prilocaine has been postulated.

[Diaplacentar transfer of local anaesthetics (author's transl)]. / Diaplazentarer Transfer von Lokalanästhetika.

A very rapid resorption of the amide-type local anaesthetic agents most commonly used in obstetrics results in detectable plasma levels in mother and fetus within 5 to 10 minutes, respectively, after peridural anaesthesia. The various local anaesthetics differ from each other as far as their protein-binding capacity and the resulting ratios of umbilical vein to maternal blood levels are concerned. The feto-maternal ratio is inversely correlated to the degree of protein-binding. Both, the protein-binding capacity and the feto-maternal ratio do not indicate the total amount of the anaesthetic transferred across the placenta. In contrast to measurements of the plasma concentrations only the measurement of the anaesthetic in the fetal tissue would give an exact information about the fetal uptake of local anaesthetics. Several observations hint at the possibility that there are interactions between local anaesthetics and other drugs: A competitive displacement of local anaesthetics from their protein binding sites resulting in an increase in the unbound fraction is suggested. Since the feto-maternal equilibration exists only for the unbound fraction interactions of local anaesthetics with simultaneously administered drugs should be taken into consideration.