Molybdenum-Iodine Cluster Loaded Polymeric Nanoparticles Allowing a Coupled Therapeutic Action with Low Side Toxicity for Treatment of Ovarian Cancer.

The ability of cancer cells to develop resistance to anti-cancer drugs, known as multidrug resistance, remains a major cause of tumor recurrence and cancer metastasis. This work explores the double mechanism of toxicity of (D, L-lactide-co-glycolide) acid (PLGA) nanoparticles encapsulating a molybdenum cluster compound, namely Cs2[{Mo6I8}(OOCC2F5)6] (CMIF). Hemocompatibility and biocompatibility assays show the safe potential of CMIF loaded nanoparticles (CNPs) as delivery systems intended for tumor targeting for PDT of ovarian cancer with a slight hemolytic activity and a lack of toxicity up to 50 µM CMIF concentration. Cellular uptake shows a preferential uptake of CNPs in lysosomes, which is not interfering with CMIF activity. The double mechanism of CNPs consists in a production of ROS and a DNA damage activity, from 5 µM and 0.5 µM respectively (CMIF concentration). The cellular death mechanism comprises 80% of necrosis and 20% of direct apoptosis by direct DNA damages. This work confirms CMIF loaded PLGA nanoparticles as an efficient and relevant delivery system for PDT.

From molecules to nanovectors: Current state of the art and applications of photosensitizers in photodynamic therapy.

Photodynamic therapy (PDT) is a concept based on a selective activation by light of drugs called photosensitizers (PS) leading to reactive oxygen species production responsible for cell destruction. Mechanisms of photodynamic reaction and cell photo-destruction following direct or indirect mechanisms will be presented as well as PS classification, from first generation molecules developed in the 1960 s to third generation vectorized PS with improved affinity for tumor cells. Many clinical applications in dermatology, ophthalmology, urology, gastroenterology, gynecology, neurosurgery and pneumology reported encouraging results in human tumor management. However, this interesting technique needs improvements that are currently investigated in the field of PS excitation by the design of new PS intended for two-photon excitation or for X-ray excitation. The former excitation technique is allowing better light penetration and preservation of healthy tissues while the latter is combining PDT and radiotherapy so that external light sources are no longer needed to generate the photodynamic effect. Nanotechnology can also improve the PS to reach the tumor cells by grafting addressing molecule and by increasing its aqueous solubility and consequently its bioavailability by encapsulation in synthetic or biogenic nanovector systems, ensuring good drug protection and targeting. Co-internalization of PS with magnetic nanoparticles in multifunctional vectors or stealth nanoplatforms allows a theranostic anticancer approach. Finally, a new category of inorganic PS will be presented with promising results on cancer cell destruction.

Molybdenum cluster loaded PLGA nanoparticles as efficient tools against epithelial ovarian cancer.

In this study, poly (lactic-co-glycolic) acid nanoparticles loading inorganic molybdenum octahedral cluster were used for photodynamic therapy (PDT) of ovarian cancer. Three cluster compounds, ((C4H9)4N)2[{Mo6Br8}Br6], Cs2[{Mo6Br8}Br6] and Cs2[{Mo6I8}(OOC2F5)6] denoted TMB, CMB and CMIF were studied following their incorporation in nanoparticles by a nanoprecipitation method. All resulting nanoparticles exhibited physico-chemical characteristics such as size and zeta potential compatible with cellular uptake. All cluster compounds tested were shown to produce singlet oxygen in vitro once released from their nanoparticulate system. Confocal images showed an internalisation of cluster loaded nanoparticles (CNPs) in A2780 ovarian cancer cell line, more efficient with CMIF compared to CMB or TMB loaded nanoparticles. In vitro cellular viability studies conducted on A2780 cell line treated with non activated CNPs did not show any sign of toxicity for concentrations up to 15 µM. Following photo-activation, CNPs were able to generate singlet oxygen resulting in a decrease of the cellular viability, compared to non-activated conditions. Nevertheless, no significant differences between IC50 with or without photo-activation were observed with TMB and CMB CNPs while for CMIF loaded nanoparticles, the photo-activation led to a significant decrease of cellular viability compared to the non activated condition and this decrease was independant of the P/C ratio. The strong photo-toxicity obtained for CMIF loaded nanoparticles with a P/C ratio of 2.5, as shown with half maximal inhibitory concentration (IC50) value near 1.8 µM suggests that PLGA nanoparticles seem to be efficient delivery systems intended for tumor management and that CMIF can be further investigated as photosensitizer for PDT of ovarian cancer.

PLGA nanoparticles embedding molybdenum cluster salts: Influence of chemical composition on physico-chemical properties, encapsulation efficiencies, colloidal stabilities and in vitro release.

We present a screening of poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles embedding a series of inorganic molybdenum octahedral clusters intended for photodynamic therapy (PDT) of cancer. Three cluster compounds from 2 cluster units, [{Mo6Br8}Br6]2- and [{Mo6I8}(OOC2F5)6]2- were studied. [{Mo6Br8}Br6]2-cluster units are found in the soluble ternary salt Cs2[{Mo6Br8}Br6] (CMB) prepared by solid state chemistry at high temperature. In solution Cs+ cations are replaced by tetrabutyl ammonium cations (C4H9)4N+) to form the salt ((C4H9)4N)2[{Mo6Br8}Br6] (TBA2). [{Mo6I8}(OOC2F5)6]2- was prepared combining solid state and solution chemistries; it is paired with Cs+ cations to form Cs2[{Mo6I8}(OOC2F5)6] (CMIF). All tested cluster-based salts could efficiently be incorporated in PLGA nanoparticles as seen with encapsulation efficiencies always higher than 60%. Cluster loaded nanoparticles (CNPs) freshly prepared by solvent displacement method showed spherical shapes, zeta potential values between -20 and -47 mV, polydispersity index in the range 0.123-0.167 and sizes in the range 75-150 nm according to the cluster compound and the polymer-to-cluster mass ratio (P/C), suggesting a good cellular uptake. CNPs colloidal stability was maintened for 3 months when they were stored refrigerated and protected from light but the chemical stability was shorter, i.e. 4 weeks, 1 week and 1 day for CMIF, TBA2 and CMB, respectively, CMIF penta-fluoropropionate apical ligands being less rapidly substituted by hydroxyles groups than TBA2 and CMB halogen apical ligands. FT-IR analysis revealed the lack of strong chemical interaction between cluster compounds and polymer within the nanoparticles. An interesting quick cluster in vitro release driven by diffusion outside the nanoparticles porous matrix was observed for all cluster compounds when P/C ratio was ≤2.5 and only a higher P/C ratio not studied in this work (i.e. >5) could significantly affect the release of the encapsulated cluster compound. Photophysical properties of cluster compounds were preserved following PLGA incorporation. This work presents PLGA nanoparticles as a stable and efficient cluster compound delivery systems for further in vitro and vivo evaluations in cancer models.

Molybdenum cluster loaded PLGA nanoparticles: An innovative theranostic approach for the treatment of ovarian cancer.

We evaluate poly (d,l-lactide-co-glycolide) (PLGA) nanoparticles embedding inorganic molybdenum octahedral cluster for photodynamic therapy of cancer (PDT). Tetrabutyl ammonium salt of Mo6Br14 cluster unit, (TBA)2Mo6Br14, presents promising photosensitization activity in the destruction of targeted cancer cells. Stable cluster loaded nanoparticles (CNPs) were prepared by solvent displacement method showing spherical shapes, zeta potential values around -30 mV, polydispersity index lower than 0.2 and sizes around 100 nm. FT-IR and DSC analysis revealed the lack of strong chemical interaction between the cluster and the polymer within the nanoparticles. In vitro release study showed that (TBA)2Mo6Br14 was totally dissolved in 20 min, while CNPs were able to control the release of encapsulated cluster. In vitro cellular viability studies conducted on A2780 ovarian cancer cell line treated up to 72 h with cluster or CNPs did not show any sign of toxicity in concentrations up to 20 µg/ml. This concentration was selected for photo-activation test on A2780 cells and CNPs were able to generate oxygen singlet resulting in a decrease of the cellular viability up to 50%, respectively compared to non-activated conditions. This work presents (TBA)2Mo6Br14 as a novel photosensitizer for PDT and suggests PLGA nanoparticles as an efficient delivery system intended for tumor targeting.

Formulation and stability study of a pediatric 2% phenylephrine hydrochloride eye drop solution.

INTRODUCTION: We present formulation and stability evaluation of a 2% (w/v) phenylephrine hydrochloride biocompatible eye drop solution, routinely prepared in hospital pharmacy under aseptic conditions, for retinal examination of neonates and premature infants. MATERIALS AND METHODS: Eye drop solution was formulated by dissolution of phenylephrine hydrochloride and disodium hydrogen phosphate as buffering agent in sterile water for injection and sodium chloride for injection as isotonic agent. The previous solution was sterile filtered through under aseptic conditions, in an iso class 5 air quality clean room under horizontal laminar airflow hood. Physical stability (visual inspection, osmolality measurements), chemical stability (pH measurement, phenylephrine assay by liquid chromatography coupled with an ultra-high resolution and accurate mass) and sterility evaluation of phenylephrine eye drop solution stored at ambient temperature were studied during 60 days. RESULTS AND DISCUSSION: The formulated eye drop solution had a pH of 6.90±0.05 and an osmolality of 285±2 mOsm/kg. Throughout the 60 days study the solutions remained clear without any precipitation or color modification, sterility was maintained, pH and osmolality were not significantly modified and no significant loss of product was detected using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument suggesting the lack of degradation. CONCLUSION: These results indicate that 2% phenylephrine hydrochloride eye drop solutions were physically, chemically and microbiologically stable for at least 60 days when stored in type I amber glass vials at room temperature, allowing the compounding of higher batch sizes.

Effect of epinephrine on epidural, intrathecal, and plasma pharmacokinetics of ropivacaine and bupivacaine in sheep.

BACKGROUND: Local vasoconstriction induced by epinephrine added to epidural local anaesthetics has been shown to improve their quality and duration of action in several clinical reports. There are several assumptions on the mechanisms. This study was designed to evaluate the influence of epinephrine on transmeningeal uptake of epidurally administered ropivacaine and bupivacaine by measuring local anaesthetic concentrations in the epidural and intrathecal spaces and in plasma. METHODS: Ropivacaine (50 mg) and bupivacaine (30 mg) were administered epidurally in sheep with and without epinephrine (75 microg). A microdialysis technique was used to simultaneously measure epidural and intrathecal drug concentrations. Resulting dialysate and plasma concentrations were used to calculate pharmacokinetic parameters for ropivacaine and bupivacaine. RESULTS: Co-administration of epinephrine decreased epidural clearance for ropivacaine [0.6 (sd 0.1) vs 0.4 (0.1) ml min(-1)] but not significantly for bupivacaine [1.2 (0.4) vs 0.8 (0.3) ml min(-1)]. The resultant increase in epidural area under the concentration-time curves (31% for ropivacaine and 52% for bupivacaine) was also observed in the intrathecal space (21% increase for ropivacaine and 37% for bupivacaine). There was no significant influence of epinephrine on ropivacaine plasma pharmacokinetics. Plasma Cmax for bupivacaine was decreased. CONCLUSIONS: These results show that epinephrine decreases the clearance and distribution processes involved in epidural disposition of ropivacaine and bupivacaine, leading to an increased uptake into the intrathecal space with an apparent more pronounced effect for bupivacaine.

Bupivacaine containing dry emulsion can prolong epidural anesthetic effects in rabbits.

To assess the prolongation of epidural bupivacaine by a novel lipid formulation, a physically stabilized bupivacaine containing dry emulsion was prepared by spray-drying. Bupivacaine release from the oil-in-water emulsion was studied using an in vitro two-phase stirred model, then the pharmacodynamic effects and the pharmacokinetics of bupivacaine from the spray-dried emulsion were evaluated and compared to a bupivacaine hydrochloride solution, following a two-period cross-over epidural administration in rabbits. The in vitro release characteristics suggested an extended release of bupivacaine from the emulsion compared to the solution. From the in vivo study, C(max) obtained with the emulsion (containing 5 mg bupivacaine) was not statistically different than from the solution (containing 2 mg bupivacaine) while T(max) was increased, suggesting a diminution of bupivacaine systemic absorption. The onset time of epidural anesthesia was similar for both formulations of bupivacaine used, while a significant blockade prolongation (360%) was observed with the emulsion compared to the solution, suggesting a controlled release of bupivacaine. Dry emulsions could be promising dosage forms to optimize the disposition of epidurally administered LAs.

Alkalinization of intra-cuff lidocaine and use of gel lubrication protect against tracheal tube-induced emergence phenomena.

BACKGROUND: We sought to determine the benefits of using alkalinized lidocaine 40 mg to fill the cuff of a tracheal tube (ETT) in combination with water-soluble gel lubrication to prevent post-intubation sore throat. METHODS: The work included an in vitro study of the diffusion of alkalinized lidocaine solution through the low-pressure, high-volume cuff of an ETT. We also performed a randomized controlled study (n=20 patients in each group) that included a group who received an alkalinized lidocaine-filled ETT cuff with lubrication of the tube using water-soluble gel (Group G), and two control groups who received an alkalinized lidocaine-filled cuff with ETT lubrication with water (Group W) or an air-filled cuff with ETT lubrication with water (Group C). RESULTS: Water-soluble gel lubrication (Group G) produced a lower incidence of sore throat during the 24-h post-extubation period than lubrication with water alone in the cuffs filled with alkalinized lidocaine (Group W), and compared with the air control group. The ability of lidocaine to pass through the cuff of an ETT when water-soluble gel and/or water alone was used as a lubricant was similar, as determined by lidocaine plasma concentrations (C(max) 45 ng x ml(-1)). Cough and restlessness before tracheal extubation were decreased in patients with the alkalinized lidocaine-filled cuffs compared with the air-filled cuffs. After extubation, nausea, vomiting, dysphonia and hoarseness were greater for patients with air-filled cuffs compared with the lidocaine-filled cuffs. No significant difference between the groups was recorded in arterial blood pressure and heart rate. In vitro data suggest that the lower the NaHCO(3) injection volume, the greater the release of lidocaine across a low-pressure, high-volume cuff. CONCLUSIONS: These data show benefits of using an alkalinized lidocaine-filled ETT cuff in combination with water-soluble gel lubrication in preventing post-intubation sore throat.

Endotracheal tube cuffs filled with lidocaine as a drug delivery system: in vitro and in vivo investigations.

The purpose of this study was to examine if lidocaine diffusion across an endotracheal tube cuff could improve post-operative tolerance, especially sore throat. The in vitro release of lidocaine from tube cuffs filled with different lidocaine formulations (base form, hydrochloride form or alkalinized lidocaine hydrochloride) was investigated. A preliminary pilot clinical study in anaesthesia for spine surgery in smoker patients was carried out to examine the pharmacokinetic (i.e. systemic uptake) and pharmacodynamic effects (i.e. incidence of sore throat) obtained with the endotracheal tube cuff filled with lidocaine solution, compared to cuffs inflated only with air. From our in vitro experiment, only the hydrophobic neutral base form of lidocaine was able to diffuse (65.1+/-1.1% released after 6 h), while for the charged hydrochloride form, only a permeation phenomenon occurred concerning only 1% of the total drug. Alkalinization of lidocaine hydrochloride (the only form available as a drug) allows smaller amounts to be used compared to previous published studies (20-40 mg vs. 200-500 mg) and no lag time for diffusion. Such a system could provide a controlled release reservoir for lidocaine to adjacent tracheal tissue. This was shown in our pilot study with sustained plasmatic profiles and improved tolerance (decreased pain scores) in the rank order: air group<

Spinal biopharmaceutics of bupivacaine and lidocaine by microdialysis after their simultaneous administration in rabbits.

The aim of the present study was to determine the intrathecal bioavailability of a mixture of lidocaine and bupivacaine in a rabbit model of spinal anesthesia by using the microdialysis technique. Catheter and microdialysis probe were inserted under control of the view either in the epidural or in the intrathecal space. First, the epidural disposition of the mixture of bupivacaine and lidocaine was studied after epidural administration. Then, the intrathecal and plasma dispositions of bupivacaine and lidocaine were investigated following intrathecal or epidural administration. The epidural clearance of bupivacaine was higher than that of lidocaine, suggesting a more significant uptake of bupivacaine into the systemic circulation and/or into the CSF. The intrathecal bioavailability of bupivacaine and lidocaine was 12.3 and 17.9%, respectively, while it was 5.5 and 17.7% following the separate administration of each agent [Clément, R., Malinovsky, J.M., Le Corre, P., Dollo, G., Chevanne, F., Le Verge, R., 1999. Cerebrospinal fluid bioavailability and pharmacokinetics of bupivacaine and lidocaine following intrathecal and epidural administrations in rabbits using microdialysis. J. Pharmacol. Exp. Ther. 289, 1015-21]. After intrathecal administration, a decrease in C(max) and AUC values was observed for bupivacaine in comparison with the separate administration. Moreover, after epidural administration, the systemic resorption was slower and lower, especially for bupivacaine. Such a reduction in the systemic absorption of bupivacaine might increase its intrathecal bioavailability, resulting from a vasoconstrictor effect of lidocaine reducing the systemic absorption of bupivacaine from the epidural space leading to an increase of its extent of absorption through meninges into CSF although its absorption rate was not modified.

[Biopharmaceutics of local anesthetic-cyclodextrin complexes following loco-regional administration]. / Biopharmacie de complexes anesthésiques locaux-cyclodextrines après administration loco-régionale.

In the research of new dosage forms improving the therapeutic index of local anesthetics, we studied cyclodextrins, cyclic oligosaccharides forming soluble inclusion complexes with various lipophilic drugs. Complexes between bupivacaine and hydroxypropyl-B-cyclodextrin, bupivacaine and sulfobutyl ether-7-B-cyclodextrin were studied in vivo, using an epidural and a perineural (sciatic) model, respectively. Biopharmaceutics and pharmacodynamics of bupivacaine were evaluated in the rabbit. In both models, only systemic absorption rate of bupivacaine was decreased upon complexation, not the quantity absorbed. Complexation with cyclodextrins could be a promising drug delivery system to improve the therapeutic index of local anesthetics.

Biopharmaceutics and pharmacokinetics of 5-phenyl-1, 2-dithiole-3-thione complexed with sulfobutyl ether-7-beta-cyclodextrin in rabbits.

The biopharmaceutics and pharmacokinetics of 5-phenyl-1, 2-dithiole-3-thione (5PDTT) were investigated in rabbits, after administration as a complex with sulfobutyl-ether-7-beta-cyclodextrin (SBE7-beta-CD) by intravenous and oral routes and as a micronized powder by oral route. 5PDTT had a rapid and large red blood cell partitioning that was not dependent on drug concentration either in vitro or ex vivo. The blood clearance was very high (354 +/- 131 mL/min) suggesting extrahepatic metabolism and/or nonrenal elimination and a significant volume of distribution (67 +/- 76 L). The renal clearance was 0.17% of total clearance. 5-phenyl-1,2-dithiol-3-one (5PDTO) was identified as a metabolite in blood and urine. The bioavailability of 5PDTT following administration of 5PDTT/SBE7-beta-CD complex was estimated to 41% while it was close to zero when 5PDTT was given as a micronized powder.

Biopharmaceutics and metabolism of yohimbine in humans.

The biopharmaceutics of yohimbine (YO) and the pharmacokinetics of 10-hydroxy-yohimbine (10-OH-YO) and 11-hydroxy-yohimbine (11-OH-YO) were investigated in healthy subjects following i.v. (5 mg) and oral (8 mg) dosing. One subject was found as a slow hydroxylator of YO. The mean (+/-S.D.) oral absolute bioavailability of YO was 22.3+/-21. 5%. Total plasma clearance (CL) and renal clearance (CL(r)) of YO following i.v. dosing were 0.728+/-0.256 ml/min and 0.001+/-0.002 ml/min, respectively. Based on the steady-state volume of distribution (V(ss)), YO had a relatively low distribution (V(ss) = 32.2+/-12.1 l). The overall renal excretion of YO, 10-OH-YO and 11-OH-YO, expressed as percent of the dose of YO administered, were not different following i.v. and oral dosing, and were around 0.1, 0.2 and 14%, respectively. Following i.v. dosing of YO, the mean apparent terminal half-life of 11-OH-YO (347+/-63 min) was almost four times higher than that of YO (91.0+/-33.6 min) suggesting an elimination rate-limited kinetics for 11-OH-YO.

Improvement in solubility and dissolution rate of 1, 2-dithiole-3-thiones upon complexation with beta-cyclodextrin and its hydroxypropyl and sulfobutyl ether-7 derivatives.

Inclusion complexes between beta-cyclodextrin derivatives and 1, 2-dithione-3-thiones were studied in aqueous solution and in the solid state. Phase solubility study was used to evaluate the complexation in solution, at 37 degrees C, of three cyclodextrins, i. e., beta-cyclodextrin (betaCD), hydroxypropyl-beta-cyclodextrin (HPbetaCD), sulfobutyl ether-7-beta-cyclodextrin (SBE7betaCD), and four 1,2-dithiole-3-thiones, i.e., the parent compound dithiolethione (DTT), dimethyldithiolethione (DMDTT), 5-phenyldithiolethione (5PDTT), and anetholetrithione (ATT). Stability constants of the DTT complexes with HPbetaCD and SBE7betaCD were also determined spectrophotometrically using a nonlinear least-squares methodology. Differential scanning calorimetry (DSC) and scanning electronic microscopy (SEM) were used to characterize spray-dried complexes formed between 5PDTT and SBE7betaCD, ATT and SBE7betaCD. Dissolution studies using the USP paddle method were carried out in water at 37 degrees C for both ATT and 5PDTT binary systems with HPbetaCD and SBE7betaCD. Solubility enhancements were much greater with the more lipophilic ATT and 5PDTT compared to DTT and DMDTT, whatever the cyclodextrin used, in the rank order SBE7betaCD > HPbetaCD >> betaCD. Stability constants obtained (between 120 and 12800 mol(-1)) were also the highest for the more lipophilic drugs and in the same rank order SBE7betaCD > HPbetaCD >> betaCD. Results obtained by UV spectrophotometry were in good agreement with those obtained by phase-solubility study. DSC thermograms of spray-dried complexes of ATT and 5PDTT with HPbetaCD and SBE7betaCD lacked the endothermal peak of pure drug peak which was found for the physical mixtures (107 degrees C and 125 degrees C for ATT and 5PDTT, respectively). Finally, dissolution profiles of spray-dried inclusion complexes studied displayed a faster dissolution rate compared to physical mixtures and pure drugs. The present study showed that complexation of 1,2-dithiole-3-thiones with beta-cyclodextrin derivatives resulted in an increase in solubility, allowing intravenous formulation for bioavailability and metabolism studies and an increase in the dissolution rate of the drugs, which should be of interest for oral absorption of these lipophilic compounds.

[Bioavailability of phloroglucinol in man]. / Biodisponibilité du phloroglucinol chez l'homme.

This work reports a bioavailability study between two oral dosage forms containing 125.2 mg active phloroglucinol. Twelve healthy volunteers subjects received a sublingual administration of both dosage forms, a flash liberation tablet and a freeze-dried reference tablet (lyoc), according to a randomized and cross-over design. An accurate, sensitive and specific high performance liquid chromatographic method was developed for the determination of free phloroglucinol as well as its conjugated metabolites, that allowed as to clarify phloroglucinol pharmacokinetic behaviour in man, specially its important metabolisation, its poor systemic bioavailability after oral administration and its total urinary elimination mainly under metabolized form. Total plasmatic phloroglucinol pharmacokinetic profiles led to pertinent parameters needed for statistical bioequivalence study, i.e. T1/2 alpha, T1/2 beta, AUC, Tmax, Cmax and MRT. The mean comparative values of these parameters showed the equivalent performances of both oral dosage forms studied and the statistical tests performed (ANOVA, Westlake and two one-sided t test) concluded to their bioequivalence.

Cerebrospinal fluid bioavailability and pharmacokinetics of bupivacaine and lidocaine after intrathecal and epidural administrations in rabbits using microdialysis.

The aim of this work was to study the cerebrospinal fluid (CSF) bioavailability and pharmacokinetics of bupivacaine (BUP) and lidocaine (LID) administered separately in rabbits using microdialysis with retrodialysis calibration. Microdialysis probe and catheters were inserted under control of the view in the intrathecal or epidural spaces. The epidural disposition of BUP and LID after epidural administration of low (0.69 microM) and high (6.9 microM) doses was studied. Then, the intrathecal and plasma dispositions after separate intrathecal (0.2 microM) and epidural administration (6.9 microM) were investigated. The CSF binding of BUP and LID was linear in a range from 50 to 500 micrograms/ml, and the mean unbound CSF fraction at a concentration of 100 micrograms/ml was 39. 3 +/- 2.3% for BUP and 75.8 +/- 7.7% for LID. Epidural and intrathecal disposition of BUP and LID showed a biexponential decline. After epidural administration, the CSF concentrations of BUP and LID were much higher than those in plasma. After intrathecal administration, the plasma concentrations were below the limit of quantitation. Although the absorption rate of BUP appeared higher than that of LID, the mean CSF bioavailability of epidural BUP and LID was 5.5 and 17.7%, respectively. The unexpectedly higher CSF bioavailability of LID, the less lipophilic drug, may result from the difference in the processes competing for drug epidural removal.

High-performance liquid chromatographic determination of ropivacaine, 3-hydroxy-ropivacaine, 4-hydroxy-ropivacaine and 2',6'-pipecoloxylidide in plasma.

A sensitive HPLC method has been developed for the determination of ropivacaine, 3-hydroxy-ropivacaine, 4-hydroxy-ropivacaine and 2',6'-pipecoloxylidide in plasma. The procedure involved extraction from plasma with a mixture of n-heptane-ethyl acetate and a back-extraction into an acidified aqueous solution. The chromatography was achieved using a LiChrospher RPB C8 column with a mobile phase consisting of a mixture of acetonitrile and pH 2.1, 0.01 M potassium dihydrogenphosphate, the latter phase containing 0.005 M 1-heptanesulfonic acid for ropivacaine metabolites analysis. The extraction yields of ropivacaine, 3-hydroxy-ropivacaine, 4-hydroxy-ropivacaine and 2',6'-pipecoloxylidide were 94.7%, 79.4%, 79.4% and 77.7%, respectively. The limits of detection of ropivacaine, 3-hydroxy-ropivacaine, 4-hydroxy-ropivacaine and 2',6'-pipecoloxylidide in plasma samples were 0.9 ng/ml, 3 ng/ml, 5 ng/ml and 1 ng/ml, respectively.

In vitro and in vivo microdialysis calibration using retrodialysis for the study of the cerebrospinal distribution of bupivacaine.

Microdialysis coupled to HPLC was used to study the disposition of local anesthetics in the cerebrospinal fluid (CSF) because of the difficulty in sampling CSF. A retrodialysis method for the microdialysis calibration was investigated in vitro and in vivo. Calibration by retrodialysis was simultaneously validated through the use of the zero net flux method. Two local anesthetics (bupivacaine and ropivacaine), which differ structurally by only one methyl group, were respectively utilized as substance of interest and as internal standard. Different parameters were tested in vitro to compare the relative recovery (RR) of bupivacaine and the relative loss (RL) of ropivacaine. Several flow rates were tried to select an optimal in vivo flow rate (1 microliter/min). the RR and RL values were not influenced by the variation of bupivacaine concentration. A significant variability among different probes within a batch was established (RR ranging from 41.1-65.3%; RL ranging from 30.7-61.0%). The K-factor values, defined as RLropivacaine/RLbupivacaine, were calculated in vitro and in vivo. This ratio decreased in vivo but was constant (K in vitro = 1.06 +/- 0.04, K in vivo = 0.87 +/- 0.03). The extracellular tissue concentration of the compound of interest was again in vitro and no deterioration of probe during the in vivo experiment was found. After administration of bupivacaine in the epidural space of rabbits, plasma and microdialysis CSF samples were simultaneously collected. Plasma and CSF disposition of bupivacaine displayed different kinetics. The maximum CSF concentration of B averaged 394 +/- 170 micrograms ml-1 with a mean Tmax of 3.8 +/- 1.8 min. The maximum CSF concentration of B averaged 0.44 +/- 0.09 micrograms ml-1 with a mean Tmax occurring at 1 min. Microdialysis, combined with accurate calibration, should be a reliable technique to gain further insight in the spinal disposition of local anesthetics.