On-line direct determination of the second virial coefficient of a natural polysaccharide using size-exclusion chromatography and multi-angle laser light scattering.

By combining a size-exclusion chromatographic (SEC) separation and an on-line multi-angle light scattering (MALLS) analysis, we have elaborated an original methodology permitting on-line direct determination of the second virial coefficient of molar mass fractions of polydisperse polysaccharides. By assimilating the SEC-MALLS data to a batch mode acquisition, we have obtained on-line the complete Zimm plot of the eluted fractions, leading to knowledge of their weight-average molar mass Mw, radius of gyration r(g) and second virial coefficient A2. Our methodology was successfully applied to a iota carrageenan sample in LiCl 100 mM, EDTA 1 g/l.

Relationship between conformation of polysaccharides -in the dilute regime and their interaction with a phospholipid bilayer.

Interactions between polysaccharides and phospholipid bilayers have already been demonstrated in the literature but little is known about the influence of macromolecule conformations related to the solvent characteristics (pH, ions, ionic strength). In this study we have investigated the conformation of iono- and thermo-sensitive polysaccharides, iota- and kappa-carrageenans, and their interaction with a dimyristoylphosphatidylcholine (DMPC) model bilayer. The study was performed in two different media (NaCl 150 mmol/L, pH 6.5, and NaCl 300 mmol/L, pH 6.5). In the first part, the iota- and kappa-carrageenan samples have been characterized by size exclusion chromatography (SEC) coupled with a multi-angle laser light-scattering detector (MALLS). The SEC-MALLS results clearly show polysaccharide chain association at high ionic strength. In the second part, the polysaccharide-membrane interaction has been studied, using fluorescent probes embedded in the membrane. The thermotropic properties of the membrane were investigated by fluorescence depolarization of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). The membrane surface accessibility was evaluated by fluorescence quenching of 2-(9-anthroyloxy) stearic acid (2-AS). Whatever the ionic strength tested, the polysaccharide presence notably enhances the membrane fluidity below the T(m). This sign of an interaction in the polar level of the membrane is more marked at low NaCl concentration. In contrast, the liposomes bilayer accessibility is drastically lowered when increasing the ionic strength. This is induced by macromolecular chain adsorption on the liposome surface, enhanced by the polysaccharide chain association. An ionic strength enhancement induces a conformational modification of the polysaccharide chains which modifies their ability to interact with the bilayer.

LC analysis of benzophenone-3: II application to determination of 'in vitro' and 'in vivo' skin penetration from solvents, coarse and submicron emulsions.

The aim of this study was to determine the skin penetration of benzophenone-3 in vitro and in vivo in order to investigate a possible influence of formulation. Six different vehicles, three solvents and three different emulsion types were evaluated in vitro and in vivo. Each vehicle was applied to the skin model at 2 mg cm(-2). First, histological studies on ear pigskin and human skin were evaluated. In vitro measurements were performed with static diffusion cells using pigskin at 1, 2, 4, and 8-h. In vivo, benzophenone-3 concentration in stratum corneum was evaluated by the stripping method after 30-min application on forearm of volunteers. It was shown that ear pigskin and human skin appear similar and in both experiments significant differences between vehicles were noticed. The six vehicles could be ranked in the same order of benzophenone-3 skin concentration. At 8-h, the highest concentration of benzophenone-3 in skin was obtained with propylene glycol, and O/W submicron emulsion. On the contrary. the two oily solvents. W/O emulsion and O/W coarse emulsion restrain the concentration of this UV-filter in the skin. At each time, permeability in vitro and in vivo were well correlated. Low concentrations were measured in the receptor fluid suggesting that percutaneous absorption of this UV-filter across the skin would be minimal. The in vitro and in vivo skin penetration capacity of benzophenone-3 from six vehicles was confirmed and quantified. A satisfactory relationship between binary in vitro and in vivo was established.

Effect of the addition of oxybenzone or octyl-methoxycinnamate on particle size of submicron emulsions.

The formulation of sunscreen products requires understanding of the solubilization of these products in different vehicles to obtain aesthetic preparations and to evaluate long-term stability. For this study, two different ultraviolet (UV) filters were selected: oxybenzone (powder) and octyl-methoxycinnamate (liquid). First, the solubility of these UV filters was tested using a three-component simplex-centroid design strategy. The mixtures were prepared with three oily phases used in this field of cosmetics: liquid paraffin, isopropyl myristate, and coconut oil. A phase diagram method was used to carry out a systematic study of submicron oil-in-water emulsions. Phase diagrams were produced by diluting fixed binary mixtures with water. The surfactant consisted of polyoxyethylene-20-sorbitan monostearate/sorbitan monostearate (50/50, w/w). The oily phase contained equal quantities of each oil studied. From this water/surfactant/oil ternary system, we selected two reference emulsions with receptively 75/5/20 and 68/7/25 proportions. Photon correlation spectroscopy (PCS) was used to investigate the influence of these two UV filters at several concentrations on droplet size and distribution of the oil droplets in the material. All emulsions were stored and checked every month for 6 months.

Solubilisation de filtres-UV dans des melanges ternaires de phases apolaires, optimisation par l'utilisation de reseaux centres.

This paper is concerned with the solubility of UV filters. Emulsions are perfect formulations used for sunscreens, but to give the skin silky feel without being greasy, the oily phase content is low. Considerations of sunscreen solubility can help to solve specific problems of stability in formulations. It is important to determine what solvent dissolves them and what mixture of solvent improves their solubility. Two water insoluble sunscreens were tested at several concentrations in various non-polar solvents. A strategy with simplex centroid design was used to optimize this work. The graphical interpretation of the data assists our understanding of the solubility of UV filters.

A preliminary study of the in vivo behaviour of an emulsion formulation of indocyanine green.

Indocyanine green (ICG) is a fluorescent dye largely used as functional indicator, fluorescent imaging contrast agent and recently as enhancer during diode laser photocoagulation. In this study, indocyanine green was incorporated in an emulsion to increase its residence time in blood. In vitro results to show that indocyanine green absorption peak is shifted towards longer wavelength (from 778 nm to 794 nm) close to the peak emission wavelength of the near infrared diode laser (805 nm) used during laser-induced photocoagulation. Plasmatic clearance is slower (in the 15-60 min time interval) compared to the plasmatic clearance of ICG administered as an aqueous solution. The absorbing capacity of ICG at 805 nm shows a two- to threefold increase when indocyanine green is incorporated in this emulsion.

Evaluation of capillary zone electrophoresis and micellar electrokinetic capillary chromatography with direct injection of plasma for the determination of cefotaxime and its metabolite.

Quantitative aspects of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were investigated for the determination of cefotaxime (C) and its deacetyl metabolite (DA) in human plasma in a concentration range of therapeutic interest. For CZE, plasma samples spiked with C and DA were injected after deproteinization with acetonitrile, and analytes were separated in a fused silica capillary using a borate buffer at pH 9.2 as electrolyte; no suitable internal standard was found. For MECC, plasma samples spiked with C, DA, and theobromine as internal standard were directly injected after dilution with water and analyzed using a phosphate buffer, pH 8.00, containing 165 mM SDS as separation electrolyte and a fused silica capillary. Both methods gave satisfactory interday precision with respect to migration times (RSD < 1%) and gave linear responses over the concentration ranges investigated (5-100 mg L-1 C and 5-20 mg L-1 DA). For CZE, intraday RSD (n = 4 graphs) between the slopes of the calibration graphs was acceptable (5.7%) for C. The corresponding figures for interday precision (n = 4 days) were fair (16.1%) in comparison to those obtained with MECC, for which the RSD was 1.49% when theobromine was used as internal standard. A satisfactory interday precision between slopes was also obtained with MECC even without the use of an internal standard (RSD = 4.38%), which demonstrated the ruggedness of this method. Detection limits (S/N = 3) were about 2 mg L-1 (CZE) and 1 mg L-1 in plasma (MECC) for C and DA. MECC was shown to be superior with regard to simplicity, rapidity, precision, and sensitivity.

Fluorescent characteristics and pharmacokinetic profiles of the fluorescent probe BCECF in various tissues: the role of blood content.

The purpose of this study was to investigate the in vitro and in vivo spectral characteristics of the fluorescent pH-sensitive probe bis-carboxyethylcarboxyfluorescein (BCECF) in different tissues and its fluorescence kinetics profiles. The in vivo study was performed on anesthetized adult Wistar rats. After intravenous administration (4.8 mg/kg), fluorescence spectra were recorded on the following tissues: skin, an isolated blood vessel and liver. Measurements performed in vitro on blood samples show modifications of the BCECF emission spectrum with a blue-shift (10 nm) and a low fluorescence emission. Blood content greatly influences the pH measurement by increasing the I(490 exc., 530 em.)/I(470 exc., 530 em.) Fluorescence ratio value (ratio of the fluorescence intensities at 530 nm following excitation at 470 nm and 490 nm) when the hematocrit is high. A 0.35 ratio difference is observed between a BCECF-buffered solution and blood samples of 44% hematocrit. The emission spectra recorded on the skin are quite similar to the emission spectrum of BCECF in aqueous solution and are consistent with an extravascular localization of the dye a few minutes after injection. On the contrary, spectra recorded on the blood vessel and the liver are more similar than those recorded in vitro on high hematocrit solutions. Kinetic profiles in skin, liver and isolated blood vessels compared to the clearance obtained by blood sampling provide information about tissue perfusion. Then the variation of in vivo spectra in different tissues may be taken into account to measure tissue pH with special regard to the blood content of the illuminated area and the time range in which the measurement is performed.

Influence du protocole opératoire sur les caractéristiques d'une émulsion L/H réalisée avec des surfactifs polymériques non ioniques.

Synopsis The mode of operation has an effect on the properties of emulsions. We have studied the influence of the method of preparation (temperature, stirring rate, time of phase introduction and method of cooling) on the rheological properties of an oil in water emulsion. The importance of factors is classified with the help of two-level full and fractional factorial designs. The most significant factor is the mode of cooling, a progressive cooling produces an increase of viscosity and the influence of the other studied factors is insignificant. Sudden cooling entails a lowering of the viscosity and a diminution of the stability, and shows the influence of the rate of mixing and the temperature of manufacture.

TEWL measurement standardization: kinetic and topographic aspects.

The aim of this study was to standardize the recording technique of the SERVO MED Ep 1C to determine the rest time required for human subjects to obtain stabilized values of transepidermal water loss (TEWL). Measurements of TEWL were performed on the flexor side of the forearm in healthy volunteers. First, different sites were tested on different days to study intra- and inter-individual variations. The recordings were carried out every 5 min for 2 h. Values reached a linear state after 15 min of rest. A time-course type curve of TEWL was established.

Evaluation expérimentale de l'hydratation et du pouvoir occlusif in vivo et in vitro d'excipients lipophiles et de leurs émulsions phase huile continue.

Synopsis A study was carried out on three water in oil emulsions and their respective oil-phases, petrolatum, liquid paraffin and sweet almond oil, in order to compare their occlusive properties and thus their influence on cutaneous hydration. An in vitro method using 'Patel'cells in a water-saturated atmosphere at controlled temperature enabled, in the first instance, the various emulsions and their oil-phases to be classified according to their permeability to water vapour. The results are in order of increasing degree of occlusion: sweet almond oil, liquid paraffin and petrolatum. In in vivo studies on human volunteers, the influence of the application of the same substances on transepidermal water loss (TEWL) and cutaneous hydration was measured with an evaporimeter and a corneometer, in a temperature and humidity-controlled room. Measurements were performed on the flexor side of the forearm after sodium lauryl sulphate 5% insult on six subjects at times of 0, 30, 120 and 270 min. The different oils, used pure, increased the hydration by an occlusive effect, and this was described by measurements of TEWL. On the other hand, after using the emulsions, the increase in hydration noted does not appear to involve an occlusive mechanism.

Displacement of thiopental from human serum albumin by associated drugs.

Displacement of thiopental from its binding sites to 4% human serum albumin solution was studied in vitro. Experimental conditions were selected to reproduce a physiological situation. Associations were studied according to the therapeutic conditions of use of the substances (drug and protein concentrations). The unbound fraction of thiopental was obtained by equilibrium dialysis at 37 degrees C and pH 7.4. Eleven drugs were associated with thiopental in 50 combinations of drugs and molar ratios. Bromhexine, citocoline, dextromoramide, dexamethasone, and methotrimeprazine had no effect on thiopental binding. The unbound fraction of thiopental significantly increased with cefamandole, cefazolin, diazepam, desmethyldiazepam, furosemide, and fentanyl. At usual therapeutic drug concentrations, the unbound fraction increase was < 5%. Higher values, however still < 10%, were found with associated drugs that were added at maximal concentrations observed in therapy. The displacement of thiopental from its albumin binding by drugs that are normally associated with the treatment of intracranial hypertension does not modify the pharmacokinetic parameters or pharmacological effect of thiopental.

[Influence of the inoculum effect on minimal inhibitory concentrations of 3 derivatives of 5-nitro-imidazole and 11 other antibiotics on Bacteroides fragilis]. / Influence de l'effet inoculum sur les concentrations minimales inhibitrices de trois dérivés du 5-nitro-imidazole et onze autres antibiotiques sur Bacteroides du groupe fragilis.

Three nitro-imidazole derivatives and eleven other antibiotics were studied for both their antimicrobial activity and the degree to which they produced inoculum effect on twenty strains of Bacteroides fragilis group. Five inocula were studied ranging from 10(5) to 10(9) CFU/ml. Statistical analysis show the significant influence of inoculum and those of strains. Nitro-imidazole derivatives exhibited a large inoculum effect while other antibiotics exhibited only a minimal to-no-inoculum effect.

[Post-antibiotic effect of ciprofloxacin alone and in combination on Streptococcus faecalis]. / Effet post-antibiotique de la ciprofloxacine seule et en association sur Streptococcus faecalis.

Post-antibiotic effect (PAE) of ciprofloxacin has been studied on 2 strains of S. faecalis in the logarithmic phase after 2 exposure times and at concentrations ranging 0.5-10 times the MIC. At the end of the exposure period the antimicrobial is removed by dilution. Similar PAE is observed for ciprofloxacin and ofloxacin, higher than for pefloxacin. Ciprofloxacin-fosfomycin or ciprofloxacin-vancomycin combinations are indifferent or even antagonistic (vancomycin) in regards to PAE. Ciprofloxacin-penicillin or ciprofloxacin-netilmicin are weakly synergistic.

[Diffusion of piperacillin in the healthy human peritoneum after administration of a prophylactic dose]. / Diffusion de la pipéracilline dans le péritoine sain de l'homme après administration à dose prophylactique.

The passage of piperacillin into the omentum was evaluated in 23 patients who received one single intravenous injection of 2 grams. Median concentrations were 7.05 micrograms.g-1 at 30 min, 7.2 micrograms.g-1 at 60 min., 4.65 micrograms.g-1 at 90 min. and 3.53 micrograms.g-1 at 120 min. The corresponding maximal values were 36 micrograms.g-1, 59.7 micrograms.g-1, 36 micrograms.g-1 and 32 micrograms.g-1 respectively. Specimens collected beyond 2 hours showed that the concentrations obtained were maintained.