European Journal of Pharmaceutics and Biopharmaceutics (v.61, #1-2)

APV diary (S1-S2).

Nano-and microparticles of poly(lactide-co-glycolide) (PLGA) were formulated using poly(vinyl alcohol) (PVA) or hydrophobically modified hydroxyethylcellulose (HMHEC) or polyethyleneimine (PEI) as stabilizers. The uptake by murine Peyer's patches (PPs) and the binding to Peyer's patches-free tissue (PPFT) of these particles was investigated using fluorescence microscopy providing qualitative information about the tissue distribution of particles. Observations of intestinal cryo-sections showed significant discrimination in the uptake by PP of nano-and microparticles. The uptake by PPs of PLGA-PVA and PLGA-HMHEC nano-and microparticles, of negative and neutral zeta potential, respectively, was comparable, whereas a smaller number was observed in the case of nano-and microparticles of PLGA-PEI, positively charged. Moreover, particle uptake by PPs appeared to be strongly size-dependent. The number of particles of mean diameter around 0.3 and 1 μm observed in PPs was much greater than that of particles of diameter average close to 3 μm. However, in all cases, particles were found in the PPFT for at least 48 h. In conclusion, regarding the tissue samples we have observed, it appeared that the uptake of particles by PPs and binding to PPFT could be influenced by the physicochemical properties of the particles but this may not have been true at all sites of the intestine and may differ between animals.
Keywords: Poly (lactide-co-glycolide); Polyethyleneimine; Gastro-intestinal uptake; Peyer's patches; Hydrophobically modified hydroxyethylcellulose; Electron spectroscopy for chemical analysis; Zeta potential;

The bioavailability of triprolidine from the ethylene vinyl acetate (EVA) matrix system containing polyoxyethylene-2-oleyl ether was studied to determine the feasibility of enhanced transdermal delivery of triprolidine in rabbits. The antihistamine effects were also confirmed to determine the percutaneous absorption of triprolidine from the EVA matrix system containing a penetration enhancer and plasticizer in rats. The triprolidine-EVA matrix (50 mg/kg) was applied to the abdominal skin of rabbits. Blood samples were collected via the femoral artery for 36 h and the plasma concentrations of triprolidine were determined by HPLC. The pharmacokinetic parameters were calculated using the LAGRAN computer program. The area under the curve(AUC) was significantly higher in the enhancer group (4582±1456 ng/mL h) than that (2958±997 ng/mL h) in the control group (P<0.05), showing an approximate 155% increased bioavailability. The average C max in the enhancer group (241±46.5 ng/mL) was significantly higher than that in the control group (198±28.9 ng/mL), (P<0.05). The mean T max in the enhancer group (8.0±2.57 h) was higher than that in the control group (6.0±2.24 h, but this was not statistically significantly. The relative bioavailability of triprolidine in the transdermal application was 35.9% in the control group and 55.6% in the enhancer group compared comparing with that after oral administration. As the triprolidine-EVA matrix, which contains polyoxyethylene-2-oleyl ether as an enhancer and triethyl citrate as a plasticizer was administered to the rabbits via the transdermal routes, the relative bioavailability increased approximately 1.55 fold compared with that in the control group, showing a relatively constant, sustained blood concentration with minimal fluctuation. The antihistamine effect was determined using the Evans blue dye procedure by comparing the changes in the vascular permeability increase following the transdermal application. The vascular permeability increase was reduced significantly by the transdermal application of the EVA-triprolidine system containing triethyl citrate and polyoxyethylene-2-oleyl ether. These results show that the plasticizer and penetration enhancer increase the skin permeation of triprolidine and the triprolidine-EVA matrix system could be developed as a transdermal delivery system providing the increased constant plasma concentration and antihistamine effects
Keywords: Bioavailability; EVA matrix; Triprolidine; Enhancer; Transdermal administration; Antihistamine effects.;

Use of nonlinear mixed effect modeling for the intestinal absorption data: Application to ritonavir in the rat by M.J. Muñoz; M. Merino-Sanjuán; R. Lledó-García; V.G. Casabó; F.J. Máñez-Castillejo; A. Nácher (20-26).
The aim of this study is to investigate in situ the mechanisms involved in the gastrointestinal absorption of ritonavir in the rat, as an animal model for preclinical studies of anti-HIV agents in vivo. Four ritonavir solutions (40, 27, 13 and 7 μM) in the presence of 1% dimethylsulfoxide (DMSO) were perfused in the small intestine of anaesthetised rats. Effects of DMSO on the intestinal permeability were investigated using solutions containing antipyrine 1.33 mM and ritonavir 7 μM with and without 1% of DMSO. Antipyrine and ritonavir transport was not modified in the presence of 1% of DMSO. The population pharmacokinetic parameters of the ritonavir intestinal transport were obtained by means of nonlinear mixed effect modelling approach according to a nonlinear absorption and nonlinear secretion. The absorption and secretion kinetic parameters for ritonavir were: Vm=47.6 μM/h; Km=8.77 μM; Vms=3.66 μM/h and Kms=0 μM. The interindividual variability found to ritonavir Vm 13.1%, and the residual variability was 8.98%. The Kms value support the saturation of the carrier at the range of concentrations of ritonavir assayed. The interindividual variability value of the Vm could explain, at least in part, the variability in absorption rate constants observed.
Keywords: Anti-HIV protease inhibitors; Ritonavir absorption; Ritonavir secretion; Carrier-mediated; Nonlinear mixed effects model (NONMEM); Bootstrap;

Uptake of fluorescein isothiocyanate-labelled dextran into the CSF after intranasal and intravenous administration to rats by Joke P.M. in 't Veen; Mascha P. van den Berg; Stefan G. Romeijn; J. Coos Verhoef; Frans W.H.M. Merkus (27-31).
With the growing number of patients suffering from central nervous system (CNS) diseases a suitable approach for drug targeting to the brain becomes more and more important. In the present study, the contribution of the nose–CSF pathway to the uptake of the model drug fluorescein isothiocyanate-labelled dextran with a molecular weight of 3.0 kDa (FD3) into the CSF was determined in rats. FD3 was administered intranasally (489 μg/rat) and by intravenous infusion (24.4 μg/ml; 119 μg/rat) in the same set of animals (n=6). Blood samples were taken from the tail vein and CSF was sampled by cisternal puncture using a stereotaxic frame. The contribution of the olfactory pathway to the uptake of FD3 into the CSF was determined by comparing the AUCCSF/AUCplasma ratios after intranasal and after intravenous application of FD3 mimicking the blood levels after intranasal delivery. No significant difference was observed between the AUCCSF/AUCplasma ratios of FD3 after intranasal administration (1.33±0.40%) and intravenous infusion (1.03±0.56%). This indicates that in rats about 1% of the amount of FD3 in plasma reaches the CSF both after nasal and intravenous administration and that no direct transport of FD3 from the nose–CSF could be found.
Keywords: Fluorescein isothiocyanate-labelled dextran (FD); Rats; Nose–CSF drug transport; Nasal delivery; Intranasal administration; Intravenous infusion;

Effects of cryoprotectants on the viability and activity of freeze dried recombinant yeasts as novel oral drug delivery systems assessed by an artificial digestive system by Stéphanie Blanquet; Ghislain Garrait; Erick Beyssac; Céline Perrier; Sylvain Denis; Géraldine Hébrard; Monique Alric (32-39).
The aim of this study was to investigate, in a gastric-small intestinal system TIM-1, the effect of cryoprotectants on the survival of freeze-dried Saccharomyces cerevisiae expressing the heterologous P450 73A1 and their ability to convert trans-cinnamic acid into p-coumaric acid. Yeasts were lyophilized in suspensions of trehalose, maltose, lactose, or a milk proteins/trehalose mix. Freeze-dried or native yeasts and trans-cinnamic acid were introduced simultaneously into TIM-1 at the beginning of digestion. Yeast survival rate was evaluated by cell counting in the ileal effluents. P450 73A1 activity was followed by HPLC assay of p-coumaric acid. Freeze-dried yeasts showed high tolerance to digestive conditions. Nevertheless, their survival rate was lower than that of non-dried cells (around 80% whatever the protective agent vs. 96%). The ability of recombinant freeze-dried S. cerevisiae to perform a bioconversion reaction in the digestive tract was shown with all the protectants. The highest trans-cinnamic acid conversion rate (24 vs. 41% for native yeasts) was obtained with the milk proteins/trehalose mix. These results show that freeze-drying might be considered for the pharmaceutical formulation of new drug delivery systems based on orally administered recombinant yeasts and that TIM-1 could be a helpful tool for the pre-screening of oral dosage forms.
Keywords: Oral formulations; Pre-screening; Freeze-drying; Cryoprotectant; Recombinant Saccharomyces cerevisiae; Artificial digestive system;

Differential effects of cyclodextrins and derivatives on the biological behavior of the myocardial perfusion imaging agent 99mTcN-NOET by Laurent Riou; Catherine Ghezzi; Denis Wouessidjewe; Ho Law; Jean-Paul Mathieu; Jacques Defaye; René Bontron; Roberto Pasqualini; Daniel Fagret (40-49).
In addition to improving drug solubilization, cyclodextrins (CDs) also affect the biological behavior of the included compound. We evaluated the effects of two natural CDs β-CD and γ-CD, and six β-CD derivatives, Dimeb, Trimeb, SBb, 2-HP, 6AD, and 6 MTU on the biological behavior of 99mTcN-NOET, a technetium-99m-labeled, lipophilic compound readily detectable through radioactivity assessment. Determination of CDs' affinities for 99mTcN-NOET indicated that the cavity size of γ-CD was not suitable for 99mTcN-NOET inclusion, and that β-CD derivatization mostly resulted in decreased CDs affinities for 99mTcN-NOET to various extents compared with the natural β-CD. In vitro and ex vivo experiments performed on newborn rat cardiomyocytes and isolated perfused rat hearts, respectively, showed 1.7- and 2.3-fold maximal differences in 99mTcN-NOET cellular and tissue activities. Regression analyzes indicated no significant correlation between these observed biological differences and the affinities of the eight CDs tested for 99mTcN-NOET or for cellular membranes. In conclusion, CD derivatization often resulted in impaired affinity of the derivatives for the lipophilic compound 99mTcN-NOET. Moreover, the in vitro and ex vivo biological behavior of 99mTcN-NOET was greatly affected depending on the CD used for inclusion of the tracer.
Keywords: Cyclodextrins; Cyclodextrin derivatives; 99mTcN-NOET; Cardiomyocytes; Isolated rat hearts; Anesthetized dogs;

Effect of chemical enhancers on the in vitro percutaneous absorption of sumatriptan succinate by A. Femenía-Font; C. Balaguer-Fernández; V. Merino; V. Rodilla; A. López-Castellano (50-55).
The effects of percutaneous enhancers on the transdermal absorption of sumatriptan succinate were investigated by in vitro permeation studies. Pretreatment of porcine skin with ethanol (vehicle), polyethylene glycol 600, Span® 20, oleic acid, R-(+)-limonene, α-bisabolol and 1,8-cineole (at 5% in ethanol, w/w) produced in all cases an increase in the flux of sumatriptan. The amount of sumatriptan retained in the skin was also determined. Ethanol has showed a low but significant increment on the drug transdermal flux. Treatment of the skin with α-bisabolol shows the same enhancer effect than ethanol. Span® 20, oleic acid, and polyethylene glycol 600 have shown a moderate enhancing activity on transdermal flux of sumatriptan. R-(+)-limonene showed the greatest ability to enhance the flux of sumatriptan.
Keywords: Sumatriptan succinate; Transdermal absorption; Percutaneous penetration; Chemical enhancers; Terpenes; Migraine treatment;

The study was conducted to investigate the effects of carrageenans, and cellulose ethers on the drug release rates of ibuprofen controlled-release tablet matrices prepared by direct compression. Polymer blends containing carrageenans or cellulose ethers were used for the formulation and the effect of varying the polymer concentration on the release of the drug was studied. Other factors such as changes in surface topography of the matrices due to hydration were observed using a cryogenic scanning electron microscopy technique. Multiple regression analysis was used to predict the time for 50% release (t 50) as a function of the concentration of the polymers used. Most of the formulations showed linear release profiles (r 2≥0.96–0.99) and sustained the release of ibuprofen over 12–16 h. The highest t 50 (9.3 h) was for the formulation that contained a blend of 1:2 ratio of Viscarin and HPMC, while the lowest (3 h) was for the matrices that contained a 2:1 ratio of methylcellulose and Gelcarin. The majority of the matrix tablets that contained 10% polymer disintegrated prematurely. Of all the polymer blends that were investigated, the combination of Viscarin and HPMC gave almost linear release profiles over the entire range of concentration that was studied. The least effective combination was methylcellulose in combination with HPMC. Most of the formulations released ibuprofen by an anomalous (non-Fickian) transport mechanism, except those matrices that contained methylcellulose and Gelcarin (in a 1:1 and 1:2 ratio), which showed zero-order release.
Keywords: Ibuprofen; Controlled-release; Zero-order; Carrageenan; Hydroxypropyl methylcellulose; Tablets;

Phospholipid hydrolysis in a pharmaceutical emulsion assessed by physicochemical parameters and a new analytical method by Laura Rabinovich-Guilatt; Catherine Dubernet; Karen Gaudin; Gregory Lambert; Patrick Couvreur; Pierre Chaminade (69-76).
The aim of this work was to develop a simple high-performance liquid chromatography (HPLC) technique with evaporative light scattering detection (ELSD) for the separation and quantification of the major phospholipid (PL) and lysophospholipid (LPL) classes contained in a pharmaceutical phospholipid-based emulsion. In the established method, phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyeline (SM), lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were separated with a PVA-Sil® stationary phase and a binary gradient from pure chloroform to methanol:water (94:6 v/v) at 3.4%/min. The ELSD detection was enhanced using 0.1% triethylamine and formic acid in each gradient mobile phases. Factors such as stationary phase and ELSD drift tube temperature were optimized, concluding in optimal temperatures of 25 °C for separation and 50 °C for evaporation. This HPLC–ELSD method was then applied to a PL-emulsion exposed to autoclaving and accelerated thermal conditions at 50 °C. Hydrolysis of PC and PE followed first-order kinetics, representing only 45% of the total lipid mass after 3 months. The chemical stability was correlated to commonly measured formulation physical and physico-chemical parameters such as droplet size, emulsion pH and ζ-potential.
Keywords: Phospholipids; Lysophospholipids; ELSD; Polyvinyl alcohol; Emulsion; Hydrolysis; ζ-potential; pH;

Solid lipid nanoparticles (SLN), an alternative colloidal drug delivery system to polymer nanoparticles, emulsions and liposomes, are generally produced by high pressure melt-emulsification. However, the harsh production process is not applicable for formulations containing shear and temperature sensitive compounds. For that reason, subsequent adsorptive SLN loading might be a promising alternative. The aim of the present study was the development and characterisation of surface-modified SLN for adsorptive protein loading by variation of both the lipid matrix and the emulsifier concentration in the continuous phase. Variations in SLN composition resulted in particle sizes between 674 and 61 nm corresponding to specific surfaces of 4.5 m2/g and 48.9 m2/g and zeta potentials between −23.4 mV and −0.9 mV. In dependence of SLN surface properties, albumin payload ranged from 2.5 to 15%. Thermoanalysis, X-ray diffraction and electron microscopy revealed anisometrical and crystalline particles. In vitro cytotoxicity was low in terms of both haemolysis, which was between 1 and 2%, and neutral red test (NRT) showing a half lethal dose between 1.1 and 4.6%.
Keywords: Haemolysis; Neutral red test; Physicochemical characterisation; Protein adsorption; Solid lipid nanoparticles; Surface modification;

Flow injection spectrophotometric determination of ofloxacin in pharmaceuticals and urine by M. Soledad García; M. Isabel Albero; Concepción Sánchez-Pedreño; Mustafa Salem Abuherba (87-93).
A sensitive and fast flow-injection spectrophotometric method for the determination of ofloxacin is proposed. The method is based on the formation of a yellow complex between ofloxacin and Fe (III), in sulphuric medium. The influence of FI and chemical variables were studied. The calibration graph resulting from measuring the absorbance at 420 nm is linear over the range 5.0×10−6  8.0×10−4  M (1.8–289 mg l−1) with a detection limit of 2.0×10−6  M (0.72 mg l−1). The method is applied to the routine analysis of ofloxacin in pharmaceuticals and human urine.
Keywords: Spectrophotometric; Flow injection; Ofloxacin; iron (III); Pharmaceuticals; Urine;

This study evaluated the utility of including superdisintegrants (croscarmellose sodium or sodium starch glycolate) in microcrystalline cellulose extrusion–spheronization pellets as a means of increasing the dissolution rate of poorly water-soluble drugs. The model drug was hydrochlorothiazide, with water or water/ethanol as wetting agent for pellet preparation. Neither disintegrant had significant effects on pellet morphology, flow properties or mechanical resistance. Neither disintegrant caused disintegration of the pellet in drug dissolution medium. Nevertheless, the disintegrants afforded a modest increase in drug dissolution rate, attributable to the observed increase in pellet micropore volume. Drug dissolution rate was slightly higher in pellets prepared with sodium starch glycolate, probably because of this disintegrant's higher swelling capacity.
Keywords: Pellets; Extrusion–spheronization; Microcrystalline cellulose; Croscarmellose sodium; Sodium starch glycolate; Hydrochlorothiazide;

Near-infrared spectroscopy (NIRS) has become a widely used analytical technique in the pharmaceutical industry, serving for example to determine the active substance or water content of tablets. Its great advantage lies in the minimal sample preparation required and speed of measurement. In a study designed to detect the effects of process on tablet dissolution, we describe the application of NIRS to the detection and identification of changes in uncoated and coated tablets in response to pilot-scale changes in process parameters during melt granulation, compression, and coating. Beginning with a qualitative comparison between pharmaceutical batches, we show that NIRS and principal component analysis can separate batches produced with different melt granulation parameters and differentiate between cores compressed with different compaction forces. Complementary infrared imaging can also explain the difference in dissolution properties between samples produced with different melt granulation parameters. NIRS is sensitive to changes in coating formulation, the quality of a coating excipient (hydroxypropyl methylcellulose), and coating time. In a concluding quantitative analysis, we demonstrate the feasibility of NIRS in a manufacturing context for predicting coating time and detecting production cores failing to meet dissolution test specifications.
Keywords: Near-infrared spectroscopy; Infrared imaging; Process analytical technology; Coated tablet; Core; Dissolution; Principal component analysis;

by Katharina Meier; Claus-Michael Lehr (111-112).

by Thomas Kissel (111).

Doctoral theses (113-114).