European Journal of Pharmaceutics and Biopharmaceutics (v.72, #2)

APV Diary (S1).

Tight junctions (TJ) are barrier forming cell–cell junctions that are found in a variety of cell types and tissues but their existence in mammalian epidermis has been shown only in the last years. A variety of TJ proteins were identified in mammalian epidermis, comprising several members of the claudin family, occludin, and JAM-A as well as ZO-1 and MUPP-1. TJ proteins exhibit complex expression and localization patterns in the epidermis. Nonetheless, even though several TJ proteins are found in various layers, typical TJ structures are only found in the stratum granulosum. TJ are important for barrier function of the skin, especially for inside–out barrier. In addition, TJ proteins might also be involved in additional functions in epidermal cells. Localization and expression of TJ proteins are altered in several skin diseases, e.g. psoriasis. Meanwhile several TJ modulators are known from simple epithelia. We discuss their putative usability for drug delivery into and through the skin.
Keywords: Claudin; Occludin; ZO-1; JAM; Barrier function; Skin; Permeation; Skin disease; Infection;

Non-invasive in vivo methods for investigation of the skin barrier physical properties by R. Darlenski; S. Sassning; N. Tsankov; J.W. Fluhr (295-303).
Skin as an organ of protection covers the body and accomplishes multiple defensive functions. The intact skin represents a barrier to the uncontrolled loss of water, proteins, and plasma components from the organism. Due to its complex structure, the epidermal barrier with its major component, stratum corneum, is the rate-limiting unit for the penetration of exogenous substances through the skin. The epidermal barrier is not a static structure. The permeability barrier status can be modified by different external and internal factors such as climate, physical stressors, and a number of skin and systemic diseases.Today, different non-invasive approaches are used to monitor the skin barrier physical properties in vivo. The quantification of parameters such as transepidermal water loss, stratum corneum hydration, and skin surface acidity is essential for the integral evaluation of the epidermal barrier status. Novel methods such as in vivo confocal Raman microspectroscopy offer the possibility for precise and detailed characterization of the skin barrier.This paper will allow the readership to get acquainted with the non-invasive, in vivo methods for the investigation of the skin barrier.
Keywords: Transepidermal water loss; Skin hydration; pH; Tape stripping; Raman microspectroscopy;

Controlled non-invasive transdermal iontophoretic delivery of zolmitriptan hydrochloride in vitro and in vivo by Sonal R. Patel; Hui Zhong; Ashutosh Sharma; Yogeshvar N. Kalia (304-309).
The objective was to investigate the transdermal delivery kinetics of zolmitriptan from an iontophoretic patch system in Yorkshire swine in vivo. Preliminary in vitro experiments showed that cumulative drug transport during a 6-h current application (0.25 mA cm−2) was independent of patch load (263.7 ± 92.7, 357.2 ± 85.9, 374.9 ± 74.3 and 335.9 ± 27.7 μg cm−2 for 7.5, 15, 45 and 90 mg patch loads, respectively; ANOVA, p  < 0.05); the steady-state flux was ∼92 μg cm−2  h−1. The in vivo studies used multistep current profiles to demonstrate (i) rapid drug uptake and (ii) the effect of superposing a bolus input on basal drug levels. In both studies, zolmitriptan was detected in the blood after 2.5 min; drug levels were 7.1   1.7 and 10.4 ± 3.5 ng ml−1 at t  = 30min in Studies 1 and 2, respectively. In Study 2, increasing current intensity from 0.2 to 1.4 mA (0.05–0.35 mA cm−2) at t  = 180 min caused zolmitriptan levels to rise from 9.38 ± 0.93 ng ml−1 at t  = 180 min to 13.57 ± 1.85 ng ml−1 at t  = 190 min; a ∼50% increase in 10 min. Extrapolation of these results to humans suggests the feasibility of delivering therapeutic amounts of zolmitriptan at faster rates than those from existing dosage forms.
Keywords: Transdermal iontophoresis; Migraine; Zolmitriptan; In vivo;

Epidermal lipids and skin morphology are assumed to substantially influence skin permeability. Although these parameters have been studied extensively, available data are hard to interpret as data have been gathered at different experimental conditions. Therefore, the aim of this study was to provide detailed information on these parameters for four different mammalian skin types.Lipids were extracted from heat separated epidermis, the total epidermal lipid content was measured and the epidermal lipid composition was quantitatively determined by high-performance thin-layer chromatography. Furthermore, vertical and horizontal cryostate skin slices were analysed by light microscopy for thickness of the horny layer, epidermal thickness, density, depth of anchorage and diameter of the hair follicles.The highest total epidermal lipid content was detected in rat epidermis, followed by bovine udder, dog and pig epidermis. Considering the amount of single lipid fractions, cholesterol, cholesteryl ester and free fatty acids were found to be the major constituents of epidermal lipids in all the examined species. However, as confirmed by hierarchical cluster analysis the epidermal lipid profile and morphology showed marked differences between all the examined species.
Keywords: Skin; Lipids; Morphology; Cattle; Dog; Rat; Pig; Stratum corneum; Epidermis; Hair follicle;

The tape stripping procedure – evaluation of some critical parameters by J. Lademann; U. Jacobi; C. Surber; H.-J. Weigmann; J.W. Fluhr (317-323).
Tape stripping is a simple and efficient method for the assessment of quality and efficacy of cosmetical and dermatological formulations. After topical application and penetration of formulations, the cell layers of the stratum corneum are successively removed from the same skin area using adhesive films. The tape strips contain the amount of corneocytes and the corresponding amount of the penetrated formulation, which can be determined by classical analytical chemical methods. Different formulations can strongly influence the amount of stratum corneum removed with every tape strip. Therefore, it is essential for the comparison of the penetration of different formulations that the amount of formulation detected on the single tape strip is not related to the tape strip number as a relative measure of the penetration depths, but to their standardized real position in the stratum corneum. Therefore, different methods are reported for the determination of the amount of stratum corneum removed with every tape strip.The tape stripping method in its standardized form is well-suited to determine the dermatopharmacokinetics of topically applied substances. Additionally, the method can be used to obtain information about the homogeneity and the distribution of formulations on the skin and in the stratum corneum. This is used, e.g., for the determination of the homogeneity of the distribution and the ex vivo determination of a universal sun protection factor (USPF) characterizing the efficacy of sunscreens.
Keywords: Penetration; Stratum corneum; Formulation; Homogeneity of the distribution; Skin structure;

There is a growing demand for skin penetration and permeation data considering toxicological and potential drug delivery aspects for an increasing number of substances. Although there are official guidelines available, results from different skin diffusion studies are often inconsistent and sometimes even controversial. The aim of our study is to address and to investigate the influence of experimental parameters as well as mathematical problems for subsequent evaluation of the permeation raw data. To create a reliable database diffusion experiments across human stratum corneum were performed under highly standardized conditions. The experimental data were evaluated using linear and non-linear regression analysis to determine the influence on the permeability coefficient and the lag-time. Additionally, the influence of two critical experimental parameters, temperature and unstirred water layers, on the permeability was investigated in silico. Based on our results we suggest that the influence of temperature on the permeability coefficient is small compared to the effect of other experimental parameters. Thickness of unstirred water layers has a tremendous effect on the permeation and may lead to underestimation of the permeability by more than 90%. Non-linear regression analysis seems to be superior compared to linear algorithms hence is advisable for evaluation of the experimental data. Our findings may help to optimize the experimental set-up and to reduce total costs for future skin diffusion testing. With regards to EU’s REACH-Initiative this will also help to create more and reliable data on safety issues of industrial materials.
Keywords: Human skin preparation; Permeation; Modelling; Unstirred water layer; Lag-time; Temperature;

The stratum corneum is the outermost layer of cells in mammalian epidermis. It is widely believed to play the key role for the barrier function of the skin. This study characterises how the cell geometry influences the permeability of the membrane. It is based on a diffusion model, which is evaluated using numerical simulation. Three different geometry concepts, i.e., ribbon, cuboid and tetrakaidekahedral type, in two and three space dimensions are compared. The results confirm that tetrakaidekahedral cells with an almost optimal surface-to-volume ratio provide a barrier, in which a minimal amount of mass is used very effectively. Additionally, the study supplies tools to quantify this and links the results to the theory of homogenization.
Keywords: Stratum corneum; Cell shape; Three-dimensional models; Permeability; Numerical simulation; Cuboid; Tetrakaidekahedra; Homogenization;

Barrier functions and paracellular integrity in human cell culture models of the proximal respiratory unit by Christine Pohl; M. Iris Hermanns; Chiara Uboldi; Michaela Bock; Sabine Fuchs; Jasmin Dei-Anang; Eckhard Mayer; Kai Kehe; Wolfgang Kummer; Charles James Kirkpatrick (339-349).
Airway epithelial cells provide a barrier to the translocation of inhaled materials. Tight (TJ) and adherens junctions (AJ) play a key role in maintaining barrier functions, and are responsible for the selective transport of various substances through the paracellular pathway. In this study we compared a bronchial cell line (16HBE14o-) and primary bronchial cells (HBEC), both cocultivated with the fibroblast cell line Wi-38, with respect to their structural differentiation and their reaction to cytokine stimulation. HBEC formed a pseudostratified epithelial layer and expressed TJ and AJ proteins after 2 weeks in coculture. Mucus-producing and ciliated cells were found within 24 days. Additionally, a beating activity of the ciliated HBEC (14–19 Hz) could be detected. 16HBE14o- in coculture showed a multilayered growth without differentiation to a pseudostratified airway epithelium. Simultaneous exposure to TNF-α- and IFN-γ-induced significant changes in barrier function and paracellular permeability in the cocultures of HBEC/Wi-38 but not in the 16HBE14o-/Wi-38. In summary, HBEC in coculture mimic the structure of native polarized bronchial epithelium showing basal, mucus-producing and ciliated cells. Our system provides an opportunity to examine the factors that influence barrier and mucociliary function of bronchial epithelium within a time frame of 3 weeks up to 3 months in an in vivo-like differentiated model.
Keywords: Primary bronchial cells; Coculture; Ciliary beat frequency; Lung; Barrier disruption;

A novel cell compatible impingement system to study in vitro drug absorption from dry powder aerosol formulations by Michael Bur; Barbara Rothen-Rutishauser; Hanno Huwer; Claus-Michael Lehr (350-357).
A modified Astra type multistage liquid impinger (MSLI) with integrated bronchial cell monolayers was used to study deposition and subsequent drug absorption on in vitro models of the human airway epithelial barrier. Inverted cell culture of Calu-3 cells on the bottom side of cell culture filter inserts was integrated into a compendial MSLI. Upside down cultivation did not impair the barrier function, morphology and viability of Calu-3 cells. Size selective deposition with subsequent absorption was studied for three different commercially available dry powder formulations of salbutamol sulphate and budesonide. After deposition without size separation the absorption rates from the aerosol formulations differed but correlated with the size of the carrier lactose particles. However, after deposition in the MSLI, simulating relevant impaction and causing the separation of small drug crystals from the carrier lactose, the absorption rates of the three formulations were identical, confirming the bioequivalence of the three formulations.
Keywords: Calu-3; Air interface deposition; Cell compatible impingement system; Multi stage liquid impinger; Transport experiment; Dry powder aerosol;

The influence of chitosan content in cationic chitosan/PLGA nanoparticles on the delivery efficiency of antisense 2′-O-methyl-RNA directed against telomerase in lung cancer cells by S. Taetz; N. Nafee; J. Beisner; K. Piotrowska; C. Baldes; T.E. Mürdter; H. Huwer; M. Schneider; U.F. Schaefer; U. Klotz; C.-M. Lehr (358-369).
Tailorable cationic chitosan/PLGA nanoparticles (CPNP) were used for the delivery of an antisense 2′-O-methyl-RNA (2OMR) directed against RNA template of human telomerase. Here, we describe the influence of the chitosan content on binding efficiency, complex stability, uptake in different human lung cell types and finally demonstrate the efficacy of this nanoplex system.CPNPs were prepared by the emulsion-solvent evaporation method using different amounts of chitosan and purified by preparative size exclusion chromatography. The characterization by photon correlation spectroscopy and zeta potential measurements showed a small increase in size and an increase of zeta potential with increasing amounts of chitosan. Binding efficiency and complex stability with 2OMR was high in water and correlated well with the chitosan content of particles but was weak in physiologically relevant media (PBS and RPMI cell culture medium). However, flow cytometry analysis showed that the uptake of 2OMR into A549 lung cancer cells was considerably higher in combination with nanoparticles and dependent on the amount of chitosan when compared to 2OMR alone. Confocal laser scanning microscopy revealed that the uptake into A549 cells is mediated via complexes of 2OMR and chitosan/PLGA nanoparticles despite the weak binding in cell culture medium. The nanoparticles were well tolerated and efficient in inhibiting telomerase activity.
Keywords: Chitosan PLGA nanoparticles; Antisense 2′-O-methyl-RNA; Telomerase; Lung cancer; A549; Calu-3; Primary human alveolar epithelial cells;

Current in vitro methods in nanoparticle risk assessment: Limitations and challenges by Alexandra Kroll; Mike H. Pillukat; Daniela Hahn; Jürgen Schnekenburger (370-377).
Nanoparticles are an emerging class of functional materials defined by size-dependent properties. Application fields range from medical imaging, new drug delivery technologies to various industrial products. Due to the expanding use of nanoparticles, the risk of human exposure rapidly increases and reliable toxicity test systems are urgently needed. Currently, nanoparticle cytotoxicity testing is based on in vitro methods established for hazard characterization of chemicals. However, evidence is accumulating that nanoparticles differ largely from these materials and may interfere with commonly used test systems. Here, we present an overview of current in vitro toxicity test methods for nanoparticle risk assessment and focus on their limitations resulting from specific nanoparticle properties. Nanoparticle features such as high adsorption capacity, hydrophobicity, surface charge, optical and magnetic properties, or catalytic activity may interfere with assay components or detection systems, which has to be considered in nanoparticle toxicity studies by characterization of specific particle properties and a careful test system validation. Future studies require well-characterized materials, the use of available reference materials and an extensive characterization of the applicability of the test methods employed. The resulting challenge for nanoparticle toxicity testing is the development of new standardized in vitro methods that cannot be affected by nanoparticle properties.
Keywords: Nanoparticles; Nanoparticle properties; In vitro toxicology; Particle assay interference; New toxicology detection systems;

Hydrosoluble polymers for muscular gene delivery by Caroline Roques; Yves Fromes; Elias Fattal (378-390).
Striated muscle tissue is an attractive target for gene delivery as it can be easily reached and can express exogenous proteins. However, administration of naked DNA results in low transfection levels, and the design and development of safe and efficient gene delivery systems are thus required.This review is focusing on the characteristics of the striated muscle tissue with regards to features possibly affecting gene transfer, as well as the different soluble polymers that have been evaluated as gene carriers. The described formulations are ranging from polymers displaying a high density of positive charges to non-ionic molecules. Nevertheless, polymers exhibiting few or no positive charges appear to our opinion as the most promising approach to achieve both safe and efficient transfection of the striated muscles.
Keywords: Gene delivery; Hydrosoluble polymers; Cationic polymers; Amphiphilic copolymers; Striated muscles;

Fluorescence in situ hybridization to monitor the intracellular location and accessibility of plasmid DNA delivered by cationic polymer-based gene carriers by K.J. Wilschut; M.A.E.M. van der Aa; R.S. Oosting; W.E. Hennink; G.A. Koning; D.J.A. Crommelin; E. Mastrobattista (391-396).
Information about the intracellular trafficking of exogenous DNA delivered by nonviral gene delivery systems is of major importance for optimization of such gene carriers. We used fluorescence in situ hybridization (FISH) as a tool to visualize polyplex-delivered pDNA inside cells. This avoids the need to directly label DNA inside the polyplexes, which may influence their cellular behavior and fate. Using FISH the introduced plasmid DNA could be detected in the cytosol and nucleus of different cell lines. The FISH probe itself did not interact with cells nor different polymers used for condensing the DNA. We further demonstrate differences in accessibility of polyplex-delivered DNA when different polymers were used for DNA complexation. Therefore, FISH is a valuable tool to detect location and accessibility of exogenous plasmid DNA delivered in the cell by cationic polymers.
Keywords: Nonviral gene delivery; Polymers; DNA; Plasmid; Fluorescence;

A high relaxivity Gd(III)DOTA-DSPE-based liposomal contrast agent for magnetic resonance imaging by Sjoerd Hak; Honorius M.H.F. Sanders; Prashant Agrawal; Sander Langereis; Holger Grüll; Henk M. Keizer; Francesca Arena; Enzo Terreno; Gustav J. Strijkers; Klaas Nicolay (397-404).
The field of molecular imaging aims to visualize and quantify (patho)physiological processes at the cellular and molecular level. Sensitive and site-targeted contrast agents are employed to visualize molecular constituents of processes of interest. The principal aim of this study was to develop a magnetic resonance imaging (MRI) detectable liposome with high relaxivity and stability. To this end, Gd(III)DOTA-DSPE was synthesized and incorporated in a liposomal formulation. The resulting liposomes were extensively characterized in vitro in terms of contrast agent efficiency and structural properties. The liposomes were shown to have a high longitudinal relaxivity, which is crucial for the detection of low concentration molecular markers in molecular imaging studies. We also demonstrated that Gd(III)DOTA-DSPE exhibits no detectable transmetallation upon incubation with Zn(II). This is important as it significantly contributes to the biocompatibility of the contrast agent. The present liposome preparation will serve as versatile and well characterized platform for molecular imaging and targeted drug delivery studies.
Keywords: Molecular imaging; Magnetic resonance imaging; Contrast agent; Liposomes; Gadolinium; Gd(III)DOTA-DSPE; Longitudinal relaxivity; cryo-TEM; Transmetallation;

Interferon-α2b (IFN-α2b) and human serum albumin (HSA) fusion protein (IFN-α2b–HSA) is a promising long acting formulation of IFN-α2b for the treatment of hepatitis C. However, accelerated mechanical and thermal stress tests revealed that IFN-α2b–HSA was prone to disulfide-linked aggregation. The formation of aggregates was associated with an increase in immunogenicity in mice. The addition of non-ionic surfactant Tween 80 increased the stability of IFN-α2b–HSA against agitation, but its thermal stability was not improved. Moreover, Tween 80 prompted the aggregation of IFN-α2b–HSA during quiescent storage. To increase the stability of IFN-α2b–HSA, the unpaired cysteine residue in this fusion protein was substituted with serine by site-directed mutagenesis. The resultant fusion protein was designated as IFN-α2b–HSA(C34S). IFN-α2b–HSA(C34S) had significant higher stability over IFN-α2b–HSA, which was evidenced by the facts that after agitation for 72 h or incubation at 60 °C for 2 h, more than 90% of IFN-α2b–HSA(C34S) remained monomeric. Consistent with its improved stability, the immunogenicity of IFN-α2b–HSA(C34S) increased less significantly after agitation. Pharmacokinetics studies in rats revealed that both fusion proteins had similar pharmacokinetic behavior, both with a half-life of about 50 h.
Keywords: Human serum albumin fusion protein; Protein aggregation; Protein engineering;

Preparation and PEGylation of exendin-4 peptide secreted from yeast Pichia pastoris by Jin Zhou; Zhong-Hua Cai; Lei Li; Chuang Kou; Yun-Feng Gao (412-417).
Exendin-4, a peptide analogue of glucagon-like peptide (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 peptide, expressed by methanol induction in Pichia pastoris, was purified to near homogeneity by Ni–NTA agarose chromatography. 103.6 mg of protein was obtained from 1 L of the supernatant and its purity was 96.1%. Subsequently, the PEGylated exendin-4 was prepared. The bioactivity of exendin-4 was determined by examining the glucose-lowering and insulin-releasing ability in plasma. Then, a safety evaluation was performed by histological examination of the main organs (liver, kidney and pancreas). PEGylated exendin-4 displayed glucose-lowering and insulin-stimulating action in vivo without obvious damage to the above organs. The results suggest that the P. pastoris expression could be used to produce large quantities of exendin-4, and PEGylation is a useful tool to maintain and enhance bioactivity of the peptide.
Keywords: Exendin-4; Pichia pastoris; PEGylation; Bioactivity; Histological;

Functionalized nanospheres loaded with anti-angiogenic drugs: Cellular uptake and angiosuppressive efficacy by Taha Hammady; Jean-Michel Rabanel; Renu Singh Dhanikula; Grégoire Leclair; Patrice Hildgen (418-427).
The objective of this study was to develop polymeric nanospheres (NPs) that are able to selectively target the activated vascular endothelium and to deliver co-encapsulated anti-angiogenic agents for improved treatment efficacy in inflammatory diseases with an angiogenic component. We evaluated a novel poly(d,l)-lactide (PLA)-based polymer, grafted with a synthetic ligand specific for selectin (PLA -g-SEL ), for the preparation of functionalized NPs. The NPs were produced according to a double emulsion–solvent diffusion/evaporation method, allowing the co-encapsulation of hydrophilic and lipophilic drugs.Incorporation of the functionalized polymer enhanced the internalization of fluorescein-labeled NPs by lipopolysaccharide-activated vascular endothelial cells relative to control NPs, as evidenced by confocal laser scanning microscopy and quantitative fluorescence measurements. Two anti-angiogenic agents, endostatin and paclitaxel, were co-loaded in the functionalized NPs. Respective drug loadings were optimized by adjusting polymer composition, as well as by the microemulsion technique.NPs loaded with either of the chosen drugs or with a combination of them were tested for their anti-angiogenic efficacy in human umbilical vascular endothelial cell (HUVEC) culture in vitro and rat aorta tissue culture ex vivo models. An enhanced anti-proliferative effect on HUVECs and heightened anti-angiogenic action on rat aorta ring cultures was observed for the loaded drugs compared to the free molecules. Moreover, combined loaded treatments were found to be more potent, evoking additive and even synergetic outcomes (at lower doses) greater than the corresponding single-loaded treatments in inhibiting new vessels sprouting in rat aortic rings.
Keywords: Functionalized nanospheres; Co-encapsulation; Internalization; HUVECs; Rat aortic ring; Angiogenesis; Selectin; Paclitaxel; Endostatin;

Physico-chemical characterisation of PLGA nanoparticles after freeze-drying and storage by Melisande Holzer; Vitali Vogel; Werner Mäntele; Daniel Schwartz; Winfried Haase; Klaus Langer (428-437).
Nanoparticles represent promising carriers for controlled drug delivery. Particle size and size distribution of the particles are important parameters for the in vivo behaviour after intravenous injection and have to be characterised precisely. In the present study, the influence of lyophilisation on the storage stability of poly(d,l lactic-co-glycolic acid) (PLGA) nanoparticles, formulated with several cryoprotective agents, was evaluated. Nanoparticles were prepared by a high pressure solvent evaporation method and freeze-dried in the presence of 1%, 2%, and 3% (m/v) sucrose, trehalose, and mannitol, respectively. Additionally, to all samples containing 3% of the excipients, l-arginine hydrochloride was added in concentrations of 2.1% or 8.4% (m/V). Dynamic light scattering (DLS), analytical ultracentrifugation and transmission electron microscopy (TEM) were used for particle characterisation before and after freeze-drying and subsequent reconstitution. In addition, glass transition temperatures were determined by differential scanning calorimetry (DSC), and the residual moisture of the lyophilisates was analysed by Karl Fischer titration. It was demonstrated that 1% sucrose or 2% trehalose were suitable to maintain particle integrity after reconstitution of lyophilised PLGA nanoparticles. The storage stability study over 3 months showed notable changes in mean particle size, size distribution, and residual moisture content, depending on the composition of the formulation.
Keywords: Poly(d,l lactic-co-glycolic acid) (PLGA) nanoparticles; Freeze-drying; Storage stability; Analytical ultracentrifugation; Dynamic light scattering; Electron microscopy; DSC; Moisture content;

Poly(vinyl alcohol) nanoparticle stability in biological media and uptake in respiratory epithelial cell layers in vitro by M. Madlova; S.A. Jones; I. Zwerschke; Y. Ma; R.C. Hider; B. Forbes (438-443).
The influence of the size and surface properties of nanoparticles (NP) upon respiratory epithelial cell uptake and translocation is difficult to study, because NP properties are often modified upon suspension in biological fluids. However, a recently developed novel fluorescently labelled poly(vinyl alcohol) (PVA) NP, which does not aggregate in simple biological fluids, is suitable for drug delivery and can be produced with a range of surface properties is a pertinent advance in this field. The aim of this study was to employ the PVA NP to investigate how surface properties influence particle uptake and translocation across Calu-3 epithelial cell layers. Several grades of PVA were synthesised, characterised and labelled covalently with carboxyfluorescein. The labelled PVA was used to fabricate trackable NP that displayed either neutral or positive charge when suspended in Hank’s Balanced Salt Solution. The NP were applied to the apical surface of Calu-3 cell layers which internalised up to 11% of the applied particle dose. The maximum fraction that translocated the Calu-3 barrier in 14 h was 1.3%.
Keywords: Nanoparticle; Airways; Drug delivery; Calu-3; Translocation; Uptake;

Formulation of ascorbic acid microemulsions with alkyl polyglycosides by N. Pakpayat; F. Nielloud; R. Fortuné; C. Tourne-Peteilh; A. Villarreal; I. Grillo; B. Bataille (444-452).
Ascorbic acid microemulsions for topical application were developed. In this study, microemulsions were prepared using HLD (hydrophilic lipophilic deviation) concept to optimise the formulation. From this optimal formulation, the realisation of dilution ternary diagrams leads to obtain microemulsion zones. In addition, the effects of composition variable on the physicochemical characteristics of each system were investigated. After optimisation of the microemulsion systems, ascorbic acid was loaded in the formulations. Surface tension and small angle neutron scattering were used to characterise the surface properties and the structure of the microemulsions. Bicontinuous structure microemulsions were identified, and the influence of ascorbic acid localisation at the interface leading to modifications of the microemulsion structure was pointed out. The solubilisation of ascorbic acid, the stabilisation and in vitro transdermal penetration “Frantz cells” of ascorbic acid microemulsions were studied. Three different microemulsions were envisaged. The results confirmed that these microemulsion systems present a real interest for formulation and protection of ascorbic acid. Regarding their transcutaneous penetration behaviour, the different microemulsions studied could be useful for different topical applications. A major location of ascorbic acid found in the epidermis where the decomposition of melanin occurred indicates that microemulsion could be considered as a suitable carrier system for application of ascorbic acid as a whitening agent. In addition, a good passage of the drug in the dermis could be interesting for the relative oxygen matrix damage.
Keywords: Microemulsions; SANS; Alkyl polyglycosides; Ascorbic acid; Microstructure; Topical application;

Glucomannan, a promising polysaccharide for biopharmaceutical purposes by M. Alonso-Sande; D. Teijeiro-Osorio; C. Remuñán-López; M.J. Alonso (453-462).
Over the last few decades, polysaccharides have gained increasing attention in the biomedical and drug delivery fields. Among them, glucomannan (GM), has become a particularly attractive polymer. In this paper, we review the physicochemical and biological properties which are decisive for the exploitation of GM as a biomaterial. These properties include the structural organization, molecular weight, solubility, viscosity, gelling properties and degradation behavior. Moreover, herein we analyze the possibilities of combining GM with other hydrophilic polymers, as well as the preparation of semisynthetic derivatives of GM, which may be of interest in the pharmaceutical context. Finally, we discuss the specific applications of GM in the drug delivery field.
Keywords: Glucomannan; Polysaccharides; Gelation; Colloidal systems; Nanoparticles; Nanocomplexes; Drug delivery;

Mini-matrices with release-sustaining properties were developed by hot-melt extrusion (diameter 3 mm, height 2 mm) using metoprolol tartrate as model drug (30%, w/w) and ethylcellulose as sustained-release agent. Polyethylene glycol or polyethylene oxide was added to the formulation to increase drug release. Changing the hydrophilic polymer concentration (0%, 1%, 2.5%, 5%, 10%, 20% and 70%, w/w) and molecular weight (6000, 100,000, 1,000,000 and 7,000,000) modified the in vitro drug release: increasing concentrations yielded faster drug release (irrespective of molecular weight), whereas the influence of molecular weight depended on concentration. Smooth extrudates were obtained when processed at 40 and 70 °C for polyethylene glycol and polyethylene oxide formulations, respectively. Raman analysis revealed that metoprolol tartrate was homogeneously distributed in the mini-matrices, independent of hydrophilic polymer concentration and molecular weight. Also drug and polymer crystallinity were independent of both parameters. An oral dose of 200 mg metoprolol tartrate was administered to dogs in a randomized order either as immediate-release preparation (Lopresor® 100), as sustained-release formulation (Slow-Lopresor® 200 Divitabs®), or as experimental mini-matrices (varying in hydrophilic polymer concentration). The sustained-release effect of the experimental formulations was limited, and relative bioavailabilities of 66.2% and 148.2% were obtained for 5% and 20% PEO 1,000,000 mini-matrices, respectively.
Keywords: Hot-melt extrusion; Sustained-release; Multiple-unit dosage form; Matrix system; Polyethylene glycol; Polyethylene oxide; Ethylcellulose; Metoprolol tartrate;

Evaluation of the transport, in vitro metabolism and pharmacokinetics of Salvinorin A, a potent hallucinogen by Zeynep S. Teksin; Insong J. Lee; Noble N. Nemieboka; Ahmed A. Othman; Vijay V. Upreti; Hazem E. Hassan; Shariq S. Syed; Thomas E. Prisinzano; Natalie D. Eddington (471-477).
Salvinorin A is an unregulated potent hallucinogen isolated from the leaves of Salvia divinorum. It is the only known non-nitrogenous kappa-opioid selective agonist, and rivals synthetic lysergic acid diethylamide (LSD) in potency. The objective of this study was to characterize the in vitro transport, in vitro metabolism, and pharmacokinetic properties of Salvinorin A. The transport characteristics of Salvinorin A were assessed using MDCK-MDR1 cell monolayers. The P-glycoprotein (P-gp) affinity status was assessed by the P-gp ATPase assay. In vitro metabolism studies were performed with various specific human CYP450 isoforms and UGT2B7 to assess the metabolic characteristics of Salvinorin A. Cohorts (n  = 3) of male Sprague Dawley rats were used to evaluate the pharmacokinetics and brain distribution of Salvinorin A (10 mg/kg, intraperitoneal (i.p.) over a 240-min period. A validated UV-HPLC and LC/MS/MS method was used to quantify the hallucinogen concentrations obtained from the in vitro and in vivo studies, respectively. Salvinorin A displayed a high secretory transport in the MDCK-MDR1 cells (4.07 ± 1.34 × 10−5  cm/s). Salvinorin A also stimulated the P-gp ATPase activity in a concentration (5 and 10 μM)-dependent manner, suggesting that it may be a substrate of (P-gp). A significant decrease in Salvinorin A concentration ranging from 14.7 ± 0.80% to 31.1 ± 1.20% was observed after incubation with CYP2D6, CYP1A1, CYP2C18, and CYP2E1, respectively. A significant decrease was also observed after incubation with UGT2B7. These results suggest that Salvinorin A maybe a substrate of UGT2B7, CYP2D6, CYP1A1, CYP2E1, and CYP2C18. The in vivo pharmacokinetic study showed a relatively fast elimination with a half-life (t 1/2) of 75 min and a clearance (Cl/F) of 26 L/h/kg. The distribution was extensive (Vd of 47.1 L/kg); however, the brain to plasma ratio was 0.050. Accordingly, the brain half-life was relatively short, 36 min. Salvinorin A is rapidly eliminated after i.p. dosing, in accordance with its fast onset and short duration of action. Further, it appears to be a substrate for various oxidative enzymes and multi-drug resistant protein, P-gp.
Keywords: Salvinorin A; Salvia divinorum; Pharmacokinetics; Metabolism; Transport; Blood–brain barrier; Hallucinogen;