European Journal of Pharmaceutics and Biopharmaceutics (v.67, #2)

APV Diary (S1-S3).

Preparation and characterization of a potent, long-lasting recombinant human serum albumin-interferon-α2b fusion protein expressed in Pichia pastoris by Yan-Shan Huang; Zhi Chen; Zhi-Yu Yang; Tong-Ying Wang; Li Zhou; Jian-Bing Wu; Lin-Fu Zhou (301-308).
A long-lasting recombinant human serum albumin-interferon-α2b fusion protein (rHSA/IFNα2b) was prepared and its structure and biological activities were studied. rHSA/IFNα2b was expressed in methylotrophic yeast Pichia pastoris with HSA’s natural signal peptide and purified by dye affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography and Sephadex G25. Purity of the prepared rHSA/IFNα2b was greater than 97% analyzed by non-reduced SDS–PAGE and RP-HPLC. Structure and biological activities of the prepared rHSA/IFNα2b were characterized by physical, chemical and biological methods. Its pI was 5.3 and showed a single band on IEF gel. Molecular weight determined by MALDI-TOF was 86004.3 ± 29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was 6.3 ± 0.8 × 105  IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNα on a molar basis. After administered in monkeys, significant increases of 2′,5′-oligoadenylate synthetase activity relative to IFN-α were maintained for 14 days in serum and the rHSA/IFNα2b showed more potent biological activity than IFN-α on a molar basis. Therefore, markedly improved in vivo biological activity of rHSA/IFNα2b could exhibit more potent antiviral activity than IFNα2b in future clinical trials.
Keywords: Bioactivity; Characterization; Peptide mapping; Pichia pastoris; Purification; Recombinant human serum albumin-interferon-α2b;

Solution formulation and lyophilisation of a recombinant fibronectin fragment by P. Pereira; S.M. Kelly; A. Cooper; H.J. Mardon; P.R. Gellert; C.F. van der Walle (309-319).
The 9th–10th type III fibronectin domain pair shows promise in tissue engineering and tumour vasculature targeting. Calorimetry and structure–function analysis were used to investigate the effects of solution formulation and lyophilisation of a mutant (9–10FNIII-P). A single endothermic transition for 9–10FNIII-P in solution was observed at pH < 8, irrespective of addition of sucrose or PEG. The temperature at the maximum heat capacity (T m) and enthalpy (ΔH) of the transition increased for increasing sucrose concentrations but decreased for increasing PEG concentrations. The transition was fitted to a single two-state unfolding mechanism (in contrast to unfolding in guanidine·HCl) and was partially reversible only at pH 4, with increasing concentrations of sucrose causing a marked fall in ΔH between scans. Circular dichroism spectra for the thermal unfolding of 9–10FNIII-P at pH 4 showed loss of native β-sheet structure and loss of aromatic contributions to the peak centred around 226 nm yielding an intermediate conformation, which in the presence of sucrose was more disordered. Despite a glass transition ( T g ′ ) for 9–10FNIII-P(aq) of −70 °C, primary drying at −30 °C did not perturb its conformation upon reconstitution or its biological activity following lyophilisation; the addition of sucrose or PEG had no influence on structure or activity. The main consideration in the formulation of 9–10FNIII-P was therefore pH.
Keywords: Fibronectin; Thermal stability; Calorimetry; Circular dichroism; Cell adhesion; Formulation;

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.
Keywords: Solid lipid nanoparticles; Freeze-drying; Gel electrophoresis; Differential scanning calorimetry; Atomic force microscopy; pEGFP-plasmid;

Long-circulating poly(ethylene glycol)-coated emulsions to target solid tumors by Joanna Rossi; Suzanne Giasson; Mohamed Nabil Khalid; Pascal Delmas; Christine Allen; Jean-Christophe Leroux (329-338).
The purpose of this study was to develop oil-in-water emulsions (100–120 nm in diameter) and to correlate the surface properties of the emulsions with blood residence time and accumulation into neoplastic tissues by passive targeting. We investigated the effect of phospholipid and sphingolipid emulsifiers, hydrogenated soybean phosphatidylcholine (HSPC) and egg sphingomyelin (ESM), in combination with polysorbate 80 (PS-80) and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE)-PEG lipids of various PEG chain lengths and structures in prolonging circulation time and enhancing accumulation into B16 melanoma or C26 colon adenocarcinoma. The relationship between amphiphile molecular packing at the air/water interface on emulsion stability upon dilution in albumin and circulation longevity in vivo was also explored for non-PEGylated emulsions. PEGylation of the droplet surface with 10–15 mol% of DSPE-PEG 2000 or 5000 enhanced the circulation time of the emulsions, however, accumulation was only observed in the C26 tumor model. The tighter molecular packing observed with ESM/PS-80 monolayers at the air/water interface compared to HSPC/PS-80 correlated with improved emulsion stability in vitro, however, enhanced circulation time in vivo was not observed. A better understanding of the relationships between composition and performance will result in improved emulsion-based drug delivery vehicles for cancer therapy.
Keywords: Long-circulating emulsions; Poly(ethylene glycol); Biodistribution; Pharmacokinetics; Drug carriers;

Cross-linking of chitosan and chitosan/poly(ethylene oxide) beads: A theoretical treatment by Leticia Martinez; Florence Agnely; Bernard Leclerc; Juergen Siepmann; Marine Cotte; Sandrine Geiger; Guy Couarraze (339-348).
The major aim of this study was to get deeper insight into the process of polymer cross-linking and the resulting structure of beads based on chitosan (CS) or chitosan/poly(ethylene oxide) (CS/PEO) semi-interpenetrating networks (semi-IPNs) as new carrier materials for oral drug delivery. Spherical hydrogels were prepared by a dropping method. The uptake kinetics of the cross-linking agent glyoxal into the beads were monitored and quantitatively described using Fick’s second law of diffusion. High-resolution synchrotron infrared microspectroscopy (SIRM) was used to characterize the inner structures of the beads. Importantly, the diffusion of glyoxal through the hydrogels was found to be much slower than the cross-linking reaction and the mesh size of the created networks to be much larger than the hydrodynamic diameter of glyoxal. The presence of PEO chains slightly decreased the diffusivity of glyoxal due to obstruction effects. However, the cross-linking reaction was not affected. Interestingly, the polymers were homogeneously cross-linked throughout the beads, except for a thin outer shell showing an elevated cross-linking density. Thus, the obtained cross-linked hydrogel-based beads exhibit well-defined polymeric structures and offer an interesting potential as novel oral drug delivery systems.
Keywords: Beads; Chitosan; Cross-linking; Diffusion; Glyoxal; Modeling; Semi-IPN;

A model describing the effect of enzymatic degradation on drug release from collagen minirods by Iris Metzmacher; Florin Radu; Markus Bause; Peter Knabner; Wolfgang Friess (349-360).
A drug delivery system, named minirod, containing insoluble non-cross-linked collagen was prepared to investigate the release of model drug compounds. To characterise the complete drug release process properly, a mathematical model was developed. Previously, a mathematical model describing water penetration, matrix swelling and drug release by diffusion from dense collagen matrices has been introduced and tested. However, enzymatic matrix degradation influences the drug release as well. Based on experimental data, a model was developed which describes drug release by collagenolytic matrix degradation based on enzyme diffusion, adsorption and cleavage. Data for swelling, collagen degradation and FITC dextran release from insoluble equine collagen type I minirods were collected. Sorption studies demonstrated a tight sorption of collagenase on collagen surfaces that follows a Freundlich sorption isotherm and results in a degradation constant of 3.8 × 10−5  mol/l for the minirods. The diffusion coefficients of FITC dextran 20 and 70 (3 × 10−3 and 2.4 × 10−3  cm2/h) in water were analyzed by fluorescence correlation spectroscopy (FCS). Using these data, the mathematical model was verified by two-dimensional simulations. The numerical results agreed well with the measurements.
Keywords: Collagen; Mathematical model; Enzymatic degradation; Enzymatic adsorption; FCS; Numerical simulation;

Coenzyme Q10 (CoQ10) is an antioxidant with well-established pharmacological activities against several chronic diseases; however, it is marketed only as a nutritional supplement without any claims of its therapeutic activity and one of the reasons for this could be the poor oral bioavailability rendering difficulties in administering this molecule to achieve therapeutic concentrations. Therefore, the present investigation was aimed at improving the oral bioavailability of CoQ10 by delivering it as nanoparticulate formulation. Biodegradable nanoparticulate formulations based on poly(lactide-co-gylcolide) (PLGA) were prepared by emulsion technique using quaternary ammonium salt didodecyldimethylammonium bromide (DMAB) as a stabilizer. The effect of initial CoQ10 loading on entrapment efficiency and the particle size was studied using 5–75% initial load resulting in good entrapment efficiency (61–83%) without any appreciable increase in the particle size for 5–30% loading (107–110 nm). However, 50% and 75% led to increase in particle size with no appreciable changes in entrapment efficiency. The intestinal uptake of CoQ10 as a suspension in carboxymethylcellulose (CMC), a commercial formulation and the developed nanoparticulate formulation was studied in male Sprague–Dawley (SD) rats and found to be 45%, 75% and 79%, respectively, suggesting that solubility and permeability related problems of CoQ10 were overcome by nanoparticulate formulation. Furthermore, the developed nanoparticulate formulation was evaluated for its therapeutic potential in renal hypertensive animals (Goldblatt 2K1C model), demonstrating improved efficacy at a 60% lowered dose as compared to CoQ10 suspension and superior efficacy than the commercial formulation at an equal dose. Together, these results indicate the potential of nanotechnology in improving the therapeutic value of molecules like CoQ10, facilitating its usage as first line therapeutic agent thus revolutionizing its role in current medical therapy.
Keywords: Antioxidants; Bioavailability; Biodegradable; Free radical; Hypertension; Nanoparticles; Oral delivery;

The aim of this study was to investigate whether mucoadhesive interactive mixtures can be created using carrier particles in a size range appropriate for nasal administration, i.e. 10–50 μm. We also used theoretical models to investigate if homogeneity measurements can be used to evaluate the formation of interactive mixtures containing carrier particles in this size range. Sodium starch glycolate (SSG) was used as carrier material and sodium salicylate (SS) as the model fine-particulate drug. The size ranges of SSG particles and amounts of SS were varied to find the smallest carrier particle size and highest amount of drug that still resulted in an interactive mixture. Visual inspection of the mixtures by scanning electron microscopy showed that interactive mixtures could be formed with carrier particles as small as 30 μm and containing up to 4% (w/w) of SS. Comparisons with theoretical models highlighted the difficulties of using homogeneity measurements to determine if interactive mixtures were formed. The measured coefficients of variation (CV) for the amount of drug in the samples were low and inferior mixtures were associated with only a slight increase. It was thus concluded that mucoadhesive interactive mixtures can be created in an appropriate size range for nasal administration, but that visual inspection of these mixtures is initially necessary to confirm the formation of an interactive mixture.
Keywords: Interactive mixtures; Ordered mixtures; Mucoadhesion; Nasal drug delivery; Sodium starch glycolate; Primojel®;

We describe a drug delivery system based on a physically cross-linked poly(vinyl alcohol) (PVA) hydrogel for the release of Theophylline (TH). A composite was created by freezing an aqueous solution of PVA/NaOH onto a PVA/poly(acrylic acid) substrate. This formed a strong interface and demonstrated greater physical strength than the hydrogel alone. Such systems have potential for a variety of localised controlled drug delivery applications, for example, as coatings for implantable devices. Importantly, the results suggest that a versatile synthetic platform is possible that may provide different functional materials or combination of such. The resultant samples were characterised using optical microscopy, modulated differential scanning calorimetry (MDSC) and dissolution testing. The microstructure of the gels was examined using micro-thermal analysis (μTA) which is a combination of atomic force microscopy and thermal analysis. TH was found to have an effect on the crystalline structure and dissolution showed a Fickian release, suggesting that swelling and crystallinity were the controlling mechanisms.
Keywords: Micro-thermal analysis; Drug release; Poly(vinyl alcohol); Freeze–thaw cycle;

The present contribution reports the use of mats of electrospun cellulose acetate (CA; acetyl content = 39.8%; M w  = 30,000 Da) nanofibers as carriers for delivery of the model vitamins, all-trans retinoic acid or vitamin A acid (Retin-A) and α-tocopherol or vitamin E (Vit-E). The amounts of Vit-E and Retin-A loaded in the base CA solution [17% w/v in 2:1 v/v acetone/N,N-dimethylacetamide (DMAc)] were 5 and 0.5 wt% (based on the weight of CA), respectively. Cross-sectionally round and smooth fibers were obtained. The average diameters of these fibers ranged between 247 and 265 nm. The total immersion of the vitamin-loaded as-spun CA fiber mats in the acetate buffer solutions containing either 0.5 vol % Tween 80 or 0.5 vol % Tween 80 and 10 vol % methanol was used to arrive at the cumulative release of the vitamins from the fiber mat samples. The same was also conducted on the vitamin-loaded solution-cast CA films for comparison. In most cases, the vitamin-loaded as-spun fiber mats exhibited a gradual and monotonous increase in the cumulative release of the vitamins over the test periods (i.e., 24 h for Vit-E-loaded samples and 6 h for Retin-A-loaded ones), while the corresponding as-cast films exhibited a burst release of the vitamins.
Keywords: Electrospinning; Nanofibers; Cellulose acetate; Transdermal drug delivery; Dermal drug delivery;

The current investigation aims to evaluate the transdermal potential of novel ethanolic liposomes (ethosomes) bearing Melatonin (MT), an anti-jet lag agent associated with poor skin permeation and long lag time. MT loaded ethosomes were prepared and characterized for vesicular shape and surface morphology, vesicular size, entrapment efficiency, stability, in vitro skin permeation and in vivo skin tolerability. Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Dynamic Light Scattering (DLS) defined ethosomes as spherical, unilamellar structures having low polydispersity (0.032 ± 0.011) and nanometric size range (122 ± 3.5 nm). % Entrapment efficiency of MT in ethosomal carrier was found to be 70.71 ± 1.4. Stability profile of prepared system assessed for 120 days revealed very low aggregation and growth in vesicular size (7.6 ± 1.2%). MT loaded ethosomal carriers also provided an enhanced transdermal flux of 59.2 ± 1.22 μg/cm2/h and decreased lag time of 0.9 h across human cadaver skin. Fourier Transform-Infrared (FT-IR) data generated to assess the fluidity of skin lipids after application of formulation revealed a greater mobility of skin lipids on application of ethosomes as compared to that of ethanol or plain liposomes. Skin permeation profile of the developed formulation further assessed by confocal laser scanning microscopy (CLSM) revealed an enhanced permeation of Rhodamine Red (RR) loaded formulations to the deeper layers of the skin (240 μm). Further, a better skin tolerability of ethosomal suspension on rabbit skin suggested that ethosomes may offer a suitable approach for transdermal delivery of melatonin.
Keywords: Melatonin; Ethosomes; Confocal laser scanning microscopy; Skin irritancy;

Effects of ethanol/l-menthol on the dynamics and partitioning of spin-labeled lipids in the stratum corneum by Jorge Luiz Vieira dos Anjos; Diógenes de Sousa Neto; Antonio Alonso (406-412).
The interaction of ethanol as well as ethanol/l-menthol mixtures with the uppermost layer of epidermis, the stratum corneum, was investigated by electron paramagnetic resonance (EPR) spectroscopy utilizing spin-labeled analogs of androstanol (ASL), stearic acid (5-DSA) and methyl stearate (5-DMS). The EPR spectra of these spin probes structured in stratum corneum tissue of neonatal rat are characterized by the coexistence of two spectral components indicating the presence of two classes of spin labels with very different states of mobility. Probably, one class of spin labels is H-bonded to the polar surface of the membrane and another class corresponds to spin labels more deeply inserted in the hydrophobic core. EPR results showed that in the ethanol range 0–70% neither fluidity in stratum corneum membranes nor the relative fractions of these two components changes were observed. Instead, ethanol only caused a selective extraction of spin labels. The removal of the steroid ASL began at 30% ethanol, reaching extraction levels over 50% at ethanol concentrations of 60–70%, whereas the more hydrophobic 5-DMS was partially removed only with 70% ethanol. Addition of 5% l-menthol to the solvent containing 20% ethanol increases both the mobility and the fraction of those spin labels situated in the hydrophobic core (more mobile spectral component). Altogether, these findings suggest that the l-menthol stabilizes mainly in the central region of stratum corneum membranes attracting the membrane lipids and causing hydrogen bond ruptures in the polar membrane interface.
Keywords: Stratum corneum; EPR; Spin label; l-Menthol; Ethanol;

Effects of cinnamene enhancers on transdermal delivery of ligustrazine hydrochloride by Chun-feng Zhang; Zhong-lin Yang; Jia-bo Luo; Quan-hong Zhu; Hui-nan Zhao (413-419).
Cinnamene compounds, cinnamic acid, cinnamaldehyde and cinnamic alcohol, were employed as enhancers. The effects and mechanisms of penetration promoters on the in vitro percutaneous absorption of ligustrazine hydrochloride across hairless porcine dorsal skin were investigated. Transdermal fluxes of ligustrazine hydrochloride through porcine skin were determined in vitro by Franz-type diffusion cells. The results indicated that the penetration flux of ligustrazine hydrochloride by cinnamic acid was the greatest. Significant statistical differences (P  < 0.05) were found between cinnamic acid and other promoters. Fourier transform-infrared (FT-IR) were carried out to analyze the effects of enhancers on the biophysical properties of the stratum corneum and the permeation enhancement mechanisms. FT-IR results revealed that the changes of peak shift and peak area due to C–H stretching vibrations in the stratum corneum lipids were associated with the selected enhancers. All of them could perturb and extract the stratum corneum lipids to different extent. Morphological changes of the skin treated with enhancers were monitored by a scanning electron microscope. It was demonstrated that the extraction of the stratum corneum lipids by the enhancers led to the disruption of stratum corneum and the desquamation of stratum corneum flake. Apparent density was newly proposed to estimate the desquamated extent of stratum corneum flake. Correlation analysis revealed that there was a linear relationship between apparent density and decrease in peak area. The results showed that the permeation enhancement mechanisms of cinnamene were pleiotropic ones, including disordering the lipids, extracting the lipids and competitive hydrogen bonding between cinnamene enhancers and amides of ceramide head groups in stratum corneum.
Keywords: Cinnamene enhancers; Permeation mechanism; Transdermal delivery; Ligustrazine hydrochloride;

Skin permeation of ketotifen applied from stick-type formulation by Chiharu Kimura; Toshihiro Nakanishi; Kakuji Tojo (420-424).
A stick-typed long lasting device for both transdermal and topical drug delivery has been developed. Ketotifen fumarate (KT) was used as a model drug. The effect of a variety of permeation enhancers was investigated using hairless mouse skin in vitro. Polyoxyethylene oleyl ether (POE), among the enhancers used, most enhanced the skin permeation of KT. The permeation enhancement was mainly due to the increase in the drug solubility in the stratum corneum and the resulting increase in the partition coefficient. The rate of skin permeation of KT was approximately proportional to the loading dose of the drug.
Keywords: Ketotifen fumarate; Stick; Transdermal delivery system; Enhancer;

Release of naltrexone on buccal mucosa: Permeation studies, histological aspects and matrix system design by Libero Italo Giannola; Viviana De Caro; Giulia Giandalia; Maria Gabriella Siragusa; Claudio Tripodo; Ada Maria Florena; Giuseppina Campisi (425-433).
Transbuccal drug delivery has got several well-known advantages especially with respect to peroral way. Since a major limitation in buccal drug delivery could be the low permeability of the epithelium, the aptitude of NLX to penetrate the mucosal barrier was assessed. Ex vivo permeation across porcine buccal mucosa 800 μm thick was investigated using Franz type diffusion cells and compared with in vitro data previously obtained by reconstituted human oral epithelium 100 μm thick. Both fluxes (Js) and permeability coefficients (K p) are in accordance, using either buffer solution simulating saliva or natural human saliva. Permeation was evaluated also in presence of chemical enhancers or iontophoresis. No significant differences in penetration rate were observed using chemical enhancers; in contrast, Js and Kp were extensively affected by application of electric fields. Tablets, designed for Naltrexone hydrochloride (NLX) administration on buccal mucosa, were developed and prepared by direct compression of drug loaded (56%) poly-octylcyanocrylate (poly-OCA) matrices. NLX is slowly discharged from buccal tablets following Higuchian kinetic. Histologically, no signs of flogosis ascribable to NLX and/or poly-OCA were observed, while cytoarchitectural changes due to iontophoresis were detected. Buccal tablets containing NLX may represent a potential alternative dosage form in addiction management.
Keywords: Transbuccal permeation; Porcine buccal mucosa; Iontophoresis; Naltrexone hydrochloride; Poly-octylcyanoacrylate matrices; Buccal tablets;

Over recent decades, the use of in vitro diffusion cell studies to assess skin permeability has evolved into a major research tool, providing key insights into the relationships between skin, drug and formulation. Sometimes, such studies involve synthetic membranes as this approach can yield useful inferences with respect to drug–skin partitioning and diffusion phenomena. Yet despite the popularity of such studies, it is still not at all known whether typical solute transport across synthetic barriers results in a normal distribution of permeability coefficients or alternatively some type of skewed distribution. The present study aims to shed light on this issue. To this end, five compounds (testosterone, oestradiol, corticosterone, aldosterone and adenosine) exhibiting a broad range of octanol–water partition coefficient values were selected as test penetrants. The protocol involved taking multiple replicate measurements of each drug’s passive steady state flux through poly(dimethylsiloxane) membrane. Each penetrant’s resultant permeability coefficient database was subjected to a Kolmogorov–Smirnov (KS) test for normality. It was found that the permeability coefficients of all five drugs were distributed in a Gaussian-normal fashion. The theoretical significance and practical impact of these findings are discussed.
Keywords: Testosterone; Oestradiol; Corticosterone; Aldosterone; Adenosine; Permeability coefficient; Variability; Gaussian distribution; Normal distribution;

Solid lipid extrusion of sustained release dosage forms by Claudia Reitz; Peter Kleinebudde (440-448).
The applicability of the solid lipid extrusion process as preparations method for sustained release dosage forms was investigated in this study. Two lipids with similar melting ranges but of different composition, glyceryl palmitostearate (Precirol ATO 5®) and glyceryl trimyristate (Dynasan 114®), and mixtures of each lipid with 50% or 75% theophylline were extruded at temperatures below their melting ranges. Extrudates were analyzed using differential scanning calorimetry, scanning electron microscopy, porosity measurements and in vitro drug dissolution studies. The possibility of processing lipids by softening instead of complete melting and without subsequent formation of low-melting, metastable polymorphs could be demonstrated. Extrudates based on formulations of glyceryl palmitostearate/theophylline (50:50) and glyceryl trimyristate/theophylline (50:50) showed sustained release properties.An influence of extrusion conditions on the matrix structure was shown for extrudates based on a mixture of glyceryl trimyristate and theophylline (50:50). Glyceryl trimyristate tended to solidify in porous structures after melting. Exceeding a material temperature of 50.5 °C led to porous extrudate matrices with a faster drug release. The production of novel, non porous sustained release matrices was possible at a material temperature of 49.5 °C. Extrudates based on glyceryl trimyristate/theophylline (50:50) only slight changes in melting enthalpy and stable drug release profiles.
Keywords: Solid lipid extrusion; Lipid matrix; Sustained release; Acylglycerides; DSC;

Characterization of the film formation of the dry coating process by Caroline Désirée Kablitz; Nora Anne Urbanetz (449-457).
As the film formation of the dry coating process differs completely from conventional coating methods it is of certain interest to define a parameter like the minimum film formation temperature (MFT) used for aqueous dispersion based processes in order to describe an efficient film formation. Film formation occurs mainly during the curing step following the coating phase. Therefore, the film formation of the dry coating process was analyzed with regard to the two process parameters influencing film formation, namely curing temperature and time.Theophylline pellets were coated using the enteric polymer HPMCAS and TEC/Myvacet® as plasticizer composition. The polymer and the plasticizer were applied to the pellets simultaneously, except for the beginning of the coating step when the plasticizer has been sprayed 30 s before powder feeding was started. The coated pellets were cured for five different time periods at eight different temperatures. Drug release was determined in 0.1 N HCl. Their surface and cross-sectional morphologies were examined by scanning electron microscopy. The glass transition temperature of the obtained films as well as of the polymer plasticizer mixture obtained by casting a film using an organic solution of the coating components was determined by thermomechanical analysis.At higher curing temperature and/or extended curing time an enhancement of acid resistance is observed. The glass transition temperature of the coating mixture was determined to be 51.7 ± 3.3 °C. It is close to the temperature needed for film formation. Enteric resistant pellets are obtained after curing for 0.75 h at 55 °C. However, enteric resistance was achieved as well slightly below the glass transition temperature at 45 °C applying long curing periods of at least 12 h. This may be caused by the plasticizer gradient along the coat which is caused by spraying the plasticizer 30 s before starting powder feeding. This results in a higher plasticizer concentration of the inner layers of the coat relatively to the outer ones. Due to this film formation starts at the inner layers even below the glass transition temperature. As the plasticizer diffuses to the outer layers during curing according to the plasticizer gradient, film formation proceeds.
Keywords: Dry coating; Glass transition temperature; Film formation; Curing;

The purpose of this study was to investigate the influence of batch size during scale-up on the abrasion of biconvex tablets. Labelled tracer tablets of six different crushing forces (23–116 N) were mixed at different times and peripheral speeds in a laboratory (4 kg) and production (360 kg) perforated pan coater. The weight loss of these tracer tablets was determined.The main factor affecting the abrasion in both scales is the tablet crushing force as a nonlinear decrease in the abrasion was observed with increasing crushing force of the tablets. An increase in mixing time results in an increase in abrasion for the laboratory scale. In contrast to the production scale an influence of the peripheral speed on the abrasion could not be observed in laboratory scale. There is no difference in total abrasion for the laboratory scale and production scale for low peripheral speed. At higher peripheral speeds the abrasion in the production scale is slightly higher than in the laboratory scale.
Keywords: Scale-up; Abrasion; Pan speed; Bohle Film Coater;

The aim of this study was to develop a dry powder coating process for chlorpheniramine maleate (CPM) tablets using Eudragit® L 100-55 as the delayed release polymer. Powder coating, a water and organic solvent-free process, was investigated as a method to prevent the migration of an ionizable, highly water soluble model drug into the polymeric film during the coating process. Eudragit® L 100-55 was pre-plasticized with triethyl citrate (TEC) using hot-melt extrusion at levels of 20%, 30%, and 40%, based on the polymer weight. The extrudate was subsequently cut into pellets and cryogenically ground into a fine powder. Talc was incorporated into the coating powder as an anti-tack agent. PEG 3350 was used as a primer for the powder coating of tablets with pre-plasticized Eudragit® L 100-55. The addition of polyethylene glycol 3350 (PEG 3350) to the pre-plasticized Eudragit® L 100-55 was necessary to enhance the adhesion of the coating powder to the tablet cores. PEG 3350 also improved film formation and coalescence of the polymeric particles due to its plasticization effects on the acrylic polymer. For comparison, theophylline tablets were also coated with pre-plasticized Eudragit® L 100-55. Theophylline was selected as a less water soluble model drug. The powder coating process was performed in a modified laboratory scale spheronizer. The drug release rate was dependent both on TEC content and the coating level. The stability of the powder-coated CPM tablets was confirmed at 25 °C/60% RH over a storage time of 12 weeks.
Keywords: Powder coating; Eudragit® L 100-55; Chlorpheniramine maleate; Theophylline; Enteric polymers; Film formation;

Stress relaxation test for the characterization of the viscoelasticity of pellets by Marco Cespi; Giulia Bonacucina; Monica Misici-Falzi; Roberto Golzi; Luigi Boltri; Giovanni F. Palmieri (476-484).
The characterization of the mechanical properties of single uncoated pellets was performed in order to verify if these parameters could be used to predict the pellets aptitude to be compressed or utilized differently.Different ratios of microcrystalline cellulose and lactose monohydrate were used for the preparation of four batches of pellets by an extrusion/spheronization process. The 0.6–0.71 mm pellet fraction was used for the tests. Crushing strength and stress relaxation tests were carried out on the single pellets. The first test provided information of both the mechanical strength and the fragmentation aptitude. The second test provided information about their deformation ability (viscous flow) and residual elasticity (stress relaxation modulus). The results obtained from these tests were then compared with those obtained from the Heckel analysis.An excellent consistency was discovered between the parameters obtained from both the stress relaxation and crushing strength tests on one side and the Heckel parameters on the other side.Tests performed on single pellets are very useful tools to predict their deformation and fragmentation aptitude under compression and can be used for early insight of the pellet aptitude to be compressed.
Keywords: Pellets; Extrusion; Spheronization; Mechanical properties; Viscoelasticity; Stress relaxation; Compression;

The present study investigates if drug diffusion through plasticized isolated ethylcellulose (EC)/hydroxypropyl methylcellulose (HPMC) films prepared by solvent casting can be used as a tool to develop spray-coated dosage forms. In particular, the importance of the level and type of plasticizers was investigated. The permeability of the model drug metoprolol tartrate through plasticized isolated films could be adjusted by selecting the type and amount of plasticizer in the films due to the different hydrophilicity of the plasticizers. The release of metoprolol tartrate from coated pellets is consistent with the drug diffusion through the films made up of the same polymer blends. This indicated that it is useful to test isolated films for early predictions and for formulation optimization.
Keywords: Ethylcellulose; Hydroxypropyl methylcellulose; Metoprolol tartrate; Diffusion; Release; Plasticizer;

Evaluation of alginate based mesalazine tablets for intestinal drug delivery by Fatmanur Tuğcu-Demiröz; Füsun Acartürk; Sevgi Takka; Öznur Konuş-Boyunağa (491-497).
The aim of this study was to develop the alginate based mesalazine tablets for intestinal delivery. Sodium alginate is a biocompatible, natural polymer with pH-sensitive gel-forming ability.Matrix tablets were prepared with two types of sodium alginate of different amounts. The in vitro release characteristics of mesalazine from alginate tablets were compared with those of the commercial product (Salofalk®). X-ray imaging was used to monitor the tablets throughout the gastrointestinal system.Although alginate tablets gave a faster release in an acidic medium compared with the commercial product (Salofalk®), the cumulative amount of released drug of the optimum formulation was found to be almost the same as that of the commercial product at the end of 4 h. The alginate type and amount in the matrices played an important role in basic media. The release of the optimum formulation containing low viscosity alginate was found to be almost identical to that of the commercial product in acidic and basic media.Tablets were visualized to determine whether they were located in the terminal ileum or cecum for 3–6 h. Mesalazine-alginate matrix tablet formulations can deliver the drug to the small and large intestine. Thus, the alginate matrix system may be a promising system for the treatment of Crohn’s disease involving both the ileum and large intestine.
Keywords: Mesalazine; Alginate; Intestinal drug delivery; X-ray imaging;

Porous hydroxyapatite tablets as carriers for low-dosed drugs by A. Cosijns; C. Vervaet; J. Luyten; S. Mullens; F. Siepmann; L. Van Hoorebeke; B. Masschaele; V. Cnudde; J.P. Remon (498-506).
The present study evaluated an innovative technique for the manufacturing of low-dosed tablets. Tablets containing hydroxyapatite and a pore forming agent (50% (w/w) Avicel PH 200/20, 37.5% and 50% corn starch/37.5% sorbitol) were manufactured by direct compression followed by sintering. The influence of pore forming agent (type and concentration), sinter temperature and sinter time on tablet properties was investigated. Sintering (1250 °C) revealed tablets with an acceptable friability (<1%). Using 50% (w/w) Avicel PH 200 as pore forming agent resulted in tablets combining the highest porosity (50%) and the highest median pore diameter (5 μm). Aqueous drug solutions (metoprolol tartrate, riboflavin sodium phosphate) were spiked on the tablet surface. The maximum volume of drug solution absorbed was limited (2 × 100μl), revealing that these porous carriers were ideal for low dosed formulations. Drug release from the tablets was slow, independent of the drug. To accelerate drug release, tablets were manufactured using a modified gelcasting technique yielding tablets with a median pore size of 60 and 80 μm. Release from these tablets was drastically increased indicating that the permeability of the tablets was influenced by the pore size, shape and connectivity of the porous network. Changing and controlling these parameters made it possible to obtain drug delivery systems providing different drug delivery behaviour.
Keywords: Hydroxyapatite; Low-dosed drugs; Sintering; Porous tablet; Modified gelcasting technique;

Application of percolation model to the tensile strength and the reduced modulus of elasticity of three compacted pharmaceutical excipients by Virginie Busignies; Bernard Leclerc; Patrice Porion; Pierre Evesque; Guy Couarraze; Pierre Tchoreloff (507-514).
Percolation theory has been applied to several mechanical properties of pharmaceutical tablets. This power law describes the change of tablet’s properties with the relative density. It defines critical tablet densities from which the mechanical properties start to change. The exponent in the law is expected to be universal for a mechanical property and numerical values are proposed in the literature. In this work, the percolation model was applied to the tensile strength and the reduced modulus of elasticity (obtained from surface indentation test) of three compacted pharmaceutical excipients (a microcrystalline cellulose, a lactose and an anhydrous calcium phosphate). Two approaches were proposed. First, the exponent was kept constant and equal to the values used in the literature (2.7 for the tensile strength and 3.9 for the reduced modulus of elasticity). Secondly, the critical tablet density (i.e. the percolation threshold) and the exponent were determined from the model. In the first approach, the percolation thresholds were higher than the relative tapped density. Using the second approach, the experimentally determined exponents were not close to the values of the literature and the critical relative densities were higher than the relative tapped density or equal to zero. Then, this study showed that the exponent seems not universal and that the model must be used carefully.
Keywords: Percolation theory; Percolation threshold; Tensile strength; Reduced modulus of elasticity; Porosity; Relative density;

In-vitro/in-vivo correlation of pulsatile drug release from press-coated tablet formulations: A pharmacoscintigraphic study in the beagle dog by Manish Ghimire; Fiona J. McInnes; David G. Watson; Alexander B. Mullen; Howard N.E. Stevens (515-523).
The aim of the current study was to investigate the in-vitro and in-vivo performance of a press-coated tablet (PCT) intended for time delayed drug release, consisting of a rapidly disintegrating theophylline core tablet, press-coated with barrier granules containing glyceryl behenate (GB) and low-substituted hydroxypropylcellulose (L-HPC). The PCTs showed pulsatile release with a lag time dependent upon the GB and L-HPC composition of the barrier layer. In-vivo γ-scintigraphic studies were carried out for PCTs containing GB:L-HPC at 65:35 w/w and 75:25 w/w in the barrier layer in four beagle dogs, in either the fed or fasted state. The in-vivo lag time in both the fed and fasted states did not differ significantly (p  > 0.05) from the in-vitro lag time. Additionally, no significant difference (p  < 0.05) between in-vivo fed and fasted disintegration times was observed, demonstrating that in-vivo performance of the PCT was not influenced by the presence or absence of food in the gastrointestinal tract. A distinct lag time was obtained prior to the appearance of drug in plasma and correlated (R 2  = 0.98) with disintegration time observed from scintigraphic images. However, following disintegration, no difference in pharmacokinetic parameters (AUC0–6 dis, K el, C max) was observed. The current study highlighted the potential use of these formulations for chronopharmaceutical drug delivery.
Keywords: Press-coated tablets; Pulsatile release; Beagle dogs; Pharmacoscintigraphy; IVIVC; Lag time;

Dissolution test for citalopram in tablets and comparison of in vitro dissolution profiles by Júlia Menegola; Martin Steppe; Elfrides E.S. Schapoval (524-530).
A dissolution test for tablets containing 20 mg of citalopram was developed and validated using a reverse-phase liquid chromatographic method and this dissolution test was applied to compare dissolution profiles. The sink conditions, filters, stability of the drug and specificity on different dissolution media were tested to choose a discriminatory dissolution method which uses USP apparatus 1 with baskets rotating at 50 rpm, 900 ml of deaerated 0.1 M hydrochloric acid (HCl) as the dissolution medium. The quantitation method was also adapted and validated. The parameters of difference factor, similar factor, according to current FDA guidelines, and dissolution efficiency were employed to compare dissolution profiles. The dissolution test developed and validated was adequate for its purposes and could be applied for quality control of citalopram tablets, since there is no monograph to citalopram in tablets, this work can be used to help pharmocopoeias.
Keywords: Citalopram; Dissolution test; HPLC; Quality control; Dissolution profile comparisons;

Solid-state characterization and dissolution profiles of the inclusion complexes of omeprazole with native and chemically modified β-cyclodextrin by Ana Figueiras; Rui A. Carvalho; Laura Ribeiro; Juan J. Torres-Labandeira; Francisco J.B. Veiga (531-539).
The aim of this work was to investigate the formation of the inclusion complex between omeprazole (OME), a benzimidazolic derivative and a methylated cyclodextrin, methyl-β-cyclodextrin (MβCD), with an average degree of substitution of 0.5. Inclusion complex between OME and β-cyclodextrin (βCD), a natural cyclodextrin, was used as reference. In aqueous media, apparent stability constants (K s), which describe the extent of formation of the complexes, have been determined by UV spectroscopy and 1H NMR experiments. The stoichiometry of the complexes was found to be 1:1 mol:mol OME:cyclodextrin (CD) and the value of K s was higher for OME:MβCD than for OME:βCD inclusion complexes. Solid binary systems of OME and CDs were prepared by different techniques, namely kneading, spray-drying and freeze-drying. The formation and physicochemical characterization of solid inclusion complexes were investigated by differential scanning calorimetry (DSC), Fourier transform-infrared (FTIR), X-ray diffractometry (XRD) and scanning electron microscopy (SEM). The results show that freeze-drying method produces true inclusion complexes between OME and both CDs. In contrast, crystalline drug was detectable in kneaded and spray-drying products. The dissolution of OME from the binary systems was studied to select the most appropriate system for the development of a buccal drug delivery formulation. It was concluded that the preparation technique played an important role in the dissolution behaviour of the drug and the inclusion complex between OME and MβCD obtained by spray-drying and freeze-drying allowed better performances.
Keywords: Omeprazole; β-Cyclodextrin; Methyl-β-cyclodextrin; Physicochemical characterization; Dissolution profile; Inclusion complex;

A formulation comparison, using a solution and different nanosuspensions of a poorly soluble compound by Kalle Sigfridsson; Sara Forssén; Paula Holländer; Urban Skantze; Jennie de Verdier (540-547).
The pharmacokinetic parameters of AZ68 administered as a solution have been compared with those from an amorphous and a crystalline nanosuspension using rats as in vivo specie. All formulations were administered intravenously (i.v.) and orally. The purpose of the study was to find out if the three different formulations were comparable and safe to administer. The results indicate that AZ68 is absorbed at a lower rate for crystalline nanosuspensions compared to amorphous nanosuspensions and solutions. However, the absorbed extent of the compound is similar. The results are a consequence of the lower solubility and the slower dissolution rate for crystalline material compared to amorphous substance in the gastrointestinal tract. The dissolution process is excluded for a solution, resulting in the fastest absorption rate. No significant difference was found between pharmacokinetic parameters when comparison was made between the formulations after i.v. administration. There were no adverse events observed after i.v. administration of the nanosuspensions.
Keywords: NK receptor; Neurokinin; Nanosuspensions; Pharmacokinetic;

Effect of cell differentiation and passage number on the expression of efflux proteins in wild type and vinblastine-induced Caco-2 cell lines by Sanna Siissalo; Leena Laitinen; Maija Koljonen; Kati-Sisko Vellonen; Hanna Kortejärvi; Arto Urtti; Jouni Hirvonen; Ann Marie Kaukonen (548-554).
The mRNA level expression of MDR1, MRP1-6, BCRP and CYP3A4 was determined by quantitative PCR in wild type (Caco-2WT) and vinblastine-treated (Caco-2VBL) Caco-2 cells at different passage levels (32–53). Differentiation increased the mRNA levels of MDR1, BCRP and all the MRPs except MRP4. Corresponding mRNA levels were observed in Caco-2WT and Caco-2VBL, except that the expression of MRD1 was higher in Caco-2VBL than in Caco-2WT cells. CYP3A4 was barely detected in either cell line.MDR1 functionality was studied using rhodamine123 and verapamil as a substrate–inhibitor pair. Corresponding to the observed differences in mRNA levels, MDR1 activity was higher in the Caco-2VBL cells. In Caco-2WT, MDR1 functionality was elevated at low passage numbers (32–35) compared to higher ones (49–53). Verapamil inhibited MDR1 efflux except at higher passage Caco-2WT cells, where no MDR1 activity could be observed.The results support the use of Caco-2VBL cells in MDR1 screening. The functional expression is higher than in Caco-2WT and remains consistent across the studied passages without major differences in mRNA levels of other efflux proteins. As both the passage number and the level of cell differentiation affect the expression profile of efflux proteins, short-term cell growth protocols should be evaluated accordingly.
Keywords: Caco-2 cell line; Efflux protein; Expression; Functionality; MDR1; Cell differentiation; Passage number;

Biological conversion of a water-soluble prodrug of cyclosporine A by F. Lallemand; E. Varesio; O. Felt-Baeyens; Leila Bossy; G. Hopfgartner; R. Gurny (555-561).
UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) designed for topical ocular delivery. The pro-moiety is grafted via an ester function to CsA and the solubilizing group is a phosphate ion. The aim of this study was to elucidate the conversion mechanisms by which UNIL088 generates CsA. UNIL088 was incubated in rabbit tears at physiological temperature to study its enzymatic and chemical conversion, respectively. Metabolites and intermediates were identified using a quadrupole-time of flight (QqTOF) mass spectrometer, which allowed biotransformation pathways to be deduced. Conversion is activated by the chemical or enzymatic hydrolysis of the terminal ester function of the pro-moiety, leading to the phospho-serine-sarcosine-cyclosporine A that spontaneously converts into CsA. In addition to the main biotransformation pathway, a secondary reaction involved hydrolysis of the phosphate ester group of the pro-moiety, probably by phosphatases present in tears.
Keywords: Cyclosporine A; Prodrug; Tears; Biotransformation; Enzymes; LC–MS; MS/MS;

Isolation of drugs from biological fluids by using pH sensitive poly(acrylic acid) grafted poly(vinylidene fluoride) polymer membrane in vitro by Jouni Karppi; Satu Åkerman; Kari Åkerman; Annika Sundell; Kristiina Nyyssönen; Ilkka Penttilä (562-568).
Isolation of acidic and basic model drugs by using pH sensitive poly(acrylic acid) grafted poly(vinylidene fluoride) (PAA–PVDF) cation-exchange membrane from biological fluids was reported. Effects of drug charge and lipophilicity on adsorption were also investigated. In the present study, basic model drugs adsorbed to a considerably greater extent onto the membrane than acidic drugs. Albumin was not adsorbed onto the membrane. Results of our study exposed, that electrostatic interactions between positively charged basic drug and negatively charged PVDF–PAA membrane were the most important factor affecting drug adsorption onto the membrane. Adsorption of acidic and basic drugs onto the PVDF–PAA membrane was not related to drug lipophilicity. The results of present study demonstrated that basic drugs adsorbed extensively onto the membrane, but albumin did not, proposing that PAA–PVDF membrane may be suitable for isolating basic drugs from proteinaceous biological fluids (i.e. serum) for subsequent monitoring and evaluation.
Keywords: Charge; Electrostatic interactions; Isolation; Lipophilicity; PDVF–PAA; Serum;

Investigation into the subambient behavior of aqueous mannitol solutions using temperature-controlled Raman microscopy by J. Renwick Beattie; Lindsay J. Barrett; John F. Malone; John J. McGarvey; M. Nieuwenhuyzen; Vicky L. Kett (569-578).
The aim was twofold; to demonstrate the ability of temperature-controlled Raman microscopy (TRM) to locate mannitol within a frozen system and determine its form; to investigate the annealing behavior of mannitol solutions at −30 °C. The different polymorphic forms of anhydrous mannitol as well as the hemihydrate and amorphous form were prepared and characterized using crystal or powder X-ray diffractometry (XRD) as appropriate and Raman microscopy. Mannitol solutions (3% w/v) were cooled before annealing at −30 °C. TRM was used to map the frozen systems during annealing and was able to differentiate between the different forms of mannitol and revealed the location of both β and δ polymorphic forms within the structure of the frozen material for the first time. TRM also confirmed that the crystalline mannitol is preferentially deposited at the edge of the frozen drop, forming a rim that thickens upon annealing. While there is no preference for one form initially, the study has revealed that the mannitol preferentially transforms to the β form with time. TRM has enabled observation of spatially resolved behavior of mannitol during the annealing process for the first time. The technique has clear potential for studying other crystallization processes, with particular advantage for frozen systems.
Keywords: Amorphous; Polymorphism; Thermal analysis; Freeze-drying; Spectroscopy; Raman; Mannitol;

Adhesion forces in interactive mixtures for dry powder inhalers – Evaluation of a new measuring method by Maike Lohrmann; Michael Kappl; Hans-Juergen Butt; Nora Anne Urbanetz; Bernhard Christian Lippold (579-586).
Dry powder inhalers mostly contain carrier based formulations where micronized drug particles are adhered to coarse carrier particles. The performance of the dry powder inhaler depends on the inhaler device, the inhalation manoeuvre and the formulation. The most important factor influencing the behaviour of the formulation is the adhesion force acting between the active ingredient and the carrier particles, which can be measured using different methods, for example the centrifuge technique or atomic force microscopy. In this study the tensile strength method, usually applied to determine cohesion forces between powder particles of one material, is optimized for adhesion force measurements between powder particles of unlike materials. Adhesion force measurements between the carrier materials lactose or mannitol and the drug substance salbutamol sulphate using the tensile strength method and the atomic force microscopy show higher values with increasing relative humidity. Consequently, the fine particle fraction determined using the Next Generation Impactor decreases with increasing relative humidity as a result of the enhanced interparticle interactions.
Keywords: Adhesion forces; Atomic force microscopy; Dry powder inhalers; Interparticle interactions; Tensile strength measurements;

by Ingfried Zimmermann (587-588).