European Journal of Pharmaceutics and Biopharmaceutics (v.70, #2)

APV Diary (S1-S2).

Irregular absorption profiles observed from diclofenac extended release tablets can be predicted using a dissolution test apparatus that mimics in vivo physical stresses by Grzegorz Garbacz; Ralph-Steven Wedemeyer; Stefan Nagel; Thomas Giessmann; Hubert Mönnikes; Clive G. Wilson; Werner Siegmund; Werner Weitschies (421-428).
The prediction of the in vivo drug release characteristics of modified release oral dosage forms by in vitro dissolution tests is a prerequisite for successful product development. A novel dissolution test apparatus that mimics the physical conditions experienced by an oral formulation during gastrointestinal transit was developed. This included the simulation of pressure forces exerted by gut wall motility, shear forces generated during propagation, and loss of water contact when the dosage form is located in an intestinal air pocket. The new apparatus was evaluated using a diclofenac extended release (ER) tablet. The in vitro dissolution profiles were compared between the novel test apparatus and a conventional dissolution apparatus (USP II). These data were compared with the profiles of plasma concentration versus time that were obtained after the administration of an ER tablet to 24 healthy volunteers under fasting conditions. Multiple peaks were observed in individual plasma concentration–time profiles after the intake of the reference ER tablet. Standard dissolution testing showed typical characteristics of an almost continuous release for this formulation; however, dissolution testing with the novel apparatus suggested that the diclofenac release from the ER tablets would be extremely variable and dependent on the applied stress. The data suggest that the observed multiple peaks of plasma concentration after dosing of the ER diclofenac tablets are most probably caused by sensitivity to physical stress events during gastrointestinal transit.
Keywords: Biorelevant dissolution test; Diclofenac; Extended release tablets; Dose dumping;

GMP production of pDERMATT for vaccination against melanoma in a phase I clinical trial by S.G.L. Quaak; J.H. van den Berg; M. Toebes; T.N.M. Schumacher; J.B.A.G. Haanen; J.H. Beijnen; B. Nuijen (429-438).
For the treatment of melanoma DNA vaccines are a promising therapeutic approach. In our institute a plasmid encoding a melanoma-associated epitope (MART-1) and an immunostimulatory sequence (tetanus toxin fragment-c) termed pDERMATT was developed. In a phase I study the plasmid will be administered intradermally using a newly developed tattoo strategy to assess the toxicity and efficacy of inducing tumor-specific T-cell immunity. To facilitate this study a Good Manufacturing Practice (GMP)-compliant plasmid manufacturing process was set up and a pharmaceutical dosage form was developed. Each batch resulted in approximately 200 mg plasmid DNA of a high purity >90% supercoiled DNA, an A260/280 ratio 1.80–1.95, undetectable or extremely low residual endotoxins, Escherichia coli host cell protein, RNA, and DNA. In the manufacturing process no animal derived enzymes like RNase or potentially harmful organic solvents are used. After sterile filtration the concentration of the plasmid solution is approximately 1.1 mg/mL. For the scheduled phase I study a concentration of 5 mg/mL is desired, and further concentration of the solution is achieved by lyophilisation. The formulation solution is composed of 1 mg/mL pDERMATT and 20 mg/mL sucrose in Water for Injections. Upon reconstitution with a five times smaller volume an isotonic sucrose solution containing 5 mg/mL pDERMATT is obtained. Lyophilised pDERMATT is sterile with >90% supercoiled DNA, an A260–280 ratio 1.80–1.95, content 90–110% of labeled, and residual water content <2% (w/w). The product yields the predicted profile upon restriction-enzyme digestion, is highly immunogenic as confirmed in an in vivo mouse model, and stable for at least six months at 5 °C. We have not only developed a reproducible process to manufacture pharmaceutical grade plasmid DNA but also a stable dosage form for the use in clinical trials.
Keywords: Clinical-grade plasmid DNA; cGMP; Gene therapy; Lyophilization; Melanoma; DNA vaccination;

The objectives of the present work were, first, to develop a new solid self-microemulsifying drug delivery system (SMEDDS) for oral poorly water-soluble drugs such as nimodipine; and second, to evaluate its oral bioavailability in healthy rabbits. The liquid SMEDDS consisted of ethyl oleate, Labrasol®, Cremophor® RH 40 and nimodipine. The solid SMEDDS was prepared by spray-drying the liquid SMEDDS in a laboratory spray dryer, using dextran as solid carrier. The imaging of TEM and photo correlation spectroscopy revealed no difference in the droplet size of reconstituted microemulsion between both SMEDDS. Solid state characterization of the solid SMEDDS was performed by SEM, DSC, and X-ray powder diffraction. The same dose of nimodipine in the solid SMEDDS and in the liquid SMEDDS resulted in similar AUC and C max values, but the maximum absorption was retarded by the solid SMEDDS. AUC and C max after oral administration of the solid SMEDDS were 2.6- and 6.6-fold higher, respectively, compared with those of the conventional tablet. These results demonstrate that the solid SMEDDS may preserve an improved bioavailability with releasing microemulsion lipid droplets from the formulation in vivo. Thus, this solid self-microemulsifying system may provide a useful solid dosage form for oral poorly water-soluble drugs.
Keywords: Self-microemulsifying; Poorly soluble drugs; Solid dosage form; Bioavailability; Spray-drying;

The local delivery and controllable release profiles make electrospun ultrafine fibers as potential implantable drug carriers and functional coatings of medical devices. There are few attempts to form acid-labile electrospun fibers, whose release behaviors respond to the local environment and fiber characteristics. In the current study a novel strategy was presented to synthesize biodegradable pH-sensitive polymers containing ortho ester groups. The acid-labile segments were synthesized through reacting 3,9-dimethylene-2,4,8,10-tetraoxaspiro [5.5] undecane with 1,10-decanediol or poly(ethylene glycol), which were further copolymerized with d,l-lactide to obtain triblock copolymers. Biodegradable acid-labile polymers were electrospun with the encapsulation of paracetamol as a model drug. In vitro release study showed that the total amount of drug released from acid-labile polymeric fibers was accelerated after incubation into acid buffer solutions, and the amount of initial burst release and sustained release rate were significantly higher for matrix polymers with hydrophilic acid-labile segments. In vitro degradation study indicated that the electrospun fibers containing acid-labile segments were stable in neutral buffer solution, but the molecular weight reduction of matrix polymers, the morphological changes and mass loss of fibrous mats were significantly enhanced under acid circumstances.
Keywords: Acid-labile polymer; Biodegradable backbone; Electrospun fiber; Ortho ester group; Controlled drug release;

Poly(N-vinylacetamide) chains enhance lectin-induced biorecognition through the reduction of nonspecific interactions with nontargets by Ken-ichiro Hiwatari; Shinji Sakuma; Kiyoko Iwata; Yoshie Masaoka; Makoto Kataoka; Hiroyuki Tachikawa; Yoshikazu Shoji; Shinji Yamashita (453-461).
Lectin-immobilized fluorescent nanospheres were designed with the aim of developing a novel endoscopic imaging agent for the detection of early colorectal cancer. Submicron-sized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) and poly(methacrylic acid) (PMAA) chains encapsulating fluorescein-labeled cholesterol were prepared as a platform of the imaging agent. Peanut agglutinin (PNA) was immobilized on the surface of fluorescent nanospheres through a chemical reaction with PMAA in order to recognize β-d-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-β(1-3)GalNAc), which is the terminal sugar of the Thomsen–Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The effect of surface structure of nanospheres on the affinity and specificity of immobilized PNA for Gal-β(1-3)GalNAc was examined. Agglutination of normal and Gal-β(1-3)GalNAc-expressed erythrocytes in the presence of nanospheres showed that PNA was immobilized actively on the nanosphere surface. Molecular weights of PNVA and PMAA affected the PNA activity most strongly. When the weight-average molecular weight of PNVA was nearly equal to that of PMAA, the affinity of PNA immobilized on the nanosphere surface for Gal-β(1-3)GalNAc was as strong as that of intact PNA; the specificity for the carbohydrate residue was higher than that of the PNA. Results indicated that PNVA enhanced the specificity of PNA through the reduction of nonspecific interactions between PNA and carbohydrates other than Gal-β(1-3)GalNAc on the erythrocyte surface without a significant decrease in the affinity.
Keywords: Endoscopic imaging agent; Nanomedicine; Colonoscopy; Colorectal cancer; Lectin; Poly (N-vinylacetamide);

Predicting blood–brain barrier penetration from molecular weight and number of polar atoms by Xu-Chun Fu; Guo-Ping Wang; Hai-Li Shan; Wen-Quan Liang; Jian-Qing Gao (462-466).
A simple three-descriptor model to predict blood–brain barrier is derived from a training set of 78 compounds: log BB = −9.880 × 10−6 M W 2  + 7.339 × 10−3M W–0.2268n pol−0.1143 (n  = 78, r 2  = 0.74), where log BB is the logarithm of the ratio of the steady-state concentration of the compound in the brain to concentration in the blood, M W is the molecular weight, n pol is the number of polar atoms (oxygen, nitrogen, and attached hydrogen), n is the number of compounds, and r is the correlation coefficient. The model is validated through use of leave-one-out procedure and an external test set (25 compounds). The model is suitable for the rapid prediction of the blood–brain barrier penetration of drug candidates because of its predictive ability and simplicity.
Keywords: Blood–brain barrier; Predictive model; Molecular weight; Polar atom;

Polypropylene grafted with smart polymers (PNIPAAm/PAAc) for loading and controlled release of vancomycin by Juan-Carlos Ruiz; Carmen Alvarez-Lorenzo; Pablo Taboada; Guillermina Burillo; Emilio Bucio; Kristof De Prijck; Hans J. Nelis; Tom Coenye; Angel Concheiro (467-477).
New smart surface-modified polypropylene (PP) was prepared for improving the loading and the sustained delivery of vancomycin and, thus, reducing the risk of biofilm formation when used as component of biomedical devices. Isothermal titration calorimetry (ITC) served for screening the most suitable monomers for grafting; the drug preferentially bonding to ionized acrylic acid (AAc). A net-PP-g-PNIPAAm-inter-net-PAAc was synthesized by first grafting and cross-linking of N-isopropylacrylamide onto PP films and then interpenetrating a second network by redox polymerization and cross-linking of AAc. PP-g-PAAc slabs were prepared by grafting AAc and, optionally, cross-linking. The amount and composition of grafted polymer (FTIR-ATR), morphology (SEM), temperature- and pH-responsiveness (swelling measurements), thermal behavior (DSC), friction coefficient (rheometry), drug loading and release rate, and effect against methicillin-resistant Staphylococcus aureus (MRSA) biofilms (modified robbins device) were evaluated. Grafting of AAc notably decreased the friction coefficient from 0.28 ± 0.03 to 0.05 ± 0.02 and enhanced the vancomycin loading (up to 2.5 mg/cm2). Drug-loaded films showed a pH-dependent release rate, sustaining the release in pH 7.4 aqueous media at 37 °C for several hours. All drug-loaded films reduced biofilm formation by MRSA; the anti-biofilm effect being statistically significant (91.7% reduction, α  < 0.05) for PP-g-PAAc with the thinnest grafting layer.
Keywords: IPN; Surface-modified polypropylene; Oxidative pre-irradiation; Gamma irradiation; Isothermal titration microcalorimetry; Dually responsive; Vancomycin local delivery; Biofilm formation; Biomedical devices;

Surface composition and contact angle relationships for differently prepared solid dispersions by Carina Dahlberg; Anna Millqvist-Fureby; Michael Schuleit (478-485).
Solid dispersions are promising drug delivery forms which offer the possibility to disperse a hydrophobic drug in a hydrophilic matrix and thereby improve the dissolution behavior and the bioavailability of the drug. One important aspect and a prerequisite in understanding the drug dissolution mechanism from solid dispersions is a better analytical monitoring of the solid dispersion surface properties, such as powder surface composition and water adsorption properties. In this paper, we have considered chemical and structural surface analysis data for solid dispersions processed by spray drying or roto-evaporation and compared these data with information obtained by contact angle measurements. Firstly, we establish the usefulness and suitability of X-ray photoelectron spectroscopy (XPS) for determination of surface chemical composition and scanning electron microscopy (SEM) for determining the structure of solid dispersions composed of different types of carriers, drugs and drug concentrations. Secondly, we measure contact angles of solid dispersions to describe wettability, to finally establish a link between the surface chemical composition, the powder structure and the wetting behavior. These experimental methods offer a rapid screening tool for the selection of carrier, drug concentration and/or process in early development. In addition, they provide a useful tool for investigating structural aspects of solid dispersions which have intrinsic relevance for drug dissolution and stability.
Keywords: Solid dispersions; Roto-evaporation; Wettability; Spray drying; Contact angle; Surface composition; XPS; Powder morphology;

Transport of valproate at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells: Mechanism and substrate specificity by Wiebke Fischer; Katrin Praetor; Linda Metzner; Reinhard H.H. Neubert; Matthias Brandsch (486-492).
To reach its target cells, the antiepileptic drug valproate has to cross both the intestinal epithelial barrier and the blood–brain barrier in intact form as well as in sufficient amounts. This study was performed to characterize the epithelial transport of valproate at intestinal (Caco-2) and at blood–brain barrier (RBE4) cells. At both cell types, uptake of [3H]valproate was independent of inwardly directed Na+, Ca2+, Mg2+, K+ or Cl gradients. Uptake was, however, strongly stimulated by an inwardly directed H+ gradient. The cells accumulated valproate against a concentration gradient and the uptake rate of valproate was saturable with K t values of 0.6 and 0.8 mM. At Caco-2 cell monolayers, the total apical-to-basolateral flux of [3H]valproate exceeded the basolateral-to-apical flux 14-fold. Various monocarboxylic acids like salicylate, benzoate, acetate, propionate, butyrate, hexanoate, diclofenac and ibuprofen inhibited [3H]valproate uptake at both cell types. Lactate and pyruvate inhibited valproate uptake at RBE4 cells but not at Caco-2 cells. We conclude that valproate is accumulated in intestinal cells against a concentration gradient by the activity of a specific H+-dependent DIDS-insensitive transport system for monocarboxylates not identical with monocarboxylate transporter 1 (MCT1). The passage of valproate across the blood–brain barrier is very likely mediated by MCT1.
Keywords: Caco-2 cells; Drug delivery; Membrane transport; RBE4 cells; Valproate; Valproic acid;

Effect of polymer type on the dissolution profile of amorphous solid dispersions containing felodipine by Hajime Konno; Tetsurou Handa; David E. Alonzo; Lynne S. Taylor (493-499).
Amorphous solid dispersions are used as a strategy to improve the bioavailability of poorly water-soluble compounds. When formulating with a polymer, it is important not only for the polymer to stabilize against crystallization in the solid state, but also to improve the dissolution profile through inhibiting crystallization from the supersaturated solution generated by dissolution of the amorphous material. In this study, the dissolution profiles of solid dispersions of felodipine formulated with poly(vinylpyrrolidone) (PVP), hydroxypropyl methylcellulose (HPMC) or hydroxypropyl methylcellulose acetate succinate (HPMCAS) were compared. In addition, concentration versus time profiles were evaluated for the supersaturated solutions of felodipine in the presence and absence of the polymers. HPMCAS was found to maintain the highest level of supersaturation for the greatest length of time for both the dissolution and solution crystallization experiments, whereas PVP was found to be the least effective crystallization inhibitor. All polymers appeared to reduce the crystal growth rates of felodipine at an equivalent supersaturation and this mechanism most likely contributes to the enhanced solution concentration values observed during dissolution of the amorphous solid dispersions.
Keywords: Solid dispersion; Amorphous; Dissolution; Concentration;

In order to improve the in vitro performance and stability of co-spray-dried itraconazole/Inutec SP1 systems, the influence of adding PVPVA 64, a polymer that is compatible with itraconazole, was evaluated. Dissolution tests were carried out on several itraconazole/PVPVA 64/Inutec SP1 compositions and spray-dried itraconazole/PVPVA 64 powders were used as references. The physicochemical properties of the samples were assessed with modulated temperature differential scanning calorimetry (MDSC), X-ray powder diffraction (XRD) and environmental scanning electron microscopy (ESEM). Physicochemical analysis revealed that there is no interaction between itraconazole and Inutec SP1 and that sufficient amount of PVPVA 64 is required to keep the drug molecularly dispersed. The improvement of the ternary solid dispersions over the binary solid dispersions was composition dependent. On one hand the increased drug/PVPVA 64 ratio in the ternary systems slowed dissolution down, on the other hand this was compensated by the solubilizing power of Inutec SP1.
Keywords: Itraconazole; Inutec SP1; PVPVA 64; Solid dispersion; Spray drying; Modulated temperature differential scanning calorimetry; X-ray diffraction; Environmental scanning electron microscopy; Dissolution;

DNA nanogels were prepared by chemically conjugating Pluronic to the surface of cationic polymer/DNA complex in order to prepare thermo-responsive nanogels with endosomal disrupting abilities. Amine-reactive Pluronic was prepared by activating hydroxyl groups of Pluronic and subsequently reacted with pre-formed PEI/DNA complex. The conjugation process was monitored by measuring liberated nitrophenyl groups during the conjugation reaction. The properties of the nanogels (size and ζ-potential) changed significantly when temperature was increased from 20 to 37 °C. The multimodal size distribution of the nanogel also confirmed the variable sizes and distribution of the nanogel upon changing temperatures. Electron microscopy and atomic force microscopy also confirmed the modulated morphologies and sizes of the thermo-responsive nanogel. Confocal microscopy revealed that the nanogel disrupted lysosomes and endosomes at low temperatures, thus confirming endosomal disrupting abilities. The survival rates results showed that the cytotoxicities of the nanogel increased as the temperature decreased from 37 to 20 °C, showing that collapsed Pluronic chain played a role in modulating cytotoxicities. In vitro transfection efficiencies of the nanogel were also measured in NIH3T3 cells. Transfection efficiencies increased as the temperature decreased to 20 °C, thus confirming that endosomal disruptions played a significant role in increasing transfection efficiencies.
Keywords: Nanogel; Pluronic; Transfection; Gene delivery; PEI; DNA;

Formulation development of freeze-dried oligonucleotide-loaded gelatin nanoparticles by Jan C. Zillies; Klaus Zwiorek; Florian Hoffmann; Angelika Vollmar; Thomas J. Anchordoquy; Gerhard Winter; Conrad Coester (514-521).
The freeze-drying properties of gelatin nanoparticles were investigated with the goal of providing practicable nanoparticle formulations for in vitro applications or clinical studies. Various excipients and rehydration protocols were assessed, and gelatin nanoparticles loaded with oligonucleotides were successfully freeze-dried and rehydrated. An NF-κB decoy oligonucleotide-loaded gelatin nanoparticle formulation was developed and applied in a drug targeting approach in an animal model. The high concentrations of nanoparticles achieved after rehydration with reduced volumes proved to be critical for the in vivo effect. Finally, short term storage stability under accelerated conditions was assessed for dried gelatin nanoparticles formulated in sucrose, trehalose, mannitol, or a mannitol/sucrose mixture. Size, size distribution, and residual moisture content were investigated. Sucrose- and trehalose-containing formulations exhibited the greatest stability, but mannitol-containing formulations also showed notable stabilization despite their crystalline nature.
Keywords: Gelatin nanoparticles; Freeze-drying; Drug delivery; Drug targeting; Oligonucleotide; NF-kB;

Effect of particle size and charge on the network properties of microsphere-based hydrogels by Sophie R. Van Tomme; Cornelus F. van Nostrum; Marjolein Dijkstra; Stefaan C. De Smedt; Wim E. Hennink (522-530).
This work describes the tailorability of the network properties of self-assembling hydrogels, based on ionic crosslinking between dextran microspheres. Copolymerization of hydroxyethyl methacrylate-derivatized dextran (dex-HEMA), emulsified in an aqueous poly(ethylene glycol) (PEG) solution, with methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA) resulted in negatively or positively charged microspheres, respectively, at physiological pH. The monomer/HEMA ratio ranged between 6 and 57, resulting in microspheres with zeta (ζ)-potentials from −6 to −34 mV and +3 to + 23 mV, for the monomers MAA and DMAEMA, respectively. By altering the emulsification procedure, microsphere batches with various sizes and size distributions were obtained. The aim of the research was to assess the effect of particle size (distribution) and charge on the network properties of the macroscopic hydrogels. The ability to tailor the mechanical properties such as strength and elasticity increases the potential of the hydrogels to be used in a variety of pharmaceutical applications. Additionally, the injectability of these self-assembling hydrogels was investigated. Injectability is an important feature of drug delivery systems, since it allows avoiding surgery. Rheological analysis showed that an increasing surface charge of the microspheres led to stronger hydrogels. Relatively small microspheres (7 μm) with a narrow size distribution (99% smaller than 14 μm) gave rise to stronger hydrogels when compared to larger microspheres of 20 μm with a broad distribution (99% smaller than 50 μm). When small microspheres were combined with large microspheres of opposite charge, it was found that the strongest gels were obtained with 75% small and 25% large microspheres, instead of equal amounts (50/50) of positively and negatively charged microspheres. Computer modeling confirmed these findings and showed that the most favorable composition, related to the lowest potential energy, comprised of 75% small microspheres. Taking both charge and size effects into account, the storage moduli (G′) of the almost fully elastic hydrogels could be tailored from 400 to 30,000 Pa. Injectability tests showed that hydrogels (G′ up to 4000 Pa) composed of equal amounts of oppositely charged microspheres (−7 and +6 mV, average particle size 7 μm) could be injected through 25G needles using a static load of 15 N, an ISO accepted value. In conclusion, a variety of options to control the network properties of macroscopic hydrogels are provided, related to the charge and particle size of the composing dextran microspheres. Furthermore, it is shown that the hydrogels are injectable, making them attractive candidates for a diversity of pharmaceutical applications.
Keywords: Particle size distribution; Zeta potential; Dextran microspheres; Hydrogels; Network properties; Injectability; Computer simulations;

Transport evaluation of salicylic acid and structurally related compounds across Caco-2 cell monolayers and artificial PAMPA membranes by Maija Koljonen; Katja Rousu; Jakub Cierny; Ann Marie Kaukonen; Jouni Hirvonen (531-538).
The purpose of this study was to evaluate passive vs. proton-dependent active transport mechanisms of salicylic acid (SA) and four structurally related anions. Transport was studied across Caco-2 cell monolayers and artificial lipid membranes (PAMPA) under pH-gradient and iso-pH conditions. Kinetic permeability parameters were provided by bidirectional Caco-2 experiments and concentration-dependency measurements. The transport route and putative transporters involved in SA transport were studied using EDTA and several inhibitors. SA and lipophilic 5-chlorosalicylic acid and 2-hydroxy-1-naphthoic acid reached saturation with increasing compound concentration indicating active transport. Permeation of 5-hydroxysalicylic acid and 5-hydroxyisophthalic acid was not saturated indicating passive transport. PAMPA with pure passive diffusion underestimated the transport of SA compared to Caco-2. Opening up the paracellular tight junctions by EDTA did not increase the transport of SA under the pH-gradient conditions confirming the hypothesis of pure transcellular transport of SA. Active transport of SA remained concentration-dependent even without the pH-gradient, and was reduced by the known MCT1 and OATP-B inhibitors and structurally related anions. Overall, several permeability test protocols are needed to obtain a more complete picture of transport properties of salicylic acid and structurally related compounds.
Keywords: Caco-2; PAMPA; Transport mechanisms; Anionic compounds; pH-dependency;

Pore shape in the sodium chloride matrix of tablets after the addition of starch as a second component by Yu San Wu; Henderik W. Frijlink; Lucas J. van Vliet; Kees van der Voort Maarschalk (539-543).
The present research aims to test the hypothesis that the addition of a minor component causes a change in pore shape in the matrix of the primary component, causing a decrease in mechanical strength. Tablets made of sodium chloride only and tablets made of a mixture of sodium chloride (97.5% v/v) and starch (2.5% v/v) were compared. Tablets were subjected to a heat treatment to remove the starch. The pore structure was evaluated with mercury porosimetry and image analysis on SEM images. At comparable porosities the tensile strength of the mixture tablets was significantly lower than that of the tablets made of NaCl only. Visual inspection of the images suggested a structure with less connectivity of the grains for the heat treated mixture tablets. This was confirmed by the results of the algorithm calculating the relative path length. Image analysis showed that the pore size distribution shifted towards larger pores after the addition of starch. It was thus concluded that the lower mechanical strength of the tablets made of the binary mixture was caused by the more open pore structure and more larger pores as could be detected with image analysis.
Keywords: Pore shape; Porosity; Tablet strength; Pore structure; Binary mixture;

Development and in vitro evaluation of a controlled release formulation to produce wide dose interval morphine tablets by M.A. Holgado; A. Iruin; J. Alvarez-Fuentes; M. Fernández-Arévalo (544-549).
In this paper, a new pharmaceutical formulation for the administration of morphine has been developed. This system is based on a polymeric complex previously characterized. After the studies performed, it has been selected the following formulation: 62.5% of morphine complex, 15% of free morphine and 22.5% of Eudragit® RS. The morphine formulation proposed has been characterized by means of the study of the influence of several parameters such as pH, ionic strength, mean particle diameter of the components and total morphine dose by means of the tablet dimensions.This assayed formulation is able to provide a specific in vitro release profile that will be no influenced by possible variations in the GIT conditions. Moreover, this formulation can reproduce the same biopharmaceutical behaviour in an independent manner of the mean diameter particle of the components and the dimension of the tablet produced with several doses inside a wide interval of doses.
Keywords: Morphine; Polymeric complexes; Controlled release tablets; pH and ionic strength influences;

Sucrose ester nanodispersions: Microviscosity and viscoelastic properties by Sebastian Ullrich; Hendrik Metz; Karsten Mäder (550-555).
Sucrose esters have the potential to enhance both drug solubility and drug absorption. They are therefore alternatives to the widely used glycerides in the formulation of lipid-based drug delivery systems. A simple production of aqueous nanosized drug carrier systems consisting of amphiphilic sucrose fatty acid esters using exclusively nontoxic materials has been achieved. By only using 2 wt% of the emulsifier a high viscosity of the sample could be reached. Diverse history of fabrication led to the differences in the macroviscosity of SE dispersions with equal chemical composition.Combining the well-established oscillating rheology with the electron paramagnetic resonance technique, three orders of magnitude difference in macroviscosity between the dispersions containing 2 wt% of the amphiphilic SE were obtained, whereas the viscosities at the molecular level were all close to the viscosity of water. Viscoelastic behaviour could also be shown for these systems. TEM experiments visualized coexisting irregular micelles and lamellar structures in the SE dispersions.The results are important to understand the complex LDDS based on amphiphilic SE.
Keywords: Sucrose ester; Sugar ester; Nanodispersion; EPR; Electron paramagnetic resonance; Viscoelastic behaviour; Microviscosity; Lipid drug delivery;

The purpose of this study was to evaluate the feasibility of using a counter polymer in polyethylene oxide (PEO)/polyethylene glycol (PEG) polymeric matrices for the sustained release of a large amount of highly water-soluble drug. PEO/PEG matrix tablets (CR-A) containing four drugs with different water solubilities were prepared to investigate the effect of drug solubility on the drug-release and diffusion properties of PEO/PEG matrices. Cross-linked carboxyvinyl polymer (CVP)/PEO/PEG matrix tablets (CR-B) containing a water-soluble drug, diltiazem hydrochloride (DTZ), were also prepared, and their in vitro characteristics were compared with those of CR-A. Their in vitro drug release properties were evaluated using a dissolution test, and the polymeric erosion and drug diffusion properties of the matrices were also calculated. Drugs with higher solubility in water were released faster for the CR-A. The drug-release rate also increased with the amount of drug loaded. CR-A containing 50% DTZ (by weight) extended drug release by only 6 h. This confirms the difficulty experienced when trying to formulate PEO/PEG matrices for the sustained release of a large amount of highly water-soluble drugs due to large drug diffusion. In an attempt to control this issue, a polymer bearing a charge opposite that of the drug was used to effectively decrease the diffusion of DTZ, resulting in sustained release for 24 h or longer. These results suggested that including counter polymer in the PEO/PEG matrix tablet is a useful tool for achieving the sustained release of a large amount of highly water-soluble drug.
Keywords: Polymeric matrix tablet; Drug release; Highly water-soluble drug; Diffusion; Counter polymer;

Physicochemical properties and biocompatibility of N-trimethyl chitosan: Effect of quaternization and dimethylation by Anchalee Jintapattanakit; Shirui Mao; Thomas Kissel; Varaporn Buraphacheep Junyaprasert (563-571).
The aim of this research was to investigate the effect of degrees of quaternization (DQ) and dimethylation (DD) on physicochemical properties and cytotoxicity of N-trimethyl chitosan (TMC). TMC was synthesized by reductive methylation of chitosan in the presence of a strong base at elevated temperature and polymer characteristics were investigated. The number of methylation process and duration of reaction were demonstrated to affect the DQ and DD. An increased number of reaction steps increased DQ and decreased DD, while an extended duration of reaction increased both DQ and DD. The molecular weight of TMC was in the range of 60–550 kDa. From the Mark–Houwink equation, it was found that TMC in 2% acetic acid/0.2 M sodium acetate behaved as a spherical structure, approximating a random coil. The highest solubility was found with TMC of an intermediate DQ (40%) regardless of DD and molecular weight. The effect of DD on the physicochemical properties and cytotoxicity was obviously observed when proportion of DD to DQ was higher than 1. TMC with relatively high DD showed reduction in both solubility and mucoadhesion and hence decreased cytotoxicity. However, the influence of DD was insignificant when DQ of TMC was higher than 40% at which physicochemical properties and cytotoxicity were mainly dependent upon DQ.
Keywords: N-trimethyl chitosan; Degree of quaternization; Degree of dimethylation; Solubility; Mucoadhesive properties; Cytotoxicity;

Targeted drug delivery: Binding and uptake of plant lectins using human 5637 bladder cancer cells by Verena E. Plattner; Maria Wagner; Gerda Ratzinger; Franz Gabor; Michael Wirth (572-576).
In an effort to detect novel strategies in bladder cancer therapy, the potential and the applicability of different plant lectins was investigated using 5637 cells as a model for human urinary carcinoma. The cell–lectin interaction studies were performed with single cells as well as monolayers using flow cytometry and fluorimetry.As a result, wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) revealed strongest interaction with single cells demonstrating a high presence of N-acetyl-d-glucosamine, sialic acid and α-l-fucose residues on the membrane surface. Considering monolayers, binding of most lectins depended on the culturing period pointing to a change in the glycocalyx composition during cultivation. However, constant binding capacities combined with a high specificity were detected for WGA. Cytoinvasion studies were performed with WGA and revealed a decreased fluorescence intensity at 37 °C as compared to 4 °C, which points to internalisation of the lectin and accumulation in acidic compartments. Intracellular localization was confirmed by addition of monensin that compensates the pH-gradient between acidic compartments and cytoplasm leading to a full reversal of the decline in fluorescence.According to these findings, some lectins, especially WGA, offer promising features for targeting drugs to bladder cancer cells. This might be interesting for the development of functionalized drug delivery systems for site specific antitumor therapy leading to reduced toxicity, prolonged exposition, and improved efficacy.
Keywords: Bladder; 5637 cells; Lectins; Bioadhesion; Drug targeting;

Non-specific translocation of peptide-displaying bacteriophage particles across the gastrointestinal barrier by Maryam Hamzeh-Mivehroud; Ali Mahmoudpour; Hasan Rezazadeh; Siavoush Dastmalchi (577-581).
Phage display technology could provide a rapid means for the discovery of novel peptides and proteins from genetically engineered variants which may act as specific vehicles for drug delivery particularly through the intestinal barrier. In this work, we utilized in vivo phage display in order to study the sequences which may be responsible for transmucosal transport. We hypothesized that the introduction of a library of peptide displaying phages into the intestine may lead to the identification of sequences that could induce transport. A biopanning protocol was performed by applying a 7-mer random amino acid phage library to mice by gavage and then assessing their absorption via phage recovery from the spleen and blood. Following the isolation of 77 different phages, the sequences of the displayed peptides were identified. Statistical treatment of the obtained sequences did not support the notion that the GI translocation depends on the presence of any particular peptide sequence fused on the pIII coat proteins of the M13 phages. There are, however, some residue types underrepresented which could be due to specific GI selection mechanisms and/or their effects on the amplification rate for phages bearing those residues.
Keywords: Phage display; Peptide library; Drug delivery; M13 phage;

A new hydrophilic tablet dosage system based on an ionic self-stabilization of a carboxylated (carboxymethyl high amylose starch, CM-HAS) and an amino (Chitosan) excipient was proposed for probiotic colon delivery. CM-HAS (protonated and compacted in acidic medium) ensures gastro-protection and Chitosan (low soluble in intestinal media) prevents early release of Lactobacillus rhamnosus bacteria. Thus, in CM-HAS:Chitosan monolithic tablets, increasing percentage and molecular weight (MW) of Chitosan generated a decrease of bacteria release rate, bacteria being the most effectively retarded by the highest MW of Chitosan (2.2 × 106  g/mol). The monolithic formulations containing high percentages of CM-HAS (80%) delivered bacteria after 2 h of incubation in gastrointestinal conditions for all the Chitosan MWs used. A combined mechanism of bacteria release is proposed for CM-HAS:Chitosan monolithic tablets, involving the swelling of the tablets (due to the Chitosan), followed by the erosion and dissolution of CM-HAS. In addition, a gel-forming barrier of Chitosan in acidic conditions also contributed to the delay of the bacteria delivery. The CM-HAS dry-coated monolithic tablets changed the effect of Chitosan molecular weight on bacteria liberation and improved the percentage of delivered bacteria in simulated intestinal conditions.
Keywords: Carboxymethyl high amylose starch; Chitosan; Tablet; Lactobacillus rhamnosus probiotic; Colon delivery;

Microcrystalline cellulose, a useful alternative for sucrose as a matrix former during freeze-drying of drug nanosuspensions – A case study with itraconazole by Van Eerdenbrugh Bernard; Vercruysse Sofie; Martens Johan A; Vermant Jan; Froyen Ludo; Van Humbeeck Jan; Van den Mooter Guy; Augustijns Patrick (590-596).
Itraconazole nanosuspensions, stabilized with 10% TPGS (relative to the weight of itraconazole), were transformed into nanoparticulate powders by freeze-drying. The crystalline itraconazole nanoparticles showed peak broadening in the X-ray powder diffraction spectra and a lower melting point as inferred from differential scanning calorimetry. As it was found that freeze-drying compromised dissolution behavior, sucrose was added as a matrix, former (50,100 and 200%, relative to the weight of itraconazole). Higher amounts of sucrose unexpectedly resulted in a decrease in the dissolution rate. After thorough evaluation of the powders, it was found that whereas higher sucrose content showed a cryoprotective effect, agglomeration during the final phase of the subsequent drying step tended to increase with higher amounts of sucrose. Therefore, microcrystalline cellulose (MCC) was evaluated as an alternative matrix former. The inclusion of MCC resulted in fast dissolution that increased with increasing amounts of MCC [for powders containing 50%,100% and 200% MCC, (relative to the weight of itraconazole), the times required for 63.2% release were 10.5 ± 0.7, 6.4 ± 1.2 and 3.1 ± 0.5 min, respectively]. The dissolution profiles showed an initial phase of burst dissolution, followed by a phase of slower release. As the fraction showing burst dissolution increased with higher MCC content, the system holds promise to maintain the dissolution enhancing properties of nanoparticles in the dry form.
Keywords: Media milling; Nanosuspensions; Freeze-drying; Sucrose; Microcrystalline cellulose; Itraconazole;

Polyelectrolyte-coated nanoparticles or microparticles interact with bioactive molecules (peptides, proteins or nucleic acids) and have been proposed as delivery systems for these molecules. However, the mechanism of adsorption of polyelectrolyte onto particles remains unsolved. In this study, cationic poly(lactide-co-glycolide) (PLGA) nanoparticles were fabricated by adsorption of various concentrations of a biodegradable polysaccharide, chitosan (0–2.4 g/L), using oil-in-water emulsion and solvent evaporation techniques. The particle diameter, zeta-potential, and chitosan adsorption of chitosan-coated PLGA nanoparticles confirmed the increase of polyelectrolyte adsorption. Five adsorption isotherm models (Langmuir, Freundlich, Halsey, Henderson, and Smith) were applied to the experimental data in order to better understand the mechanism of adsorption. Both particle diameter and chitosan adsorption increased with chitosan concentration during adsorption. A good correlation was obtained between PLGA-chitosan nanoparticle size and adsorbed chitosan on the surface, suggesting that the increased particle size was primarily due to the increased chitosan adsorption. The zeta-potential of chitosan-coated PLGA nanoparticles was positive and increased with chitosan adsorbed until a maximum value (+55 mV) was reached at approximately 0.4–0.6 g/L; PLGA nanoparticles had a negative zeta-potential (−20 mV) prior to chitosan adsorption. Chitosan adsorption on PLGA nanoparticles followed a multilayer adsorption behavior, although the Langmuir monolayer equation held at low concentrations of chitosan. The underlying reasons for adsorption of chitosan on PLGA nanoparticles were thought to be the cationic nature of chitosan, high surface energy and microporous non-uniform surface of PLGA nanoparticles.
Keywords: Nanoparticle; PLGA; Cationic nanoparticles; Chitosan; Adsorption isotherm; Surface modification;

Influence of plasticizers on the stability and release of a prodrug of Δ9-tetrahydrocannabinol incorporated in poly (ethylene oxide) matrices by Sridhar Thumma; Mahmoud A. ElSohly; Shuang-Qing Zhang; Waseem Gul; Michael A. Repka (605-614).
The objective of this research was to stabilize a heat-labile novel prodrug of Δ9-tetrahydrocannabinol (THC), THC-hemiglutarate (THC-HG), in polyethylene oxide (PEO) [PolyOx® WSR N-80 (PEO N-80), MW 200,000 Daltons] polymeric matrix systems produced by hot-melt fabrication for systemic delivery of THC through the oral transmucosal route. For this purpose, the effects of processing conditions (processing temperature and heating duration), plasticizer type and concentration and storage conditions on the stability of the prodrug were investigated. The selected plasticizers studied included vitamin E succinate (VES), acetyltributyl citrate (ATBC), triethyl citrate (TEC), triacetin and polyethylene glycol 8000 (PEG 8000). Furthermore, the influence of plasticizer concentration on drug release was also studied. The stability of THC-HG in PEO matrices was influenced by all the aforementioned variables. Films processed at 110 °C for 7 min were found to be favorable for hot-melt processing with a post-processing drug content of 95%, while significant degradation of THC-HG (∼42%) was observed in those processed at 200 °C for 15 min. The degradation of the prodrug during hot-melt fabrication and also upon storage was considerably reduced in the presence of the plasticizers investigated, VES being the most effective. Modulation of the microenvironmental pH to an acidic range via incorporation of citric acid in PEO-plasticizer matrices significantly improved the stability of the prodrug, with almost 90% of the theoretical drug remaining as opposed to only 15% remaining in PEO-only matrices when stored at 40 °C for up to 3 months. The release of drug from PEO matrices was influenced both by the plasticizer type and concentration. A faster release resulted from water-soluble plasticizers, PEG 8000 and triacetin, and with increasing concentration. However, a slower release was observed with an increase in concentration of water-insoluble plasticizers, VES and ATBC.
Keywords: THC; Plasticizers; Stability; Hot-melt; Poly (ethylene oxide); Release; Microenvironmental pH; Prodrug;

Reversible lipidization of somatostatin analogues for the liver targeting by Liyun Yuan; Jeff Wang; Wei-Chiang Shen (615-620).
Tyr3-octreotide (TOC), a somatostatin analogue, is reversibly lipidized for passive delivery to the liver with the aim of increasing its association with hepatocytes. The reversibly lipidized TOC (REAL-TOC) was formed by the conjugation of the N-palmitoyl cysteinyl moiety to the cysteinyl residues of reduced TOC through disulfide linkages and characterized by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) analysis. The measured mass of REAL-TOC (M+H)+ is 1752.31 Da (calculated mass: 1752.78), confirming that two molecules of N-palmitoyl cysteines are linked to TOC via disulfide bonds. TOC and REAL-TOC were radioiodinated and administered to mice. Their biodistribution and intrahepatic distribution were subsequently investigated. The area under the curve (AUC) of 125I-REAL-TOC in the liver was 3.8-fold greater than that of 125I-TOC, with 20.5% and 5.8% of the injected dose (ID)/g of 125I-REAL-TOC remaining in the liver at 2 and 24 h post injection, respectively. Within the liver, TOC was primarily distributed to parenchymal cells (PC). Nevertheless, TOC was quickly excreted out and only 2.4% ID per 100 mg protein remained in the PC at 2 h post injection. 125I-REAL-TOC was retained in PC for up to 2 h with a constant concentration of around 6% ID/100 mg protein. 125I-REAL-TOC was also highly associated with nonparenchymal cells (NPC) at significantly higher levels than 125I-TOC at 10 min, 1 h and 2 h post injection. Since somatostatin analogues have been evaluated for treating late-stage hepatocellular carcinoma (HCC), the reversibly lipidized conjugates may possess enhanced therapeutic efficacy due to the liver-targeting effect.
Keywords: Tyr3-octreotide; Reversible lipidization; Liver targeting; Parenchymal cells; Nonparenchymal cells; Hepatocellular carcinoma;

PEGylated conjugated linoleic acid stimulation of apoptosis via a p53-mediated signaling pathway in MCF-7 breast cancer cells by Ji-Hye Seo; Hyun-Seuk Moon; In-Yong Kim; Ding-Ding Guo; Hong-Gu Lee; Yun-Jaie Choi; Chong-Su Cho (621-626).
The objective of this study was to investigate whether PEGylated conjugated linoleic acid (PCLA), as compared with conjugated linoleic acid (CLA) alone, displays anti-cancer properties in MCF-7 breast cancer cells. To generate PCLA, CLA was simply coupled to poly(ethylene glycol) (PEG) at the melting state of PEG without a solvent or a catalyst. The coupling reaction generated an ester linkage between the carboxyl group of CLA and hydroxyl one of PEG. The half-life of the generated PCLA was 52 h at pH 7.4 at 37 °C, indicating that PCLA potentially acts as a pro-drug. Apoptosis of MCF-7 breast cancer cells treated with PCLA showed a dose response to PCLA concentration during treatment. In addition, pro-apoptotic proteins such as Bax were up-regulated, whereas anti-apoptotic proteins, such as Bcl-2, were down-regulated by treatment with both CLA and PCLA. The tumor suppressor gene p53 was significantly up-regulated by treatment with increasing concentrations of PCLA, suggesting that PCLA-induced apoptosis is regulated by a p53-mediated signaling pathway. Overall, the anti-cancer effects of PCLA on MCF-7 breast cancer cells may have therapeutic significance.
Keywords: Poly (ethylene glycol) (PEG); PEGylation; Conjugated linoleic acid (CLA); Nanoparticle; Anti-cancer; Pro-drug;

Delivery of nanoparticles to the brain detected by fluorescence microscopy by Isolde Reimold; Diana Domke; Joe Bender; Christoph A. Seyfried; Hans-Eckhard Radunz; Gert Fricker (627-632).
This study aimed to explore and extend the application potential of poly(n-butylcyano-acrylate) (PBCA) nanoparticles to cross the blood–brain barrier (BBB) and to deliver their content into the central nervous system. PBCA particles were prepared by a new and efficient mini-emulsion method with excellent yield and reproducibility. These nanoparticles were loaded with 1.5% (w/v) fluorescein-isothio-cyanate-dextran (FITC-dextran), 1.5% rhodamine-123 or 7.3% doxorubicin. Particles were characterized by dynamic light scattering determining particle size, polydispersity index and ζ-potential. They were coated with 10% w/v polysorbate 80 and administered to rats. Cryosections of the brain were prepared and time-dependent distribution of fluorescence was studied. After the administration of polysorbate 80-coated particles by carotic injection, fluorescence could first be detected in capillary lumina with a progressive shift to capillary endothelial cells at 30 min and a rather evenly spread distribution across the brain tissue at 60 min after administration. 60 min after administration into the tail vein, fluorescent particles could be assigned to endothelial cells, whereas after 2 h a rather evenly spread distribution across the brain tissue was seen. These observations indicate that surface-coated PBCA nanoparticles are able to cross the blood–brain barrier and to serve as a drug-delivery system to the central nervous system.
Keywords: Poly(n-butylcyano-acrylate) (PBCA) nanoparticles; Blood–brain barrier; Drug delivery;

Solid lipid nanoparticles (SLN) were developed by using Precirol ATO 5 as the solid core of the particles for topical psoralen delivery. Nanostructured lipid carriers (NLC) consisting of Precirol and squalene, a liquid lipid, were also prepared for comparison. SLN and NLC showed respective mean particle sizes of ∼300 and 200 nm, respectively. Viscosity, polarity, and differential scanning calorimetry (DSC) studies were performed to characterize the physicochemical properties of the SLN and NLC. The viscosity of all nanoparticulate systems exhibited Newtonian behavior except the NLC with Tween 80 and soybean phospholipids as the emulsifiers (NLC-Tw). According to the DSC thermograms, the melting peak of Precirol shifted from 58 to 55 °C after incorporating squalene into the solid lipid cores (of NLC), which suggests defects in the crystalline lattice of the lipid cores and smaller particle sizes. Three psoralen derivatives for psoriasis treatments were loaded in SLN and NLC to examine their ability to permeate skin. The permeability of psoralens increased in the order of 8-methoxypsoralen (8-MOP) > 5-methoxypsoralen (5-MOP) > 4,5,8-trimethylpsoralen (TMP). Enhanced permeation and controlled release of psoralen delivery were both achieved using the NLC. The in vitro permeation results showed that NLC-Tw increased the 8-MOP flux 2.8 times over that of a conventional emulsion. Hyperproliferative or psoriasis-like skin produced by repeated strippings in the dorsal skin of nude mouse was also used as a permeation barrier. The results showed that the entrapment of 8-MOP in nanoparticulate systems could minimize the permeation differentiation between normal and hyperproliferative skin compared to the free drug in an aqueous control.
Keywords: Solid lipid nanoparticles; Nanostructured lipid carriers; Psoralen; Topical delivery; Psoriasis;

Bioavailability of amoxicillin and clavulanic acid from extended release tablets depends on intragastric tablet deposition and gastric emptying by W. Weitschies; C. Friedrich; R.S. Wedemeyer; M. Schmidtmann; O. Kosch; M. Kinzig; L. Trahms; F. Sörgel; W. Siegmund; S. Horkovics-Kovats; F. Schwarz; J. Raneburger; H. Mönnikes (641-648).
The rate and extent of amoxicillin and clavulanic acid absorption from pharmacokinetically enhanced extended release (ER) tablets is strongly influenced by the intake conditions. In order to investigate the cause of the food effects, a pharmacokinetic study with simultaneous imaging of the in vivo behaviour of the ER tablets by magnetic marker monitoring (MMM) was performed. Under fasting conditions the amoxicillin AUC (1854 ± 280 μg min ml−1) was significantly lower than after intake at the beginning of the breakfast (2452 ± 354 μg min ml−1) or after the breakfast (2605 ± 446 μg min ml−1). In contrast, clavulanic acid AUC was well comparable after tablet intake under fasting conditions and intake at the beginning of a breakfast (191 ± 46 and 189 ± 44 μg min ml−1, respectively) but significantly lower following a breakfast (126 ± 71 μg min ml−1). The localization data showed that the reduced bioavailability of amoxicillin under fasting conditions is due to early gastric emptying in combination with poor absorption from deeper parts of the small intestine. Prolonged gastric residence of clavulanic acid caused by intragastric tablet deposition in the proximal stomach was identified as the reason for the decreased bioavailability of clavulanic acid after tablet intake following the meal.
Keywords: Magnetic marker monitoring; Food effect; Absorption window; Pulsatile drug release;

The aim of this work was to develop and compare two formulations of melarsoprol (nanosuspension and hydroxypropyl-β-cyclodextrin inclusion complex). The arsenic concentrations in the organs have been assessed on a mouse model. Since this organoarsenical drug has been proposed for the treatment of cerebral trypanosomiasis and refractory leukaemias, special emphasis has been put on the bone marrow and on the brain.The organic solution of melarsoprol (Mel B, 0.039 mmol/kg), injected intravenously as control formulation, was found to concentrate significantly in the bone marrow (C max  = 1.64 mmol/g), though, not surprisingly, the brain concentration was quite high (C max  = 0. 093 mmol/g) and the LD50 was 0.12 mmol/kg.The hydroxypropyl-β-cyclodextrin inclusion complex was found to concentrate much more in the brain (C max  = 0.25 mmol/g) leading to a higher acute toxicity (i.e., lower LD50; 0.056 mmol/kg). Nevertheless, even if the encephalopathy risk has to be taken in to account, this could be considered as a positive point for the treatment of the cerebral trypanosomiasis, which is the main indication for this drug.On the contrary, the use of nanosuspensions allowed us to reduce the cerebral concentration (C max  = 0.02 μmol/g) and the acute toxicity (LD50  = 0.25 mmol/kg). Moreover, nanosuspensions, especially those prepared with polxamer 407, preserved a good in vitro antileukemic activity (IC50  = 3.34 ± 0.33 after 48 h on K562) with high bone marrow concentrations (C max  = 1.85 μmol/g). As a consequence this formulation could be proposed for the treatment of refractory leukaemias.
Keywords: Cyclodextrin; Nanosuspensions; Leukemia; Melarsoprol; Arsenic;

Encapsulation of mitoxantrone into pegylated SUVs enhances its antineoplastic efficacy by ChunLei Li; JingXia Cui; CaiXia Wang; YinGui Li; HongWu Zhang; JinXu Wang; YanHui Li; Lan Zhang; Li Zhang; WenMin Guo; YongLi Wang (657-665).
Mitoxantrone (MIT) was encapsulated into 60, 80 and 100 nm pegylated hydrogenated soy phosphatidylcholine/cholesterol (HSPC/chol) vesicles using a transmembrane (NH4)2SO4 gradient. In-vitro release studies revealed that small-sized formulation had fast drug-release rate. Acute toxicity studies performed in c57 mice proved that all pegylated liposomal MIT (plm) formulations could be well-tolerated at a dose of 9 mg/kg, significantly compared to severe toxicity induced by free mitoxantrone (f-M). In KM mice, plm60 was at least 2- to 3-fold less toxic than f-M. After intravenous injection, plm60 was slowly eliminated from plasma relative to f-M, resulting in about 6459-fold increase in AUC and its plasma kinetics exhibited dose dependence. In S-180 bearing KM mice, plm60 preferentially accumulated into tumor zone, with a ∼12-fold increase in AUC and ∼10-fold increase in C max Furthermore, the accumulation of plm60 in almost all normal tissues markedly decreased. The antitumor efficacy of plm60 was also considerably enhanced. In L1210 ascitic tumor model, plm60 was the most efficacious which led to a ∼70% long-term survival, significantly compared to 16–33% survival rate in plm80, plm100 and f-M groups at the same dose level (4 mg/kg). The antitumor efficacy of plm60 was more encouraging in L1210 liver metastasis model. At a dose of 6 mg/kg, ∼90% animals receiving plm60 treatment could survive 60 days; however, in f-M group at the same dose, all the mice died at ∼14 days post inoculation. Similarly, plm60 could effectively inhibit the growth of RM-1 tumor in BDF1 mice, resulting in marked increase in tumor doubling time at different dose levels relative to f-M. The improved antineoplastic effects could be ascribed to its small vesicle size, which allowed more drug release after the accumulation into tumor zone. Theoretical considerations revealed that the reduction of vesicle size could increase the specific area of MIT/sulfate precipitate inside the vesicle and the release constant K, which is inversely proportional to vesicle volume ( K = pA m k 2 k 2 ′ / ( [ H + ] i 2 V i ) ) .
Keywords: Mitoxantrone; Liposomes; Vesicle size; Drug release; Antitumor efficacy; Toxicity;

In vitro/in vivo characterization of melt-molded gabapentin-loaded poly(epsilon-caprolactone) implants for sustained release in animal studies by Ángel M. Carcaboso; Diego A. Chiappetta; Christian Höcht; Mariano G. Blake; Mariano M. Boccia; Carlos M. Baratti; Alejandro Sosnik (666-673).
Gabapentin (GBP) is a water soluble low molecular weight drug with anticonvulsivant and antinociceptive activity. In animal models, systemic administration regimes resembling chronic exposure to this drug (50 mg/kg, twice a day during one week), induce memory impairment. Aiming to gain further insight on the mechanisms involved in this process, a monolithic implant that releases constant plasma levels during one-week was designed. GBP-loaded poly(epsilon-caprolactone) matrices were produced by means of a simple and reproducible melt-molding/compression procedure. In vitro release studies firstly comprised uncoated implants that displayed release profiles according to a pseudo-first order model. In order to further regulate the release, two-sided coated implants where drug-free layers would perform as membranes controlling the delivery rate were prepared. A more moderated burst effect and a relatively linear (zero-order) release between days 1 and 7 were apparent. Implants were investigated in vivo and the plasma levels monitored during 10 days. Findings indicated that after a more pronounced release during day 1 and the achievement of the levels in blood comparable to a twice-a-day intraperitoneal management, relatively constant levels were attained until day 7. Overall results support the usefulness of this manufacturing method for the production of implants to attain more prolonged GBP release profiles in memory animal studies.
Keywords: Poly(epsilon-caprolactone) implants; Melt-molding/compression method; Gabapentin in vitro release; In vivo pharmacokinetics towards memory studies;

Preparation and, in vitro, preclinical and clinical studies of aceclofenac spherical agglomerates by Achutha Nayak Usha; Srinivas Mutalik; Meka Sreenivasa Reddy; Averineni Kumar Ranjith; Pralhad Kushtagi; Nayanabhirama Udupa (674-683).
Aceclofenac agglomerates were prepared by spherical crystallization technique using a three solvent system comprising acetone: dichloromethane (DCM): water (bridging liquid, good solvent and bad solvent, respectively). Hydroxypropyl methylcellulose-50 cps (HPMC) in different concentrations was used as hydrophilic polymer. The effect of speed of rotation and amount of bridging liquid on spherical agglomeration were studied. The agglomerates were subjected to various physicochemical evaluations such as practical yield, drug content, particle size, loss on drying, porosity, IR spectroscopy, differential scanning calorimetry, X-ray diffraction studies, relative crystallinity, scanning electron microscopy, micromeritic properties, solubility and dissolution studies. The agglomerates showed improved micromeritic properties as well as dissolution behaviour in comparison to conventional drug crystals. The optimized agglomerates (F-9) showed good sphericity as well as high drug release, and hence they were compressed into tablets by direct compression. The tablets were found within the limits with respect to various physicochemical parameters. The dissolution rate of prepared tablets was better than that of marketed tablet and pure drug. The optimized agglomerates and tablet formulations were found to be stable for 6 months under accelerated conditions. The in vivo studies (preclinical pharmacokinetics, pharmacodynamics and toxicity studies, and clinical pharmacokinetics) of optimized agglomerates were carried out. The results of preclinical studies revealed that the agglomerates provided improved pharmacodynamic and pharmacokinetic profiles of drug besides being nontoxic. The results of pharmacokinetic studies of optimized tablet in human subjects indicated improved pharmacokinetic parameters of drug in comparison with that of marketed tablet.
Keywords: Aceclofenac; Spherical crystallization; Analgesic; Anti-inflammatory; Pharmacokinetics; Toxicity; Tablets; Direct compression;

The aim of the study was to measure transonychial water loss (TOWL) in order to identify the extent of inter-individual, intra-individual inter-finger, inter-hand, and inter-day variabilities, and the influence of nail wetting, filing and varnishing on TOWL, with a view to determine parameters for the measurement of TOWL and its possible applications. Fingernail and toenail TOWL was measured using the condenser-chamber AquaFlux (Biox) and a specially designed Nail Adaptor supplied by Biox. A wide range of TOWL values (28–75 g/m2  h for fingernails and 26–48 g/m2  h for toenails) were found, with significant inter-individual variability. Intra-individual variability was lower; however, in the same individual, inter-finger, inter-hand/foot and inter-day variabilities were found, as well as a strong correlation between nail plate thickness and TOWL. Wetting the nails, even briefly, resulted in a significant rise in TOWL, which subsequently took much longer to return to control values. Filing the nail plate surface with a pharmaceutical file caused large increases in TOWL, whose profile (with number of filing strokes) was fairly different among individuals. As expected, nail varnish application reduced TOWL; the different extent of TOWL reduction by different varnishes suggests a potential use of TOWL measurements for product comparisons.
Keywords: Nail; Ungual; Transonychial water loss; TOWL; Topical therapy;

Correlation of in vivo and in vitro release data for rh-INFα lipid implants by M. Schwab; B. Kessler; E. Wolf; G. Jordan; S. Mohl; G. Winter (690-694).
Previous in vitro experiments had shown that rh-INFα releasing tristearin implants feature promising properties making them an excellent tool for the delivery of therapeutic proteins. Sustained release for periods up to one month could be achieved, associated with high protein stabilization. The objective of this study was to investigate for the first time the in vivo release properties of these implants in rabbits and to establish an in vivo–in vitro correlation. Computer modeling was used to simulate rh-INFα serum levels based on pharmacokinetic data. Protein serum concentrations on therapeutically relevant nearly constant levels could be detected for 9 days. Modeling revealed that in vivo release correlated closely with the release monitored in vitro.
Keywords: Protein release; Lipid implants; Computer modeling; In vivo–in vitro correlation;

by Sebastian Rudolf; Wolfgang Frieß (695).